Idea Transcript
Structure determination of small and large molecules using single crystal X-ray crystallography
A thesis submitted to The University of Manchester for the degree of Master of Science by Research in the Faculty of Engineering and Physical Sciences
2010
Richard Prendergast School of Chemistry
Structure determination of small and large molecules using single crystal X-ray crystallography List of figures
4
List of tables
9
Abstract
10
Declaration
11
Copyright statement
12
Acknowledgements
13
Part A - Small molecule X-ray crystallography Chapter 1 - A review of the single crystal method
Page
1.1
Basic Principles of X-ray crystallography
15
1.2
Diffraction of X-rays by crystals
16
1.3.1
Crystal structure and symmetry
17
1.3.2
The Bragg equation
22
1.3.3
Miller Indices
23
1.4.1
Nature, production and generation of X-rays
24
1.4.2
X-ray tube source
24
1.4.3
Synchrotron source
26
1.4.4
Detection of X-rays
27
1.5
Crystal growth
28
1.6.1
Structure determination procedure
30
The measurement of intensities
30
1.6.2.1 1.6.2.2
Preparation and mounting of the crystal
30
1.6.2.3
The collection of the X-ray intensities
32
1.6.2.4
The diffraction images data reduction process
32
1.6.3.1
The phase problem and possible solutions
33
1.6.3.2
The Patterson synthesis
34
1.6.3.3
Direct methods
35
1.6.4
36
Refining the structure
Chapter 2 - Structure determination of a small molecule – C26H36N8018Cl2Co
1
2.1
Introduction to C26H36N8018Cl2Co
38
2.2
X-ray diffraction data collection and processing procedure
38
2.3
Crystal structure analysis
40
2.4
Crystal structure implications
43
Chapter 3 - Structure determination of a small molecule – C26H36N8010F12P2Co 3.1
Introduction to C26H36N8010F12P2Co
44
3.2
X-ray diffraction data collection and processing procedure
44
3.3
Crystal structure analysis
46
3.4
Crystal structure implications
48
3.5
Comparison of the crystal structures
48
Chapter 4 - Structure determination of two small molecules – C30H24N04Sn & C30H20Sn 4.1
Introduction to C30H24N04Sn & C30H20Sn
52
4.2
X-ray diffraction data collection and processing procedure for C30H20Sn
53
4.3
X-ray diffraction data collection and processing procedure for C30H24N04Sn
55
4.4
Crystal structure analysis for C30H20Sn
56
4.5
Crystal structure analysis for C30H24N04Sn
59
4.6
Crystal structure implications of C30H20Sn
63
4.7
Crystal structure implications of C30H24N04Sn
64
Part B - Macromolecular X-ray crystallography Chapter 5 - Macromolecular X-ray crystallography 5.1
Introduction
67
5.2.1
Crystallisation techniques
68
5.2.2
The batch method
69
5.2.3
Dialysis
69
5.2.4
Vapour diffusion methods
69
5.2.5
Hot box technique
70
5.3.1
Solving the phase problem in macromolecular X-ray crystallography
70
5.3.2
Isomorphous replacement
70
5.3.3
Anomalous scattering
74
2
5.3.4
Molecular replacement
76
5.4
Rigid body and restrained refinement
77
5.5
The R free factor
78
Chapter 6 - Crystal structure determination and model refinement of a cocrystallisation of HEWL and TA6Br12 6.1
Introduction
79
6.1.2
Introduction to lysozyme
79
6.1.3
Introduction to Ta6Br12
80
6.2
Co-crystallisation procedure of HEWL and Ta6Br12
81
6.3
X-ray diffraction data collection procedure
82
6.4
The model refinement procedure
84
6.5
Refinement of the occupancies of the Ta6Br12 binding sites using SHELX
88
6.6
Analysis of the three dimensional structure
91
6.7
Implications of the three dimensional structure
98
Chapter 7 - Crystal structure determination and model refinement of a cocrystallisation of HEWL and Carboplatin 7.1.1
Introduction to carboplatin
99
7.1.2
Previous work by Casini et al
101
7.1.3
Previous work by the Helliwell group
101
7.2
Co-crystallisation procedure and optimisation of the conditions
102
7.3
X-ray diffraction data collection procedure
106
7.4
The model refinement procedure
108
7.5.1
Analysis of the three dimensional structure
111
7.5.2
Comparison with HEWL and cisplatin crystal structure
114
7.5.3
Comparison with previous HEWL and carboplatin crystal structure
116
7.5.4
Comparison with HEWL and NAG trisaccaride crystal structure
116
7.6
Implications of the three dimensional structure
117
Chapter 8 - Conclusions and future work
120
References Bibliography
121 126 Word count = 26,292 3
List of figures Chapter 1
Page
1.1
An example of a diffraction pattern.
16
1.2
An example of the lattice of a crystal.
18
1.3
An example of a unit cell with the constituent axes and angles
18
labelled.
1.4
The fourteen Bravais lattices.
20
1.5
A 21 screw axis.
22
1.6
A pictorial representation of the Bragg equation.
23
1.7
The 111 Miller plane.
24
1.8
The 010 Miller plane.
24
1.9
A schematic representation of an X-ray tube.
26
1.10
The vapour diffusion method for small molecule crystallisation.
29
1.11
The vapour diffusion method for macromolecular crystallisation.
30
1.12
A crystal mounted within a loop.
32
Chapter 2
2.1
Page
An ORTEP diagram of C26H36N8018Cl2Co with 50% ellipsoid
41
probability.
2.2
Figure to show hydrogen bonding arrangement between cobalt
42
malonate molecules to form one dimensional chains.
2.3
A figure to show the crystal packing arrangement of C26H36N8018Cl2Co.
4
43
Chapter 3
3.1
Page
An ORTEP diagram of C26H36N8O10F12P2Co with 50% ellipsoid
46
probability.
3.2
A figure to show the crystal packing arrangement of
47
C26H36N8O10F12P2Co.
3.3
A figure illustrating the common hydrogen bonding motif which is
49
present in both structures C26H36N8O18Cl2Co and C26H36N8O10F12P2Co.
3.4
A figure to illustrate the difference in the hydrogen bonding
50
arrangements around the PF6 and perchlorate counter ions.
Chapter 4
4.1
Page
The expected chemical structure of the molecule in the crystal
52
MHB7.
4.2
The expected chemical structure of the molecule in the crystal
52
MHB8.
4.3
An ORTEP diagram of C24H20Sn with 50% ellipsoid probability
57
4.4
A figure to show the location of eight weak H…C-H interactions that
58
each C24H20Sn molecule forms.
4.5
A figure to show the stacking of the C24H20Sn molecule within the
58
crystal..
4.6
A figure to show the stacking of layers of C24H20Sn molecules
59
stabilised by weak van der Waals interactions.
4.7
An ORTEP diagram of C30H24NO4Sn with 50% ellipsoid probability.
60
4.8
A figure to show the arrangement of the polymeric chains in
61
C30H24NO4Sn with weak van der Waals interactions shown as blue
5
lines. Hydrogen atoms are omitted for clarity.
4.9
A figure to show the distance between aromatic phenyl rings in
61
C30H24NO4Sn. Hydrogen atoms are omitted for clarity.
4.10
A figure to show the intramolecular hydrogen bond present within
62
the monomeric units.
4.11
A figure to show how SHELX views the molecules as discrete units
63
and not as a polymeric structure.
4.12
The crystallographically determined structure has this chemical
65
diagram with the highlighted area corresponding to the deviation from the expected chemical structure.
Chapter 5
Page
5.1
The crystal growth phases.
68
5.2
A vectorial representation of the isomorphous replacement method.
72
5.3
A Harker construction for a native protein and a single heavy atom
73
derivative.
5.4
A Harker construction for a native protein and a second heavy atom
74
derivative.
Chapter 6
Page
6.1
The structure of the Ta6Br12 cluster.
80
6.2
A picture of the Ta6Br12 and HEWL crystals.
82
6.3
An X-ray diffraction pattern image from the Ta6Br12 & HEWL data
83
collection.
6.4
A figure to illustrate the gradual reduction of the conventional R factor with each step of refinement performed.
6
88
6.5
A figure to show the evolution of the occupancies of the four binding
91
sites versus each step of refinement
6.6
A figure showing the electron density around the first Ta6Br12 to
92
lysozyme binding site.
6.7
A figure to show the distances between the tantalum atoms in the first
92
Ta6Br12 to lysozyme binding site.
6.8
A figure showing the electron density around the second Ta6Br12 to
94
lysozyme binding site.
6.9
A figure to show the distances between the tantalum atoms in the
94
second Ta6Br12 to lysozyme binding site.
6.10
A figure showing the electron density around the third Ta6Br12 to
95
lysozyme binding site.
6.11
A figure to show the distances between the tantalum atoms in the
96
third Ta6Br12 to lysozyme binding site.
6.12
A figure showing the electron density around the fourth Ta6Br12 to
97
lysozyme binding site.
6.13
A figure to show the distances between the tantalum atoms in the
98
fourth Ta6Br12 to lysozyme binding site.
Chapter 7
Page
7.1
The chemical structure of cisplatin.
99
7.2
The chemical structure of carboplatin.
99
7.3
A picture of the carboplatin and HEWL crystals.
105
7.4
A picture of an aggregate of carboplatin and HEWL crystals.
106
7.5
A picture of a carboplatin and HEWL crystal mounted onto a loop.
106
7.6
An X-ray diffraction pattern image from the carboplatin and HEWL
108
data collection.
7
7.7
A figure to illustrate the gradual reduction of the conventional R
111
factor with each step of refinement performed.
7.8
A figure to show the distances from the platinum atom to the two
112
ammonia groups in both carboplatin to lysozyme binding sites.
7.9
A figure to show the distances from the platinum atom to the nearest
113
nitrogen atom on the histidine 15 residue for both binding sites
7.10
A figure to show the electron density around the binding site present
113
on the left hand side of histidine 15.
7.11
A figure to show the electron density around the binding site present
114
on the right hand side of histidine 15.
7.12
A figure showing the superimposition of the histidine 15 residue in a
115
crystal of cisplatin and HEWL and a crystal of carboplatin and HEWL.
7.13
A figure displaying the location of the DMSO molecule present
116
within the lysozyme active site for both the cisplatin and carboplatin models.
7.14
Figure 7.13 – A figure showing the location of a NAG trisaccharide and the DMSO molecules present in the cisplatin and carboplatin models.
8
117
List of tables Table
1
Page
The essential symmetry and unit cell restrictions of the seven crystal
19
systems.
2
A summary of the X-ray diffraction and crystal data for
40
C26H36N8018Cl2Co.
3
The hydrogen bonding details for structure C26H36N8O18Cl2Co.
41
4
A summary of the X-ray diffraction and crystal data for
45
C26H36N8O10F12P2Co.
5
The hydrogen bonding details for structure C26H36N8O10F12P2Co.
46
6
A summary of the X-ray diffraction and crystal data for C24H20Sn.
53
7
A summary of the X-ray diffraction and crystal data for
55
C30H24NO4Sn.
8
A comparison of the details of the original 1970 C24H20Sn structure
64
and the structure reported in this thesis.
9
A summary of the X-ray diffraction data collection of a Ta6Br12 and
84
HEWL crystal.
10
A summary of the conditions attempted in the crystallisation of
103
HEWL in the presence of DMSO.
11
A summary of the X-ray diffraction data collection of a carboplatin and HEWL crystal.
9
107
The University of Manchester Richard Prendergast Msc. in Chemistry by Research - Structure determination of small and large molecules using single crystal X-ray crystallography 06/09/2010
Abstract Single crystal X-ray crystallography can be applied to the entire spectrum of molecular size. If performed correctly the result is an unambiguous, three dimensional image of all the atoms located within a molecule. This applies to small chemical structures all the way through to biological macromolecules. In this thesis the method is used to solve the crystal structures of four small molecules and in addition to two macromolecular adducts. The first two molecules studied were believed to be closely isomorphous cobalt containing structures. The first small molecule was found to be C26H36N8018Cl2Co and crystallised in the triclinic space group P 1 . The structure was solved with an R factor of 0.0309. The second small molecule was found to be C26H36N8O10F12P2Co and crystallised in the triclinic space group P 1 . The structure was solved with an R factor of 0.0313. The remaining two small molecules were believed to be closely isomorphous tin containing structures. The third small molecule was found to be Ph4Sn and crystallised in the tetragonal space group P 4 21/c. The structure was solved with an R factor of 0.0353. The fourth small molecule was found to be C30H24NO4Sn and crystallised in the triclinic space group P 1 . The structure was solved with an R factor of 0.0245. In addition the crystal packing of all four small molecules were analysed. The implications of the determined crystal structures are discussed in terms of the relevant literature in each case. The method was also used to determine the structure of two macromolecular adducts. The first was a co-crystallisation of hen egg white lysozyme and Ta6Br12. The model refinement and a description of the Ta6Br12 binding sites are included. The second was a co-crystallisation of hen egg white lysozyme and carboplatin with the solubility of the carboplatin optimised using DMSO, whilst still obtaining crystals. The model refinement and a description of the carboplatin binding sites are included. Finally conclusions and possible routes for future work are offered.
10
Declaration I declare that no portion of the work referred to in this thesis has been submitted in support of an application for another degree or qualification at this or any other university or other institute of learning.
11
Copyright
i. The author of this thesis (including any appendices and/or schedules to this
thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trade marks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright
works
in
the
thesis,
for
example
graphs
and
tables
(“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University
IP
Policy
(see
http://www.campus.manchester.ac.uk/medialibrary/policies/intellectualproperty.pdf), in any relevant Thesis restriction declarations deposited in the University
Library,
The
University
Library’s
http://www.manchester.ac.uk/library/aboutus/regulations)
12
regulations
(see
Acknowledgements
I would like to begin by thanking Professor John R. Helliwell for his supervision and for allowing me this opportunity. I am extremely grateful to Dr Madeline Helliwell and Dr George Habash for their help and patience regarding small molecule and protein X-ray crystallography respectively. I would also like to thank Dr Jim Raftery for his advice and for stimulating discussions regarding subjects ranging from crystallography to politics.
Finally, I would like to thank my parents. Their loving support has made this possible.
RJP Manchester 2010
13
Part A – Small molecule X-ray crystallography
14
Chapter 1 A review of the single crystal method
1.1 – Basic principles of X-ray crystallography A simple analogy to help visualise the basic principles that underpin X-ray crystallography is that of a simple optical microscope. In both microscopy and crystallography it is useful to view radiation in terms of a travelling wave of energy as opposed to a particle. In the case of the optical microscope a light source provides visible light waves which pass through the sample under study and are subsequently diffracted. Each of these diffracted waves has a characteristic intensity and phase associated with it. These intensities and phases are then recombined by a lens in order to form an image. As the name suggests X-ray crystallography utilises X-rays as opposed to visible light. They are used as they are easily accessible and possess wavelengths comparable to bond lengths allowing for visualisation down to the atomic level. However the use of X-rays poses a problem – there is no known method capable of recombining the scattered X-rays and thus forming an image. The intensity of the diffracted waves can easily be determined by using an X-ray sensitive detector or photographic plate. Unfortunately the phase information of the waves has been lost. This is the physical basis of the phase problem that is inherently present within crystallography. Instead a branch of mathematics known as Fourier series are used in place of a lens to recombine the scattered# X-rays. #
Technically the terms “scattered” and “diffracted” describe different wave-obstacle
phenomenon. Scattering is results in the wave changing direction with no form of interference produced.
15
In comparison diffraction wavelength results in a change of direction of the wave as well as the production of constructive and destructive interference. Therefore technically it is diffraction and not scattering that produces the patterns observed in crystallography.
1.2 – Diffraction of X-rays by crystals In theory a single molecule could be irradiated in order to produce a diffraction pattern. However in practice this would lead to an immeasurably weak pattern and rapid degradation of the molecule by the X-rays. Crystals are highly ordered structures which are composed of a regular arrangement of units (these units could be atoms, molecules or ions) that is repeated infinitely in three dimensions. Therefore instead of having one unit in a particular orientation there are now in effect an infinite number – this leads to “reinforcement” of the diffraction pattern and hence an averaged data set. In addition due to the huge amount of identical units radiation damage is usually negligible.
Figure 1.1 – An example of a diffraction pattern. The particular position and symmetry of the spots is illustrated in addition to the varying intensities of the spots.
To create a diffraction pattern a crystal is bathed in a beam of X-rays. The regular arrangement of the atoms present in the crystal acts as a three dimensional diffraction 16
grating .The incident X-rays interact with the electrons of the crystal via inelastic collisions which causes diffraction. The result is a pattern consisting of spots which possesses three important properties directly related to the crystal under study (Figure 1.1). The position, symmetry and intensity of the spots all hold information that must be extracted. However, one diffraction pattern is not sufficient to allow for structure determination. This is because that only a small number of reflections will be excited at the particular angle of the stationary crystal. As a result the crystal must be slowly rotated (through small increments) whilst still fully immersed within the X-ray beam. In modern day diffractometers this a a fully automated, computer controlled process which results in the maximum number of reflections being recorded The X-rays most commonly used in “home” laboratory based experiments are monochromated MoKα (λ = 0.71Ǻ) and CuKα (λ = 1.54 Ǻ). These particular wavelengths are favoured as they are comparable with the distances under study. (E.g. C-C = 1.54 Ǻ). This helps to ensure appreciable diffraction occurs.
1.3.1 - Crystal structure and symmetry
As a consequence of their highly ordered structure, crystals also display a high degree of symmetry. This symmetry is described by a number of different concepts which are subsequently defined. As previously mentioned crystals are composed of a regular, repeating arrangement of units. If each of the constituent units was represented by a single point then the resulting array would be representative of the repeating nature of the crystal. This array of points (related to each other by translational symmetry) is known as the lattice of the crystal (Figure 1.2).
17
Figure 1.2 – A demonstration of the crystal lattice, created by representing the constituent units with points
An extension upon the theme of lattice points is the unit cell. A unit cell is a parallelogram consisting of four lattice points. Crystals are defined by their unit cells – they describe the simplest “building block” that is repeated in three dimensions to produce the bulk crystal. A unit cell is characterised by three vectors a, b and c which lie along the x, y and z directions respectively. Also of importance are the angles between these vectors – alpha, beta and gamma. Convention dictates that alpha is the angle between vectors b and c, beta is the angle between vectors a and c whilst gamma is the angle between a and b (Figure 1.3).
Figure 1.3 – An example of a unit cell with the axes and angles labelled.
