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Electron-density Maps (electron-density + map)
Kinds of Electron-density Maps Selected AbstractsCrystallization of a pentapeptide-repeat protein by reductive cyclic pentylation of free amines with glutaraldehydeACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Matthew W. Vetting The pentapeptide-repeat protein EfsQnr from Enterococcus faecalis protects DNA gyrase from inhibition by fluoroquinolones. EfsQnr was cloned and purified to homogeneity, but failed to produce diffraction-quality crystals in initial crystallization screens. Treatment of EfsQnr with glutaraldehyde and the strong reducing agent borane,dimethylamine resulted in a derivatized protein which produced crystals that diffracted to 1.6,Å resolution; their structure was subsequently determined by single-wavelength anomalous dispersion. Analysis of the derivatized protein using Fourier transform ion cyclotron resonance mass spectrometry indicated a mass increase of 68,Da per free amino group. Electron-density maps about a limited number of structurally ordered lysines indicated that the modification was a cyclic pentylation of free amines, producing piperidine groups. [source] Structure of BthA-I complexed with p -bromophenacyl bromide: possible correlations with lack of pharmacological activityACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2005Angelo J. Magro The crystal structure of an acidic phospholipase A2 isolated from Bothrops jararacussu venom (BthA-I) chemically modified with p -bromophenacyl bromide (BPB) has been determined at 1.85,Å resolution. The catalytic, platelet-aggregation inhibition, anticoagulant and hypotensive activities of BthA-I are abolished by ligand binding. Electron-density maps permitted unambiguous identification of inhibitor covalently bound to His48 in the substrate-binding cleft. The BthA-I,BPB complex contains three structural regions that are modified after inhibitor binding: the Ca2+ -binding loop, ,-wing and C-terminal regions. Comparison of BthA-I,BPB with two other BPB-inhibited PLA2 structures suggests that in the absence of Na+ ions at the Ca2+ -binding loop, this loop and other regions of the PLA2s undergo structural changes. The BthA-I,BPB structure reveals a novel oligomeric conformation. This conformation is more energetically and conformationally stable than the native structure and the abolition of pharmacological activities by the ligand may be related to the oligomeric structural changes. A residue of the `pancreatic' loop (Lys69), which is usually attributed as providing the anticoagulant effect, is in the dimeric interface of BthA-I,BPB, leading to a new hypothesis regarding the abolition of this activity by BPB. [source] X-ray investigation of gene-engineered human insulin crystallized from a solution containing polysialic acidACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010V. I. Timofeev Attempts to crystallize the noncovalent complex of recombinant human insulin with polysialic acid were carried out under normal and microgravity conditions. Both crystal types belonged to the same space group, I213, with unit-cell parameters a = b = c = 77.365,Å, , = , = , = 90.00°. The reported space group and unit-cell parameters are almost identical to those of cubic insulin reported in the PDB. The results of X-ray studies confirmed that the crystals obtained were cubic insulin crystals and that they contained no polysialic acid or its fragments. Electron-density maps were calculated using X-ray diffraction sets from earth-grown and microgravity-grown crystals and the three-dimensional structure of the insulin molecule was determined and refined. The conformation and secondary-structural elements of the insulin molecule in different crystal forms were compared. [source] A complicated quasicrystal approximant ,16 predicted by the strong-reflections approachACTA CRYSTALLOGRAPHICA SECTION B, Issue 1 2010Mingrun Li The structure of a complicated quasicrystal approximant ,16 was predicted from a known and related quasicrystal approximant ,6 by the strong-reflections approach. Electron-diffraction studies show that in reciprocal space, the positions of the strongest reflections and their intensity distributions are similar for both approximants. By applying the strong-reflections approach, the structure factors of ,16 were deduced from those of the known ,6 structure. Owing to the different space groups of the two structures, a shift of the phase origin had to be applied in order to obtain the phases of ,16. An electron-density map of ,16 was calculated by inverse Fourier transformation of the structure factors of the 256 strongest reflections. Similar to that of ,6, the predicted structure of ,16 contains eight layers in each unit cell, stacked along the b axis. Along the b axis, ,16 is built by banana-shaped tiles and pentagonal tiles; this structure is confirmed by high-resolution transmission electron microscopy (HRTEM). The simulated precession electron-diffraction (PED) patterns from the structure model are in good agreement with the experimental ones. ,16 with 153 unique atoms in the unit cell is the most complicated approximant structure ever solved or predicted. [source] Performance of phased rotation, conformation and translation function: accurate protein model building with tripeptidic and tetrapeptidic fragmentsACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2010Franti, ek Pavel The automatic building of protein structures with tripeptidic and tetrapeptidic fragments was investigated. The oligopeptidic conformers were positioned in the electron-density map by a phased rotation, conformation and translation function and refined by a real-space refinement. The number of successfully located fragments lay within the interval 75,95% depending on the resolution and phase quality. The overlaps of partially located fragments were analyzed. The correctly positioned fragments were connected into chains. Chains formed in this way were extended directly into the electron density and a sequence was assigned. In the initial stage of the model building the number of located fragments was between 60% and 95%, but this number could be increased by several cycles of reciprocal-space refinement and automatic model rebuilding. A nearly complete structure can be obtained on the condition that the resolution is reasonable. Computer graphics will only be needed for a final check and small corrections. [source] Features of the secondary structure of a protein molecule from powder diffraction dataACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010Sebastian Basso Protein powder diffraction is shown to be suitable for obtaining de novo solutions to the phase problem at low resolution via phasing methods such as the isomorphous replacement method. Two heavy-atom derivatives (a gadolinium derivative and a holmium derivative) of the tetragonal form of hen egg-white lysozyme were crystallized at room temperature. Using synchrotron radiation, high-quality powder patterns were collected in which pH-induced anisotropic lattice-parameter changes were exploited in order to reduce the challenging and powder-specific problem of overlapping reflections. The phasing power of two heavy-atom derivatives in a multiple isomorphous replacement analysis enabled molecular structural information to be obtained up to approximately 5.3,Å resolution. At such a resolution, features of the secondary structure of the lysozyme molecule can be accurately located using programs dedicated to that effect. In addition, the quoted resolution is sufficient to determine the correct hand of the heavy-atom substructure which leads to an electron-density map representing the protein molecule of proper chirality. [source] X-ray structure of the metcyano form of dehaloperoxidase from Amphitrite ornata: evidence for photoreductive dissociation of the iron,cyanide bondACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010V. S. De Serrano X-ray crystal structures of the metcyano form of dehaloperoxidase-hemoglobin (DHP A) from Amphitrite ornata (DHPCN) and the C73S mutant of DHP A (C73SCN) were determined using synchrotron radiation in order to further investigate the geometry of diatomic ligands coordinated to the heme iron. The DHPCN structure was also determined using a rotating-anode source. The structures show evidence of photoreduction of the iron accompanied by dissociation of bound cyanide ion (CN,) that depend on the intensity of the X-ray radiation and the exposure time. The electron density is consistent with diatomic molecules located in two sites in the distal pocket of DHPCN. However, the identities of the diatomic ligands at these two sites are not uniquely determined by the electron-density map. Consequently, density functional theory calculations were conducted in order to determine whether the bond lengths, angles and dissociation energies are consistent with bound CN, or O2 in the iron-bound site. In addition, molecular-dynamics simulations were carried out in order to determine whether the dynamics are consistent with trapped CN, or O2 in the second site of the distal pocket. Based on these calculations and comparison with a previously determined X-ray crystal structure of the C73S,O2 form of DHP [de Serrano et al. (2007), Acta Cryst. D63, 1094,1101], it is concluded that CN, is gradually replaced by O2 as crystalline DHP is photoreduced at 100,K. The ease of photoreduction of DHP A is consistent with the reduction potential, but suggests an alternative activation mechanism for DHP A compared with other peroxidases, which typically have reduction potentials that are 0.5,V more negative. The lability of CN, at 100,K suggests that the distal pocket of DHP A has greater flexibility than most other hemoglobins. [source] Multivariate phase combination improves automated crystallographic model buildingACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010Pavol