Jane S. Richardson

PHENIX: a comprehensive Python-based system for macromolecular structure solution

The PHENIX software for macromolecular structure determination is described.

MolProbity: all-atom structure validation for macromolecular crystallography

MolProbity structure validation will diagnose most local errors in macromolecular crystal structures and help to guide their correction.

The Anatomy and Taxonomy of Protein Structure

Publisher Summary This chapter investigates the anatomy and taxonomy of protein structures. A protein is a polypeptide chain made up of amino acid residues linked together in a definite sequence. Amino acids are “handed,” and naturally occurring proteins contain only L-amino acids. A simple mnemonic for that purpose is the “corncrib.” The sequence of side chains determines all that is unique about a particular protein, including its biological function and its specific three-dimensional structure. The major possible routes to knowledge of three-dimensional protein structure are prediction from the amino acid sequence and analysis of spectroscopic measurements such as circular dichroism, laser Raman spectroscopy, and nuclear magnetic resonance. The analysis and discussion of protein structure is based on the results of three-dimensional X-ray crystallography of globular proteins. The basic elements of protein structures are discussed. The most useful level at which protein structures are to be categorized is the domain, as there are many cases of multiple-domain proteins in which each separate domain resembles other entire smaller proteins. The simplest type of stable protein structure consists of polypeptide backbone wrapped more or less uniformly around the outside of a single hydrophobic core. The outline of the taxonomy is also provided in the chapter.

MolProbity: all-atom contacts and structure validation for proteins and nucleic acids

MolProbity is a general-purpose web server offering quality validation for 3D structures of proteins, nucleic acids and complexes. It provides detailed all-atom contact analysis of any steric problems within the molecules as well as updated dihedral-angle diagnostics, and it can calculate and display the H-bond and van der Waals contacts in the interfaces between components. An integral step in the process is the addition and full optimization of all hydrogen atoms, both polar and nonpolar. New analysis functions have been added for RNA, for interfaces, and for NMR ensembles. Additionally, both the web site and major component programs have been rewritten to improve speed, convenience, clarity and integration with other resources. MolProbity results are reported in multiple forms: as overall numeric scores, as lists or charts of local problems, as downloadable PDB and graphics files, and most notably as informative, manipulable 3D kinemage graphics shown online in the KiNG viewer. This service is available free to all users at http://molprobity.biochem.duke.edu.

Structure validation by Cα geometry: ϕ,ψ and Cβ deviation

Geometrical validation around the Cα is described, with a new Cβ measure and updated Ramachandran plot. Deviation of the observed Cβ atom from ideal position provides a single measure encapsulating the major structure‐validation information contained in bond angle distortions. Cβ deviation is sensitive to incompatibilities between sidechain and backbone caused by misfit conformations or inappropriate refinement restraints. A new ϕ,ψ plot using density‐dependent smoothing for 81,234 non‐Gly, non‐Pro, and non‐prePro residues with B < 30 from 500 high‐resolution proteins shows sharp boundaries at critical edges and clear delineation between large empty areas and regions that are allowed but disfavored. One such region is the γ‐turn conformation near +75°,−60°, counted as forbidden by common structure‐validation programs; however, it occurs in well‐ordered parts of good structures, it is overrepresented near functional sites, and strain is partly compensated by the γ‐turn H‐bond. Favored and allowed ϕ,ψ regions are also defined for Pro, pre‐Pro, and Gly (important because Gly ϕ,ψ angles are more permissive but less accurately determined). Details of these accurate empirical distributions are poorly predicted by previous theoretical calculations, including a region left of α‐helix, which rates as favorable in energy yet rarely occurs. A proposed factor explaining this discrepancy is that crowding of the two‐peptide NHs permits donating only a single H‐bond. New calculations by Hu et al. [Proteins 2002 (this issue)] for Ala and Gly dipeptides, using mixed quantum mechanics and molecular mechanics, fit our nonrepetitive data in excellent detail. To run our geometrical evaluations on a user‐uploaded file, see MOLPROBITY (http://kinemage.biochem.duke.edu) or RAMPAGE (http://www‐cryst.bioc.cam.ac.uk/rampage). Proteins 2003;50:437–450.