These vectors and the angles between them give rise to the seven crystal systems which are used to describe the geometry of the unit cell. Rotational and reflection symmetry place restrictions of the allowed vector lengths and angles. These restrictions allow for 18
classification into seven groups – triclinic, monoclinic, orthorhombic, tetragonal, trigonal, hexagonal and cubic. The aforementioned restrictions are given in Table 1.
Crystal system
Unit cell restrictions
Essential symmetry of crystal
Triclinic
None
None
Monoclinic
One diad axis (2 fold rotation)
a≠b≠c
or mirror plane (inverse diad
β ≠ α = γ = 90°
axis) Orthorhombic
Tetragonal
Trigonal
Hexagonal
Cubic
Three orthogonal diad axes or
a≠b≠c
inverse diad axes
α = β = γ = 90°
One tetra axis (four fold
a=b=c
rotation) or inverse tetrad axis
α = β = γ = 90°
One triad (three fold rotation)
a=b=c
axis or inverse triad axis
α = β = γ ≠ 90°
One hexad (five fold rotation)
a=b≠c
axis or inverse hexad axis
α = β = 90°, γ = 120°
Four triad axes or inverse triad
a=b=c
axes
α = β = γ = 90°
Table 1 – The essential crystal symmetry and unit cell restrictions of the seven crystal systems.
Introducing translational symmetry into the seven crystal systems (which only include rotational and reflection symmetry) forms the Bravais (or space) lattices. There are 14 possible Bravais lattices which involve four different ways of centring the lattice points (Figure 1.4). The possible lattice centrings are – -
Primitive (P) – Lattice points are located at the corners of the unit cell.
-
Body Centred (I) – All primitive points included plus an additional point at the centre of the unit cell.
-
Face Centred (F) – All primitive points included plus additional points at the centre of each face of the unit cell.
19
-
Centred (C) – All primitive points included plus an additional point at the centre of one face of the unit cell.
Figure 1.4 – The fourteen Bravais lattices with the lattice points displayed1.
A point group is a mathematical descriptor for a group of symmetry operations that pass through a central point. These symmetry operations must leave at least one point unchanged and the appearance of the object unaltered. There are four symmetry operations associated with point groups -
n-fold rotation axes – a rotation through (360°/n) which leaves the object unaltered (where n is an integer).
-
Mirror planes – involves a reflection which takes place with respect to a mirror plane.
-
Inversions – involves moving every point x,y,z to –x,-y,-z.
-
Improper rotations - a rotation followed by an inversion.
Compared to the point groups for an isolated object (such as a single molecule) there are 230 possible crystallographic space groups. This is because there are 32 possible ways to combine the point group symmetry operations with the translational symmetry inherently present within crystals (crystallographic restriction theorem). For point groups the Schoenflies notation is most commonly used. For example the molecule SF6 belongs to
20
the octahedral point group (in Schoenflies notation represented by Oh) which contains 31 associated symmetry elements2.
The specific method of describing the symmetry present in a crystal is that of space groups. Space groups describe the symmetry operations present in an infinitely repeating three dimensional pattern (crystals are as an approximation to infinite repeating structures). Therefore each space group is a combination of the point group symmetry operations with translational (or space) symmetry operations. There are 230 space groups which completely describe all possible combinations of the aforementioned symmetry operations.
Typically the internationally recognised Hermann and Magiun notation is used. A typical example is P 21/m –Where P is the type of Bravais lattice – Primitive in this example. The letters following the P represent the symmetry operations which lie along a special direction in the crystal. In this example 21 represents a 21 screw axis in the direction of the unique axis of the monoclinic crystal system. The ‘/m’ represents an ordinary reflection plane which is perpendicular to the unique 21 axis. The space groups and their associated symmetry operations are systematically detailed in the International Tables for Crystallography3.
In addition to the symmetry operations possessed by point groups there are two space symmetry operations which may be contained within space groups. These operations are termed glide planes and screw axes. A screw axis is a combination of a rotation of (360/n) followed by an appropriate translation parallel to the axis of rotation to preserve the translational repetition (where n is an integer). For example a 21 screw axis consists of a twofold rotation axis (360°/2) followed by a translation along half of the lattice axis that is parallel to the rotation (Figure 1.5).
21
Figure 1.5 – The effect a 21 screw axis has upon a particular point4. A glide plane is a combination of a reflection in a mirror plane followed by a translation. There are five possible glide planes – denoted a, b, c, n and d. For example a c glide plane consists of a reflection in the xy plane followed by a translation along half of the c axis. Screw axes and glide planes can cause the systematic absence of certain reflections in a diffraction pattern. These systematic absences can help in the assignment of space groups as the absences are well known and are listed in the International Tables for Crystallography3 (although space group ambiguities do exist).
1.3.2 - The Bragg equation In 1913 W. L. Bragg derived his now eponymous equation5 following on from work conducted by Freidrich, Knipping and Laue. This work proved a proposal by Laue which was stimulated by Ewald that crystals were capable of diffracting X-rays. The Bragg equation is still used today to mathematically explain the diffraction geometry of X-rays by crystals. The equation treats crystals as being composed of a series of parallel planes of atoms separated by a small distance. The planes are assumed to be capable of reflecting the Xrays in a manner which results in the angle of incidence equalling the angle of reflection (Figure 1.6). The contributions from successive planes will be in phase (i.e. the difference in path length between successive waves must be an integer number of wavelengths) only for certain angles. As a result constructive interference and the production of diffraction maxima can only occur if the Bragg equation (Equation 1) is satisfied.
22
Where – n=
An integer
λ=
Wavelength of the radiation (m)
d=
Interplanar spacing (m)
sin θ = Angle of incidence of radiation
Equation 1 – The Bragg equation
If the waves are out of phase destructive inference will occur. Indeed since most of a diffraction pattern consists of empty space this the common situation. The absence of diffraction spots can provide as much information as their presence. For example space groups can be assigned on the basis of systematically absent reflections . Laue also derived a set of three equations that describe the same effect but these are less widely used6.
Figure 1.6 – A pictorial depiction of the relationships that constitute the Bragg equation7.
1.3.3 - Miller Indices Named after W. H. Miller these indices are an unambiguous way of defining crystal planes. They consist of three numbers (hkl) which correspond to the inverse of the ratio
23
of the intercepts on the a, b and c axes of the unit cell (for examples see Figures 1.7 & 1.8). Z
Y
X
Figure 1.7 – A pictorial representation of the 111 Miller plane. Z
Y X
Figure 1.8 – A pictorial representation of the 010 Miller plane.
1.4.1 - Nature, production and detection of X-rays
X-rays are a form of electromagnetic radiation which possess wavelengths within the range of 0.01nm (0.1 Ǻ) to 10nm (100 Ǻ) with wavelengths in the range 0.2 – 3 Ǻ being useful in crystallography. As such they consist of an electric field and magnetic field vector which are perpendicular to each other. These vectors oscillate in a sinusoidal manner perpendicular to the direction of propagation. X-rays are the favoured form of radiation in crystallography as they possess wavelengths comparable to bond lengths and can also be easily generated in a “home” laboratory setting. A more recent method of generating much more intense and finely tuneable Xrays using a synchrotron source is also now widely used.
1.4.2 - X-ray tubes – For X-ray generation in the “home” laboratory 24
The X-ray tubes used in modern day crystallography are known as filament (or Coolidge) tubes and date back to 1913. They consist of an evacuated glass enclosure which contains a tungsten filament and a disk of a target metal (Figure 1.9). The target metal is responsible for the production of the characteristic wavelength of the X-rays. The most commonly used target metals are Molybdenum (wavelength = 0.71 Ǻ), Copper (1.54 Ǻ) and Silver (0.56 Ǻ). To initiate the production of X-rays the tungsten filament is heated by passing an electric current through it. This results in the production of electrons which are accelerated by a potential difference and directed towards the target metal. If the potential difference is sufficiently high (typically 40kV) the electrons will possess enough energy to cause ionisation of inner core electron`s of the target metal. To compensate an electron in a higher atomic energy level for the metal will drop in energy to take the place of the ejected electron. This results in the emission of a photon with a characteristic wavelength. The characteristic wavelength produced is dependent upon the metal atom energy levels from which each electron is ejected. For example MoKα emission corresponds to electrons moving between the L and M shells (λ = 0.71Ǻ) of Molybdenum whilst MoKβ emission corresponds to a movement between the L and K shells (λ = 0.63λ). This characteristic wavelength created is defined by Equation 2. Where – h = Plancks constant (6.6261 x 10-34 J s) c = Speed of light (2.9989 x 108 m/s) E1 = Lower energy level of target atom E2 = Higher energy level of target atom
Equation 2 – The equation for the characteristic wavelength generated from a particular target material.
The spectra purity of the X-ray beam onto the crystal is created by using filters to remove background and other unwanted wavelengths whilst a beryllium window allows the Xrays to leave the tube head with minimum absorption. 25
This method of X-ray production can be considered quite inefficient as the vast majority of the energy carried by the electrons is converted into heat rather than X-rays (literature sources mention 1% X-ray conversion8). The heating of the target is largely compensated by a water cooling system which prevents melting of the target material up to certain current limits. An additional disadvantage of this method is that the X-rays generated are quite divergent which may pose a problem if small crystals are under study.
Figure 1.9 – A schematic diagram of an X-ray tube9.
An improved method of generating X-rays in the home laboratory is known as a rotating anode. In this apparatus the target metal is cylindrical and is spun about its axis. This allows the energy of the X-rays to be spread out over a larger overall area thereby reducing the heating problem. As a result much higher electrical currents can be introduced which creates a much higher flux density. This method of X-ray generation is important especially for molecules which possess large unit cells such as proteins which are often in large complexes.
1.4.3 - Synchrotron source X-ray generation Synchrotrons were initially developed as a tool in particle physics to accelerate beta particles (electrons and positrons). It is observed that when such particles are accelerated through magnetic fields at relativistic speeds they lose energy in the form of electromagnetic radiation (this radiation covers the entire EM spectrum not just X-rays).
26
When the beta particles pass through the magnetic fields they change direction. This causes the tangential emission of radiation. Although emission of radiation occurs at non relativistic speeds, a feature of relativity known as the Lorentz transformation means that the radiation is emitted in a highly collimated fashion at speeds approaching that of the speed of light. This emission of radiation was first observed in 1946 at a 70MeV synchrotron in Schenectady by F. R. Elder et al10. Today many synchrotron sources are now operational as nationally and internationally shared facilities. Synchrotrons consist of a linear accelerator (LINAC) which creates high energy electrons (around 10MeV). These electrons are subsequently injected into a small accelerator (known as a booster synchrotron) which increases the energy of the electrons to around 500MeV. Once this point has been reached the electrons are injected into the main synchrotron ring where the energy is further increased via multiple passes through radio frequency cavities. This produces X-rays which extends to the necessary short wavelengths and are much more intense and well collimated than laboratory based sources. This allows for extremely fast data collection times and smaller crystals to be studied. The continuous spectrum allows for the fine tuning of the selected wavelengths using monochromators. Alternatively the whole ‘white’ X-ray spectrum may be used in Laue diffraction experiments.
1.4.4 - Detection of X-rays In the beginnings of X-ray crystallography intensities were often measured by using photographic films coated in silver halide. Exposure to X-rays causes silver halide to darken. The darkness of the spots is related to the intensity of the absorbed radiation in a given reflection (spot). A vast improvement was the appearance of computer controlled diffractometers in the 1960`s. These routinely utilised an X-ray sensitive electronic device known as a scintillation counter. A scintillation counter consists of a crystal mounted onto a photomultiplier tube. A commonly used crystal is sodium iodide doped with a small amount of Thallium (around 1%)11. These crystals produce light when irradiated by Xrays. This light can then enter the photomultiplier which results in the ejection of electrons and thus the generation of an electric current. This process results in the 27
production of an electric pulse for each individual X-ray allowing for the measurement of intensities. There are disadvantages associated with scintillation counters. Foremost is that the diffracted beams are measured one at a time which often translates to long data collection times. A more recently developed method of X-ray detection is known as a charge coupled device (CCD). These detectors have the advantage of being able to record a number of diffracted beams at the simultaneously, thereby reducing data collection times. A CCD detector employs a semiconductor in which the incident X-rays induce the production of free electrons and electron holes12. The electrons produced are trapped in potential wells, and in addition to the electron holes, are read out as a current. The magnitude of this current is proportional to the intensity of the diffracted beams .The various designs of CCD detectors can be roughly divided into two groups depending on how the intensity of the radiation is detected. This may be done by either measuring the intensity of the Xrays directly or by conversion of the X-rays to visible light using a phosphor conversion mechanism13. Diffractometers that utilise a CCD detector are often known as three circle diffractometers. This is because they possess three rotation axes (one in relation to the detector and two in relation to the crystal). Scintillation counter based diffractometers possess four rotation axes as the detector is smaller and can only record reflections which occur in the horizontal plane. As a result an additional crystal rotation axes is required.
1.5 - Crystal growth
Crystals are formed as a result of chemical systems seeking to minimise the Gibbs free energy. On the one hand the formation of crystals results in an unfavourable loss of entropy. This arises because the individual molecules which constitute the crystal are in effect “locked” in place. As a result they lose rotational and translational degrees of freedom. Conversely this is coupled with a favourable increase in the enthalpy of a system. This increase arises because the crystallisation process involves the formation of many new, stable non covalent chemical bonds. This increase in enthalpy more than
28
counterbalances the unfavourable decrease in entropy and overall favourably decreases the Gibbs free energy. Small molecule and macromolecule crystal growth utilise different apparatus, although common pricincples. The initial aim is to tailor the experimental conditions so that the solution is just saturated. At this moment the saturation point should be very slowly lowered whilst the rate of nucleation is limited. In theory this should yield well formed and decently sized crystals possessing a high degree of regularity. In practice however obtaining crystals of a suitable size and quality is often a major rate limiting step in the structure determination process. The crystallisation process in a given case is often poorly understood whilst the high number of variables involved (e.g. temperature, pH, concentrations) further complicates the process. However there are exceptions, for example the crystal growth of silicon is exceedingly well understood. Techniques for inducing the crystallisation of small molecules are often much simpler than those used in macromolecular crystallisation. Commonly used techniques to induce small molecule crystallisation are the slow evaporation of a solution, slow precipitation by vapour diffusion and sublimation. Macromolecular crystallisation techniques are discussed in Chapter 5 although both areas often use vapour diffusion (although the apparatus differs slightly as illustrated by Figures 1.10 & 1.11).
Figure 1.10 – The vapour diffusion method for small molecule crystallisation.
29
Figure 1.11 – The hanging drop vapour diffusion method for macromolecular crystallisation.
1.6.1 - Structure determination procedure
Once crystals of a suitable size have been grown the crystal structure determination procedure can begin. This procedure can be thought of as being divided into three main stages – -
The first stage involves the measurement of the intensities of the Bragg reflections and the application of corrections to take into account various geometrical and physical phenomena.
-
The second stage involves using mathematical and computer program methods to imitate the behaviour of a microscope lens to solve the phase problem.
-
The final stage involves refining the initial structure so that there is an optimum agreement between the observed and calculated structure factors.
The steps involved in each of these stages are further explained below.
1.6.2.1 - Stage 1 – Measurement of X-ray intensities 1.6.2.2 - Step 1 - The first step towards measuring X-ray intensities is the selection and preparation of a suitable single crystal. For use in home laboratory experiments single crystals in the order of 0.2-0.4mm are routinely required. This is because the X-ray beam generated is relatively weak in 30
intensity (compared to a synchrotron source) and the diameter is less than 1mm; using such a small size ensures that the crystal is fully immersed in the X-ray beam. It is important to inspect crystals beforehand using a microscope to ensure that no visible defects such as cracks or twinning are apparent. In addition crossed polarisers can be used to ensure that the crystals extinguish. This can help to reveal defects within the crystal that were previously not apparent. However should not be considered a conclusive test as some crystals (depending upon their symmetry) do not extinguish. Once a crystal of a suitable size and quality has been selected it can be mounted onto a small loop or mesh (Figure 1.12). The crystal is held in place by using a small amount of amorphous glue. If the data is to be collected at a cryogenic temperature then a viscous oil can be used. The oil will freeze in the cryogenic stream thereby again fixing the crystal in place. Data collection at cryogenic temperatures is advantageous as it reduces the rate of radiation damage caused by the incident X-rays. In addition cryogenic temperatures reduce atomic mobility which in turn enhances the diffraction spot intensities (as this minimises disorder). In special cases such as if the sample is air sensitive the crystal can be contained within a thin walled glass capillary. The glass has an amorphous structure and hence does not appreciably contribute to the diffraction pattern. Finally, the loop, mesh or capillary containing the crystal is mounted onto a goniometer head and placed onto the diffractometer. The goniometer head is a device that allows the crystal to be easily centred in the X-ray beam. Additionally in modern day diffractometers a high magnification video camera is used to ensure that the crystal is in the correct position and to record a digital picture of the crystal for size determination.
31
Figure 1.12 – A crystal mounted within a loop held in place with a viscous oil. Pictured via a high magnification video camera present on the diffractometer.
1.6.2.3 - Step 2 – The collection of the X-ray intensities Once the crystal has been correctly centred with respective to the X-ray beam irradiation can begin. This irradiation will produce a diffraction pattern that is commonly recorded by a CCD detector. The CCD diffraction images collected then need to be integrated to produce a list of reflections i.e. spots (hkl values) each with an associated intensity. It is possible to determine the unit cell dimensions from the first few images. Other factors such as the quality of the crystal (the mosaic spread and/or splitting) are also obvious from the first few images obtained.
1.6.2.4 - Step 3 – The diffraction images data reduction process This step includes the application of corrections to the measured intensities which take into account various geometrical and physical phenomena. A common geometrical correction applied is known as the Lorentz-polarisation factor. The Lorentz factor is related to the amount of time the reflection is in a diffraction position and is instrument dependent. The polarisation factor is required because the reflected X-rays are partially polarised. A commonly applied physical correction concerns the absorption of X-rays by crystals (this is particularly true for inorganic crystals). Absorption corrections are needed for crystals that are not approximately spherical and are calculated by analysing systematic 32
variations in the intensities of symmetry related reflections. This is because the amount of absorption is dependent upon the path length the X-rays travel through the crystal. Absorption of X-rays also increases the larger the crystal is; using a crystal as small as possible helps to minimise this error. Finally absorption varies with elemental composition; often heavy atoms strongly absorb X-rays.
