Crystallographic protein model-building on the web

X-ray crystallography is the most widely used method to determine the 3D structure of protein molecules. One of the most difficult steps in protein crystallography is model-building, which consists of constructing a backbone and then amino acid side chains into an electron density map. Interpretation of electron density maps represents a major bottleneck in protein structure determination pipelines, and thus, automated techniques to interpret maps can greatly improve the throughput. We have developed WebTex, a simple and yet powerful web interface to TEXTAL, a program that automates this process of fitting atoms into electron density maps. TEXTAL can also be downloaded for local installation. Availability: Web interface, downloadable binaries and documentation at http://textal.tamu.edu     

A database method for automated map interpretation in protein crystallography.

A significant portion of new protein structures contain folds that are related to those seen before. During the development of a computer program that can accurately position, in electron density maps, large protein domains with large structural deviations, it became apparent that the redundancy in protein folds could be used in a non trivial manner during a protein structure determination. As a result a computational procedure, Database Assisted Density Interpretation (DADI), was developed and tested to aid in the building of models in protein crystallography and to assist in interpreting electron density maps. The initial tests of the DADI procedure using a small database of protein domains are described. The philosophy is to first work with entire domains then with the secondary structure elements of these domains and finally with individual residues of the secondary structure elements via Monte Carlo, "chopping" and "clipping" procedures, respectively. The first test case was a traceable 3.2 A multiple isomorphous replacement with anomalous scattering (MIRAS) electron density map of a human topoisomerase I-DNA complex. The second test case uses poor electron density for the third domain of the diphtheria toxin repressor resulting from a molecular replacement solution with the first two domains. Despite the fact that a fairly small database was employed in these test cases, the DADI procedure was able to find a large portion of the protein backbone with very few errors. In the first case nearly 45% of the backbone and more than 80% of the secondary structure was placed automatically. In the second test case nearly 50% of the third domain was automatically detected. A particular encouraging result was that in both cases more than 75% of the beta sheet secondary structure was found automatically by the DADI procedure. Clearly, the procedures employed are promising avenues to exploit the current explosion of protein structures for the determination of future structures. Proteins 1999;36:526-541.     

Sharpening high resolution information in single particle electron cryomicroscopy

Advances in single particle electron cryomicroscopy have made possible to elucidate routinely the structure of biological specimens at subnanometer resolution. At this resolution, secondary structure elements are discernable by their signature. However, identification and interpretation of high resolution structural features are hindered by the contrast loss caused by experimental and computational factors. This contrast loss is traditionally modeled by a Gaussian decay of structure factors with a temperature factor, or B-factor. Standard restoration procedures usually sharpen the experimental maps either by applying a Gaussian function with an inverse ad hoc B-factor, or according to the amplitude decay of a reference structure. EM-BFACTOR is a program that has been designed to widely facilitate the use of the novel method for objective B-factor determination and contrast restoration introduced by Rosenthal and Henderson [Rosenthal, P.B., Henderson, R., 2003. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721-745]. The program has been developed to interact with the most common packages for single particle electron cryomicroscopy. This sharpening method has been further investigated via EM-BFACTOR, concluding that it helps to unravel the high resolution molecular features concealed in experimental density maps, thereby making them better suited for interpretation. Therefore, the method may facilitate the analysis of experimental data in high resolution single particle electron cryomicroscopy.     

A rapid method for positioning small flexible molecules, nucleic acids, and large protein fragments in experimental electron density maps.

A Monte Carlo procedure, encoded in the program Blob, has been developed and tested for the purpose of positioning large molecular fragments or small flexible molecules in electron density maps. The search performed by the algorithm appears to be sufficiently thorough to accurately position a small flexible ligand in well-defined density while remaining sufficiently random to offer interesting alternate suggestions for density representing disordered binding modes of a ligand. Furthermore, the algorithm is shown to be efficient enough to accurately position large rigid molecular fragments. In the first of the test cases with large molecular fragments, Blob was surprisingly effective in positioning a poly-alanine model of a 53-residue domain in poor electron density resulting from molecular replacement with a partial model. At 3.0 A resolution the domain was positioned consistently within 0.2 A of its experimentally determined position. Even at 6.0 A resolution Blob could consistently position the domain to within 0.75 A of its actual position. A second set of tests with large molecular fragments revealed that Blob could correctly position large molecular fragments with quite significant deviations from the actual structure. In this test case, fragments ranging from a 170-residue protein domain with a 3.8 A rms deviation from the actual structure to a 22-base pair ideal B-form DNA duplex were positioned accurately in a 3.2 A electron density map derived from multiple isomorphous replacement methods. Even when decreasing the quality of the maps, from a figure of merit of 0.57 to as low as 0. 35, Blob could still effectively position the large protein domain and the DNA duplex. Since it is efficient, can handle large molecular fragments, and works in poor and low resolution maps, Blob could be a useful tool for interpreting electron density maps in de novo structure determinations and in molecular replacement studies. Proteins 1999;36:512-525.     

Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7

ARP/wARP is a software suite to build macromolecular models in X-ray crystallography electron density maps. Structural genomics initiatives and the study of complex macromolecular assemblies and membrane proteins all rely on advanced methods for 3D structure determination. ARP/wARP meets these needs by providing the tools to obtain a macromolecular model automatically, with a reproducible computational procedure. ARP/wARP 7.0 tackles several tasks: iterative protein model building including a high-level decision-making control module; fast construction of the secondary structure of a protein; building flexible loops in alternate conformations; fully automated placement of ligands, including a choice of the best-fitting ligand from a 'cocktail'; and finding ordered water molecules. All protocols are easy to handle by a nonexpert user through a graphical user interface or a command line. The time required is typically a few minutes although iterative model building may take a few hours.     

The aquaporin sidedness revisited

Aquaporins are transmembrane water channel proteins, which play important functions in the osmoregulation and water balance of micro-organisms, plants, and animal tissues. All aquaporins studied to date are thought to be tetrameric assemblies of four subunits each containing its own aqueous pore. Moreover, the subunits contain an internal sequence repeat forming two obversely symmetric hemichannels predicted to resemble an hour-glass. This unique arrangement of two highly related protein domains oriented at 180 degrees to each other poses a significant challenge in the determination of sidedness. Aquaporin Z (AqpZ) from Escherichia coli was reconstituted into highly ordered two-dimensional crystals. They were freeze-dried and metal-shadowed to establish the relationship between surface structure and underlying protein density by electron microscopy. The shadowing of some surfaces was prevented by protruding aggregates. Thus, images collected from freeze-dried crystals that exhibited both metal-coated and uncoated regions allowed surface relief reconstructions and projection maps to be obtained from the same crystal. Cross-correlation peak searches along lattices crossing metal-coated and uncoated regions allowed an unambiguous alignment of the surface reliefs to the underlying density maps. AqpZ topographs previously determined by AFM could then be aligned with projection maps of AqpZ, and finally with human erythrocyte aquaporin-1 (AQP1). Thereby features of the AqpZ topography could be interpreted by direct comparison to the 6 A three-dimensional structure of AQP1. We conclude that the sidedness we originally proposed for aquaporin density maps was inverted.     

Determining relevant features to recognize electron density patterns in x-ray protein crystallography

High-throughput computational methods in X-ray protein crystallography are indispensable to meet the goals of structural genomics. In particular, automated interpretation of electron density maps, especially those at mediocre resolution, can significantly speed up the protein structure determination process. TEXTAL(TM) is a software application that uses pattern recognition, case-based reasoning and nearest neighbor learning to produce reasonably refined molecular models, even with average quality data. In this work, we discuss a key issue to enable fast and accurate interpretation of typically noisy electron density data: what features should be used to characterize the density patterns, and how relevant are they? We discuss the challenges of constructing features in this domain, and describe SLIDER, an algorithm to determine the weights of these features. SLIDER searches a space of weights using ranking of matching patterns (relative to mismatching ones) as its evaluation function. Exhaustive search being intractable, SLIDER adopts a greedy approach that judiciously restricts the search space only to weight values that cause the ranking of good matches to change. We show that SLIDER contributes significantly in finding the similarity between density patterns, and discuss the sensitivity of feature relevance to the underlying similarity metric.     

Refined atomic model of the four-layer aggregate of the tobacco mosaic virus coat protein at 2.4-A resolution.

Previous x-ray studies (2.8-A resolution) on crystals of tobacco mosaic virus coat protein grown from solutions containing high salt have characterized the structure of the protein aggregate as a dimer of a bilayered cylindrical disk formed by 34 chemically identical subunits. We have determined the crystal structure of the disk aggregate at 2.4-A resolution using x-ray diffraction from crystals maintained at cryogenic temperatures. Two regions of interest have been extensively refined. First, residues of the low-radius loop region, which were not modeled previously, have been traced completely in our electron density maps. Similar to the structure observed in the virus, the right radial helix in each protomer ends around residue 87, after which the protein chain forms an extended chain that extends to the left radial helix. The left radial helix appears as a long alpha-helix with high temperature factors for the main-chain atoms in the inner portion. The side-chain atoms in this region (residues 90-110) are not visible in the electron density maps and are assumed to be disordered. Second, interactions between subunits in the symmetry-related central A pair have been determined. No direct protein-protein interactions are observed in the major overlap region between these subunits; all interactions are mediated by two layers of ordered solvent molecules. The current structure emphasizes the importance of water in biological macromolecular assemblies.     

