Structure of form III crystals of bovine pancreatic trypsin inhibitor

The structure of bovine pancreatic trypsin inhibitor has been solved in a new crystal form III. The crystals belong to space group P2(1)2(1)2 with a = 55.2 A, b = 38.2 A, c = 24.05 A. The structure was solved on the basis of co-ordinates of forms I and II of the inhibitor by molecular replacement, and the X-ray data extending to 1.7 A were used in a restrained least-squares refinement. The final R factor was 0.16, and the deviation of bonded distances from ideality was 0.020 A. Root-mean-square discrepancy between C alpha co-ordinates of forms III and I are 0.47 A, whilst between forms II and III the discrepancy is 0.39 A. These deviations are about a factor of 3 larger than the expected experimental errors, showing that true differences exist between the three crystal forms. Two residues (Arg39 and Asp50) were modeled with two positions for their side-chains. The final model includes 73 water molecules and one phosphate group bound to the protein. Sixteen water molecules occupy approximately the same positions in all three crystal forms studied to date, indicating their close association with the protein molecule. Temperature factors also show a high degree of correlation between the three crystal forms.     

Building protein backbones from C alpha coordinates.

An automatic procedure for building polyalanine backbones from guiding alpha-carbon positions is presented. Polyalanine backbones are built based on the geometric restraints of angle N-C alpha-C and the knowledge of main-chain dihedral angle distributions. A building module constructs a list of polyalanine backbones that follow exactly the C alpha trace. Then a selection module selects one backbone with the largest portion of phi-psi pairs in favoured regions. Several test cases on C alpha coordinates from X-ray refined structures give acceptable results. Less than 10% of the peptide planes are incorrectly built, and the result is not sensitive to random shift up to 0.5 A of C alpha coordinates.     

Modelling the polypeptide backbone with 'spare parts' from known protein structures

An automatic procedure for building a protein polyalanine backbone from C alpha positions and 'spare parts' retrieved from a data base of 66 high-resolution protein structures is described. Protein backbones are constructed from overlapping fragments of variable length, which allows the backbone of regular secondary structure elements to be built in one block. The procedure is shown to yield backbones which compare very favourably with those from highly refined X-ray structures (r.m.s. deviation between generated and crystal structures less than 1A). The method is furthermore quite insensitive to experimental errors in C alpha positions as well as to the size of the data base, and is seen to yield valuable insight into the relationships between sequence and 3-D structure: one example on triose phosphate isomerase, a beta-barrel protein, shows that beta alpha loops can be considered as structurally more uncommon than alpha beta loops. The 'spare parts' approach is also found to be useful for general-purpose modelling of local structural changes produced by insertion or deletion of residues. It should, however, be used with caution. Crude selection criteria based solely on fragment length and geometric fit to the loop base regions yield realistic backbones in about two-thirds of the test cases (r.m.s. deviations from refined crystal structure approximately 1A). In the remaining cases, sequence information, in particular the presence of glycine residues which tend to adopt more unusual backbone conformations, must be considered to obtain comparable results.     

Protein structure prediction constrained by solution X-ray scattering data and structural homology identification

Here we perform a systematic exploration of the use of distance constraints derived from small angle X-ray scattering (SAXS) measurements to filter candidate protein structures for the purpose of protein structure prediction. This is an intrinsically more complex task than that of applying distance constraints derived from NMR data where the identity of the pair of amino acid residues subject to a given distance constraint is known. SAXS, on the other hand, yields a histogram of pair distances (pair distribution function), but the identities of the pairs contributing to a given bin of the histogram are not known. Our study is based on an extension of the Levitt-Hinds coarse grained approach to ab initio protein structure prediction to generate a candidate set of C(alpha) backbones. In spite of the lack of specific residue information inherent in the SAXS data, our study shows that the implementation of a SAXS filter is capable of effectively purifying the set of native structure candidates and thus provides a substantial improvement in the reliability of protein structure prediction. We test the quality of our predicted C(alpha) backbones by doing structural homology searches against the Dali domain library, and find that the results are very encouraging. In spite of the lack of local structural details and limited modeling accuracy at the C(alpha) backbone level, we find that useful information about fold classification can be extracted from this procedure. This approach thus provides a way to use a SAXS data based structure prediction algorithm to generate potential structural homologies in cases where lack of sequence homology prevents identification of candidate folds for a given protein. Thus our approach has the potential to help in determination of the biological function of a protein based on structural homology instead of sequence homology.     