Skubák Density modification is a standard technique in macromolecular crystallography that can significantly improve an initial electron-density map. To obtain optimal results, the initial and density-modified map are combined. Current methods assume that these two maps are independent and propagate the initial map information and its accuracy indirectly through previously determined coefficients. A multivariate equation has been derived that no longer assumes independence between the initial and density-modified map, considers the observed diffraction data directly and refines the errors that can occur in a single-wavelength anomalous diffraction experiment. The equation has been implemented and tested on over 100 real data sets. The results are dramatic: the method provides significantly improved maps over the current state of the art and leads to many more structures being built automatically. [source] Recent developments in classical density modificationACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2010Kevin Cowtan Classical density-modification techniques (as opposed to statistical approaches) offer a computationally cheap method for improving phase estimates in order to provide a good electron-density map for model building. The rise of statistical methods has lead to a shift in focus away from the classical approaches; as a result, some recent developments have not made their way into classical density-modification software. This paper describes the application of some recent techniques, including most importantly the use of prior phase information in the likelihood estimation of phase errors within a classical density-modification framework. The resulting software gives significantly better results than comparable classical methods, while remaining nearly two orders of magnitude faster than statistical methods. [source] Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoproteinACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2009Jeffrey E. Lee The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008), Nature (London), 454, 177,182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glycoprotein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B -value-sharpened electron-density maps and the proper sequence register was confirmed by building alternate sequences using N-linked glycan sites as anchors and secondary-structural predictions. [source] A description of the structural determination procedures of a gap junction channel at 3.5,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2009Michihiro Suga Intercellular signalling is an essential characteristic of multicellular organisms. Gap junctions, which consist of arrays of intercellular channels, permit the exchange of ions and small molecules between adjacent cells. Here, the structural determination of a gap junction channel composed of connexin 26 (Cx26) at 3.5,Å resolution is described. During each step of the purification process, the protein was examined using electron microscopy and/or dynamic light scattering. Dehydration of the crystals improved the resolution limits. Phase refinement using multi-crystal averaging in conjunction with noncrystallographic symmetry averaging based on strictly determined noncrystallographic symmetry operators resulted in an electron-density map for model building. The amino-acid sequence of a protomer structure consisting of the amino-terminal helix, four transmembrane helices and two extracellular loops was assigned to the electron-density map. The amino-acid assignment was confirmed using six selenomethionine (SeMet) sites in the difference Fourier map of the SeMet derivative and three intramolecular disulfide bonds in the anomalous difference Fourier map of the native crystal. [source] Decision-making in structure solution using Bayesian estimates of map quality: the PHENIX AutoSol wizardACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2009Thomas C. Terwilliger Estimates of the quality of experimental maps are important in many stages of structure determination of macromolecules. Map quality is defined here as the correlation between a map and the corresponding map obtained using phases from the final refined model. Here, ten different measures of experimental map quality were examined using a set of 1359 maps calculated by re-analysis of 246 solved MAD, SAD and MIR data sets. A simple Bayesian approach to estimation of map quality from one or more measures is presented. It was found that a Bayesian estimator based on the skewness of the density values in an electron-density map is the most accurate of the ten individual Bayesian estimators of map quality examined, with a correlation between estimated and actual map quality of 0.90. A combination of the skewness of electron density with the local correlation of r.m.s. density gives a further improvement in estimating map quality, with an overall correlation coefficient of 0.92. The