Using known substructures in protein model building and crystallography.

Retinol binding protein can be constructed from a small number of large substructures taken from three unrelated proteins. The known structures are treated as a knowledge base from which one extracts information to be used in molecular modelling when lacking true atomic resolution. This includes the interpretation of electron density maps and modelling homologous proteins. Models can be built into maps more accurately and more quickly. This requires the use of a skeleton representation for the electron density which improves the determination of the initial chain tracing. Fragment-matching can be used to bridge gaps for inserted residues when modelling homologous proteins.     

Refinement of Protein Structures into Low-Resolution Density Maps Using Rosetta

We describe a method based on Rosetta structure refinement for generating high-resolution, all-atom protein models from electron cryomicroscopy density maps. A local measure of the fit of a model to the density is used to directly guide structure refinement and to identify regions incompatible with the density that are then targeted for extensive rebuilding. Over a range of test cases using both simulated and experimentally generated data, the method consistently increases the accuracy of starting models generated either by comparative modeling or by hand-tracing the density. The method can achieve near-atomic resolution starting from density maps at 4-6 Å resolution.     

Structural basis of the drastically increased initial electron transfer rate in the reaction center from a Rhodopseudomonas viridis mutant described at 2.00-A resolution

It has previously been shown that replacement of the residue His L168 with Phe (HL168F) in the Rhodopseudomonas viridis reaction center (RC) leads to an unprecedented drastic acceleration of the initial electron transfer rate. Here we describe the determination of the x-ray crystal structure at 2.00-A resolution of the HL168F RC. The electron density maps confirm that a hydrogen bond from the protein to the special pair is removed by this mutation. Compared with the wild-type RC, the acceptor of this hydrogen bond, the ring I acetyl group of the "special pair" bacteriochlorophyll, D(L), is rotated, and its acetyl oxygen is found 1.1 A closer to the bacteriochlorophyll-Mg(2+) of the other special pair bacteriochlorophyll, D(M). The rotation of this acetyl group and the increased interaction between the D(L) ring I acetyl oxygen and the D(M)-Mg(2+) provide the structural basis for the previously observed 80-mV decrease in the D(+)/D redox potential and the drastically increased rate of initial electron transfer to the accessory bacteriochlorophyll, B(A). The high quality of the electron density maps also allowed a reliable discussion of the mode of binding of the triazine herbicide terbutryn at the binding site of the secondary quinone, Q(B).     

Crystal structure of rubredoxin from Desulfovibrio gigas to ultra-high 0.68 A resolution

Rubredoxin (D.g. Rd) is a small non-heme iron-sulfur protein shown to function as a redox coupling protein from the sulfate reducing bacteria Desulfovibrio gigas. The protein is generally purified from anaerobic bacteria in which it is thought to be involved in electron transfer or exchange processes. Rd transfers an electron to oxygen to form water as part of a unique electron transfer chain, composed by NADH:rubredoxin oxidoreductase (NRO), rubredoxin and rubredoxin:oxygen oxidoreductase (ROO) in D.g. The crystal structure of D.g. Rd has been determined by means of both a Fe single-wavelength anomalous dispersion (SAD) signal and the direct method, and refined to an ultra-high 0.68 A resolution, using X-ray from a synchrotron. Rd contains one iron atom bound in a tetrahedral coordination by the sulfur atoms of four cysteinyl residues. Hydrophobic and pi-pi interactions maintain the internal Rd folding. Multiple conformations of the iron-sulfur cluster and amino acid residues are observed and indicate its unique mechanism of electron transfer. Several hydrogen bonds, including N-H...SG of the iron-sulfur, are revealed clearly in maps of electron density. Abundant waters bound to C-O peptides of residues Val8, Cys9, Gly10, Ala38, and Gly43, which may be involved in electron transfer. This ultrahigh-resolution structure allows us to study in great detail the relationship between structure and function of rubredoxin, such as salt bridges, hydrogen bonds, water structures, cysteine ligands, iron-sulfur cluster, and distributions of electron density among activity sites. For the first time, this information will provide a clear role for this protein in a strict anaerobic bacterium.     