Errors in three dimensions

Now that some protein X-ray structures have been proved to contain major errors, the question of the precision of 3-dimensional structures is taken seriously by crystallographers and NMR spectroscopists. Errors which cannot be avoided during model building in electron density maps, should correct themselves during crystallographic refinement, and the precision of the refined model should reach 0.15 to 0.25 A depending on the resolution of the data. Independent estimates based on homologous protein structures confirm that better than 0.5 A precision is commonly achieved, at least for C alpha and main chain atoms. The precision of NMR structures is less easily evaluated, but it should be better than 2 A when a sufficient number of NOE distance constraints are available. One may deplore the fact that not all published structures meet these standards, but possible errors should not be an excuse for not depositing atomic co-ordinates in data banks.     

Improved side-chain prediction accuracy using an ab initio potential energy function and a very large rotamer library

Accurate prediction of the placement and comformations of protein side chains given only the backbone trace has a wide range of uses in protein design, structure prediction, and functional analysis. Prediction has most often relied on discrete rotamer libraries so that rapid fitness of side-chain rotamers can be assessed against some scoring function. Scoring functions are generally based on experimental parameters from small-molecule studies or empirical parameters based on determined protein structures. Here, we describe the NCN algorithm for predicting the placement of side chains. A predominantly first-principles approach was taken to develop the potential energy function incorporating van der Waals and electrostatics based on the OPLS parameters, and a hydrogen bonding term. The only empirical knowledge used is the frequency of rotameric states from the PDB. The rotamer library includes nearly 50,000 rotamers, and is the most extensive discrete library used to date. Although the computational time tends to be longer than most other algorithms, the overall accuracy exceeds all algorithms in the literature when placing rotamers on an accurate backbone trace. Considering only the most buried residues, 80% of the total residues tested, the placement accuracy reaches 92% for chi(1), and 83% for chi(1 + 2), and an overall RMS deviation of 1 A. Additionally, we show that if information is available to restrict chi(1) to one rotamer well, then this algorithm can generate structures with an average RMS deviation of 1.0 A for all heavy side-chains atoms and a corresponding overall chi(1 + 2) accuracy of 85.0%.     

Hierarchic organization of domains in globular proteins

An automatic procedure is developed for the identification of domains in globular proteins from X-ray elucidated co-ordinates. Using this tool, domains are shown to be iteratively decomposable into subdomains, leading to a hierarchic molecular architecture.There is no convenient geometry that will fully characterize the atom by atom interdigitation at an interface between domains, and the strategy adopted here was devised to reduce this unwieldy three-dimensional problem to a closely approximating companion analysis in a plane. These analytically derived domain choices can be used subsequently to construct computer-generated, space-filling, color-coded views of the domains; and when this is done, the derived domains are seen to be completely resolved.The number of domains in a protein is a mathematically well-behaved function of the chain length, lending support to the supposition that the domains are an implicit structural consequence of the folding process. A spectrum of domains ranging in size from whole protein monomers to the individual units of secondary structure is apparent in each of the 22 proteins analyzed here.The hierarchic organization of structural domains is evidence in favor of an underlying protein folding process that proceeds by hierarchic condensation. In this highly constrained model, every pathway leading to the native state can be described by a tree of local folding interactions.     

Microsecond hydrophobic collapse in the folding of Escherichia coli dihydrofolate reductase, an alpha/beta-type protein

Using small-angle X-ray scattering combined with a continuous-flow mixing device, we monitored the microsecond compaction dynamics in the folding of Escherichia coli dihydrofolate reductase, an alpha/beta-type protein. A significant collapse of the radius of gyration from 30 A to 23.2 A occurs within 300 micros after the initiation of refolding by a urea dilution jump. The subsequent folding after the major chain collapse occurs on a considerably longer time-scale. The protein folding trajectories constructed by comparing the development of the compactness and the secondary structure suggest that the specific hydrophobic collapse model rather than the framework model better explains the experimental observations. The folding trajectory of this alpha/beta-type protein is located between those of alpha-helical and beta-sheet proteins, suggesting that native structure determines the folding landscape.     