PHENIX AutoSol wizard carries out automated structure solution based on any combination of SAD, MAD, SIR or MIR data sets. The wizard is based on tools from the PHENIX package and uses the Bayesian estimates of map quality described here to choose the highest quality solutions after experimental phasing. [source] Structure of SRP14 from the Schizosaccharomyces pombe signal recognition particleACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Mark A. Brooks The signal recognition particle (SRP) Alu domain has been implicated in translation elongation arrest in yeasts and mammals. Fission yeast SRP RNA is similar to that of mammals, but has a minimal Alu -domain RNA lacking two stem-loops. The mammalian Alu -domain proteins SRP9 and SRP14 bind their cognate Alu RNA as a heterodimer. However, in yeasts, notably Saccaromyces cerevisiae, SRP14 is thought to bind Alu RNA as a homodimer, the SRP9 protein being replaced by SRP21, the function of which is not yet clear. Structural characterization of the Schizosaccharomyces pombe Alu domain may thus help to identify the critical features required for elongation arrest. Here, the crystal structure of the SRP14 subunit of S. pombe SRP (SpSRP14) which crystallizes as a homodimer, is presented. Comparison of the SpSRP14 homodimer with the known structure of human SRP9/14 in complex with Alu RNA suggests that many of the protein,RNA contacts centred on the conserved U-turn motif are likely to be conserved in fission yeast. Initial attempts to solve the structure using traditional selenomethionine SAD labelling failed. However, two As atoms originating from the cacodylate buffer were found to make cysteine adducts and strongly contributed to the anomalous substructure. These adducts were highly radiation-sensitive and this property was exploited using the RIP (radiation-damage-induced phasing) method. The combination of SAD and RIP phases yielded an interpretable electron-density map. This example will be of general interest to crystallographers attempting de novo phasing from crystals grown in cacodylate buffer. [source] Structure of a fatty acid-binding protein from Bacillus subtilis determined by sulfur-SAD phasing using in-house chromium radiationACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Jie Nan Sulfur single-wavelength anomalous dispersion (S-SAD) and halide-soaking methods are increasingly being used for ab initio phasing. With the introduction of in-house Cr X-ray sources, these methods benefit from the enhanced anomalous scattering of S and halide atoms, respectively. Here, these methods were combined to determine the crystal structure of BsDegV, a DegV protein-family member from Bacillus subtilis. The protein was cocrystallized with bromide and low-redundancy data were collected to 2.5,Å resolution using Cr,K, radiation. 17 heavy-atom sites (ten sulfurs and seven bromides) were located using standard methods. The anomalous scattering of some of the BsDegV S atoms and Br atoms was weak, thus neither sulfurs nor bromides could be used alone for structure determination using the collected data. When all 17 heavy-atom sites were used for SAD phasing, an easily interpretable electron-density map was obtained after density modification. The model of BsDegV was built automatically and a palmitate was found tightly bound in the active site. Sequence alignment and comparisons with other known DegV structures provided further insight into the specificity of fatty-acid selection and recognition within this protein family. [source] De novo sulfur SAD phasing of the lysosomal 66.3,kDa protein from mouseACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2009Kristina Lakomek The 66.3,kDa protein from mouse is a soluble protein of the lysosomal matrix. It is synthesized as a glycosylated 75,kDa preproprotein which is further processed into 28 and 40,kDa fragments. Despite bioinformatics approaches and molecular characterization of the 66.3,kDa protein, the mode of its maturation as well as its physiological function remained unknown. Therefore, it was decided to tackle this question by means of X-ray crystallography. After expression in a human fibrosarcoma cell line, the C-terminally His-tagged single-chain 66.3,kDa variant and the double-chain form consisting of a 28,kDa fragment and a 40,kDa fragment were purified to homogeneity but could not be separated during the purification procedure. This mixture was therefore used for crystallization. Single crystals were obtained and the structure of the 66.3,kDa protein was solved by means of sulfur SAD phasing using data collected at a wavelength of 1.9,Å on the BESSY beamline BL14.2 of Freie Universität Berlin. Based on the anomalous signal, a 22-atom substructure comprising 21 intrinsic S atoms and one Xe atom with very low occupancy was found and refined at a resolution of 2.4,Å using the programs SHELXC/D and SHARP. Density modification using SOLOMON and DM resulted in a high-quality electron-density