A machine learning approach for the identification of protein secondary structure elements from electron cryo-microscopy density maps

The accuracy of the secondary structure element (SSE) identification from volumetric protein density maps is critical for de-novo backbone structure derivation in electron cryo-microscopy (cryoEM). It is still challenging to detect the SSE automatically and accurately from the density maps at medium resolutions (～5-10 ?). We present a machine learning approach, SSELearner, to automatically identify helices and β-sheets by using the knowledge from existing volumetric maps in the Electron Microscopy Data Bank. We tested our approach using 10 simulated density maps. The averaged specificity and sensitivity for the helix detection are 94.9% and 95.8%, respectively, and those for the β-sheet detection are 86.7% and 96.4%, respectively. We have developed a secondary structure annotator, SSID, to predict the helices and β-strands from the backbone Cα trace. With the help of SSID, we tested our SSELearner using 13 experimentally derived cryo-EM density maps. The machine learning approach shows the specificity and sensitivity of 91.8% and 74.5%, respectively, for the helix detection and 85.2% and 86.5% respectively for the β-sheet detection in cryoEM maps of Electron Microscopy Data Bank. The reduced detection accuracy reveals the challenges in SSE detection when the cryoEM maps are used instead of the simulated maps. Our results suggest that it is effective to use one cryoEM map for learning to detect the SSE in another cryoEM map of similar quality.     

Low-resolution density maps from atomic models: how stepping "back" can be a step "forward"

Atomic-resolution structures have had a tremendous impact on modern biological science. Much useful information also has been gleaned by merging and correlating atomic-resolution structural details with lower-resolution (15-40 A), three-dimensional (3D) reconstructions computed from images recorded with cryo-transmission electron microscopy (cryoTEM) procedures. One way to merge these structures involves reducing the resolution of an atomic model to a level comparable to a cryoTEM reconstruction. A low-resolution density map can be derived from an atomic-resolution structure by retrieving a set of atomic coordinates editing the coordinate file, computing structure factors from the model coordinates, and computing the inverse Fourier transform of the structure factors. This method is a useful tool for structural studies primarily in combination with 3D cryoTEM reconstructions. It has been used to assess the quality of 3D reconstructions, to determine corrections for the phase-contrast transfer function of the transmission electron microscope, to calibrate the dimensions and handedness of 3D reconstructions, to produce difference maps, to model features in macromolecules or macromolecular complexes, and to generate models to initiate model-based determination of particle orientation and origin parameters for 3D reconstruction.     

Low dose electron microscopy of the crotoxin complex thin crystal

The crotoxin complex from Crotalus d. terrificus rattlesnake venom was crystallized in the form of thin platelets. These crystals were prepared by the glucose embedding technique and examined by low dose electron microscopy. Electron diffraction patterns and images have been recorded to 2.2 and 4.5 A, respectively. By a combination of electron and X-ray diffraction techniques, the space group of this crystal was determined to be P4(2)22 with eight crotoxin complex molecules in one unit cell with dimensions of 38.8 A x 38.8 A x 256.8 A. The Patterson maps and the symmetry reliability factors calculated from the electron diffraction intensities clearly showed the existence of three types of electron diffraction patterns in different crystals. The phases in the computer-calculated transform of the low dose images also show the variation in symmetry among crystals. These phenomena are explained by the presence of crystals consisting of one-half, three-quarter and one unit cell in thickness. The interpretation of the computer reconstructed two-dimensional density map was limited, partly because of the similarity in density between the protein and the embedding glucose and partly because of the non-uniqueness in relating projected structure to the three-dimensional structure.     

Structural analysis of membrane protein complexes by single particle electron microscopy

Single particle electron microscopy (EM) is an increasingly important tool for the structural analysis of macromolecular complexes. The main advantage of the technique over other methods is that it is not necessary to precede the analysis with the growth of crystals of the sample. This advantage is particularly important for membrane proteins and large protein complexes where generating crystals is often the main barrier to structure determination. Therefore, single particle EM can be employed with great utility in the study of large membrane protein complexes. Although the construction of atomic resolution models by single particle EM is possible in theory, currently the highest resolution maps are still limited to approximately 7-10A resolution and 15-30 A resolution is more typical. However, by combining single particle EM maps with high-resolution models of subunits or subcomplexes from X-ray crystallography and NMR spectroscopy it is possible to build up an atomic model of a macromolecular assembly. Image analysis procedures are almost identical for micrographs of soluble protein complexes and detergent solubilized membrane protein complexes. However, electron microscopists attempting to prepare specimens of a membrane protein complex for imaging may find that these complexes require different handling than soluble protein complexes. This paper seeks to explain how high-quality specimen grids of membrane protein complexes may be prepared to allow for the determination of their structure by EM and image analysis.     