Comparison of backbone structures of glucose isomerase from Streptomyces and Arthrobacter

The C alpha backbones of the glucose isomerase molecules of Streptomyces rubiginosus and Arthrobacter have been determined by X-ray crystallography and compared. Each molecule is a tetramer of eight-stranded alpha/beta barrels, and the mode of association of the tetramers is identical in each case. The Arthrobacter electron density shows four additional amino acids at the carboxyl terminus. There is also an insertion of six amino acids at position 277, and two individual insertions at about positions 348 and 357 (numbering according to the Streptomyces structure). There is a close structural homology throughout the whole molecule, which is most accurate up to position 325. The r.m.s. displacement for 315 homologous C alpha positions up to this position is 0.92 A.     

The building of protein structures from alpha-carbon coordinates

A procedure for the construction of complete protein structures from only alpha-carbon coordinates is described. This involves building the backbone by sequential addition of Pro, Gly, or Ala residues. This main chain structure is then refined using molecular dynamics. Side chains are constructed by sequential addition of atoms with intermediate molecular dynamics refinement. For alpha lytic protease (a structure that is mostly beta sheet) a backbone root mean square deviation (RMSD) of 0.19 A and an overall RMSD of 1.24 A from the crystallographic coordinates are attained. For troponin C (67% alpha-helix), where the coordinates are available only for the alpha-carbons, a backbone RMSD of 0.41 A and an overall RMSD of 1.68 A are attained (fits kindly provided by Dr. Michael James and Natalie Strynadka). For flavodoxin a backbone RMSD of 0.49 A and an overall RMSD of 1.64 A were attained.     

Structure of myoglobin-ethyl isocyanide. Histidine as a swinging door for ligand entry

The structure of myoglobin(Fe II)-ethyl isocyanide has been solved at 1.68 A resolution by X-ray crystallography. The isocyano group of the ligand is distorted from the linear conformation observed in solution and in model compounds. Local changes in the protein conformation are also seen. The side-chain of Arg-CD3 moves out into the solvent, and the side-chain of His-E7 swings up and away from the ligand. Both of these side-chains show disorder indicative of dynamic behavior. These outward movements of His-E7 and Arg-CD3 side-chains clear a path from the solvent to the heme iron, suggesting a mechanism for ligand entry.     

Calculation of protein backbone geometry from alpha-carbon coordinates based on peptide-group dipole alignment.

An algorithm is proposed for the conversion of a virtual-bond polypeptide chain (connected C alpha atoms) to an all-atom backbone, based on determining the most extensive hydrogen-bond network between the peptide groups of the backbone, while maintaining all of the backbone atoms in energetically feasible conformations. Hydrogen bonding is represented by aligning the peptide-group dipoles. These peptide groups are not contiguous in the amino acid sequence. The first dipoles to be aligned are those that are both sufficiently close in space to be arranged in approximately linear arrays termed dipole paths. The criteria used in the construction of dipole paths are: to assure good alignment of the greatest possible number of dipoles that are close in space; to optimize the electrostatic interactions between the dipoles that belong to different paths close in space; and to avoid locally unfavorable amino acid residue conformations. The equations for dipole alignment are solved separately for each path, and then the remaining single dipoles are aligned optimally with the electrostatic field from the dipoles that belong to the dipole-path network. A least-squares minimizer is used to keep the geometry of the alpha-carbon trace of the resulting backbone close to that of the input virtual-bond chain. This procedure is sufficient to convert the virtual-bond chain to a real chain; in applications to real systems, however, the final structure is obtained by minimizing the total ECEPP/2 (empirical conformational energy program for peptides) energy of the system, starting from the geometry resulting from the solution of the alignment equations. When applied to model alpha-helical and beta-sheet structures, the algorithm, followed by the ECEPP/2 energy minimization, resulted in an energy and backbone geometry characteristic of these alpha-helical and beta-sheet structures. Application to the alpha-carbon trace of the backbone of the crystallographic 5PTI structure of bovine pancreatic trypsin inhibitor, followed by ECEPP/2 energy minimization with C alpha-distance constraints, led to a structure with almost as low energy and root mean square deviation as the ECEPP/2 geometry analog of 5PTI, the best agreement between the crystal and reconstructed backbone being observed for the residues involved in the dipole-path network.     