The data reduction process also involves the merging of symmetry related reflections and the calculation and application of scale factors to the measured reflections. The result is a unique, scaled data set. The data reduction process is a ‘black box’ method that is performed by computers.
1.6.3.1 - Stage 2 – The crystallographic phase problem and possible solutions The phase problem is intrinsic to X-ray crystallography. Each of the diffracted X-rays will have a particular phase and amplitude associated with it. X-ray sensitive detection methods such as photographic film or CCD detectors are able to measure intensities from which the amplitudes are easily obtained (intensities = ampltiude2). However the relative phases of the waves are lost during the experiment. This is a problem because in order to elucidate the crystal structure both the intensities and relative phases are required. Therefore a method of obtaining approximate phases and hence solving the phase problem is required. In small molecule crystallography two methods are almost exclusively used which both utilise a branch of mathematics known as Fourier series.
Fourier series arise from Fourier’s theorem which states that any periodic function can be represented by a summation of sine and cosine terms. The diffraction pattern and the electron density of a crystal are related by a Fourier series. In addition a diffraction pattern consists of well defined individual spots. Therefore a summation must be used as opposed to integration which would be performed if the pattern was diffuse.
Crystals can be described by a Fourier series as the structure of a crystal is a periodically repeating, (effectively) infinite array. 33
The structure factor equation is used to describe how the incident X-rays are diffracted by the constituent atoms of a crystal. This equation takes into account the scattering power of each atom (which is described by fj which is the scattering factor for the jth atom) and is dependent upon electron density. This is described by Equation 3. Where – N=
The number of atoms
within the structure fj =
Atomic scattering factor
for the jth atom
Equation 3 – The structure factor equation.
The electron density calculation must be performed in three dimensions in order for a three dimensional structure to be produced. The unit cell volume (V) must also be taken into account. The equation used to calculate the electron density at a particular point (xyz) is given by Equation 4.
Equation 4 – The equation used to calculate the electron density at point ρ(xyz). The two commonly used methods used to solve the phase problem in small molecule crystallography are described below.
1.6.3.2 – The Patterson Synthesis This method is commonly employed when there is one or a small number of heavy atoms present in the structure. In 1934 A. L. Patterson presented a synthesis (or Patterson map) that is obtained by performing a Fourier series on the square of the amplitudes with all waves taken in 34
phase14. If there are an N number of atoms in a unit cell then there is a N2 number of vectors running between these atoms. Therefore a Patterson map shows where atoms are located relative to each other but not where they are located with respective to the unit cell origin. The result is a map that has an appearance similar to that of an electron density map in that it contains peaks of positive density located in particular positions. However this is not a map of electron density, instead it is a map of the vectors between pairs of atoms in the structure. The Patterson synthesis is described mathematically by Equation 5.
Where – V=
The unit cell volume
(in Ȧ ). 3
Equation 5 – The mathematical representation of the Patterson synthesis.
1.6.3.3 – Direct Methods This method was developed for equal atom structures i.e. those that contain no heavy atoms. Direct methods also use the measured intensities but takes advantage of the fact that electron density within a crystal can not be negative. This places restrictions on the possible phase angles between reflections. The process is almost a trial and error approach – the reflections which contribute most to the Fourier transform are selected as are approximations that appear promising (assessed by a numerical factor). The Fourier series are calculated using the measured intensities and these approximate phases. Sensible looking chemical fragments can be used to assess the different trial structures. The resulting trial structure is only an initial approximation of the true structure and must undergo further refinement. Direct methods are often described as black box as the process is automated and performed by computers.
35
Methods used to solve the phase problem in macromolecular crystallography differ and are detailed in Chapter 5.
1.6.4 - Stage 3 – Refining the structure The final stage involves refining the initial structure so that there is a optimum agreement between the observed structure factor amplitudes and the structure factor amplitudes calculated for the current structure. The measure by which these factors agree is described by the conventional residual factor (commonly known as the R factor). The R factor is defined by Equation 6.
Where – yO =
Observed structure factor amplitude
yC=
Calculated structure factor amplitude from model
Equation 6 – The conventional residual factor.
As illustrated by Equation 6 the lower the R factor the better the agreement and the more correct the structure is. That the earlier stages of the structure determination procedure were performed correctly is essential if a low R factor is to be obtained. Refinement uses a mathematical technique known as least squares analysis which adjusts parameters such as atom positions and atomic displacement parameters in order to produce the maximum agreement between two sets of data (in this case the observed and calculated amplitudes). The refinement on F2 was used in this thesis and is defined by Equation 7. Where – FO =
Observed structure factor amplitude
FC=
Calculated structure factor amplitude from model
36
Equation 7 – The least squares refinement of the square of the structure factor amplitudes.
Several cycles of refinement are required as the data used is calculated using Fourier series which are non linear equations. Consequently cycles of refinement are required until the adjustments of the parameters are insignificant (a process known as convergence). The refinement parameters can be split into two groups based on the mathematical detail used to describe the atoms. Isotropic refinement uses three positional coordinates (x,y,z) as well as a single vibrational parameter to approximate vibrating atoms as spheres. Anisotropic refinement uses the same three positional coordinates as well as six vibrational parameters to describe atoms in terms of ellipsoids. Although a perfect ellipsoid can be described by three vibrational parameters atoms typically possess distorted ellipsoids which are described by require six vibrational parameters .This results in a significantly more accurate and realistic model structure. In addition, once anisotropic refinement has been carried out small peaks can often be observed, corresponding to hydrogen atom positions. The process of refinement is complete when convergence is achieved and the electron density map contains no undefined peaks or holes. For small molecule X-ray crystallography a final R factor in the range 0.02 – 0.07 is an indicator of a good quality structure.
37
Chapter 2 Structure determination of a small molecule – C26H36N8018Cl2Co 2.1 - Introduction to C26H36N8O18Cl2Co This complex was synthesised by the group of Professor Subrata Mukhopadhyay of the University of Jadavpur, India for study into the field of crystal engineering. The field of crystal engineering seeks to utilise intermolecular interactions to aid molecular recognition through the identification and control of recognition motifs. The field is still relatively new and its full potential has yet to be realised. This is because there are problems present which are poorly understood, for example weak interactions can prove especially unpredictable and therefore difficult to control. However it is hoped that molecular recognition may prove useful in the design and synthesis of future functional materials. In order for molecular recognition to be successfully implemented a full appreciation of the intermolecular interactions present is required. This can be achieved using single crystal X-ray crystallography to provide a complete unambiguous three dimensional structure. The probable composition of the complex was determined by the synthetic chemists as [Co(mal)2(H2O)2](ClO4)2(LH)4 where mal indicates malonate and LH4 indicates protonated 2-amino pyridine.
2.2 – X-ray diffraction data collection and processing procedure
The crystals provided were approximately 1-2 mm in length and pink in colour. The crystals appeared to be of good quality and extinguished well under crossed polarisers. As a result a single crystal was cut using a razor blade to around 0.5mm in length. The crystal was then immersed in a viscous oil and fitted onto a loop, which was placed onto a goniometer head and fitted onto the diffractometer. The data collection was performed 38
at a cryogenic temperature (100K), which caused freezing of the viscous oil and thus fixation of the crystal. This is in addition to being beneficial by reducing atomic displacement parameters. The crystal was centred using rotating screws on the goniometer head and a video camera as a visual aide. Firstly the X-axis was adjusted at phi 0˚ to ensure the crystal was centred. Once complete the screw was rotated to phi 180˚ and the crystal centred again. This process was repeated in the same manner for the Yaxis but with phi angles 90˚ and 270˚. Once the crystal was completely centred it was irradiated with monochromated MoKα radiation. The X-ray generator settings were 40kV and 40 mA. The computer program SAINT15 was used to collect and integrate the CCD frame images in order to produce integrated intensities. This produced files with filename extensions .p4p and .RAW. These files were then introduced into SHELX16 XPREP for determination of the crystal system and space group. SHELX SADABS was used to produce an X-ray absorption corrected data set which took into account absorption of Xrays by the crystal, although this was a symmetrically sized crystal (crystal dimensions listed in Table 2).
It was found that SHELX XS was unable to satisfactorily solve the structure using both direct methods or a Patterson synthesis. This sort of unexplained failure of SHELX can occur. As a result the direct methods program SIR 200417 was used to solve the structure. Further refinement was carried out within the SHELX XSHELL program. All non hydrogen atom positions were refined anisotropically. The hydrogen atom positions were clearly visible using difference Fourier methods and were refined isotropically.
Empirical formula
C26H36N8018Cl2Co
Chemical formula weight
878.46 g mol-1
Crystal system
Triclinic
Space group
P1
39
Unit cell dimensions
a = 7.1122(7) Ǻ
α = 86.908(2)°
b = 11.2696(10) Ǻ
β = 84.168(2)°
c = 11.7951(11) Ǻ
γ = 72.440(2)°
Unit cell volume
896.41(15) Ǻ3
Z
1
Data collection temp
100 K
Radiation
MoKα, graphite monochromator
Diffractometer
Bruker AXS Apex (3 circle)
Detector
CCD area detector
Crystal size
0.5 x 0.4 x 0.4 mm
Tmin & Tmax
0.1756 & 0.8698
F (000)
453
Theta range
2.54 to 26.38°
Total reflections measured
5183
Independent reflections
3548 (Rint = 0.0242)
R indices [F2 > 2σ(F2)]
0.0309
R indices (F2)
0.0363
Largest diff. peak and hole
0.423 and -0.374 e.Å-3
Number of refined parameters
322
Table 2 – A summary of the X-ray diffraction and crystal data for C26H36N8O18Cl2Co.
2.3 - Crystal structure analysis
It was found that the small molecule C26H36N8O18Cl2Co crystallises in the triclinic space group P
1
. The asymmetric unit contains half of the cobalt malonate anion
[Co(C3H2O4)2(H2O)2]- in addition to two protonated 2-amino pyridine cations (C5H7N2+) and a perchlorate counter ion. The cobalt atom is located on an inversion centre bonded to two oxygens on each of the equatorial malonates, as well as to a single oxygen on each of the trans, axial, waters (Figure 2.1).
40
Figure 2.1 – An ORTEP diagram of C26H36N8018Cl2Co with 50% ellipsoid probability. Atoms labelled b are symmetry generated. The equatorial Co-O bonds are 2.0335(12) Ǻ and 2.0540(12) Ǻ respectively whilst the axial Co-O bonds are 2.1257(14) Ǻ. This gives a distorted octahedral environment with malonate and water present as the primary ligands. The secondary ligands are 2-amino pyridine and perchlorate. This environment in the crystal produces an extensive hydrogen bonding network comprising nine unique hydrogen bonds, four of which involve the perchlorate counter ion (Table 3).
D-H...A
D-H
H...A
D...A
D-H...A (°)
O(5)-H(1O5)...O(2)#2
0.90(3)
1.78(3)
2.6867(19)
178(3)
N(4)-H(1N4)...O(4)
#3
0.85(2)
1.93(2)
2.768(2)
169(2)
N(1)-H(1N1)...O(2)
#4
0.87(2)
2.10(2)
2.952(2)
169.9(19)
N(3)-H(2N3)...O(3)
#3
0.89(3)
1.93(3)
2.818(2)
177(2)
N(2)-H(1N2)...O(1)
#4
0.87(2)
1.93(2)
2.788(2)
169(2)
N(1)-H(2N1)...O(7)
#5
0.88(3)
2.16(3)
3.001(2)
160(2)
O(5)-H(2O5)...O(6)
0.79(3)
2.05(3)
2.839(2)
177(3)
O(5)-H(2O5)...Cl(1)
0.79(3)
2.93(3)
3.6677(16)
155(2)
0.85(2)
2.18(2)
3.024(2)
171(2)
N(3)-H(1N3)...O(8)
#6
Table 3 – The hydrogen bonding details for structure C26H36N8018Cl2Co. 41
Symmetry transformations used to generate equivalent atoms: #1 -x+2,-y+1,-z+2 #2 x+1,y,z #3 x-1,y,z #4 -x+1,-y+1,-z+2 #5 x,y-1,z #6 -x+1,-y+1,-z+1
The cobalt malonate anions are linked by a hydrogen bond of length 1.78(3) Ȧ between a axial water molecule and an equatorial carboxylate oxygen (Figure 2.2). As a result the cobalt malonate molecules form one dimensional chains which run parallel to each other.
Figure 2.2 – A figure to show the hydrogen bonding arrangement linking two cobalt malonate units to form a one dimensional chain.
The 2-amino pyridine and perchlorate ions form alternating layers between the chains of cobalt malonate anions (Figure 2.3). This arrangement is further stabilised by π-π interactions between 2-amino pyridine cations. Each pair of 2-amino pyridines that is joined to a particular cobalt malonate anion can interact but pairs belonging to different cobalt malonate anions are unable to interact due to ring slippage (different pairs do not lie directly above each other and are therefore unable to interact).
42
Figure 2.3 – A figure to show the crystal packing arrangement of C26H36N8018Cl2Co . The 2-amino pyridine and perchlorate molecules form alternating layers between the parallel cobalt malonate chains.
2.4 - Crystal structure implications
The structure was solved with a low R factor of 0.031 (as described by Equation 6) and was consistent with the expected chemical composition. The presence of the electron accepting oxygen and chlorine atoms of the perchlorate anion help in the formation of an extensive hydrogen bonding network in the crystal. Further stabilisation is provided via π-π interactions between aromatic rings of the 2amino pyridine cations. These intermolecular interactions help to minimise the free energy by decreasing the enthalpy and therefore help promote crystallisation.
43
Chapter 3
Structure determination of a small molecule – C26H36N8O10F12P2Co 3.1 Introduction to C26H36N8O10F12P2Co Presented in this chapter is the data collection procedure and structural analysis of a cobalt containing complex. This complex was again synthesis by the group of Professor Subrata Mukhopadhyay of the University of Jadavpur, India, for study into the field of crystal engineering. The complex and the previous example were expected to be closely related with both expected to contain 2-amino pyridine and malonate ions. The probable composition of the complex was determined by the synthetic chemists as [Co(mal)2(H2O)2](PF6)2(LH)4 where mal indicates malonate and LH4 indicates protonated 2-amino pyridine.
3.2 – X-ray diffraction data collection and processing procedure
The crystals provided were pink in colour and had an average size of approximately 5mm to 1cm in length. The crystals appeared to be of good quality and extinguished well under crossed polarisers. As a result a single crystal was cut using a razor blade to around 0.3mm in length. The crystal was then immersed in a viscous oil then fitted and mounted as described in chapter 2. Once centred the crystal was irradiated with monochromated MoKα radiation. The X-ray generator settings were 40 kV and 40 mA. The computer program SAINT15 was used to collect and integrate the CCD frame images in order to produce integrated intensities. This produced files with filename extensions .p4p and .RAW. These files were then introduced into SHELX16 XPREP for determination of the crystal system and space group. SHELX SADABS was used to produce an absorption corrected data set which took into account absorption of X-rays by the crystal, although this was a symmetrically sized crystal (crystal dimensions listed in 44
Table 4). The direct methods program SHELX XS was used to solve the structure with further refinement being performed in the SHELX XSHELL program All non hydrogen atom positions were refined anisotropically. The hydrogen atom positions were clearly visible using difference Fourier methods and were refined isotropically.
C26H36N8O10F12P2Co
Empirical formula
969.50 g mol
Chemical formula weight Crystal system
Triclinic
Space group
P1
Unit cell dimensions
-1
a = 7.1433(5) Ǻ
α = 84.3130(10)°
b = 11.7421(9) Ǻ
β = 84.2630(10)°
c = 11.8894(9) Ǻ
γ = 72.3190(10)°
Unit cell volume
9421.90(12) Ǻ3
Z
1
Data collection temp
100K
Radiation
MoKα, graphite monochromator
Diffractometer
Bruker AXS Apex (3 circle)
Detector
CCD area detector
Crystal size
0.50 x 0.50 x 0.20 mm
Tmin & Tmax
0.7328 & 0.8788
F (000)
493
Theta range
2.42 to 28.31°
Total reflections measured
8224
Independent reflections
4299
R indices [F2 > 2σ(F2)]
0.0313
R indices (F2)
0.0325
Largest diff. peak and hole
0.502 and -0.276 e.Å-3
Number of refined parameters
340
Table 4– A summary of the X-ray diffraction and crystal data for C26H36N8O10F12P2Co.
45
3.3 - Crystal structure analysis
It was found that the small molecule C26H36N8O10F12P2Co crystallises in triclinic space group P
1
. The asymmetric unit contains half of the cobalt malonate anion
[Co(C3H2O4)2(H2O)2]- in addition to two protonated 2-amino pyridine cations (C5H7N2+) and a PF6 counter ion (Figure 3.1). Like the previous structure the cobalt atom is located on an inversion centre bonded to two oxygens on each of the equatorial malonates as well as to a single oxygen on each of the trans axial waters.
Figure 3.1– An ORTEP diagram of C26H36N8O10F12P2Co with 50% ellipsoid probability. Atoms labelled b are symmetry generated. The equatorial Co-O bonds are 2.03275(10) Ǻ and 2.0554(10) Ǻ respectively whilst the axial Co-O bonds are 2.1232(12) Ǻ . This gives a distorted octahedral environment with malonate and water present as the primary ligands. The secondary ligands are 2-amino pyridine and PF6. Again this environment in the crystal gave rise to an extensive hydrogen bonding network comprising of nine unique hydrogen bonds, four of which involve the PF6 counter ion (Table 5).
D-H…A
D-H (Ǻ)
H…A (Ǻ)
D…A (Ǻ)
D-H…A (º)
N(4)-H(1)…O(1)
0.83(2)
2.01(2)
2.8286(16)
167.1(18)
46
N(3)-H(2)...O(2)
0.85(2)
2.10(2)
2.9409(17)
175(2)
N(3)-H(1)...F(4)
0.89(2)
2.12(2)
2.9828(16)
162.6(18)
N(2)-H(1)...O(4)
0.84(2)
1.93(2)
2.7737(17)
176(2)
N(1)-H(2)...F(1)
0.83(2)
2.13(2)
2.9238(18)
161(2)
N(1)-H(1)...O(3)
0.86(2)
1.96(2)
2.8155(19)
171(2)
O(5)-H(2)...O(2)
0.80(3)
1.88(3)
2.6737(16)
172(2)
O(5)-H(1)...F(2)
0.78(2)
2.36(2)
3.0681(19)
152(2)
O(5)-H(1)...F3
0.78(2)
2.24(2)
2.9381(17)
149(2)
Table 5 – The hydrogen bonding details for structure C26H36N8O10F12P2Co. Like the previous crystal structure the cobalt malonate anions are linked by a hydrogen bond of length 1.88(3) Ǻ between a axial water and a equatorial carboxylate oxygen (Figure 2.2). The cobalt malonate anions form one dimensional chains which run parallel to each other. The 2-amino pyridine and PF6 ions form alternating layers between the chains (Figure 3.2). This arrangement is further stabilised by π-π interactions between 2-amino pyridine cations. Each pair of 2-amino pyridines that is joined to a particular cobalt malonate anion can interact but pairs belonging to different cobalt malonate anions are unable to interact due to ring slippage (different pairs do not lie directly above each other and are therefore unable to interact).