Sequence information within the lamB genes in required for proper routing of the bacteriophage lambda receptor protein to the outer membrane of Escherichia coli K-12

The structure of Satellite tobacco necrosis virus (STNV) has been determined to 3.0 Å resolution by X-ray crystallography. Electron density maps were obtained with phases based on one heavy-atom derivative and several cycles of phase refinement using the 60-fold non-crystallographic symmetry in the particle. A model for one protein subunit was built using a computer graphics display. The subunit is constructed mainly of a β-roll structure forming two β-sheets, each of four antiparallel strands. The N-termini of the subunits form bundles of three α-helices extending into the RNA region of the virus at the 3-fold axis. The topology of the polypeptide chain is the same as, and the conformation clearly similar to, that of the shell domains of the Tomato bushy stunt virus (TBSV) and Southern bean mosaic virus (SBMV) protein subunits. The subunit packing in the T = 1 STNV structure is, however, significantly different from the packing of these T = 3 viruses: parts of some of the structural elements facing the RNA in TBSV and SBMV are utilized for subunit-subunit contacts in STNV. No RNA structure is obvious in the present icosahedrally averaged electron density maps. The protein surface facing the RNA contains mainly hydrophilic residues, especially lysine and arginine.     

经各向异性温度因子修正后的1.2A分辨率的猪胰岛素晶体结构——温度因子的结构信息,水分子,氢原子和氢键体系

本文主要报道在1.2(?)高分辨率利用限制立体化学参数的倒易空间最小二乘技术对胰岛素的非氢原子进行各向异性温度因子修正后的结果。在修正后的电子密度图上,少数原子电子密度的各向异性分布有明显表现;80％的氢原子的电子密度有不同程度的反映;修正建立的包括水和溶剂体系,氢原子和氢键在内的胰岛素分子的精确结构模型,为各种胰岛素类似物结构的分析和比较,以及结构与功能关系的研究提供了可靠的,精度的参照和基础。     

A probabilistic approach to protein backbone tracing in electron density maps

One particularly time-consuming step in protein crystallography is interpreting the electron density map; that is, fitting a complete molecular model of the protein into a 3D image of the protein produced by the crystallographic process. In poor-quality electron density maps, the interpretation may require a significant amount of a crystallographer's time. Our work investigates automating the time-consuming initial backbone trace in poor-quality density maps. We describe ACMI (Automatic Crystallographic Map Interpreter), which uses a probabilistic model known as a Markov field to represent the protein. Residues of the protein are modeled as nodes in a graph, while edges model pairwise structural interactions. Modeling the protein in this manner allows the model to be flexible, considering an almost infinite number of possible conformations, while rejecting any that are physically impossible. Using an efficient algorithm for approximate inference--belief propagation--allows the most probable trace of the protein's backbone through the density map to be determined. We test ACMI on a set of ten protein density maps (at 2.5 to 4.0 A resolution), and compare our results to alternative approaches. At these resolutions, ACMI offers a more accurate backbone trace than current approaches.     

Statistical and conformational analysis of the electron density of protein side chains

Protein side chains make most of the specific contacts between proteins and other molecules, and their conformational properties have been studied for many years. These properties have been analyzed primarily in the form of rotamer libraries, which cluster the observed conformations into groups and provide frequencies and average dihedral angles for these groups. In recent years, these libraries have improved with higher resolution structures and using various criteria such as high thermal factors to eliminate side chains that may be misplaced within the crystallographic model coordinates. Many of these side chains have highly non-rotameric dihedral angles. The origin of side chains with high B-factors and/or with non-rotameric dihedral angles is of interest in the determination of protein structures and in assessing the prediction of side chain conformations. In this paper, using a statistical analysis of the electron density of a large set of proteins, it is shown that: (1) most non-rotameric side chains have low electron density compared to rotameric side chains; (2) up to 15% of chi1 non-rotameric side chains in PDB models can clearly be fit to density at a single rotameric conformation and in some cases multiple rotameric conformations; (3) a further 47% of non-rotameric side chains have highly dispersed electron density, indicating potentially interconverting rotameric conformations; (4) the entropy of these side chains is close to that of side chains annotated as having more than one chi(1) rotamer in the crystallographic model; (5) many rotameric side chains with high entropy clearly show multiple conformations that are not annotated in the crystallographic model. These results indicate that modeling of side chains alternating between rotamers in the electron density is important and needs further improvement, both in structure determination and in structure prediction.