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Review
. 2000 Feb;53(1):8-14.
doi: 10.1136/mp.53.1.8.

x ray crystallography

M S Smyth  1 , J H Martin
Affiliations
Review

x ray crystallography

M S Smyth et al. Mol Pathol. 2000 Feb.

Abstract

x Ray crystallography is currently the most favoured technique for structure determination of proteins and biological macromolecules. Increasingly, those interested in all branches of the biological sciences require structural information to shed light on previously unanswered questions. Furthermore, the availability of a protein structure can provide a more detailed focus for future research. The extension of the technique to systems such as viruses, immune complexes, and protein-nucleic acid complexes serves only to widen the appeal of crystallography. Structure based drug design, site directed mutagenesis, elucidation of enzyme mechanisms, and specificity of protein-ligand interactions are just a few of the areas in which x ray crystallography has provided clarification.

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Figures

Figure 1
Figure 1
Hanging drop vapour diffusion is usually set up with a drop of 1–20 μl suspended from a glass coverslip over the reservoir solution. The drop is a 50/50 (vol/vol) mixture of the protein solution and the reservoir solution. Hence, the vapour pressure of water around the drop is greater than that over the reservoir. The pressure gradient across the vapour space leads to a net loss of water in the drop. Sitting drop vapour diffusion differs only in that the drop sits in a depression on a specially constructed raised platform within the diffusion chamber.
Figure 2
Figure 2
A crystal of a bovine picornavirus measuring 0.2 mm in the longest dimension grown, using ammonium sulphate as precipitant and using microdialysis apparatus. These crystals take 48 hours to grow at room temperature, but are very fragile because the virus particles in the crystal are roughly spherical and 300 Å in diameter, making the solvent content very high and the particle contacts very low. This is common with many protein crystals and great care must be taken during manipulation.
Figure 3
Figure 3
A typical synchrotron data collection station: PX 9.6 at Daresbury, Cheshire, UK. A charged coupled device detector (1) is mounted on a motorised system, which enables the “crystal to detector” distance to be altered. This is protected from the direct beam by the backstop, a small lead pellet mounted on plastic film supported by a ring (2). Frozen crystals are cooled by a stream of nitrogen at 100 K from the nozzle (3). The diameter of the beam incident on the crystal is adjusted manually using two horizontal and two vertical slits on the panel (4). The spindle (6) on which the crystal is mounted can be rotated manually (5) to ensure perfect alignment. The complete assembly is housed within a lead walled “hutch” and the x ray beamline (7) runs tangentially from the storage ring. Photograph courtesy of M Papiz, CLRC Daresbury Laboratory.
Figure 4
Figure 4
x Ray diffraction photograph taken at PX7.2, Daresbury Laboratory, Cheshire, UK, of bovine enterovirus with 0.5° oscillation range and maximum resolution at the edge of the film of 2.8 Å. The x ray wavelength was 1.488 Å.
Figure 5
Figure 5
Processing of x ray diffraction data using the program DENZO for autoindexing. The program searches for peaks of intensity on the image and, using the “crystal to detector” distance and the wavelength, determines the unit cell dimensions and crystal system. It then calculates a prediction of what the image will look like at this crystal orientation and superimposes this on the real image. The enlarged portion shows the coloured circles of the predicted spots surrounding real spots. The user refines various parameters until the fit is optimised and then the program measures the intensities within the circles.
Figure 6
Figure 6
A portion of an electron density map calculated by molecular replacement to 3 Å resolution. The photograph represents a slice through a virus structure at the point of fivefold non-crystallographic symmetry. Five tyrosine residues from symmetry related subunits can be seen in their entirety, but the complete model has been built into the density. This natural fivefold symmetry axis also serves as a centre for crystallographic averaging. Because this particular virus has 12 such points, none of which lie along crystallographic symmetry axes, 60-fold averaging was possible.
Figure 7
Figure 7
The format of a protein data bank (PDB) file. Structural data may be downloaded from the Brookhaven protein data bank in files containing the following information: the first column indicates that the line contains atom data rather than—for example, a remark; the second column is the atom number; the third, the atom type, CA representing an α carbon, and so on; the fourth, the residue type in three letter code; the fifth, the chain identifier, if the structure has more than one subunit; the sixth, the residue number; the seventh, eighth, and ninth, the coordinates of the atom within the unit cell; the tenth, the occupancy—usually assigned to 1.00; the eleventh, the B factor, a measure of how much an atom vibrates around its equilibrium position.
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References

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