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The fact that protein structures are dynamic by nature and that structure models determined by X-ray crystallography, electron microscopy (EM) and nuclear magnetic resonance (NMR) spectroscopy have limited accuracy limits the precision with which derived properties can be reported. Here, the issue of the precision of calculated solvent-accessible surface areas (ASAs) is addressed. A number of protein structures of different sizes were selected and the effect of random shifts applied to the atomic coordinates on ASA values was investigated. Standard deviations of the ASA calculations were found to range from ∼10 to ∼80  Å². Similar values are obtained for a handful of cases in the Protein Data Bank (PDB) where `ensembles' of crystal structures were refined against the same data set. The ASA values for 69 hen egg-white lysozyme structures were calculated and a standard deviation of the ASA of 81  Å² was obtained (the average ASA value was 6571  Å²). The calculated ASA values do not show any correlation with factors such as resolution or overall temperature factors. A molecular-dynamics (MD) trajectory of lysozyme was also analysed. The ASA values during the simulation covered a range of more than 800  Å². If different programs are used, the ASA values obtained for one small protein show a spread of almost 600  Å². These results suggest that in most cases reporting ASA values with a precision better than 10  Å² is probably not realistic and a precision of 50–100  Å² would seem prudent. The precision of buried surface-area calculations for complexes is also discussed.

Human glutathione transferase A1-1 is a well studied enzyme, but despite a wealth of structural and biochemical data a number of aspects of its catalytic function are still poorly understood. Here, five new crystal structures of this enzyme are described that provide several insights. Firstly, the structure of a complex of the wild-type human enzyme with glutathione was determined for the first time at 2.0 angstroms resolution. This reveals that glutathione binds in the G site in a very similar fashion as the glutathione portion of substrate analogues in other structures and also that glutathione binding alone is sufficient to stabilize the C-terminal helix of the protein. Secondly, we have studied the complex with a decarboxylated glutathione conjugate that is known to dramatically decrease the activity of the enzyme. The T68E mutant of human glutathione transferase A1-1 recovers some of the activity that is lost with the decarboxylated glutathione, but our structures of this mutant show that none of the earlier explanations of this phenomenon are likely to be correct. Thirdly, and serendipitously, the apo structures also reveal the conformation of the crucial C-terminal region that is disordered in all previous apo structures. The C-terminal region can adopt an ordered helix-like structure even in the apo state, but shows a strong tendency to unwind. Different conformations of the C-terminal regions were observed in the apo states of the two monomers, which suggests that cooperativity could play a role in the activity of the enzyme.

All naturally occurring amino acids with the exception of glycine contain one or more chiral carbon atoms and can therefore occur in two different configurations, L (levo, left-handed) and D (dextro, right-handed). Proteins are almost exclusively built from L-amino acids. The stereochemical bias of nature is further reflected at the secondary structure level where right-handed helices are strongly preferred over left-handed helices. The handedness of helices has not received much attention in the past and is often overlooked during the analysis, description and deposition of experimentally solved protein structures. Therefore, an extensive survey of left-handed helices in the Protein Data Bank (PDB) was undertaken to analyse their frequency of occurrence, length, amino acid composition, conservation and possible structural or functional role. All left-handed helices (of four or more residues) in a non-redundant subset of the PDB, were identified using hydrogen-bonding analysis, comparison of related structures, and experimental electron density assessment to filter out likely spurious and artefactual hits. This analysis yielded 31 verified left-handed helices in a set of 7284 proteins. The phi angles of the residues in the left-handed helices lie between 30 degrees and 130 degrees and the psi angles lie between -50 degrees and 100 degrees . Most of the helices are short (four residues) and for 87% of them, it was possible to determine that they are important for the stability of the protein, for ligand binding, or as part of the active site. This suggests that, even though left-handed helices are rare, when they do occur, they are structurally or functionally significant. Four secondary structure assignment programs were tested for their ability to identify the handedness of the helices. Of these programs, only DSSP correctly assigns the handedness.

When a new protein structure has been determined, comparison with the database of known structures enables classification of its fold as new or belonging to a known class of proteins. This in turn may provide clues about the function of the protein. A large number of fold comparison programs have been developed, but they have never been subjected to a comprehensive and critical comparative analysis. Here we describe an evaluation of 11 publicly available, Web-based servers for automatic fold comparison. Both their functionality (e.g., user interface, presentation, and annotation of results) and their performance (i.e., how well established structural similarities are recognized) were assessed. The servers were subjected to a battery of performance tests covering a broad spectrum of folds as well as special cases, such as multidomain proteins, Calpha-only models, new folds, and NMR-based models. The CATH structural classification system was used as a reference. These tests revealed the strong and weak sides of each server. On the whole, CE, DALI, MATRAS, and VAST showed the best performance, but none of the servers achieved a 100% success rate. Where no structurally similar proteins are found by any individual server, it is recommended to try one or two other servers before any conclusions concerning the novelty of a fold are put on paper.