Figure 3.2– A figure to show the crystal packing arrangement of C26H36N8O10F12P2Co . The 2-amino pyridine and PF6 molecules form alternating layers between the parallel cobalt malonate chains. 47
3.4 - Crystal structure implications
The structure was solved with a low R factor of 0.031 (as described by Equation 6) and was consistent with the expected composition. The presence of the electron accepting fluorine atoms of the PF6 anion help in the formation of an extensive hydrogen bonding network. Further stabilisation is provided via π-π interactions between aromatic rings of the 2-amino pyridine cations. These interactions help to minimise the free energy by decreasing the enthalpy and therefore allowing crystallisation to occur.
3.5 - Comparison of the crystal structures
X-ray analysis revealed that the crystal structures are closely isomorphous. It was found that both crystallised in triclinic P
1
space group with almost identical atomic
arrangements and unit cell dimensions. Due to the similar atomic composition and arrangement both structures contained a common hydrogen bonding motif composed of 2-amino pyridine and cobalt malonate molecules (Figure 3.3) which accounted for five of the nine bonds present within the two structures.
48
Figure 3.3 – A figure illustrating the common hydrogen bonding motif which is present in both structures C26H36N8O18Cl2Co and C26H36N8O10F12P2Co . Differences arise when the hydrogen bonding arrangements around the respective counter ions are examined (Figure 3.4). In the crystal structure of C26H36N8O18Cl2Co the central chorine of the perchlorate molecule is involved in hydrogen bonding. However in the crystal structure of C26H36N8O10F12P2Co the central phosphorous of the counter ion is not involved in the hydrogen bonding network. This may be expected due to the difference in electro negativity between the two atoms (phosphorus has a value of 2.19 whilst chlorine has a value of 3.16 on the Pauling scale of electro negativity). In addition the respective counter ions form hydrogen bonds with different ions. As illustrated the perchlorate counter ion forms hydrogen bonds with two 2-amino pyridine cations and two hydrogen bonds with a malonate anion (the chlorine to malonate interaction is not pictured). In comparison the PF6 counter ion forms hydrogen bonds with three 2-amino pyridine cations and only one hydrogen bond with the cobalt malonate anion. The lengths of the hydrogen bonds between the two structures are comparable except for the case involving the chlorine atom of the perchlorate. This bond is anomalously long in comparison to the others at 2.93(3) Ǻ with the remaining hydrogen bonds in the two 49
complexes all with hydrogen to acceptor distances in the range of 1.78 Ǻ – 2.36 Ǻ. It is unlikely that the electronegativity fully accounts for this anomaly as the fluorine atoms form shorter hydrogen bonds (in the range of 2.12 – 2.36 Ǻ) even though they possess a greater electronegativity.
Figure 3.4 – A figure to illustrate the differences in the hydrogen bonding arrangements around the perchlorate and PF6 counter ions. A fourth interaction not shown is present between the chlorine of the perchlorate counter ion and an oxygen of the axial water. The equatorial Co-O bond lengths of the two complexes reported here (2.0335(12) Ǻ, 2.03275(12) Ǻ, 2.03275(10) Ǻ, 2.0554(10) Ǻ) are consistent with Co-O bond lengths observed in previously reported cobalt malonate complexes18 (2.034(1) Ǻ, 2.063(1) Ǻ) synthesised by the group of Professor Subrata Mukhapadhyay. In addition the type of intermolecular interactions seen here are known as previously reported examples of transition metal, malonate complexes with 2-amino pyridine have displayed the same hydrogen bonding motif as described here (Figure 12). This molecular recognition phenomenon appears to be responsible for driving the crystal packing 50
arrangement in the solid state. Formation of this motif apparently requires the presence of 2-amino pyridine. Previous work by the by the group of Professor Subrata Mukhapadhyay found no such motif was formed when 4-amino pyridine was used in place of 2-amino pyridine18. The hydrogen bond lengths reported here are consistent with those previously reported cobalt malonate complexes. However differences arise due to the presence of the perchlorate and PF6 counter ions. It appears that introducing varying counter ions into the complex can subtly alter the hydrogen bonding arrangement and therefore the crystal packing arrangement. This may hold useful implications for crystal engineering where the tight control of intermolecular interactions is essential.
51
Chapter 4 Structure Determination of two Small Molecules – C30H24NO4Sn & C24H20Sn (SnPh4) 4.1 - Introduction to C30H24NO4Sn & C24H20Sn (SnPh4) A set of two tin containing, crystalline compounds were submitted to the university by a Pakistani research group for structure determination. The two compounds were expected to be closely related. The expected chemical structures were determined by synthetic chemists with the predicted structures shown in Figures 4.1 & 4.2.
CF3 O Sn
O
N H
Figure 4.1 – The expected chemical structure of the molecule in the crystal MHB7.
O Sn
O
O N
CH3
H
Figure 4.2 – The expected chemical structure of the molecule in the crystal MHB8.
Sample MHB7 consisted of colourless, needle shaped crystals which were extremely thin (around 0.08mm across). In contrast sample MHB8 consisted of colourless, block shaped crystals with fairly symmetric dimensions. Both samples appeared to be of good quality when viewed under a microscope and both extinguished well under crossed polarisers.
52
4.2 – X-ray diffraction data collection and processing procedure for C24H20Sn (SnPh4) Sample MHB7 was the first to be analysed. A single needle shaped crystal measuring approximately 0.30 x 0.08 x 0.08mm was selected and immersed in a viscous oil. The crystal was fitted on a loop, which was then placed onto a goniometer head and fitted onto the diffractometer. The data collection was performed at a cryogenic temperature (100K), which caused freezing of the viscous oil and thus fixation of the crystal as well as being beneficial to reduce atomic displacement parameters. The crystal was centred using rotating screws on the goniometer head and a video camera as a visual aide. Firstly the Xaxis was adjusted at phi 0˚ to ensure the crystal was centred. Once complete the screw was rotated to phi 180˚ and the crystal centred again. This process was repeated in the same manner for the Y-axis but with phi angles 90˚ and 270˚. Once the crystal was completely centred it was irradiated with monochromated MoKα radiation. The X-ray generator settings were 40kV and 40 mA. After a sufficient number of frames had been collected using the SAINT15 computer program the unit cell dimensions were determined (using the SMART15 computer program) whilst data collection was still continuing. It was found that the dimensions of the unit cell were 12.0079(14) x 12.0079(14) x 6.3934(16)Ǻ with angles 90 x 90 x 90˚. These dimensions correspond to a tetragonal crystal system, which is fairly unusual for small molecules (most small molecules crystallise in monoclinic or triclinic). A search of the unit cell dimensions and space group of the Cambridge Crystallographic Data Centre (CCDC) 19 using CONQUEST20 software revealed the dimensions to correspond to SnPh4. This compound was used as a starting material in the synthesis and typically forms needle shaped crystals.
Empirical formula
C24H20Sn
Chemical formula weight
427.09 g mol-1
Crystal system
Tetragonal
53
Space group
P 4 21/c
Unit cell dimensions
a = 12.0079(14) Ǻ
α = 90.00 °
b = 12.0079(14) Ȧ
β = 90.00 °
c = 6.3934(16) Ȧ
γ = 90.00 °
Unit cell volume
921.9(3) Ǻ 3
Z
2
Data collection temp
100 K
Radiation
MoKα, graphite monochromator
Diffractometer
Bruker AXS Apex (3 circle)
Detector
CCD area detector
Crystal size
0.30 x 0.08 x 0.08 mm
Tmin & Tmax
0.6808 & 0.8971
F (000)
428
Theta range
2.40 to 26.35°
Total reflections measured
4976
Independent reflections
684 (Rint = 0.0205)
R indices [F2 > 2σ(F2)]
0.0353
R indices (F2)
0.0386
Largest diff. peak and hole
0.564 and -0.365 e.Ǻ-3
Number of refined parameters
77
Table 6 – Summary of the X-ray diffraction data and refinement for C24H20Sn. The computer program SAINT was used to collect and integrate the CCD frame images in order to produce integrated intensities. This produced files with filename extensions .p4p and .RAW. These files were then introduced into SHELX16 XPREP for determination of the crystal system and space group. As the crystal contained a heavy tin atom X-ray absorption corrections were applied using SHELX SADABS. These corrections were also required because the crystal had quite unsymmetrical dimensions (crystal dimensions listed in Table 6). The direct methods program SHELX XS was used to solve the structure with further refinement being performed in the SHELX XSHELL program. 54
All non hydrogen atom positions were refined anisotropically. The hydrogen atom positions were clearly visible using difference Fourier methods and were refined isotropically.
4.3 - X-ray diffraction data collection and processing procedure for C30H24NO4Sn Sample MHB8 was subsequently analysed. A single colourless, block shaped crystal measuring approximately 0.40 x 0.30 x 0.30mm was selected and mounted as described for the previous crystal (Chapter 4.2). Once the crystal was completely centred it was irradiated with monochromated MoKα radiation. The X-ray generator settings were 40kV and 40 mA. The computer program SAINT15 was used to collect and integrate the CCD frame images in order to produce integrated intensities. This produced files with filename extensions .p4p and .RAW. These files were then introduced into SHELX16XPREP for determination of the crystal system and space group. Although this was a symmetrical sized crystal (crystal dimensions listed in Table 7) the presence of a heavy tin atom required the application of X-ray absorption corrections to the data set, which was done using SHELX SADABS The direct methods program SHELX XS was used to solve the structure with further refinement being performed in the SHELX XSHELL program. All non hydrogen atom positions were refined anisotropically. The hydrogen atom positions were clearly visible using difference Fourier methods and were refined isotropically.
Empirical formula
C30H24NO4Sn
Chemical formula weight
581.19 g mol-1
Crystal system
Triclinic
Space group
P1 a = 9.7556(5) Ǻ
Unit cell dimensions
55
α = 73.1870(10) °
b = 11.3298(6) Ǻ β = 87.0820(10) ° c =12.0571(6) Ǻ
γ = 79.8410(10) °
Unit cell volume
1255.69(11) Ǻ 3
Z
2
Data collection temp
100K
Radiation
MoKα, graphite monochromator
Diffractometer
Bruker AXS Apex (3 circle)
Detector
CCD area detector
Crystal size
0.40 x 0.30 x 0.30 mm
Tmin & Tmax
0.6916 & 0.9023
F (000)
586
Theta range
1.91 to 26.35°
Total reflections measured
10025
Independent reflections
5034 (Rint = 0.0236)
R indices [F2 > 2σ(F2)]
0.0245
R indices (F2)
0.0250
Largest diff. peak and hole
0.938 and -0.516 e.Ǻ-3
Number of refined parameters
421
Table 7 – Summary of the X-ray diffraction data and refinement for C30H24NO4Sn.
4.4 - Crystal structure analysis of C24H20Sn (SnPh4) It was found that the small molecule SnPh4 crystallises in the tetragonal space group P 4 21/c. The asymmetric unit consists of the central tin atom bonded to a single phenyl (C6H6) ring. The central tin atom is present in a tetrahedral coordination environment with four Sn-C bonds of length 2.148(5) Ǻ (Figure 4.3).
56
Figure 4.3 – An ORTEP diagram of C24H20Sn with 50% ellipsoid probability. Atoms labelled a,b or c are symmetry generated. Using both the PLATON21 and SHELX computer programs it was found that no classical hydrogen bonds were present within the crystal structure. This is due to the complete absence of both suitable hydrogen bond donors and acceptors. There is however the presence of weak van der Waals interactions (between carbon and hydrogen atoms) with each SnPh4 molecule forming eight weak interactions (Figure 4.4) with eight other neighbouring SnPh4 molecules.
57
Figure 4.4 – A figure to show the location of eight weak H…C-H interactions that each C24H20Sn molecule forms. It is found that the aromatic phenyl rings do not lie above each other in the crystal packing arrangement. The adopted arrangement prevents the aromatic phenyl rings interacting with each other via π-π stacking (Figure 4.5).
Figure 4.5 – A figure to show the stacking of the C24H20Sn molecule within the crystal. Such an arrangement prevents the formation of π-π interactions.
58
Instead the molecules appear to form layers that are stabilised by the weak H…C-H interactions running between them (Figure 4.6).
Figure 4.6 – A figure to show the stacking of layers of C24H20Sn molecules stabilised by weak van der Waals interactions.
4.5 - Crystal structure analysis of C30H24NO4Sn It was found that the small molecule C30H24NO4Sn crystallises in the triclinic space group P 1 . The asymmetric unit contains two molecules of C30H24NO4Sn, which are joined to
each other via a O-Sn bond.
59
Figure 4.7 – An ORTEP diagram of C30H24NO4Sn with 50% ellipsoid probability. Atoms labelled a or b are symmetry generated. Using PLATON21 to analyse the crystal structure the molecules are shown to adopt a polymeric structure (Figure 4.7) with the monomeric units linked by an unusually long OSn bond of length 2.6534(15) Ǻ. In this arrangement the central tin atom is involved in a trigonal bipyrimidal coordination environment with the remaining four bonds (to the three phenyl groups and to an oxygen) possessing more reasonable lengths of 2.113(2), 2.117(2), 2.119(2) and 2.1152(15) Ǻ (with the final value corresponding to the Sn-O bond).
The polymeric chains form layers which run parallel to each other. This allows the bulky triphenyl groups to be arranged so as to minimise the steric clashes between the group present on adjacent molecules (Figure 4.8).
60
Figure 4.8 – A figure to show the arrangement of the polymeric chains in C30H24NO4Sn with weak van der Waals interactions shown as blue lines. Hydrogen atoms are omitted for clarity.
It is found that the aromatic phenyl groups are arranged in a fashion which prevented the formation of π-π stacking. Although Figure 4.8 appears to show aromatic phenyl rings stacked above each other the distances involved (around 20Ǻ) are obviously far too great for any significant interaction to occur (Figure 4.9).
Figure 4.9 – A figure to show the distance between aromatic phenyl rings in C30H24NO4Sn. Hydrogen atoms are omitted for clarity.
61
Instead stabilisation of the crystal packing appears to be entirely dependent upon weak van der Waals type interactions with each monomeric unit able to form twelve of these weak interactions. Using both the SHLEX and PLATON computer programs it was found that no hydrogen bonds were present in the crystal packing. This is due to the absence of suitable hydrogen bond donors although suitable hydrogen bond acceptors are present in the form of carboxylate groups. However, the presence of an intermolecular hydrogen bond within the monomeric units is observed (Figure 4.10). This interaction is around 2.700 Ǻ long and is between a carboxylate group and the nitrogen atom.
Figure 4.10 – A figure to show the intramolecular hydrogen bond present within the monomeric units.
62
An interesting observation is that SHELX does not detect the presence of the unusually long O-Sn bond. As a result molecules are displayed as discrete units with the central tin atom present in a tetrahedral environment (Figure 4.11). This difference arises because the SHELX and PLATON computer programs use different values for the atomic radius of the tin atom, which affects the bonds displayed.
Figure 4.11 – A figure to show how SHELX views the molecules as discrete units and not as a polymeric structure.
4.6 - Crystal structure implications of C24H20Sn (SnPh4) The structure was solved with a low R factor of 0.0353 (as described by Equation 6) which indicates a good quality structure. The complete absence of hydrogen bond donors and acceptors precluded the formation of hydrogen bonds whilst the arrangement of the aromatic phenyl rings was not favourable to allow for the formation of π-π interactions. The presence of weak van der Waals type interactions was detected (of distance 3.062 Ǻ) and appears to be the major force in the stabilisation of the crystal packing. As the crystals provided consisted of starting material the obvious major implication is that the attempted chemical synthesis was unsuccessful. As a result the synthesis must be modified and improved upon if the desired product is to be obtained. This information has been fed back to the synthetic chemist concerned. 63
A search of the CCDC revealed that C24H20Sn was a previously determined structure which was first reported in 1970 by Chieh and Trotter22. This original structure was obtained using a CuKα source and the measurement of 366 independent reflections. The structure was solved using a Patterson synthesis. A comparison of the results between the original structure and the structure presented in this thesis reveal a favourable agreement. The same space group (P 4 21/c) was assigned in both cases as well as almost identical unit cell dimensions (listed in Table 8).
Unit cell dimensions
Original 1970 structure
Structure reported in this
(Chieh & Trotter22)
thesis
a = 12.058(1) Ǻ
α = 90.00°
a = 12.0079(14) Ǻ
α = 90.00°
b = 12.058(1) Ǻ
β = 90.00°
b = 12.0079(14) Ǻ
β = 90.00°
c = 6.568(1) Ǻ
γ = 90.00°
c =6.3934(16) Ǻ
γ = 90.00°
R indices [F2 > 2σ(F2)]
0.078
0.0309
Independent reflections
366
3548
Length of Sn-C bond
2.14 Ǻ
2.148(5) Ǻ
Table 8 – The differences between the original 1970 SnPh4 structure and the structure determined in this thesis.
The R factor of the two structures differs with the structure reported in this thesis possessing a significantly lower R factor. This improvement is likely to be a consequence of improvements in instrumentation as well as the substantially increased number of reflections measured for the structure reported in this thesis.
4.7 - Crystal structure implications of C30H24NO4Sn The presence of the heavy tin atom and the relatively high number of reflections allowed the structure to be solved with a low R factor of 0.0245 (as described by Equation 6).
64
No classical hydrogen bonds were present in the crystal packing arrangement. It is likely that the presence of an intramolecular hydrogen bond is not relevant to the crystal packing arrangement. Although the expected chemical structure and crystallographically three dimensional structure are similar there are clear differences (Figure 4.12). The crystallographically determined structure contains an extra two C-H units in addition to an extra carboxylate unit. These differences imply that the attempted chemical synthesis was unsuccessful and therefore requires modification if the desired product is to be obtained, and which has been relayed to the synthetic chemist concerned.
