Protein crystallography data collection

Once a suitable crystal is obtained and the X-ray diffraction data are collected, the calculation of the electron density map from the data has to overcome a hurdle inherent to X-ray analysis. The X-rays scattered by the electrons in the protein crystal are defined by their amplitudes and phases, but only the amplitude can be calculated from the intensity of the diffraction spot. Different methods have been developed in order to obtain the phase information. Two approaches, commonly applied in protein crystallography, should be mentioned here. In case the structure of a homologous protein or of a major component in a protein complex is already known, the phases can be obtained by molecular replacement. The other possibility requires further experimentation, since crystals and diffraction data of heavy atom derivatives of the native crystals are also needed. Heavy atoms may be introduced by covalent attachment to cystein residues of the protein prior to crystallization, by soaking of heavy metal salts into the crystal, or by incorporation of heavy atoms in amino acids (e.g., Se-methionine) prior to bacterial synthesis of the recombinant protein. Determination of the phases corresponding to the strongly scattering heavy atoms allows successive determination of all phases. This method is called isomorphous replacement. 

Muchmore, S. W., Olson, J., Jones, R., Pan, J., Blum, M., Greer, J., Merrick, S. M., Magdalinos, P and Nienaber, V. L. (2000). Automated crystal mounting and data collection for protein crystallography. Structure 8, R243-R246. 

Muchmore, S. W.,etal. (2000). Automated crystal mounting and data collection for protein crystallography. Structure Fold Des. 8, R243-246. 

All the above techniques use incident monochromatic radiation, usually focus in one or two dimensions. However for cases a) and d) the reduction of radiation damage and more particularly in kinetic crystallography the use of polychromatic data collection is yielding promising results. This technique makes combined use of the intensity and collimation of the SR beam with a large wavelength spread for Laue data colla tion from protein single crystals. 

It took the short time of one year or so to solve the structure of rhinovirus which causes the common cold. This relied on two major advances in methods. The first was the use of synchrotron radiation in data collection. Nearly a million reflections were collected on the protein crystallography facility at the Cornell Synchrotron source in a matter of days. This conveyed a speed advantage over data collection on a conventional source and also ameliorated an otherwise impossible problem of radiation damage when long exposure times were used. The far greater rate of radiation damage in the X-ray beam in relation to plant viruses is symptomatic of an inherently less stable protein capsid and the absence of quasi-symmetry. The capsid consists of 60 copies each of four proteins and the virus with about 30 % RNA has a total molecular weight of about 8.5 million. 

The indirect chemical evidence described above was masterfully interpreted to suggest the dithiolene chelate and substituents of molybdopterin. Nevertheless, it was protein crystallography that provided definitive proof of the intact dithiolene chelate in the molybdenum and tungsten enzymes. Improvements both in protein crystal growth, diffraction data collection, and in computation... 

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