Structure validation by Calpha geometry: phi,psi and Cbeta deviation.

Geometrical validation around the Cα is described, with a new Cβ measure and updated Ramachandran plot. Deviation of the observed Cβ atom from ideal position provides a single measure encapsulating the major structure‐validation information contained in bond angle distortions. Cβ deviation is sensitive to incompatibilities between sidechain and backbone caused by misfit conformations or inappropriate refinement restraints. A new ϕ,ψ plot using density‐dependent smoothing for 81,234 non‐Gly, non‐Pro, and non‐prePro residues with B < 30 from 500 high‐resolution proteins shows sharp boundaries at critical edges and clear delineation between large empty areas and regions that are allowed but disfavored. One such region is the γ‐turn conformation near +75°,−60°, counted as forbidden by common structure‐validation programs; however, it occurs in well‐ordered parts of good structures, it is overrepresented near functional sites, and strain is partly compensated by the γ‐turn H‐bond. Favored and allowed ϕ,ψ regions are also defined for Pro, pre‐Pro, and Gly (important because Gly ϕ,ψ angles are more permissive but less accurately determined). Details of these accurate empirical distributions are poorly predicted by previous theoretical calculations, including a region left of α‐helix, which rates as favorable in energy yet rarely occurs. A proposed factor explaining this discrepancy is that crowding of the two‐peptide NHs permits donating only a single H‐bond. New calculations by Hu et al. [Proteins 2002 (this issue)] for Ala and Gly dipeptides, using mixed quantum mechanics and molecular mechanics, fit our nonrepetitive data in excellent detail. To run our geometrical evaluations on a user‐uploaded file, see MOLPROBITY (http://kinemage.biochem.duke.edu) or RAMPAGE (http://www‐cryst.bioc.cam.ac.uk/rampage). Proteins 2003;50:437–450.

The Penultimate Rotamer Library

All published rotamer libraries contain some rotamers that exhibit impossible internal atomic overlaps if built in ideal geometry with all hydrogen atoms. Removal of uncertain residues (mainly those with B‐factors ≥40 or van der Waals overlaps ≥0.4 Å) greatly improves the clustering of rotamer populations. Asn, Gln, or His side chains additionally benefit from flipping of their planar terminal groups when required by atomic overlaps or H‐bonding. Sensitivity to skew and to the boundaries of χ angle bins is avoided by using modes rather than traditional mean values. Rotamer definitions are listed both as the modal values and in a preferred version that maximizes common atoms between related rotamers. The resulting library shows significant differences from previous ones, differences validated by considering the likelihood of systematic misfitting of models to electron density maps and by plotting changes in rotamer frequency with B‐factor. Few rotamers now show atomic overlaps in ideal geometry; those overlaps are relatively small and can be understood in terms of bond angle distortions compensated by favorable interactions. The new library covers 94.5% of examples in the highest quality protein data with 153 rotamers and can make a significant contribution to improving the accuracy of new structures. Proteins 2000;40:389–408.

Determination and analysis of the 2 A-structure of copper, zinc superoxide dismutase.