O
O Sn
N
O
CH3
H H O
Figure 4.12– The crystallographically determined structure has this chemical diagram with the highlighted area corresponding to the deviation from the expected chemical structure. The monomer units are linked by a O-Sn bond measuring 2.6534(15) Ǻ. This is an unusually long bond with regular tin to oxygen bonds expected to be around 2.10 Ǻ. A search of the CCDC19 using CONQUEST20 software revealed 198 previously reported structures contained a O-Sn bond of at least 2.65 Ǻ with bond lengths of up to 2.9 Ǻ being reported. However none of these reported crystal structures possessed a significant structural similarity to the structure of C30H24NO4Sn.
65
Part B – Macromolecular X-ray crystallography
66
Chapter 5 Macromolecular X-ray Crystallography 5.1 - Introduction
The principles of small molecule X-ray crystallography (as detailed in chapter one) are equally applicable to the study of macromolecules such as proteins and viruses. However, there are differences in procedure for the crystal structure determination and refinement. The field of macromolecular X-ray crystallography is still relatively new with the first protein crystal structures (those of myoglobin and haemoglobin) being solved in 1958 by Kendrew and Perutz respectively. However thanks to advances in instrumentation as well as technique development larger and more complex structures are now routinely studied. Foremost amongst these developments is the advent of synchrotron radiation which allows for the production of much more intense, well collimated and finely tuneable Xrays. These properties are important as proteins consists of light, poorly scattering elements primarily carbon, nitrogen and hydrogen as well as containing a high solvent content. In addition methods of solving the phase problem specifically for use in macromolecular crystallography have been developed. Recently the 2009 Nobel Prize in chemistry was awarded to Ramakrishnan, Steitz and Yonath for “Studies of the structure and function of the ribosome”23. This involved the determination of a bacterial 70S ribosome consisting of two subunits with molecular weights 800,000 and 1,500,000 using synchrotron X-ray crystallography. Obtaining high resolution three dimensional structures of such macromolecules is important as it helps to elucidate the mechanisms by which they operate. A dedicated repository for the three dimensional structure of biological macromolecules now exists24 and is free to access (available at www.pdb.org). As of 13/07/10 there were 66,324 structures deposited within the Protein Data Bank (PDB). The vast majority of these being solved via X-ray crystallography (approx 57,000)
67
Although the number of structures solved by X-ray crystallography is growing annually at an exponential rate problems are still prevalent. Foremost is the issue of growing suitably sized diffraction grade single crystals. As the measured resolution is dependent upon the crystal quality, well ordered crystals must be obtained if accurate structures are to be obtained.
5.2.1 - Macromolecular crystallisation techniques
Small molecule crystallisation is a relatively simple process in comparison to macromolecular crystallisation. This is because macromolecular crystallisation involves a larger number of complicated interactions and is still poorly understood. Obtaining diffraction grade, single crystals is a notorious ‘bottleneck’ and is often the rate limiting step in the crystal structure determination. For example the pH at which crystallisation is attempted will affect the net charge of a protein (via the protonation states of titratable side chains). The resulting net charge will affect the solubility of the protein and therefore the crystallisation process. A wide range of experimental conditions must be considered and tailored, which may involve many attempts to perfect. The techniques used to induce macromolecular crystallisation can be divided into four broad categories25. All four methods involve different steps that crystal growth passes through26(Figure 5.1) such as super saturation (which involves the formation of nuclei) and nucleation (which leads to the formation of larger crystals.
Figure 5.1 – A phase diagram for crystal growth26. 68
5.2.2 – The batch method This is the simplest method of crystallisation and is most useful when the conditions of crystallisation have been narrowed down to a small range. A number of small glass vials containing a protein and a precipitant (which is present at a level slightly less than at which the protein precipitates) are prepared. The level of the protein and the precipitant is varied between the vials therefore allowing the effect that the different concentrations have on the crystallisation process to be observed. Usually, only microlitre volumes are required. For example in the research behind this thesis crystallisations of 1ml and 2ml were set up using this method. 5.2.3 - Dialysis Dialysis uses a semi permeable membrane the pore sizes of which permit the passage of solvent and small molecules. However as macromolecules are significantly larger they are unable to pass through the pores. The macromolecule is slowly brought towards supersaturation and its precipitation point by dialysis against a concentration of a precipitating agent. It is also possible to induce crystallisation by altering the pH (achieved by altering the concentration of the buffer). Like the batch method dialysis can be performed on a bulk or a microlitre scale.
5.2.4 - Vapor diffusion methods There are a number of variations of this method. Examples include the sitting drop, hanging drop (Figure 1.11) and sandwich drop methods. The basic idea behind them is that a small amount of the macromolecule is mixed with a small amount of a precipitating agent. This drop is then allowed to equilibrate with a reservoir of the precipitating agent contained within a closed system. An equilibrium will form which will result in the water present in the sample diffusing out. Conversely the concentration of the protein will increase, eventually reaching the supersaturation and precipitation points.
69
5.2.5 - Hot box technique This technique uses a temperature gradient to induce crystallisation. In this technique the protein is dissolved at a low ionic strength in a test tube. The test tube is then suspended in a thermos at high temperature (around 60°)27. The high temperature seeks to render the protein more soluble and, as it slowly cools, a supersaturation point should be reached.
5.3.1 - Solving the phase problem in macromolecular crystallography
Owing to the increased complexity of proteins with respect to small molecules different methods have been developed to solve the phase problem in macromolecular X-ray crystallography. A conventional Patterson synthesis cannot be performed as proteins often contain no heavy atoms. In addition even small proteins contain a large number of atoms which would lead to an uninterpretable Patterson map. For example lysozyme contains 2303 (non hydrogen) atoms, which would correspond to 5,303,809 vectors between the atoms!. This results in far too many peaks for meaningful information to be extracted from the Patterson map. Direct methods are not applied to macromolecules as they require a relatively low number of reflections for the computations to be effective. The large number of reflections from a protein crystal would require an unrealistic amount of calculation to retrieve the phases, for the computations to be effective. As a result phase retrieval methods specific to macromolecular crystallography have been developed, which are described below.
5.3.2 - Isomorphous replacement method The isomorphous replacement method is based on the variation in intensities of the diffraction spots belonging to two or more isomorphous crystals. Crystals can be described as isomorphous if they have the same space group and almost identical unit cell dimensions and atomic arrangements.
70
One of the crystals must be the native protein with one or more derivatives containing at least one heavy atom. The heavy atom can be introduced by soaking a pre formed crystal in a solution containing the heavy atom. Alternatively the heavy atom may be introduced via a co-crystallisation. An essential condition is that the binding of the heavy atom must not result in a significant conformational change of the protein. Usually this is not a problem as the heavy atom will bind at specific sites within a protein. As a result only the local area where the metal binds will be disturbed. As proteins are such large structures the overall conformation of the native protein and its derivatives is largely identical allowing for effective computational comparisons to be made. The light atoms, which constituent a protein, scatter with different phases and essentially cancel. In contrast a heavy atom contains a large number of electrons concentrated within a small sphere (the atomic radius). As a result these electrons scatter in phase relative to each other. As the diffraction pattern is composed of contributions from all atoms in the unit cell the addition of even a single heavy atom results therefore in a change in the intensities of the spots. Under isomorphous conditions the difference in intensities between the native and its heavy atom derivatives can be attributed solely to the contribution of the heavy atom present within the derivative, expressed as vector structure factor amplitudes (Equation 9 & Figure 5.2).
Where – FPH =
Vector representing the structure factor
amplitude of the heavy atom derivative FP =
Vector representing the structure factor
amplitude of the native protein
FPH = FP + FH
F H=
Vector representing the structure factor
amplitude of the heavy atom
Equation 9 – The basic principle of the isomorphous replacement method.
71
FH αH
FPH FP Figure 5.2 – A vectorial representation of the isomorphous replacement method
The differences in structure factor amplitudes can be used to calculate a difference Patterson map which will consist of just the vectors between the heavy atoms. Combining the difference map with the crystal symmetry should allow the heavy atom positions to be determined. Once the heavy atom positions are known their contributions to the structure factors can be determined. The phase angles may be determined graphically by considering structure factors as vectors. The vectors possess a length equal to the amplitude of the structure factor and a direction corresponding to the estimated phase. If only one derivative is considered and taking the assumption that FPH = FP + FH and that FH can be calculated then there are two possible values that the phase may possess If a second derivative is prepared there are again two possible values for the phase. However only one value will be consistent with the first case thereby providing one possible solution for the phase angle A graphical representation of the phase angle calculation is known as a Harker construction. The case when only one derivative is available will be considered first. In this method a circle with a radius corresponding to the amplitude of the native protein structure factor (FP) is drawn centred on the origin. A line corresponding to the position and phase angle of the heavy atom (calculated from the difference Patterson map and crystal symmetry) is then drawn. A second circle corresponding to this heavy atom derivative is drawn (FPH) with the origin cantered on the end of the heavy atom line. The
72
two circles will intersect at two points giving two possible values for the phase angle (Figure 5.3).
90º Circle FP with origin as centre and radius corresponding to amplitude.
α P(1)
0º FH
Circle FPH with FH as origin and radius corresponding to amplitude.
α P(2)
Figure 5.3 – A Harker construction for a native protein and a heavy atom derivative. The two possible phase angles are labelled as α (P1) and α (P2).
If a second heavy atom derivative is available the above process is repeated and extended to include the second derivative. This will result in three circles with only one point where all three circles intersect. This point of intersection corresponds to the value of the phase angle (Figure 5.4).
73
90º Circle (blue) FP with origin as centre and radius corresponding to amplitude. Circle (black) FPH2 with FH2 as centre and radius corresponding to amplitude.
α P(1)
FH
FH2
0º
Circle (red) FPH with FH as centre and radius corresponding to amplitude. Figure 5.4 - A Harker construction for a native protein and two heavy atom derivatives. The one possible phase angle is labelled as α (P1). An inherent disadvantage of the method is the basically unavoidable introduction of some level of errors through non-isomorphism. This is because crystals will never be completely isomorphous. Therefore performing and comparing measurements of multiple crystals will lead to the introduction of some level of errors. The effect of some errors will progressively affect the higher resolution X-ray diffraction data and electron density map details will become blurred. 5.3.3 - Anomalous scattering Anomalous scattering is also dependent upon the presence of a heavy atom within a protein. Conventional X-ray diffraction is a result of coherent scattering whereby the incident X-rays cause electrons to vibrate. This effect generates radiation of a frequency equal to the frequency of the incident radiation. However in the case of anomalous scattering this is no longer true. To enhance this effect the wavelength of the incident X-rays is tuned to correspond to an absorption edge of a heavy atom present within the protein. An absorption edge involves a small energy range and corresponds to an atomic transition, and which promotes the 74
heavy atom to an electronically excited state. This effect may involve the simple promotion of a core electron to an unoccupied higher energy level or the complete ejection of the electron from the atom (ionisation). This promotion or ejection of an electron will alter the phase of the scattered radiation with respect to the scattering from the light atoms. The effect of this phase change is equivalent to altering the path length of the scattered radiation. This results in a change in intensities of the diffraction spots. The increase in the anomalous scattering is coupled with a decrease in the coherent scattering. This is because a proportion of the energy of the incident X-rays is used to create transitions within the heavy atom. An important use of anomalous scattering is in the determination of absolute configurations. This can be achieved because anomalous scattering leads to a violation of a condition known as Friedel`s law (which is assumed in what might be called conventional X-ray crystallography). Friedel`s law states that a pair of symmetry related reflections will have the same intensity and phases of equal magnitude but opposite in sign (i.e. one will be positive the other negative). However at wavelengths close to an absorption edge this condition is violated. This is because the heavy atoms will behave in a different manner to the light atoms (in terms of how the phases are effected by the scattering). This can be observed in the diffraction pattern as the intensities of the two symmetry related spots being different e.g. F(hkl) will no longer equal F(-h,-k,-l). The phases can be calculated graphically in the same way as in isomorphous replacement using either a Harker construction or a vectorial representation (shown by Figures 5.2, 5.3, 5.4). The expected structure factors for a pair of enantiomers can then be calculated and compared with the observed structure factors which should then allow for assignment of a specific enantiomer. Anomalous scattering is now often the method of choice for solving the phase problem in macromolecular X-ray crystallography. It is considered a more accurate method than isomorphous replacement as it involves performing measurements on only one crystal as opposed to two or three. This means that there are no errors introduced through nonisomorphism. Isomorphous replacement can also be used in conjunction with anomalous scattering resulting in an overall powerful method of phase determination. 75
5.3.4 - Molecular replacement28 The previous two methods of phase retrieval are required when the structure under study is completely unknown. In contrast molecular replacement can be used when a suitably related structure has been previously reported (known as the model). For example if a model of oxyhaemoglobin is available it would assist in elucidating the structure of deoxyhaemoglobin. As a rough guide a model can be considered suitable if the amino acid sequence is greater than 30% identical to that of the structure under study. In the case of oxyhaemoglobin and deoxyhaemoglobin this is obviously true (being 100%) but for other examples it may be unclear as the success of the method is not guaranteed. If this is the case the sequence identities may have to be determined. If a suitable model is available then the phases from the model are used as initial approximations for the phases of the structure under study. For this to be done the model must firstly be correctly orientated and positioned in the unit cell of the structure under study. This is done by systematically comparing predicted and observed structure factors and thereby finding the orientation and position where there is an optimum correlation. The orientation and positioning involves six positional parameters (three rotational angles and three translational parameters). Performing calculations using all six parameters at once presents an extremely large problem. This is because for N atoms in the asymmetric unit there will be 6N parameters required to describe the solution. However Patterson functions can be used, which allows the rotational and translational parameters to be separated thereby simplifying the calculation convergence. Likelihood based methods may also be used and are increasingly used in place of Patterson functions. These use statistical methods in reciprocal space and can be divided into rotational and translational functions in the same manner as for the Patterson methods. As a result many molecular replacement programs choose a relatively small number of good quality solutions provided by the rotational parameters and test these using the translational parameters to finally provide a solution. The rotational Patterson function involves calculating the Patterson map of the model and rotating it over the observed Patterson map. The most probable orientation is found when there is a close agreement between the two maps.
76
The translational Patterson function involves placing the centre of the model at all positions in the unit cell of the structure under study. For each position attempted the Patterson map can be calculated and compared to the observed Patterson map of the structure under study. Where the two will agree yields the most likely position. Once the correct orientation and position has been identified an electron density map of the structure under study can be calculated. This electron density map is calculated using the measured X-ray diffraction structure factor amplitudes from the structure under study, and the estimated phases obtained form the model (that is correctly positioned and orientated in the unit cell). The difference map can be calculated, which will include areas of negative density (corresponding to areas which are present in the model but do not fit the real density) and areas of positive density (corresponding to areas which are not included in the model but are present in the structure under study). The molecular replacement method is increasingly popular as it is relatively quick to perform. A high degree of automation is also involved with the rotation and translation function computer programs now available. An example of a popular molecular replacement computer program is PHASER.
5.4 – Rigid body and restrained refinement
Rigid body refinement is used as a first step in the refinement process for the macromolecular adducts studied in this thesis. In rigid body refinement the distances between the constituent atoms of a protein are fixed. In the simplest possible case this means that the entire protein is treated as one large, rigid molecule. Alternatively the protein may be divided into a small number of subunits (e.g. beta sheets or alpha helices). The rigid blocks of the structure are then placed to match the experimentally determined electron density. In restrained refinement the bond lengths and angles of the protein are allowed a certain degree of freedom. This means that the bond lengths and angles are allowed to vary within a small range but not but a large amount i.e. they are restrained to within a certain range. Although bond lengths and angles are perhaps the most important restraint used, a 77
large number of other restraints are possible (such as forcing peptide bonds to adopt a planar conformation). The aim is the same as small molecule refinement refinement, which is the optimal correlation between the observed and calculated structure factor amplitudes.
5.5 - The R free factor
In addition to the conventional R factor detailed in Chapter one macromolecular crystallography uses another criterion to describe the correctness of a structure. The additional criterion is known as the R free factor and is calculated using the same equation as the conventional R factor (Equation 6). However the R free factor is instead calculated exclusively from a small percentage of reflections (typically around 5%) which are excluded from the refinement process. This avoids using the same data to perform refinement as well as measuring the correctness of the structure. The R free factor is normally higher than the conventional R factor although both should possess values relatively close together. A difference of up to 6% is usually tolerated. In addition to monitoring the progress of the model refinement the R free factor is used to validate that the conventional R factor is not being artificially lowered by the addition of an increased number of parameters.
78
Chapter 6
Crystal structure determination and model refinement of a cocrystallisation of HEWL and TA6Br12 6.1.1 - Introduction
This chapter details the data collection procedure and subsequent model refinement of a co-crystallisation of hen egg white lysozyme (HEWL) and Ta6Br12. The motivation for conducting this work lies in facilitating technique development. It is hoped that the crystal structure determination will lead to a well resolved structure which is solved to a satisfactory resolution. This model structure can then be compared to models obtained using data gathered from newly developed methods. Ideally this will allow for the accuracy of methods to be accessed and weaknesses identified and improved upon. The advent of the free electron laser has catalysed technique development in a number of areas. These include protein powder diffraction and the possibility of using nanoclusters or even single molecules as opposed to crystals. Developments such as these which remove the need for crystals (which are often difficult or sometimes impossible to obtain) could yield new possibilities.
6.1.2 - Introduction to lysozyme Lysozyme is an enzyme that catalyses the cleaving of polysaccharide chains present in the cell walls of bacteria29, 30. This has the effect of causing the cell wall to rupture. Without the rigidity supplied by the cell wall the bacteria burst as a result of intolerable osmotic pressure. As a result of this antibacterial function lysozyme is often termed as a natural antibiotic. It is commonly found in tears, saliva and in hen egg white (the form used in this thesis). Lysozyme is a commonly used test enzyme within crystallography for a variety of reasons. It is cheap and easy to obtain. Its structure has been previously well studied
79
(which allows for molecular replacement) and it is relatively small in size (129 amino acids). In addition it crystallises easily in a wide range of experimental conditions. It is hoped that if a molecule binds in a particular site in lysozyme then this may act as a model for a more complicated enzyme.
