Protein crystallography methods

Perutz, M. Early Days of Protein Crystallography. Methods in Enzymology 114A, 3-18 (1985). 

Spurlino JC (2011) Fragment screening purely with protein crystallography. Methods Enzymol 493 321-356... 

The 1980s saw many important developments in the scientific disciplines that underpin the use of protein crystallography in the pharmaceutical and biotechnology industries. Molecular biology and protein chemistry methods... 

Babine RE, Abdel-Meguid SS. Protein crystallography in drug discovery (Vol. 20 of Mannhold R, Kubinyi H, Folkers G, editors. Methods and Principles in Medicinal Chemistry). Weinheim Wiley-VCH, 2004. 

Methods that utilize structural data of the target, generally identified by protein crystallography, to look for molecules that complement the binding site through favorable protein-ligand interactions (protein structure-based VS or SBVS). 

Power and Limits of the SAXS Methods. This field of SAXS is in competition with the field of protein crystallography. The spatial resolution of the SAXS method is limited (> 0.5 nm), whereas structures determined by protein crystallography are exact up to fractions of Angstrpms. On the other hand, the protein crystallography is unable to study living proteins under almost physiological conditions. Moreover, kinetic processes can be monitored by SAXS but cannot be studied by means of protein crystallography. 

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. 

The method we will not spend much time discussing, as it is currently not of much use in protein crystallography, is the direct method. This is the method of choice for determining the structures of small molecules, but as yet is only of limited value in protein crystallography. Much work is being carried out on this particular problem and advances have been made, but are not enough to make it practical for most protein crystallography applications. 

Jin, L. and Babine, R. E. (2004). Engineering proteins to promote crystallization. In Protein Crystallography in Drug Discovery. Methods and Principles in Medicinal Chemistry, Babine, R. E. and Abdel-Meguid, S. S., eds, Vol. 20, Wiley-VCH, pp. 209-216. 

The problem of phase determination is the fundamental one in any crystal structure analysis. Classically protein crystallography has depended on the method of multiple isomorphous replacement (MIR) in structure determination. However lack of strict isomorphism between the native and derivative crystals and the existence of multiple or disordered sites limit the resolution to which useful phases may be calculated. 

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. 

In the literature Raman spectroscopy has been used to characterize protein secondary structure using reference intensity profile method (Alix et al. 1985). A set of 17 proteins was studied with this method and results of characterization of secondary structures were compared to the results obtained by x-ray crystallography methods. Deconvolution of the Raman Amide I band, 1630-1700 cm-1, was made to quantitatively analyze structures of proteins. This method was used on a reference set of 17 proteins, and the results show fairly good correlations between the two methods (Alix et al. 1985). 

A major recent innovation has been to use substrates that are unreactive but may be activated by, for example, photolysis (see Chapter 6). This has necessitated the introduction of crystallographic procedures that can gather data in fractions of a second, rather than the minutes or hours conventionally used. Conventional protein crystallography uses a beam of monochromatic x-rays. An older technique that has been reintroduced is that of von Laue, which uses a spectrum of polychromatic radiation. An intense beam from a synchrotron, spanning wavelengths from 0.25 to 2.5 A, enables data for the Laue method to be taken over a fraction of a second.46,47... 

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