Protein crystallography resolution

Obtaining large single crystals that diffract to high resolution remains the primary bottleneck of protein crystallography. The most widely used... 

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. 

In summary, there are three important generalizations about error estimation in protein crystallography. The first is that the level of information varies enormously as a function primarily of resolution, but also of sequence knowledge and extent of refinement. The second generalization is that no single item of information is completely immune from possible error. If the electron density map is available or indicators such as temperature factors are known from refinement, then it is possible to tell which parameters are most at risk. The third important generalization is that errors occur at a very low absolute rate 95% of the reported information is completely accurate, and it represents a detailed and objective storehouse of knowledge with which all other studies of proteins must be reconciled. 

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. 

In the early years we had to depend on a relatively low-intensity x-ray source. High-resolution data was obtained through collaboration with John R. Helliwell and his group at the Daresbury Laboratory Synchrotron Radiation Source in England. Today greatly improved equipment and more synchrotron facilities are available for protein crystallography. 

The X-ray crystal structure of virus sialidase was first solved in early 1980s.24,25 While the active site could be identified, resolution was insufficient to determine the orientation of the bound ligand (Neu5Ac) within the catalytic center. Subsequent efforts in protein crystallography allowed further refinement... 

In particular, Sukumar etal. used the Protein Crystallography Station (PCS) at LANSCE to investigate the structure of amicyanin, a Type I blue copper protein. The study was used to identify the positions of key hydrogens within the protein, something that even high resolution and high intensity studies using X rays cannot properly determine. 

The data are at present recorded on video tape for later digitization and processing. The scintillator screen, deposited on a fiber optics face plate is either ZnS(Ag) or Gd S 0(Tb). A spatial resolution of 0,5 mm FWHM has been measured. The system has been used in diffraction measurements of polymer samples and in protein crystallography as well. At present a quantitative analysis of the collected data is in progress. 

Until recently, structure determination by protein crystallography was a time-consuming method accessible to a few privileged skilled practitioners. X-ray crystallography was reserved to tackle questions requiring atomic resolution details of a demonstrably important protein, often a drug target. Indeed, to... 

Rux is much less of a problem at X-ray sources indeed sometimes it needs to be reduced to avoid detector saturation. The whole field of protein crystallography is heavily dependent on the use of synchrotron sources, an enormous area of application which has made a spectacular impact - and no doubt will continue to do so. The high flux additionally means that the application of real time studies is much further advanced for both the wide and small angle regimes. Additionally, X-rays are much more easily focussed, and can therefore be used at high spatial resolution to produce microfocus data. For biological systems this has already led to some interesting studies, e.g. on starch " and flax. ... 

The other, technically quite different application area, is protein crystallography which deals with very large structures, even virus structures, where VF , can be as high as 100000. However these structures cannot be considered to be crystallographically very accurate, often the resolution is not high enough to determine individual atom positions, and instead amino acid residues are located. Protein R-values are seldom less than 0.15 and the ratio of the number of observed reflections and number of refined parameters is very low (<5). However protein X-ray structures are considered to be reliable structural information about the conformation and overall structure of the protein in the crystalline state. 

Petrova, T., Podjarny, A. Protein crystallography at subatomic resolution. Rep. Prog. Phys. 2004, 67, 1565-1605. 

For a random structure, / = 0.83 for a centric distribution and R = 0.59 for an acentric distribution (which is always the case with proteins in three dimensions) [112,113]. In a small molecule structure R values of <0.10 are routine and many have R <0.05. For proteins an R 0.30 at 2.5 A resolution usually indicates that most of the structure is correct but several errors may remain. An R < 0.2 is usually satisfactory. Luzzati [114] has shown that if the errors in position are normally distributed and that if these errors are the sole cause of differences between observed and calculated structure factors, then at 2 A resolution a mean error in atomic position of 0.2 A gives rise to an R = 0.23, and an error of 0.1 A gives rise to an R of 0.12. The Luzzati estimate of errors, which is frequently used in protein crystallography, is usually an overestimate because other sources of error also contribute to the residual R. 

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