map, enabling automatic model building with ARP/wARP. The initial model contained 85% of the amino-acid residues expected to be present in the asymmetric unit of the crystal. Subsequently, the model was completed and refined to an Rfree factor of 19.8%. The contribution of the single Xe atom to the anomalous signal was analyzed in comparison to that of the S atoms and was found to be negligible. This work should encourage the use of the weak anomalous scattering of intrinsic S atoms in SAD phasing, especially for proteins, which require both expensive and time-consuming expression and purification procedures, preventing extensive screening of heavy-atom crystal soaks. [source] Crystal packing of the c6 -type cytochrome OmcF from Geobacter sulfurreducens is mediated by an N-terminal Strep-tag IIACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2008Peer Lukat The putative outer membrane c -type cytochrome OmcF from Geobacter sulfurreducens contains a single haem group and shows homology to soluble cytochromes c6, a class of electron-transfer proteins that are typically found in cyanobacterial photosynthetic electron-transfer chains. OmcF was overexpressed heterologously in Escherichia coli as an N-terminal Strep-tag II fusion protein and isolated using streptactin-affinity chromatography followed by size-exclusion chromatography. The structure was solved by Fe SAD using data collected to a resolution of 1.86,Å on a rotating copper-anode X-ray generator. In the crystal, packing interactions in one dimension were exclusively mediated through the Strep-tag II sequence. The tag and linker regions were in contact with three further monomers of OmcF, leading to a well defined electron-density map for this engineered and secondary-structure-free region of the molecule. [source] Pattern-recognition-based detection of planar objects in three-dimensional electron-density mapsACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2008Johan Hattne A pattern-recognition-based method for the detection of planar objects in protein or DNA/RNA crystal structure determination is described. The procedure derives a set of rotation-invariant numeric features from local regions in the asymmetric unit of a crystallographic electron-density map. These features, primarily moments of various orders, capture different aspects of the local shape of objects in the electron density. Feature classification is achieved using a linear discriminant that is trained to optimize the contrast between planar and nonplanar objects. In five selected test cases with X-ray data spanning 2.0,3.0,Å resolution, the procedure identified the correct location and orientation for almost all of the double-ring and a majority of the single-ring planar groups. The accuracy of the location of the plane centres is of the order of 0.5,Å, even in moderately noisy density maps. [source] Structure of the ribosomal protein L1,mRNA complex at 2.1,Å resolution: common features of crystal packing of L1,RNA complexesACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2006S. Tishchenko The crystal structure of a hybrid complex between the bacterial ribosomal protein L1 from Thermus thermophilus and a Methanococcus vannielii mRNA fragment containing an L1-binding site was determined at 2.1,Å resolution. It was found that all polar atoms involved in conserved protein,RNA hydrogen bonds have high values of density in the electron-density map and that their hydrogen-bonding capacity is fully realised through interactions with protein atoms, water molecules and K+ ions. Intermolecular contacts were thoroughly analyzed in the present crystals and in crystals of previously determined L1,RNA complexes. It was shown that extension of the RNA helices providing canonical helix stacking between open,open or open,closed ends of RNA fragments is a common feature of these and all known crystals of complexes between ribosomal proteins and RNAs. In addition, the overwhelming majority of complexes between ribosomal proteins and RNA molecules display crystal contacts formed by the central parts of the RNA fragments. These contacts are often very extensive and strong and it is proposed that they are formed in the saturated solution prior to crystal formation. [source] HKL -3000: the integration of data reduction and structure solution , from diffraction images to an initial model in minutesACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2006Marcin Cymborowski A new approach that integrates data collection, data reduction, phasing and model building significantly accelerates the process of structure determination and on average minimizes the number of data sets and synchrotron time required for structure solution. Initial testing of the HKL -3000 system (the beta version was named HKL -2000_ph) with more than 140 novel structure determinations has proven its high value for MAD/SAD experiments. The heuristics for choosing the best computational strategy at