Glycera dibranchiata hemoglobin. Structure and refinement at 1.5 A resolution

The coelomic cells of the common marine bloodworm Glycera dibranchiata contain several hemoglobin monomers and polydisperse polymers. We present the refined structure of one of the Glycera monomers at 1.5 A resolution. The molecular model for protein and ordered solvent for the deoxy form of the Glycera monomer has been refined to a crystallographic R-factor of 12.7% against an X-ray diffraction dataset at 1.5 A resolution. The positions of 1095 protein atoms have been determined with a maximum root-mean-square (r.m.s.) error of 0.13 A, and the r.m.s. deviation from ideal bond lengths is 0.015 A and from ideal bond angles is 1.0 degree. The r.m.s. deviation of planar groups from their least-squares planes is 0.007 A, and the r.m.s. deviation for torsion angles is 1.2 degrees for peptide groups and 16.8 degrees for side-chains. A total of 153 water molecules has been located, and they have been refined to a final average occupancy of 0.80. Multiple conformations have been found for five side-chains, and a change has been suggested for the sequence at five residues. The heme group is present in the "reverse" orientation that differs only in the positions of the vinyl beta-carbons from the "normal" orientation. The doming of the heme towards the proximal side, and the bond distances and angles of the heme and proximal histidine are typical of most deoxy globin structures. The substitution of leucine for the distal histidine residue (E7) creates an unusually hydrophobic heme pocket.     

Reconstruction of protein conformations from estimated positions of the C alpha coordinates.

Protein C alpha coordinates are used to accurately reconstruct complete protein backbones and side-chain directions. This work employs potentials of mean force to align semirigid peptide groups around the axes that connect successive C alpha atoms. The algorithm works well for all residue types and secondary structure classes and is stable for imprecise C alpha coordinates. Tests on known protein structures show that root mean square errors in predicted main-chain and C beta coordinates are usually less than 0.3 A. These results are significantly more accurate than can be obtained from competing approaches, such as modeling of backbone conformations from structurally homologous fragments.     

Automated amino acid side-chain NMR assignment of proteins using <Superscript>13</Superscript>C- and <Superscript>15</Superscript>N-resolved 3D [<Superscript>1</Superscript>H,<Superscript>1</Superscript>H]-NOESY

ASCAN is a new algorithm for automatic sequence-specific NMR assignment of amino acid side-chains in proteins, which uses as input the primary structure of the protein, chemical shift lists of (1)H(N), (15)N, (13)C(alpha), (13)C(beta) and possibly (1)H(alpha) from the previous polypeptide backbone assignment, and one or several 3D (13)C- or (15)N-resolved [(1)H,(1)H]-NOESY spectra. ASCAN has also been laid out for the use of TOCSY-type data sets as supplementary input. The program assigns new resonances based on comparison of the NMR signals expected from the chemical structure with the experimentally observed NOESY peak patterns. The core parts of the algorithm are a procedure for generating expected peak positions, which is based on variable combinations of assigned and unassigned resonances that arise for the different amino acid types during the assignment procedure, and a corresponding set of acceptance criteria for assignments based on the NMR experiments used. Expected patterns of NOESY cross peaks involving unassigned resonances are generated using the list of previously assigned resonances, and tentative chemical shift values for the unassigned signals taken from the BMRB statistics for globular proteins. Use of this approach with the 101-amino acid residue protein FimD(25-125) resulted in 84% of the hydrogen atoms and their covalently bound heavy atoms being assigned with a correctness rate of 90%. Use of these side-chain assignments as input for automated NOE assignment and structure calculation with the ATNOS/CANDID/DYANA program suite yielded structure bundles of comparable quality, in terms of precision and accuracy of the atomic coordinates, as those of a reference structure determined with interactive assignment procedures. A rationale for the high quality of the ASCAN-based structure determination results from an analysis of the distribution of the assigned side chains, which revealed near-complete assignments in the core of the protein, with most of the incompletely assigned residues located at or near the protein surface.     