6.1.3 - Introduction to Ta6Br12 Ta6Br12 is a cluster used in the heavy atom derivatisation of macromolecules. Examples present in the literature detail how the cluster has been used for phase determination involving macromolecular structures31, 32. For example a paper by Szczepanowski et al33 published in 2005 details how crystals of mouse ubiquitin activating enzyme were soaked in a solution containing the Ta6Br12 cluster. This produced promising heavy atom derivatives that were used in a multiple anomalous scattering experiment using synchrotron X-ray radiation. The cluster is used because it contains two anomalous scatters with the tantalum L-ІІІ edge at 1.2548 Ǻ and the bromine K edge at 0.9202 Ǻ34. In addition the cluster can also be used to produce derivatives for use in the isomorphous replacement method. The cluster is highly symmetrical consisting of six tantalum atoms in an octahedral environment with twelve bridging bromine atoms located along the twelve edges of the tantalum octahedron (Figure 6.1). Each tantalum is bonded to four other tantalum atoms with a Ta-Ta bond length of 2.898 Ǻ. In addition each tantalum is bonded to four bromine atoms with a Ta-Br bond length of 2.604 Ǻ.
Figure 6.1 – A figure of the crystallographically determined structure of the Ta6Br12 cluster with bromines in yellow and tantalums in purple. . 80
The Ta6Br12 was supplied by Jena Bioscience in the form of a fine green powder. The cluster has a +2 charge which is countered by two bromine ions in the preparation supplied. It is a possibility that the positive charge on the cluster will cause it to bind to side chains in the protein which possess negative charges (a Coulombic interaction). The binding of Ta6Br12 to lysozyme was first investigated using single crystal X-ray crystallography by Corey et al in 196235. However due to the technological restrictions of the time no three dimensional structure was produced. It is hoped that the subsequently vast improvements in instrumentation and technique development will enable the three dimensional structure to be determined.
6.2 – Co-crystallisation procedure of HEWL and Ta6Br12 The crystals of hen egg white lysozyme and Ta6Br12 were grown using a batch method co-crystallisation (adopted from Blundell and Johnson25). A sodium acetate buffer was used to regulate the pH. This was prepared by dissolving 0.54g of sodium acetate trihydrate (CH3COONa.3H2O) in 50ml of distilled water in a volumetric flask. Once all solids had dissolved 229µl of acetic acid (CH3COOH) was added to the solution which was then stirred for five minutes. The volume of the solution was then accurately increased to 100ml. The resulting solution was pH 4.7 with an acetate concentration of 0.04M. The precipitating agent used was a 10% salt solution. This was prepared by adding 10g of salt (NaCl) to a volumetric flask. Distilled water was then added to accurately bring the volume to 100ml.
For the co-crystallisation 0.04M acetate buffer (1ml) was added to lysozyme (50mg) in a small glass vial. The solution was stirred for 5 minutes to ensure the lysozyme powder had fully dissolved. At this point one aliquot (1mg) of Ta6Br12 was added. Stirring the solution for five minutes with the end of a Finn pipette was essential to ensure the Ta6Br12 had fully dissolved. The solution was now pale green in colour. Finally, 10% salt solution (1ml ) was added over a five minute period to help induce crystallisation. The final 81
solution was stirred for five minutes. The vial was then left in an undisturbed position at room temperature. After three days it was found that a large number of single crystals were present on the bottom of the vial. The crystals appeared to be of good quality with no visible defects. In addition the crystals extinguished well under crossed polarisers. The crystals were green in colour, like Ta6Br12 (Figure 6.2).
Figure 6.2 – A picture of the Ta6Br12 & HEWL crystals as viewed under a microscope after 3 days. Crystals were approximately 0.1mm in length at this point in time.
6.3 – X-ray diffraction data collection procedure Glycerol (4µl) was used as a cryoprotectant and was added to mother liquor (12µl) which contained the crystals. A low level of glycerol (25%) was required as it appeared to slowly interact with the crystals. From this a single, green crystal measuring 0.2mm across was selected. The crystal was fitted onto a fibre mesh and then mounted onto an RAxis imaging plate diffractometer with a rotating copper anode source. The detector to crystal distance was carefully considered. This is because moving the detector further away from the crystal will reduce the amount of incoherent scattering from the crystal and thus improve the accuracy of the data. However moving the detector further away will also result in a smaller range of θ angles being recorded which will 82
cause a reduction in the measured resolution. In this case the crystal to detector distance was set at 120mm and the data collection temperature at 100K.
A full 360º of data were collected with an exposure time of seven minutes per degree. Figure 6.3 is one of the X-ray diffraction images obtained. A summary of the data collection statics is listed in Table 9.
Figure 6.3 – An X-ray diffraction pattern image from the Ta6Br12 & HEWL data collection. The resulting data was processed, merged and scaled using the d*trek program36 (part of the Rigaku suite of programs). It was decided to remove the images in the ranges 1-74º and 342 - 360º from the processing as removing these images improved the value of Rmerge. An initial model structure was obtained using the model replacement method. This was done using the PHASER computer program which is part of the CCP4i suite37. The resolution of the model was solved to 1.95Ȧ.
83
Crystal system
Tetragonal
Space group
P 43 21 2
Unit cell dimensions
a = 78.9964 Ǻ
α = 90.00°
b = 78.9964 Ǻ
β = 90.00°
c = 36.8507 Ǻ
γ = 90.00°
Unit cell volume
229964 Ǻ3
Data collection temperature
100 K
Radiation
CuKα rotating anode
Diffractometer
R-Axis
Detector
Image plate
Crystal size
0.20 x 0.20mm
Crystal mosaicity
1.434°
Total reflections measured
177143
Independent reflections
16885
Data completeness
100% (100%)
13.2 (3.8)
Average redundancy
10.49 (10.44)
Rmerge
0.085 (0.433)
Resolution range
55.86 - 1.95 (2.02 - 1.95)
Table 9 – The summary of the X-ray diffraction data collection of HEWL and Ta6Br12 crystal. Values in parentheses indicate the last resolution shell
6.4 – Model refinement procedure The following steps were performed to move from an initial model to a final structure. All the refinement steps were performed in the refmac5 program which is part of the CCP4i suite. Map inspection and model building was performed in the COOT38 program.
Step 1
84
A previously reported lysozyme structure was used as an initial model (PDB file 2W1Y)39. This was deemed a suitable starting model as it was obtained using the same wavelength of X-ray radiation (1.54 Ǻ). To begin with a twenty cycle rigid body refinement was performed on the model protein coordinates with overall refinement of the temperature factor. This was done to avoid any model bias on the R free reflections of the experimentally determined results.
Initial R factor
0.3171
Initial RFree
0.2928
R factor after
RFree after
refinement
refinement
0.3170
0.2910
Step 2 The COOT program was used to inspect the electron density map. This revealed a good correlation between the model and the experimentally obtained electron density. The model was subsequently subjected to ten cycles of restrained refinement with isotropic refinement of temperature factors.
Initial R factor
0.2960
Initial RFree
0.2730
R factor after
RFree after
refinement
refinement
0.2296
0.2850
Step 3 The electron density map revealed two groups of six peaks in a roughly octahedral environment with significant sigma values of 2.52 and 3.12. These peaks were assigned as tantalum atoms with an initial occupancy set at 0.30 and an initial temperature factor of 50.00. A further 10 cycles of restrained refinement with isotropic refinement of temperature factors were performed.
Initial R factor
0.2862
Initial RFree
0.3124 85
R factor after
RFree after
refinement
refinement
0.2250
0.2697
Step 4 The 178 water molecules present in the model structure were systematically checked to see if they correlated with the experimentally determined electron density. This process resulted in the removal of 61 water molecules. A further ten cycles of restrained refinement were performed with isotropic refinement of temperature factors.
Initial R factor
0.2554
Initial RFree
0.2966
R factor after
RFree after
refinement
refinement
0.2317
0.2922
Step 5 A further group of six peaks in a roughly octahedral environment were identified with a sigma value of 3.22. The six peaks were assigned as tantalum atoms. A further ten cycles of restrained refinement was performed with isotropic refinement of temperature factors. Five cycles of COOT: Findwater were performed after the isotropic refinement.
Initial R factor
0.2623
Initial RFree
0.3113
R factor after
RFree after
refinement
refinement
0.2267
0.2868
Step 6 The tantalum positions were slightly altered to give distances corresponding to those observed in the small molecule crystal structure of the cluster. The tantalum positions had apparently shifted during the last cycle of refinement. This was attributed to incorrect occupancy values. As a result the occupancy of the tantalums of the three sites was reduced to 0.12 with temperature factors of 30.00. A further ten cycles of restrained refinement were performed with isotropic refinement of temperature factors.
Initial R factor
Initial RFree
86
R factor after
RFree after
refinement
refinement
0.2687
0.3297
0.2273
0.2904
Step 7 The occupancy of the three groups of tantalum atoms was reduced from 0.12 to 0.10. A further seven waters were removed. A further ten cycles of restrained refinement were performed with isotropic refinement of temperature factors.
Initial R factor
0.2397
Initial RFree
0.3095
R factor after
RFree after
refinement
refinement
0.2252
0.2830
Step 8 A fourth group of six peaks in a roughly octahedral environment with a sigma value of 3.32 was located. The six peaks were assigned as tantalum atoms with an occupancy of 0.10 and a temperature factor of 30.00. A further ten cycles of restrained refinement were performed with isotropic refinement of temperature factors.
Initial R factor
0.2226
Initial RFree
0.2796
R factor after
RFree after
refinement
refinement
0.2194
0.2731
Step 9 A group of eight bromine atoms was added to the best binding site by inspecting the difference map. A further ten cycles of restrained refinement were performed with isotropic refinement of temperature factors.
Initial R factor
0.2179
Initial RFree
0.2713
Step 10 87
R factor after
RFree after
refinement
refinement
0.2178
0.2679
A further nine waters were removed. Two water molecules were added manually by inspecting the electron density. This gave 75 waters in the final structure. One of the binding sites was assigned a final occupancy value of 10% whilst the remaining three were assigned final occupancy values of 8%.
Initial R factor
0.2355
Initial RFree
0.2860
R factor after
RFree after
refinement
refinement
0.2241
0.2764
The gradual reduction of the R factor is illustrated clearly by Figure 6.4.
Figure 6.4 – A figure to illustrate the gradual reduction of the conventional R factor with each step of refinement performed.
6.5 - Refinement of the occupancies of the Ta6Br12 binding sites using the SHELX computer program
As the occupancy values of the binding sites were altered a number of times (using refmac5 and COOT) it was decided to use the SHELX computer program to obtain occupancy values. The occupancy values obtained from the SHELX program could then
88
be compared to the occupancy values already being used. It was hoped that this may provide an indication of the accuracy of the values being used. The SHELX program required a model structure (known as a fragment). This was provided in the form of a crystallographically determined structure40 of Ta6Br12.6H2O from the ICSD database. This structure contained the ideal bond lengths present within the molecule as well as accurate unit cell dimensions. The coordinated waters molecules were removed from this cluster as they were not present in the form of the cluster which is bound to lysozyme. The idea cluster was then fixed one at a time into the four binding sites within lysozyme. These locations had been determined by inspecting the electron density map using the COOT computer program. Although all six of the tantalum atoms in the four binding sites had been located using COOT the bromine atoms had not. Therefore the bromine atom positions were approximated using the position of a tantalum atom to which a particular bromine was attached to. After one Ta6Br12 cluster had been fixed into place, ten cycles of refinement were performed in the SHELX computer program. After the refinement another Ta6Br12 cluster was fixed followed by 10 cycles of refinement and so on. After each cycle of refinement the resulting R factor and occupancy values were recorded. The SHELX computer program calculates theoretical electron density from the ideal cluster and attempts to match it and the unit cell dimensions to the experimentally determined electron density. It refines the position of the cluster and its occupancy value based on the minimisation of the difference between the observed and calculated electron density.
After one Ta6Br12 fragment has been placed into a binding site. R factor
Occupancy of binding site
24.61
9.9%
After two Ta6Br12 fragments have been placed into binding sites. R factor
Occupancy of binding site 1
Occupancy of binding site 2
0.2446
8.7%
8.2%
89
After three Ta6Br12 fragments have been placed into binding sites R factor
Occupancy of binding site 1
0.2439
Occupancy of binding
Occupancy of binding
site 2
site 3
10.7%
10.2%
9.4%
After four Ta6Br12 fragments have been placed into binding sites R factor
0.2431
Occupancy of
Occupancy of
Occupancy of
Occupancy of
binding site 1
binding site 2
binding site 3
binding site 4
9.2%
10.0%
10.0%
10.0%
After all four Ta6Br12 clusters had been placed, ten cycles of refinement were carried out which yielded the results above. However the refinement process did not converge satisfactorily. As a result the number of refinement cycles was increased from ten to thirty in the hope of obtaining more accurate results. Convergence was achieved once the number of cycles was increased, the results of which should be more accurate than previous results.
After four Ta6Br12 fragments have been placed into binding sites. (30 cycle refinement) R factor
0.2318
Occupancy of
Occupancy of
Occupancy of
Occupancy of
binding site 1
binding site 2
binding site 3
binding site 4
10.1%
12.4%
9.6%
16.4%
The values of 10.1% and 9.6% are relatively similar to those obtained using the refmac5 and COOT computer programs. However the values of 12.4% and 16.4% are significantly larger. Inspection of the .res file obtained from SHELX revealed that the clusters in these positions had been placed too close to the protein. It is possible that this may account for the high occupancy values observed.
The evolution of the occupancies of each of the four binding sites with each step of refinement is clearly illustrated in a graphically format by Figure 6.5. 90
Figure 6.5 – A figure to show the evolution of the occupancies of the four binding sites versus each step of refinement
6.6 - Analysis of the three dimensional structure Initial inspection of the electron density map using the COOT computer program revealed that the Ta6Br12 had bound at four distinct sites within lysozyme. A description of each of the sites is given below.
Analysis of the first binding site The position and octahedral nature of the tantalums atoms was visible in the electron density map at 2.52 sigma in the form of six distinct peaks. It was also possible to locate the positions of eight bromine atoms by inspection of the electron density and difference maps. However the remaining four bromine atoms could not be located due to poor electron density. The occupancy of the site was set at 10% which produced realistic temperature factors for both the bromine and tantalum atoms.
91
Figure 6.6 – The electron density surrounding the first Ta6Br12 to lysozyme binding site.
The tantalum atoms are arranged in a fairly regular octahedral environment with the equatorial tantalums joined by bonds with distances of 2.49 Ǻ , 2.30 Ǻ, 3.11 Ǻ and 3.36 Ǻ. The two axial tantalums are separated by 4.47 Ǻ. These distances compare favourably with the actual values40 of .2.901(7) Ǻ and 4.096(10) Ǻ respectively.
Figure 6.7 – A figure to show the distances between the tantalum atoms in the first binding site. Also shown are the distances to the nearest residues. 92
There are two possible residues with which the cluster may interact (as shown in Figure 6.7). The closest residue is arginine 125, the amine groups of which are 1.95 Ǻ and 3.24 Ǻ away from the closest tantalum atoms. It is likely that at the crystallisation pH of 4.7, arginine would be protonated which would generate a positive charge. As the cluster is also positively charged it is surprising to find the two so close together. The next nearest residue is aspartic acid 119 which is 2.67 Ǻ away. It is likely that at the crystallisation pH of 4.7 aspartic acid would possess a negative charge. As a result it is a possible that there is a columbic interaction between the cluster and this residue. The third nearest amino acid is glutamine 121 which is 4.12 Ǻ away which appears to be too great a distance for an interaction to occur.
Analysis of the second binding site The position and octahedral environment of the six tantalum atoms was clearly visible at 3.12 sigma. However the position of the bromine atoms could not be determined due to poor electron density .The tantalum atoms are arranged in a poor octahedral environment with the equatorial tantalum atoms joined by bonds with distances of 2.70 Ǻ, 3.80 Ǻ, 3.52 Ǻ and 2.09 Ǻ. The two axial tantalum atoms are separated by a distance of 5.54 Ǻ. These distances display substantial deviation from the ideal values. In addition it was found that the tantalum positions appeared to shift significantly with each cycle of refinement. This is probably due to the low occupancy of the binding site, which was set at 8%. The temperature factors of the bromine atoms were consistent with those of the previous site.
93
Figure 6.8 – The electron density surrounding the second Ta6Br12 to lysozyme binding site. The site is most closely located to a carbon atom of a proline residue which is 2.92 Ǻ away. As the carbon will possess no charge at the crystallisation pH (pH = 4.7) it is unclear how the cluster interacts at this particular location.
Figure 6.9 – A figure to show the distances between the tantalum atoms in the second binding site. Also shown is the distance to the nearest residue.
94
Analysis of the third binding site The position and octahedral environment of the six tantalum atoms was clearly visible at 3.22 sigma. However the position of any of the bromine atoms could not be determined due to poor electron density .The tantalum atoms are arranged in a poor octahedral environment with the equatorial tantalums joined by bonds with distances of 1.96 Ǻ, 4.34 Ǻ, 3.05 Ǻ and 3.39 Ǻ. The two axial tantalum atoms are separated by a distance of 5.38 Ǻ. These distances display substantial deviation from the ideal values. In addition it was found that the tantalum positions appeared to shift significantly with each cycle of refinement. This is probably due to the low occupancy of the binding site which was set at 8%. The temperature factors of the tantalum atoms appeared consistent with the previous binding sites.
It is likely that the cluster interacts with a symmetry related lysozyme molecule that is not displayed. This is because the closest residues, glycine 71 and arginine 61 are 5.21 and 4.45 Ǻ away respectively. These distances appear to be too great for any significant interaction to occur.
Figure 6.10 – The electron density surrounding the third Ta6Br12 to lysozyme binding site.
95
Figure 6.11 – A figure to show the distances between the tantalum atoms in the third binding site. Also shown are the distances to the nearest residues.
Analysis of the fourth binding site The position and octahedral environment of the six tantalum atoms was clearly visible at 3.32 sigma. However the position of any of the bromine atoms could not be determined due to poor electron density. The tantalum atoms are arranged in a poor octahedral environment with the equatorial tantalum atoms joined by bonds of distances 4.60 Ǻ, 3.11 Ǻ, 3.38 Ǻ and 3.88 Ǻ. The two axial tantalum atoms are separated by a distance of 5.07 Ǻ. The tantalum atoms appeared to shift during the refinement process probably due to the low occupancy of the site which was set at 8%. The temperature factors of the tantalum atoms appeared consistent with the previous binding sites.
96
Figure 6.12 – The electron density surrounding the fourth Ta6Br12 to lysozyme binding site.