different data resolution limits of phasing signal and crystal diffraction are being optimized. The typical end result is an interpretable electron-density map with a partially built structure and, in some cases, an almost complete refined model. The current development is oriented towards very fast structure solution in order to provide feedback during the diffraction experiment. Work is also proceeding towards improving the quality of phasing calculation and model building. [source] The structure of the hexagonal crystal form of hen egg-white lysozymeACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2006M. S. Weiss The three-dimensional structure of hen egg-white lysozyme (HEWL) in a hexagonal crystal form has been determined and refined to 1.46,Å resolution. This hexagonal crystal form crystallizes from a saturated sodium nitrate solution at pH 8.4. The crystals belong to space group P6122, with unit-cell parameters a = b = 85.64, c = 67.93,Å. A total of 165 water molecules, 16 nitrate ions and five sodium ions were located in the electron-density map. The hexagonal crystal form exhibits a higher solvent content and a higher degree of disorder than other crystal forms of lysozyme. The flexibility of the protein depends on the crystal packing, although some residue ranges are flexible in all native HEWL crystal forms. [source] Real-space protein-model completion: an inverse-kinematics approachACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2005Henry Van Den Bedem Rapid protein-structure determination relies greatly on software that can automatically build a protein model into an experimental electron-density map. In favorable circumstances, various software systems are capable of building over 90% of the final model. However, completeness falls off rapidly with the resolution of the diffraction data. Manual completion of these partial models is usually feasible, but is time-consuming and prone to subjective interpretation. Except for the N- and C-termini of the chain, the end points of each missing fragment are known from the initial model. Hence, fitting fragments reduces to an inverse-kinematics problem. A method has been developed that combines fast inverse-kinematics algorithms with a real-space torsion-angle refinement procedure in a two-stage approach to fit missing main-chain fragments into the electron density between two anchor points. The first stage samples a large number of closing conformations, guided by the electron density. These candidates are ranked according to density fit. In a subsequent refinement stage, optimization steps are projected onto a carefully chosen subspace of conformation space to preserve rigid geometry and closure. Experimental results show that fitted fragments are in excellent agreement with the final refined structure for lengths of up to 12,15 residues in areas of weak or ambiguous electron density, even at medium to low resolution. [source] Making the most of two crystals: structural analysis of a conserved hypothetical protein using native gel screening and SAD phasingACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2003J. Shaun Lott The protein PAE2307 is a member of a protein family of unknown function which is conserved among a number of bacterial and archaeal species. The protein was overexpressed in Escherichia coli, purified and crystallized in two crystal forms. The prevalent form was twinned, but the other diffracted to 1.45,Å resolution. The non-twinned crystals proved difficult to reproduce, so screening of potential heavy-atom derivatives by native polyacrylamide gel electrophoresis was used to establish suitable derivatization conditions. This process enabled the production of a K2Pt(NO2)4 derivative that was used to collect a single-wavelength anomalous diffraction (SAD) data set from the only available crystal. Phase information of high quality was obtained, enabling the calculation of an interpretable electron-density map. [source] Statistical density modification using local pattern matchingACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2003Thomas C. Terwilliger A method for improving crystallographic phases is presented that is based on the preferential occurrence of certain local patterns of electron density in macromolecular electron-density maps. The method focuses on the relationship between the value of electron density at a point in the map and the pattern of density surrounding this point. Patterns of density that can be superimposed by rotation about the central point are considered equivalent. Standard templates are created from experimental or model electron-density maps by clustering and averaging local patterns of electron density. The clustering is based on correlation coefficients after rotation to maximize the correlation. Experimental or model maps are also used to create histograms relating the value of electron density at the central point to the correlation coefficient of the density surrounding