Solution of the phase problem in the X-ray diffraction method for proteins with the nuclear magnetic resonance solution structure as initial model. Patterson search and refinement for the alpha-amylase inhibitor tendamistat

Patterson search calculations using the three-dimensional structure of the alpha-amylase inhibitor from Streptomyces tendae obtained from experimental nuclear magnetic resonance (n.m.r.) data were performed to study the possibility of solving the phase problem in the X-ray diffraction method with protein structures determined by n.m.r. Using all heavy atoms (C, N, O, S) of the residues 5 to 73 in the best n.m.r. structure of the alpha-amylase inhibitor (520 out of the 558 heavy atoms in the complete polypeptide chain), the maximum of the rotation function corresponded to the correct solution obtained by the previous independent determination of the crystal structure. However, additional local maxima, which are not significantly lower than the global maximum, also showed up. Performing the Patterson search with a model containing the backbone atoms and the heavy atoms of only the interior side-chains (399 atoms), which are much better defined by the n.m.r. data, the correct maximum was significantly higher than all other maxima. A translation search for the best orientation of the latter model yielded the correct solution. The energy-restrained crystallographic refinement was performed with this model to an R-factor of 26%. This corresponds approximately to the R-factor calculated for the X-ray crystal structure previously determined using the isomorphous replacement technique, if the residues 1 to 4 and 74 and all localized solvent molecules were removed from this structure. During the refinement the root-mean-square deviation between the two structures decreased from 1.03 A to 0.26 A for the polypeptide backbone and from 1.64 A to 0.73 A for all heavy atoms. There are no major local conformational differences between the two structures, with the single exception of the side-chain of Gln52.     

Cryo‐EM map interpretation and protein model‐building using iterative map segmentation

A procedure for building protein chains into maps produced by single‐particle electron cryo‐microscopy (cryo‐EM) is described. The procedure is similar to the way an experienced structural biologist might analyze a map, focusing first on secondary structure elements such as helices and sheets, then varying the contour level to identify connections between these elements. Since the high density in a map typically follows the main‐chain of the protein, the main‐chain connection between secondary structure elements can often be identified as the unbranched path between them with the highest minimum value along the path. This chain‐tracing procedure is then combined with finding side‐chain positions based on the presence of density extending away from the main path of the chain, allowing generation of a C_α model. The C_α model is converted to an all‐atom model and is refined against the map. We show that this procedure is as effective as other existing methods for interpretation of cryo‐EM maps and that it is considerably faster and produces models with fewer chain breaks than our previous methods that were based on approaches developed for crystallographic maps.     

Structure of rubredoxin from Desulfovibrio vulgaris at 1.5 A resolution

The X-ray model of rubredoxin from Desulfovibrio vulgaris has been refined against 1.5 A X-ray diffraction data collected on a diffractometer. The final model comprises 395 non-hydrogen protein atoms, and 180 solvent O atoms. The final R-value for the model with calculated H atom positions included as fixed contributions is 0.098 over all reflections greater than 2 sigma I from infinity to 1.5 A. The error in co-ordinates is estimated to be 0.08 A. The solvent model was twice redetermined during the later stages of refinement and was instrumental in its success. One sequence error has been detected and corrected (Thr21----Asp). The iron-sulfur site bond angles are distorted from true tetrahedral symmetry, as found in other rubredoxin structures. A significant deviation from tetrahedral angles is seen at C alpha atoms 9, 10, 42 and 43, interior angles of the loops binding the iron atom. The planes of two aromatic groups, Tyr4 and Trp37, are nearly parallel to, and lie under, an extended system of atoms that includes the peptide bonds preceding the first cysteine residue of each cysteine loop as well as the cysteine side-chain, the iron, and the cysteine side-chain of the opposite loop, forming a previously unrecognized extended system that may function in electron transfer.     

Comparison of two highly refined structures of bovine pancreatic trypsin inhibitor

The high resolution structures of bovine pancreatic trypsin inhibitor refined in two distinct crystal forms have been compared. One of the structures was a result of new least-squares X-ray refinement of data from crystal form I, while the other was the joint X-ray/neutron structure of crystal form II. After superposition, the molecules show an overall root-mean-squares deviation of 0.40 A for the atoms in the main chain, while the deviations for the side-chain atoms are 1.53 A. The latter number decreases to 0.61 A when those side-chains that adopted drastically different conformations are excluded from comparison. The discrepancy between atomic temperature factors in the two models was 6.7 A2, while their general trends are highly correlated. About half of the solvent molecules occupy similar positions in the two models, while the others are different. As expected, solvents with the lowest temperature factors are most likely to be common in the two crystal forms. While the two models are clearly similar, the differences are significantly larger than the errors inherent in the structure determination.