There are three possible residues which are close enough for the cluster to interact with (as shown in Figure 6.13). The closest residue is aspartic acid 18 which is 1.43 Ǻ away. This would possess a negative charge at the crystallisation ph of 4.7 which would complement the positive charge of the cluster. The second closet residue is lysine 13 which is 2.34 Ǻ away. This would possess a positive charge at ph 4.7 which would be expected to repel the positive charge of the cluster. The third nearby residue is leucine 129 which is 3.18 Ǻ away. This side chain will not be charged at ph 4.7. However this particular residue is relatively far from the cluster which may prevent the formation of a significant interaction.
97
Figure 6.13 – A figure to show the distances between the tantalum atoms in the fourth binding site. Also shown are the distances to the nearest residues.
6.7 - Implications of the three dimensional structure Four Ta6Br12 binding sites to lysozyme were clearly identified at 1.95 Ȧ. The tantalum atoms present in all four binding sites were located with bond angles and positions roughly corresponding to those of an octahedral environment. It was possible to locate eight bromine atoms in only one of the binding sites. The occupancy of one of the binding sites was around 10% with the remaining three binding sites around 8%. The high amount of electron density present within the Ta6Br12 cluster allows for it to be easily located using the electron density map, even at the low occupancies reported here. In contrast to this the binding of another bromine containing, transition metal cluster, K2PtBr6 to HEWL has been previously reported41. The cluster was introduced using a soaking method which resulted in largest occupancies of around 50% for the longest soak time used of 170 minutes. For the shorter time used of ten minutes this occupancy was 33%. Future work on this cluster may include focusing on its potential susceptibility to radiation damage with possible loss of the bromine atoms. The three dimensional structure obtained should provide a model to allow for comparison with models obtained using new methodologies such a s protein powder X-ray diffraction
98
Chapter 7 Crystal structure determination and model refinement of a cocrystallisation of HEWL and carboplatin
7.1.1 – Introduction to carboplatin Carboplatin (cis diammine-1,1-cyclobutanedicarboxylate platinum (ΙΙ)) is a second generation, platinum containing anti cancer medication. A second generation of platinum anti cancer medications was deemed necessary, owing to the severe side effects attributed to the administration of the parent compound, cisplatin (cis – diamminedichloro platinum (ΙΙ)) the most notable of which was nephrotoxicity. Both are commonly used to treat a variety of cancers including ovarian, testicular and cancers of the head and neck. The original interest in platinum drugs for anti cancer applications originally arose from a 1965 observation by Rosenberg et al42 who reported that certain transition metal compounds inhibited bacterial division. The most effective compound was cisplatin and after successful results in animal models it entered clinical trials in the early 1970`s.
NH3
Cl Pt Cl
NH3
Figure 7.1 – The chemical structure of the anti cancer drug cisplatin O NH3
O Pt
NH3
O O
Figure 7.2 – The chemical structure of the anti cancer drug carboplatin 99
The cis isomers of both compounds are used for therapeutic applications as the trans isomers display no biological activity. Cisplatin contains two labile cis chloride ions which act as leaving groups. This is in addition to two relatively stable ammonia groups which in conjugation with the chloride ions are arranged in a square planar geometry (Figure 7.1). In contrast carboplatin contains a more stable bidentate dicarboxylate ligand in place of the chloride ions in addition to the two ammonia groups (Figure 7.2). It is widely accepted that the labile chlorine ions of cisplatin are exchanged for nucleophilic groups which result in the formation of chemically stable links. The administered form of carboplatin reacts with water to form an active hydrated intermediate. This intermediate is only formed inside cells as the chloride ion concentration outside cells is sufficiently high enough to prevent hydrolysis. However, inside cells the chloride ion concentration is low enough for hydrolysis to take place. This results in the displacement of one chloride ion by a water molecule. The water molecule is subsequently displaced which allows the platinum atom to coordinate with nucleophiles present within the DNA helix. The displacement of the second chloride ion allows the formation of interstrand cross links43. Cisplatin displays little affinity for the sugars and phosphates but instead reacts with the purine and pyridimine base pairs of DNA. The primary target of cisplatin at physiological pH is the N7 atoms of guanine and adenine. The reaction most commonly results in intrastrand cross linking of two neighbouring guanines which accounts for the majority of the cross linking seen (around 60%). The cross linking caused by cisplatin causes a major bending of the DNA helix towards the major groove of DNA44. It is possible that this structural change is sufficient to inhibit further DNA synthesis resulting in cell death. The crystal structure of cisplatin and duplex DNA has been determined at 2.6 Ǻ resolution by Takahara et al45. The structure was solved using the multiple isomorphous derivative method. This crystal structure support the formation of guanine-guanine cross links within the DNA. Carboplatin appears to act via a similar mechanism owing to the more stable nature of the bidentate carboxylate ligand it acts at a slower rate. In addition for carboplatin the drug
100
passing through the kidneys is not aquated. Therefore it does not react with the kidney tissue which is the cause of the nephrotoxicity displayed by cisplatin. 7.1.2 – Introduction to work by Casini et al (2006) The interest in carboplatin originally arose from a paper by Casini et al46 which was published in 2006. In this paper the authors studied the adducts of anticancer platinum drugs with hen egg-white lysozyme (HEWL) using both electron spray ionisation mass spectrometry (ESI-MS) and single crystal X-ray crystallography. The results obtained using ESI-MS indicated that the protein platination had only partially taken place. This result was confirmed using ICP-OES (inductively coupled optical emission spectroscopy) which indicated that platination levels were around 50% for cisplatin and less than 15% for carboplatin. This proved that the cisplatin and carboplatin had bound to HEWL albeit at seemingly low binding occupancies. It was surprising that the platination was of such low levels as high excesses of the anticancer drugs were used (three fold excess of anticancer drug with respect to lysozyme) in addition to long soaking times. In order to gain a more precise idea of the adduct structures and the location of the metal binding sites single crystal X-ray crystallography was employed. Two sets of lysozyme crystals were grown using the hanging drop method and separately soaked in solutions containing excesses of carboplatin and cisplatin. It was found that diffraction quality crystals were only obtained in the case of cisplatin. The adduct was subsequently solved at a resolution of 1.9Ǻ.
7.1.3 – Previous work by the Helliwell group As the soaking method had previously proved to be unsuccessful it was decided by us to pursue a co-crystallisation of HEWL and carboplatin. The initial attempt was performed by Joanne Meredith for a MChem dissertation47. For the crystallisation hen egg white lysozyme (49mg) was transferred to a glass vial to which a 0.04m acetate buffer solution (1ml) was also added (preparation as described in Chapter. The mixture was gently stirred for five minutes to ensure the lysozyme powder had fully dissolved. Once completed carboplatin (3.713mg, 5mM) was added followed by a further five minutes of 101
gentle stirring. Finally 10% salt solution (1ml) was added gradually over a period of five minutes after which the solution was stirred for a further five minutes. At this point the glass vial was sealed and left undisturbed at room temperature. The amounts of carboplatin and lysozyme were chosen to give a three fold excess of carboplatin with respect to lysozyme (in terms of moles). After inspection after 72 hours a larger number of colourless block like crystals with a typical size of 0.3-0.35mm were present. Unfortunately after data collection and processing it was discovered that the occupancy of the carboplatin was low. There was insufficient electron density to provide a satisfactory structure so it was decided to find a method to improve the occupancy. However from this initial wok it appeared that carboplatin bound to the sugar binding, active site of lysozyme. This was a promising result as it may indicate the possibility of using carboplatin as an inhibitor of sugar binding enzymes.
7.2 – Co-crystallisation method and optimisation of the conditions
Following on from the work carried out by Joanne Meredith it was decided to attempt use a chemical additive to attempt to improve the occupancy of the carboplatin binding. A search of literature sources indicated that both lysozyme and carboplatin are soluble in dimethyl sulfoxide (DMSO)48,49. It was envisaged that if the solubility of both components is increased then the occupancy of the carboplatin binding will also increase. In addition it was decided to increase the carboplatin concentration to 10mM in the hope this would promote increased binding. A paper by Lu et al50 published in 2002 describes the growth of lysozyme crystals from a binary mixture consisting of 12.5% DMSO and water. It was therefore decided to attempt to recreate these conditions with the addition of carboplatin. Therefore, a batch co-crystallisation was attempted using the same method and the following amounts – lysozyme (24.5mg), 0.04M acetate buffer (0.438ml), 10% salt solution (0.438ml), DMSO (0.125ml) and carboplatin (3.713mg, 10mM). The amounts of carboplatin and lysozyme were chosen to give a six fold excess of carboplatin with respect to lysozyme (in terms of moles). In all the crystallisation attempts carboplatin was 102
obtained from Calbiochem in the form of a white powder. Hen egg white lysozyme was obtained from Sigma Aldrich in the form of a white powder The vial was then left undisturbed at room temperature for a one week period. Unfortunately after this time it was discovered that no crystals had grown. A further 15.5mg of lysozyme was added which again resulted in no crystals. As a final step the lysozyme concentration was increased to 60 mg/ml. After five days this resulted in precipitation of the lysozyme suggesting the concentration was too high for crystallisation to take place. Initially the lack of crystals was attributed to the presence of carboplatin. In order to test this theory the crystallisation conditions listed in the Lu et al50 paper were recreated. These conditions were lysozyme (20mg), 0.04M acetate buffer (0.438ml), 10% salt solution (0.438ml), DMSO (0.125ml). After two days it was found that no crystals were present and that the published conditions could not be recreated. As a result it was decided to systematically vary the lysozyme and DMSO concentrations in order to find the optimum conditions for crystal growth. This process required multiple attempts as many cases resulted in a complete absence of crystals. The conditions attempted are listed in Table 10.
Attempt 1
2
3
Crystallisation conditions
Result
•
Lysozyme – 40mg
After four days a few extremely
•
DMSO – 0.060ml
few small crystals were present.
•
10% salt solution – 0.470 ml
Full magnification of the
•
Buffer – 0.470 ml
microscope was required.
•
Lysozyme – 45mg
After four days only a handful of
•
DMSO – 0.060ml
crystals had formed. Crystals too
•
10% salt solution – 0.470 ml
small to be used at around
•
Buffer – 0.470 ml
0.003mm in length.
•
Lysozyme – 50mg
A large number of square plate
•
DMSO – 0.060ml
crystals were present that
•
10% salt solution – 0.470 ml
extinguished well under crossed
103
•
polarisers. No precipitated
Buffer – 0.470 ml
lysozyme was present. Crystal dimensions around 0.1mm x 0.1mm. 4
•
Lysozyme – 55mg
After four days a small number of
•
DMSO – 0.060ml
crystals were present. Lysozyme
•
10% salt solution – 0.470 ml
concentrations appears to be too
•
Buffer – 0.470 ml
high as significant amounts have precipitated.
5
•
Lysozyme – 60mg
After four days a small number of
•
DMSO – 0.060ml
crystals were present. Lysozyme
•
10% salt solution – 0.470 ml
concentrations appears to be too
•
Buffer – 0.470 ml
high as large amounts have precipitated.
Table 10 – The condition attempted in the crystallisation of HEWL in the presence of DMSO.
As a result of the findings from the condition optimisation a final crystallisation was set up. The conditions best suited to crystal growth from Table 10 were used in the presence of carboplatin (3.713mg, 10mM), lysozyme (50mg), DMSO (0.060ml), 10% salt (0.0470 ml) and 0.04M acetate buffer (0.0470 ml). After a period of one week it was found that two different crystal morphologies had formed. A square plate form with dimensions of around 0.1mm in length in addition to elongated plates of around 0.15mm in length were present (Figure 7.3). Crystals were present on the liquid surface as well as on the bottom of the vial. An interesting observation was that after a period of 7 days, it was found that extremely thin needle shaped crystals were present (Figure 7.3). These crystals were much too thin to be used and were not observed in the carboplatin free, HEWL and DMSO crystallisations.
104
Figure 7.3 – A picture of the carboplatin & HEWL crystals as viewed under a microscope after 4 days. Crystals were approximately 0.1mm to 0.15mm in length at this point in time. The presence of extremely thin needle shaped crystals is also shown.
In addition a number of crystals were found to be grouped together to form an aggregate (Figure 7.4). These were not found in the carboplatin free, HEWL and DMSO crystallisations. This suggests that carboplatin has some chemical effect upon the crystallisation process.
105
Figure 7.4 – An aggregate of lysozyme crystals observed in the carboplatin and HEWL co-crystallisation in the presence of DMSO.
7.3 – X-ray diffraction data collection procedure
Glycerol (10µl) was used as a cryoprotectant and was added to mother liquor (40µl) containing the crystals. The mother liquor and glycerol were allowed to mix for a two minute period before a suitable crystal was selected. A single colourless crystal 0.10mm in length was selected and fitted into a 50-100 µM loop (Figure 7.5)
Figure 7.5 – A crystal of carboplatin and HEWL mounted onto a loop. Pictured using high magnification video camera present on diffractometer.
106
and mounted onto an R-Axis imaging plate diffractometer with a rotating copper anode. The crystal to detector distance was set at 120mm and the data collection temperature at 100K. A full 360º of data were collected with an exposure time of six and a half minutes per degree. Figure 7.6 is one of the X-ray diffraction images obtained. A summary of the data collection statics is listed in Table 11. The resulting data was processed, merged and scaled using the d*trek program36 (part of the Rigaku suite of programs). An initial model structure was obtained using the model replacement method. This was done using the PHASER computer program which is part of the CCP4i suite37. The resolution of the model was solved to 2 Ǻ.
Crystal system
Tetragonal
Space group
P 43 21 2
Unit cell dimensions
a = 77.0869 Ǻ
α = 90.00°
b = 77.0869 Ǻ
β = 90.00°
c = 36.4220 Ǻ
γ = 90.00°
Unit cell volume
216433 Ǻ3
Data collection temperature
100 K
Radiation
CuKα rotating anode
Diffractometer
R-Axis
Detector
Image plate
Crystal size
0.10mm x 0.10mm
Crystal mosaicity
1.737°
Total reflections measured
265965
Independent reflections
21439
Data completeness
82.7% (18.6%)
12.7 (2.4)
Average redundancy
12.41 (4.17)
Rmerge
0.084 (0.512)
Resolution range
54.51 – 1.65 (1.71 – 1.65)
Table 11 – The summary of the X-ray diffraction data collection of HEWL and carboplatin crystal. Values in parentheses indicate the last resolution shell 107
Figure 7.6 – An X-ray diffraction pattern image from the carboplatin & HEWL data collection.
7.4 – Model refinement procedure
The following steps were performed to move from an initial model to a final structure. All the refinement steps were performed in the refmac 5 program which is part of the CCP4i suite. Map inspection and model building was performed in the COOT program38.
Step 1 A previously reported lysozyme structure was used as an initial model (PDB file 1BWJ51). To begin with a twenty cycle rigid body refinement was performed on the protein coordinates with overall refinement of the temperature factor. Initial R factor
0.3335
Initial RFree
0.3341 108
R factor after
RFree after
refinement
refinement
0.3335
0.3359
Step 2 The COOT program was used to inspect the electron density map. This revealed a good correlation between the model and the experimentally obtained electron density. Two peaks of 10.49 sigma and 8.29 sigma were present in the electron density map and were assigned as platinum atoms. The occupancy of the atoms was set at 30% with a temperature factor of 50.00. The model was then subjected to 20 cycles of restrained refinement with isotropic refinement of temperature factors.
Initial R factor
0.3189
Initial RFree
0.3161
R factor after
RFree after
refinement
refinement
0.2030
0.2624
Step 3 Two ammonia groups were added to one of the platinum sites in place of water molecules. This was because the bond lengths are similar to those reported for the length of the Pt-NH3. The occupancy of the nitrogen atoms was set at 30% and the temperature factor at 50.00. A further ten cycles of restrained refinement was performed with isotropic refinement of temperature factors.
Initial R factor
0.2036
Initial RFree
0.2662
R factor after
RFree after
refinement
refinement
0.2016
0.2678
Step 4 One ammonia group was added to the other platinum site in place of a water molecule. Again the bond length appeared consistent with the reported value of the Pt-NH3 bond. The occupancy of the nitrogen atom was set at 30% and the temperature factor at 50.00. A further ten cycles of restrained refinement was performed with isotropic refinement of temperature factors. 109
Initial R factor
0.2014
Initial RFree
0.2676
R factor after
RFree after
refinement
refinement
0.2012
0.2697
Step 5 The remaining ammonia group was added to the other platinum site in place of a water molecule. The occupancy of the nitrogen atom was set at 30% and the temperature factor at 50.00. A further ten cycles of restrained refinement was performed with isotropic refinement of temperature factors.
Initial R factor
0.2033
Initial RFree
0.2728
R factor after
RFree after
refinement
refinement
0.2020
0.2695
Step 6 The electron density around each water molecule was inspected at one sigma to make sure the waters contained within the starting model correlated with the experimentally determined electron density. This process resulted in the removal of 52 water molecules. In addition 5 cycles of COOT : Find water were performed.
Initial R factor
0.2104
Initial RFree
0.2720
R factor after
RFree after
refinement
refinement
0.2069
0.2677
Step 7 A further sixteen water molecules were deleted as well as a sodium atom. This was because these species displayed no electron density. The final occupancy value of both sites was set at 30% which is consistent with the cisplatin binding to HEWL as reported by Casini et al.
110
Initial R factor
0.2149
Initial RFree
0.2778
R factor after
RFree after
refinement
refinement
0.2133
0.2809
Further optimisation of the structure was not possible and it is noted that the RFree has increased by around 1% from the last refinement step.
The evolution of the R factor with each step of refinement is illustrated clearly by Figure 7.7.
Figure 7.7 – A figure to illustrate the gradual reduction of the conventional R factor with each step of refinement performed.
7.5.1 - Analysis of the three dimensional structure From initial inspection of the electron density map it was observed that carboplatin had bound at two distinct sites within lysozyme. The two sites were on either side of the 111
histidine 15 residue. Electron density peaks of 12.19 sigma and 9.39 sigma corresponding to the platinum atom positions were observed on either side of histidine 15. For the site located on the left hand side of histidine 15 it was possible to locate the position of the platinum atom and the two amine groups of carboplatin. The ammonia groups were joined to the platinum atom by bonds of distance 1.89 Ǻ and 1.98 Ǻ. The angle between the three was 77.30°. The bond length values compare favourably with those measured in the crystal structure of carboplatin at 100K47 displays (Pt-N bond lengths of 2.024(3) Ǻ and an N-Pt-N angle of 95.6°(2)). Similarly, for the site located on the right hand side of histidine 15 it was possible to locate the position of the platinum atom and the two amine groups of carboplatin. The ammonia groups were joined to the platinum atom by bonds of distance 1.86 Ǻ and 2.01 Ǻ. The angle between the three was 95.28° (Figure 7.8). This bond angle is almost identical to that observed in the crystal structure of carboplatin at 100K. The discrepancy between the values of the two sites may be down to the platinum atom dominating the Xray scattering with respect to the relatively light carbon, nitrogen and oxygen atoms.