this point with each member of the set of standard patterns. These histograms are then used to estimate the electron density at each point in a new experimental electron-density map using the pattern of electron density at points surrounding that point and the correlation coefficient of this density to each of the set of standard templates, again after rotation to maximize the correlation. The method is strengthened by excluding any information from the point in question from both the templates and the local pattern of density in the calculation. A function based on the origin of the Patterson function is used to remove information about the electron density at the point in question from nearby electron density. This allows an estimation of the electron density at each point in a map, using only information from other points in the process. The resulting estimates of electron density are shown to have errors that are nearly independent of the errors in the original map using model data and templates calculated at a resolution of 2.6,Å. Owing to this independence of errors, information from the new map can be combined in a simple fashion with information from the original map to create an improved map. An iterative phase-improvement process using this approach and other applications of the image-reconstruction method are described and applied to experimental data at resolutions ranging from 2.4 to 2.8,Å. [source] Recursive direct phasing with reference-beam diffractionACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2003Qun Shen The reference-beam diffraction technique provides a practical way to measure a large number of triplet phases in a standard oscillating-crystal diffraction experiment for protein crystals. The triplet-phase data set from such reference-beam measurements contains a unique phase-ocurrence pattern that leads to a new recursive phasing algorithm for the individual structure-factor phases. Application of the new algorithm is demonstrated for tetragonal lysozyme using 7360 triplet phases measured in a reference-beam experiment with a median phase discrepancy of 45°. An electron-density map obtained using this phasing algorithm and the measured triplet phases shows good agreement with the known protein structure. [source] Phasing power at the K absorption edge of organic arsenicACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2003Pascal Retailleau Single/multiple-wavelength anomalous dispersion (SAD/MAD) experiments were performed on a crystal of an organic arsenic derivative of hen egg-white lysozyme. A para -arsanilate compound used as a crystallizing reagent was incorporated into the ordered solvent region of the lysozyme molecule. Diffraction data were collected to high resolution (,2.0,Å) at three wavelengths around the K edge (1.04,Å) of arsenic at beamline BM30A, ESRF synchrotron. Anomalous Patterson maps clearly showed the main arsanilate site to be between three symmetry-related lysozyme molecules, at a location previously occupied by a para -toluenesulfonate anion. MAD phases at 2,Å derived using the program SHARP led to an electron-density map of sufficient quality to start manual building of the protein model. Amplitudes from a second crystal measured to a resolution of 1.8,Å at the peak wavelength revealed two additional heavy-atom sites, which reinforced the anomalous subset model and therefore dramatically improved the phasing power of the arsenic derivative. The subsequent solvent-flattened map was of such high accuracy that the program ARP/wARP was able to build a nearly complete model automatically. This work emphasizes the great potential of arsenic for de novo structure determination using anomalous dispersion methods. [source] Generating isomorphous heavy-atom derivatives by a quick-soak method.ACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2002Part II: phasing of new structures A quick-soak method has been applied to generate de novo heavy-atom phasing to solve two new protein structures, a type II transforming growth factor , receptor (TBRII) and a natural killer cell receptor,ligand complex, NKG2D,ULBP3. In the case of TBRII, a crystal derivatized for only 10,min in saturated HgCl2 provided adequate phasing for structure determination. Comparison between HgCl2 derivatives generated by 10,min soaking and by 12,h soaking revealed similar phasing statistics. The shorter soak, however, resulted in a derivative more isomorphous to the native than the longer soak as judged by changes in the unit-cell parameter a upon derivatization as well as by the quality of a combined SIRAS electron-density map. In the case of the NKG2D,ULBP3 structure, all overnight soaks in heavy-atom solutions resulted in crystal lattice disorder and only the quick soaks preserved diffraction. Despite fragile lattice packing, the quick-soaked K2PtCl4 derivative was isomorphous with the native crystal and the electron-density map calculated from combined SIR and