The structure of bovine erythrocyte Cu, Zn superoxide dismutase has been determined to 2 A resolution using only the larger structure factors beyond 4 A. The enzyme crystallizes in space group C2 with two dimeric enzyme molecules per asymmetric unit. All four crystallographically independent subunits were fitted separately to the electron density map at 2 A resolution on the University of North Carolina GRIP-75 molecular graphics system. Atomic co-ordinates were refined using the Hendrickson & Konnert (1980) program for stereochemically restrained refinement against structure factors, which allowed the use of non-crystallographic symmetry. The crystallographic residual error for the refined model was 25.5% with a root-mean-square deviation of 0.03 A from ideal bond lengths and an average atomic temperature factor of 12 A2. Each enzyme subunit is composed primarily of eight antiparallel β strands that form a flattened cylinder, plus three external loops. The β barrel is asymmetrical and can be viewed as having two distinct sides; β strands 5 to 8 are shorter with fewer hydrogen bonds, less regular side-chain alternation, and greater twist than strands 1 to 4. The main-chain hydrogen bonds primarily link β strand residues; side-chain to main-chain hydrogen bonds are extensively involved in the formation of tight turns, which form a major structural element of the three loops. The largest loop includes both a disulfide region and a Zn-liganding region, each of which resembles one of the other two loops in overall structure. The second largest loop includes a short section of α helix. The smallest loop forms a Greek key connection across one end of the β barrel. The single disulfide bond, which forms a left-handed spiral, covalently joins the largest loop to the beginning of β strand 8. Symmetrically related β bulge pairs fold the two large loops back against the external surface of the β barrel to surround the active channel. The active site Cu(II) and Zn(II) lie 6.3 A apart at the bottom of this long channel; the Zn is buried, while the Cu is solvent-accessible. The side-chain of His61 forms a bridge between the Cu and Zn and is coplanar with them within the current accuracy of the data. The Cu ligands ND1 of His44 and NE2 of His46, −61 and −118 show an uneven tetrahedral distortion from a square plane. The Cu has a fifth axial coordination position exposed to solvent. Zn ligands ND1 of His61, −69 and −78 and OD1 of Asp81 show tetrahedral geometry with a strong distortion toward a trigonal pyramid having the buried Asp81 at the apex. Both the side-chains and mainchains of the metal-liganding residues are stabilized in their orientation by a complex network of hydrogen bonds.

MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes.

MolProbity is a general-purpose web service offering quality validation for three-dimensional (3D) structures of proteins, nucleic acids and complexes. It provides detailed all-atom contact analysis of any steric problems within the molecules and can calculate and display the H-bond and van der Waals contacts in the interfaces between components. An integral step in the process is the addition and full optimization of all hydrogen atoms, both polar and nonpolar. The results are reported in multiple forms: as overall numeric scores, as lists, as downloadable PDB and graphics files, and most notably as informative, manipulable 3D kinemage graphics shown on-line in the KiNG viewer. This service is available free to all users at http://kinemage.biochem.duke.edu.

Natural β-sheet proteins use negative design to avoid edge-to-edge aggregation

The fact that natural β-sheet proteins are usually soluble but that fragments or designs of β structure usually aggregate suggests that natural β proteins must somehow be designed to avoid this problem. Regular β-sheet edges are dangerous, because they are already in the right conformation to interact with any other β strand they encounter. We surveyed edge strands in a large sample of all-β proteins to tabulate features that could protect against further β-sheet interactions. β-barrels, of course, avoid edges altogether by continuous H-bonding around the barrel cylinder. Parallel β-helix proteins protect their β-sheet ends by covering them with loops of other structure. β-propeller and single-sheet proteins use a combination of β-bulges, prolines, strategically placed charges, very short edge strands, and loop coverage. β-sandwich proteins favor placing an inward-pointing charged side chain on one of the edge strands where it would be buried by dimerization; they also use bulges, prolines, and other mechanisms. One recent β-hairpin design has a constrained twist too great for accommodation into a larger β-sheet, whereas some β-sheet edges are protected by the bend and reverse twist produced by an Lβ glycine. All free edge strands were seen to be protected, usually by several redundant mechanisms. In contrast, edge strands that natively form β H-bonded dimers or rings have long, regular stretches without such protection. These results are relevant to understanding how proteins may assemble into β-sheet amyloid fibers, and they are especially applicable to the de novo design of β structure. Many edge-protection strategies used by natural proteins are beyond our current abilities to constrain by design, but one possibility stands out as especially useful: a single charged side chain near the middle of what would ordinarily be the hydrophobic side of the edge β strand. This minimal negative-design strategy changes only one residue, requires no backbone distortion, and is easy to design. The accompanying paper [Wang, W. & Hecht, M. H. (2002) Proc. Natl. Acad. Sci. USA 99, 2760–2765] makes use of the inward-pointing charge strategy with great success, turning highly aggregated β-sandwich designs into soluble monomers.