Figure 7.8 – A figure to show the distances from the platinum atom to the two ammonia groups in both binding sites.
It is possible that the platinum atom of the left hand site coordinates to the NE atom of histidine 15 (Figure 7.9). This interaction involves a distance of 3.40 Ǻ. It is possible that the platinum atom of the right hand side site coordinates to the ND atom of histidine 15 (Figure 7.9). This interaction involves a similar distance of 3.35 Ǻ.
112
Figure 7.9 - A figure to show the distance from the platinum atom to the nearest nitrogen atom on the histidine 15 residue for both binding sites.
Both sites displayed significant amounts of electron density (Figure 7.10 & Figure 7.11) but with shapes that made the location of additional atoms difficult. The binding site on the left hand side of histidine 15 appeared to more strongly defined suggesting a higher occupancy than the site on the right hand side of histidine 15.
Figure 7.10- A figure to show the electron density around the binding site on the left hand side of histidine 15.
113
Figure 7.11 - A figure to show the electron density around the binding site on the right hand side of histidine 15.
7.5.2 - Comparison with HEWL and cisplatin crystal structure The structure of HEWL and cisplatin as reported by Casini et al46 was deposited as a PDB file under the deposition code 216Z. This PDB file was superimposed with respect to the final version of the HEWL and carboplatin PDB. The superimposition was done with respect to the alpha carbon atoms of lysozyme and was performed using the Lsqkab program (which is part of the CCP4i suite). This ensured that both the structures were arranged in the same orientation within the unit cell. The PDB was then subjected to twenty cycles of restrained refinement with overall refinement of the temperature factor using refmac 5. Finally, a FO-FC map was generated with peaks greater than 3sigma listed. This was done using the FFT program (part of the CCP4i suite). The superimposition (Figure 7.12) revealed that the positions of the majority of the amino acid sequences, of the two PDB files closely agreed. However it appears that the histidine 15 residue is noticeably displaced (0.75 Ǻ ) in the HEWL and carboplatin model (with respect to the HEWL and cisplatin model). It is possible that this displacement allows the binding of the two carboplatin molecules on either side of histidine 15 as opposed to a single cisplatin molecule. 114
Figure 7.12 – A figure showing the superimposition of the histidine 15 residue in a crystal of cisplatin and HEWL (shown in yellow & blue) and a crystal of carboplatin and HEWL (shown entirely in blue). A noticeable displacement (0.75 Ȧ) in the case of carboplatin and HEWL is displayed.
The PDB file deposited by Casini et al contains a single DMSO molecule (that is not mentioned in the paper) in the active site of lysozyme. This molecule is in an almost identical location to the one observed in the HEWL and carboplatin study described here (Figure 7.13).
115
Catalytic residues of lysozyme Top arrow indicates glutamic acid 135. Bottom arrow indicates aspartic acid 52.
Left hand arrow indicates the location of the sulphur atom of DMSO present in the carboplatin and HEWL model. The right hand arrow indicates the location of the DMSO molecule present in the cisplatin and HEWL model. Figure 7.13 – A figure displaying the location of the DMSO molecule present within the lysozyme active site for both the cisplatin and carboplatin models.
7.5.3 - Comparison with previous HEWL and carboplatin crystal structure This work was conducted by Joanne Meredith for award of an MChem degree47. The crystals of HEWL and carboplatin were grown using a batch method co-crystallisation in the absence of DMSO. In this case it was found that the carboplatin had bound at four distinct sites within lysozyme with four peaks visible at 3.10 sigma. Interestingly, it appeared one of the binding sites was in the sugar binding, active site of lysozyme. This is an intriguing result as it suggests the possibility of using carboplatin to inhibit enzymes where sugar binding is involved in the catalytic process.
116
However this result was not observed in the presence of DMSO. This may be because DMSO has had some kind of chemical effect that has altered the binding behaviour of carboplatin.
7.5.4 - Comparison with HEWL and NAG (N-acetyl-D-glucosamine) crystal structure The crystal structure of HEWL and NAG (N-acetyl-D-glucosamine) was deposited in the PDB under the deposition code 3A3Q52. This study showed that the NAG trisaccharide bound to the active site of lysozyme. It was therefore decided to compare how the location of the trisaccharide compared to the location of the DMSO molecules observed in the HEWL cisplatin and HEWL carboplatin studies. The 3A3Q PDB file was superimposed with respect to the final version of the HEWL and carboplatin PDB. The superimposition was done with respect to the alpha carbon atoms of lysozyme and was performed using the Lsqkab program (which is part of the CCP4i suite). This ensured that both the structures were arranged in the same orientation within the unit cell
Figure 7.14 – A figure showing the location of a NAG trisaccharide and the DMSO molecules present in the cisplatin and carboplatin models. The location of all three species is almost identical with the DMSO molecules indicated by arrows.
117
The superimposition revealed that one end of the NAG trisaccharide is in the same position as the DMSO molecule in both the HEWL and cisplatin and HEWL and carboplatin studies. This may mean that the DMSO prevents the carboplatin from binding at the active site in the manner observed in the DMSO free work conducted by Joanne Meredith.
7.6 – Implications of the three dimensional structure
X-ray diffraction analysis of a DMSO free co-crystallisation of carboplatin and HEWL (conducted by Joanne Meredith) revealed that the carboplatin had bound to the sugar binding, active site of lysozyme. Binding to this site was thought to be feasible as the bidentate dicarboxylate ligand possess a reasonable structural resemblance to that of a sugar ring. This raised the possibility that carboplatin might act as an inhibitor for sugar binding enzymes such as heparanase. In an effort to increase the occupancy of the carboplatin, DMSO was introduced into a co-crystallisation of carboplatin and HEWL. X-ray diffraction analysis revealed that the presence of DMSO had apparently altered the binding behaviour of carboplatin. It was found that although DMSO had improved the occupancy of the carboplatin it had also bound to the active site. This raises the possibility that the presence of DMSO prevented the carboplatin binding to the sugar binding, active site of lysozyme. Indeed it was found that the DMSO bound to the same site in lysozyme as a NAG trisaccharide. From this work it is impossible to verify if carboplatin displays binding behaviour similar to that of sugars, possibly due to the interference of DMSO. Instead it was found that the carboplatin bound in a similar manner to that of cisplatin (as reported by Casini et al). However, where cisplatin had only bound to a single side of histidine 15, it was found that carboplatin had bound to both sides. A superimposition of the protein coordinates from the co-crystallisation of carboplatin and HEWL and those of cisplatin and HEWL reported by Casini et al was performed. This revealed that in the cocrystallisation of carboplatin and HEWL the histidine 15 residue was noticeably displaced (displacement of 0.75 Ǻ ) with respect to the histidine 15 observed in the Casini et al coordinates. This displacement may be essential in allowing the carboplatin to bind 118
at both sides of the residue as opposed to one side. However, Casini et al used a soaking method as opposed to a co-crystallisation which may have had some bearing upon the cisplatin and HEWL results. To confirm these apparent differences in carboplatin and cisplatin binding to HEWL, X-ray diffraction analysis of a co-crystallisation of cisplatin and HEWL would need to be performed. In both of the carboplatin binding sites only the platinum atom and the two ammonia groups could be located (achieving the same level of detail as reported by Casini et al).
119
Chapter 8 Future work In the HEWL and Ta6Br12 co-crystallisation the location of the tantalum atoms was relatively easy by inspection of the electron density map. This is owing to the high amount of electron density possessed by the tantalum atoms. In contrast it was difficult to locate the bromine positions which have a much lower electron density and scatter X-rays more weakly. The difficulty in determining the location of the bromine atoms was probably at least partly due to the low occupancy of the binding sites. A possible solution would be to perform the co-crystallisation of the Ta6Br12 cluster and HEWL in the presence of a solvent such as DMSO. This could possibly increase the solubility of the Ta6Br12 cluster and promote increased binding. An additional idea would be to perform the co-crystallisation at a different pH. As the Ta6Br12 cluster is positively charged it may bind to different locations in HEWL. These derivatives could possibly be used in the multiple isomorphous replacement method in order to obtain a more detailed three dimensional structure.
In the carboplatin and HEWL co-crystallisation the presence of DMSO apparently chemically altered the binding behaviour of carboplatin. The obvious suggestion would be to repeat the co-crystallisation of carboplatin and HEWL in the presence of a different additive. However as carboplatin is insoluble in solvents such as ethanol and acetone this may prove difficult. If crystals in the presence of a different additive could be obtained the possible sugar binding behaviour of carboplatin could be properly assessed.
120
References 1. Bravais lattice types image (2010), [Online] Available at: www.schielo.cl/fbpe/img/bscq/v46n3/fig04.gif
2. Molloy, C. K., (2004), Group theory for chemists: fundamental theory and applications, Horwood 3. Hahn, T. (Ed)., (1983), International tables for crystallography (volume A), Published for the International Union of Crystallography by Reidel 4. 21 screw axis image (2010), [Online] Available at: www.axxel.iqfr.csic.es/Cristalographica/archives-07/helicodial.jpg 5. Bragg, L. W., (1983), The diffraction of short electromagnetic waves by a crystal, Proc. Camb. Phil. Soc. 17 part 1, 43-57 6. Woolfson, M. M., (1997), An introduction to X-ray crystallography (2nd edition), Cambridge University Press 7. Bragg pictorial representation (2010), [Online] Available at: http://serc.carleton.edu/images/research_education/geochemsheets/braggslaw.jpg 8. Suryanarayna, C., Norton, M. G., (1998), X-ray diffraction: A practical approach, Plenium Press 9. X-ray tube image (2010), [Online] Available at: http://pubs.usgs.gov/of/2001/of01-041/htmldocs/images/xrdtube.jpg 10. Elder, R. F., Gurewitsch, M. A., Langmui,r V. R., Pollock, H. C., (1947), Radiation from electrons in a synchrotron, Physical review, 71, 829-830 11. Ramakanth, Hebbar, K., (2007), Basics of X-ray diffraction and its applications, I.K. International 12. Clegg, W., Blake, J. A., (2009), Crystal structure analysis: Principles and practice (2nd edition), Oxford University Press
121
13. Helliwell, R. J., (1992), Macromolecular crystallography with synchrotron radiation, Cambridge University Press 14. Patterson, L. A., (1935), A direct method for the determination of the component of interatomic distances in crystals, Z. Krist, 90, 517-542 15. Bruker, (2002), Saint version 6.36a, Bruker AXS Inc. 16. Sheldrick, H. G., Schneider, R. T., (1997), SHELXL: High resolution refinement, Methods in enzymology, 277, 319-343 17. Burla, C. M., Caliandro, R., Camalli, M., Carrozzini, B., Cascarano, L. G., De Caro, L., Giacovazzo, C., Polidori, G., Spagna, R., (2005), SIR2004: An improved tool for crystal structure determination and refinement, J. Appl. Cryst, 38, 381-388 18. Das, A., Dey, B., Jana Dipankar, A., Hemming, J., Helliwell, M., Lee Man, H., Hsiao, T., Suresh, E., Colacio, E., Choudhury, R. S., Mukhopadhyay, S., (2010), Effect of protonated aminopyridines on the structural divergences of M(ΙΙ)malonate complexes, Polyhedron, 29, 1317-1325 19. Allen H. F, (2002), The Cambridge structural database: a quarter of a million structures and rising, Acta. Cryst. B., 58 ,380-388 20. Bruno, J. I., Cole, C. J., Edgington, R. P., Kessler, M., Macrae, F. C., McCabe, P., Pearson, J., Taylor, R., (2002), New software for searching the Cambridge Structural Database and visualizing crystal structures, Acta. Cryst. B., 58, 389397. 21. Spek, L. A., (2003), Single crystal structure validation with the program PLATON, J. Appl. Cryst., 36, 7-13. 22. Chieh, C. P., Trotter, J., (1970), Crystal structure of tetraphenyltin, J. Chem. Soc. 6, 911-914. 23. (2010), [Online] Available at: http://nobelprize.org/nobel_prizes/chemistry/laureates/2009/ 24. (2010), [Online] Available at www.pdb.org
122
25. Blundell, L. T., Johnson, N. L., (1976), Protein crystallography, Academic Press, 69-77 26. Chayen, N., Comparative studies of protein crystallization by vapour diffusion and micro batch techniques, Acta Cryst D, 54(1), 8-15
27. King, V. M., (1965), A low resolution structural model for cubic glucagon based on the packing of cylinders, J. Mol. Biol, 11, 549-561 28. Evans, P., McCoy, A., (2008), An introduction to molecular replacement, Acta Cryst D, 64, 1-10 29. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P., (2002), Molecular biology of the cell (4th edition), Garland Science 30. Stryer, L., Berg, M. J., Tymoczko, L. J., (2006), Biochemistry (6th edition), W. H. Freeman 31. Yonath, A et al., (2005), Crystallographic studies on the ribosome, a large macromolecular assembly exhibiting severe nonisomorphoism, extreme beam sensitivity and no internal symmetry, Acta. Cryst A. 54, 945-955 32. Knablein, J et al., (1997), Ta6Br122+, a tool for phase determination of large biological assemblies by X-ray crystallography, J. Mol. Biol. 270, 1-7. 33. Szcezepanowski, H. R., Filipek, R., Bochtler, M., (2005), Crystal structure of mouse ubiquitin activating enzyme, J. Biol. Chem. 280, 22006-22011 34. (2010) Jena Bioscience tantalum cluster derivatization user guide, [Online] Available at: www.jenabioscience.com 35. Corey, B. R., Stanford, H. R., Marsh, E. R., Leung, C. Y., Kay, M. L., (1962), An X-ay investigation of wet lysozyme chloride crystals, Acta Cryst, 15, 1157-1163 36. Pflugrath, W. J., (1999), The finer things in X-ray diffraction data collection, Acta Cryst D, 55, 1718-1725 37. Collaborative, (1994), The collaborative computational project number 4: programs for protein crystallography, Acta. Cryst. D., 50(5), 760-763 123
38. Emsley, P., Lohkamp, P., Scott, W., Cowtan, K., (2010), Features of Coot, Acta. Cryst. D., In press. 39. PDB ID = 2W1Y Cianci, M., Helliwell R. J., Suzuki, A., (2008), The interdependence of wavelength. Redundancy and dose is sulphur SAD experiments, Acta Cryst D, 64, 1196-1209 40. Vojnovic, M., Brnicevic, N., Basic, I., Planinic, P., Giester, G., (2002), The Co crystallization of the Cubic [Ta6Br12(H2O)6][CuBr2X2]·10H2O and Triclinic [Ta6Br12(H2O)6]X2·trans-[Ta6Br12(OH)4(H2O)2]·18H2O (X = Cl, Br, NO3) Phases with the Coexistence of [Ta6Br12]2+ and [Ta6Br12]4+ in the Latter, ZAAC, 628, 401408 41. Kumar, Y. S., (2009), X-ray crystal structure analyses of small and large molecules (Msc thesis), The University of Manchester 42. Rosenberg, B., Van Camp, L., Krigas, T., (1965), Inhibition of cell division in Escherichia Coli by electrolysis products from a platinum electrode, Nature, 205, 698-699 43. Skeel, T. R. (Ed), Handbook of cancer chemotherapy (6th edition), Lippincott Williams and Wilkins. 44. Pratt, B. W., Ruddon, W. R., (1979), The anticancer drugs, Oxford University press. 45. Takahara, T., Rosenweig, C. A., Frederick, A. C., Lippard, J. S., (1995), Crystal structure of double-stranded DNA containing the major adduct of the anticancer drug cisplatin, Nature, 377, 649-652 46. Casini, A., Mastrobuoni, G., Temperini, C., Gabbiani, C., Francese, S., Moneti, G., Supuran, T. C., Scozzfava, A., Messori, L., (2006), ESI mass spectrometry and X-ray diffraction studies of adducts between anticancer platinum drugs and hen egg white lysozyme, Chem. Commun, 2,156-158, 47. Meredith, J., (2010), The University of Manchester (MChem dissertation), 48. Carboplatin (2010), [Online] Available at: 124
www.tocris.com/dispprod.php?ItemId=5205 49. Lehmann, S. M,. Stansfield, D. F. R., (1989), Binding of dimethyl sulfoxide to lysozyme crystals, studied with neutron diffraction, Biochemistry, 28, 7028-7033 50. Lu, J., Wang, J. X,. Ching, B. C., (2002), Effect of additives on the crystallization of lysozyme and chymotrypsinogen A, Crystal growth & design, 3(1), 83-87 51. PDB ID = 1BWJ Dong, J., Boggon, J. T., Chayen, E. N., Raftery, J., Bi, C. R., Helliwell, R. J., (199), Bound solvent structures for microgravity, ground control, gel and microbatch grown hen egg white lysozyme crystals at 1.8Ȧ resolution, Acta Cryst D. 55 745-752 52. PDB ID = 3A3Q Ose, T., Kuroki, K., Matsushima, M., Maenaka, K., Kumagai, I., (2009), Importance of the hydrogen bonding network including Asp 52 for catalysis, as revealed by Asn 59 mutant hen egg white lysozyme, J Biochem, 146, 651-657
125
Bibliography •
Blow, D., (2002), Outline of crystallography for biologists, Oxford University Press
•
Clegg, W., (1998), Crystal structure determination, Oxford University Press
•
Glusker Pickworth, J., Trueblood, N. K., (1985), Crystal structure analysis, Oxford University Press
•
Arndt, W. U., Willis, M. T. B., (1996), Single crystal diffractometery, Cambridge University Press
•
Woolfson, W. M., (1997), An introduction to X-ray crystallography, Cambridge University Press
•
Blundell, T. L., Johnson, N. L., (1976), Protein crystallography, Academic Press Inc
•
McPherson, A., (1982), Preparation and analysis of protein crystals, John Wiley & Sons
•
Ramakanth, Hebbar, K., (2007), Basics of X-ray diffraction and its applications, I.K. International
•
Helliwell, R. J., (1992), Macromolecular crystallography with synchrotron radiation, Cambridge University Press
126