MAD phases is better than that calculated from MAD phases alone. Combined with mass-spectrometry-assisted solution heavy-atom derivative screening and the use of synchrotron radiation, the quick-soak derivatization has the potential to transform the time-consuming conventional heavy-atom search into a real-time `on-the-fly' derivatization process that will benefit high-throughput structural genomics. [source] Pattern-recognition methods to identify secondary structure within X-ray crystallographic electron-density mapsACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2002Thomas Oldfield The interpretation of macromolecular crystallographic electron-density maps is a difficult and traditionally a manual step in the determination of a protein structure. The visualization of information within an electron-density map can be extremely arduous owing to the amount and complexity of information present. The ability to see the overall fold and structure of the molecule is usually lost among all the detail, particularly with larger structures. This paper describes a novel method of analysis of electron density in real space that can determine the secondary structure of a protein within minutes without any user intervention. The method is able to work with poor data as well as good data at resolutions down to 3.5,Å and is integral to the functionality of QUANTA. This article describes the methodology of the pattern recognition and its use with a number of sets of experimental data. [source] Gd-HPDO3A, a complex to obtain high-phasing-power heavy-atom derivatives for SAD and MAD experiments: results with tetragonal hen egg-white lysozymeACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2002Éric Girard A neutral gadolinium complex, Gd-HPDO3A, is shown to be a good candidate to use to obtain heavy-atom derivatives and solve macromolecular structures using anomalous dispersion. Tetragonal crystals of a gadolinium derivative of hen egg-white lysozyme were obtained by co-crystallization using different concentrations of the complex. Diffraction data from three derivative crystals (100, 50 and 10,mM) were collected to a resolution of 1.7,Å using Cu,K, radiation from a rotating anode. Two strong binding sites of the gadolinium complex to the protein were located from the gadolinium anomalous signal in both the 100 and 50,mM derivatives. A single site is occupied in the 10,mM derivative. Phasing using the anomalous signal at a single wavelength (SAD method) leads to an electron-density map of high quality. The structure of the 100,mM derivative has been refined. Two molecules of the gadolinium complex are close together. Both molecules are located close to tryptophan residues. Four chloride ions were found. The exceptional quality of the SAD electron-density map, only enhanced by solvent flattening, suggests that single-wavelength anomalous scattering with the Gd-HPDO3A complex may be sufficient to solve protein structures of high molecular weight by synchrotron-radiation experiments, if not by laboratory experiments. [source] Crystallization and secondary-structure determination of a protein of the Lrp/AsnC family from a hyperthermophilic archaeonACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2001Norio Kudo A protein belonging to the Lrp/AsnC transcription-factor family, pot1216151, from the hyperthermophilic archaeon Pyrococcus sp. OT3 was crystallized. In Escherichia coli, leucine-responsive protein (Lrp) and AsnC regulate a number of metabolic genes. The crystals of pot1216151 diffracted to 2.3,Å using a conventional X-ray source and to 1.8,Å using a synchrotron-radiation source. The space group was identified to be P3121 or P3221, with unit-cell parameters a = b = 96.9, c = 98.5,Å. In combination with diffraction data obtained from K2[Pt(CN)6] and K(AuCl4) derivatives, an electron-density map was calculated at a resolution of 3.0,Å. Four monomers were identified in the asymmetric unit, with four ,-strands and two ,-helices in each monomer. [source] Low-resolution phase extension using wavelet analysisACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2000Peter Main A method to extend low-resolution phases is presented which uses histogram matching not only of the electron density, but also of histograms obtained from the different levels of detail provided by the wavelet transform of the electron density. Statistical values for the wavelet coefficients can be predicted and depend only on the resolution and solvent content. Therefore, new details can be added to an electron-density map by matching the values of the wavelet coefficients to those predicted for an increased resolution. The positions of the new details are also guided by the diffraction pattern. In this way, the resolution can be increased gradually; on a number of trial structures of different size, solvent percentage and space group, it has been possible to extend the phasing from 10,Å to around 6,7,Å. [source] | ||||||||||