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1.
Hum Mol Genet ; 29(3): 444-458, 2020 02 01.
Article in English | MEDLINE | ID: mdl-31915829

ABSTRACT

Inherited optic neuropathies are rare eye diseases of optic nerve dysfunction that present in various genetic forms. Previously, mutation in three genes encoding mitochondrial proteins has been implicated in autosomal recessive forms of optic atrophy that involve progressive degeneration of optic nerve and retinal ganglion cells (RGC). Using whole exome analysis, a novel double homozygous mutation p.L81R and pR212W in malonyl CoA-acyl carrier protein transacylase (MCAT), a mitochondrial protein involved in fatty acid biosynthesis, has now been identified as responsible for an autosomal recessive optic neuropathy from a Chinese consanguineous family. MCAT is expressed in RGC that are rich in mitochondria. The disease variants lead to structurally unstable MCAT protein with significantly reduced intracellular expression. RGC-specific knockdown of Mcat in mice, lead to an attenuated retinal neurofiber layer, that resembles the phenotype of optic neuropathy. These results indicated that MCAT plays an essential role in mitochondrial function and maintenance of RGC axons, while novel MCAT p.L81R and p.R212W mutations can lead to optic neuropathy.


Subject(s)
Acyl-Carrier Protein S-Malonyltransferase/genetics , Genes, Recessive , Mitochondria/pathology , Optic Nerve Diseases/pathology , Optic Nerve/pathology , Retinal Ganglion Cells/pathology , Acyl-Carrier Protein S-Malonyltransferase/chemistry , Acyl-Carrier Protein S-Malonyltransferase/metabolism , Amino Acid Sequence , Animals , Child , Female , Humans , Male , Mice , Mice, Knockout , Mitochondria/metabolism , Mutation , Optic Nerve/metabolism , Optic Nerve Diseases/etiology , Optic Nerve Diseases/metabolism , Pedigree , Protein Conformation , Retinal Ganglion Cells/metabolism , Sequence Homology , Exome Sequencing
2.
Nature ; 515(7525): 147-50, 2014 Nov 06.
Article in English | MEDLINE | ID: mdl-25118175

ABSTRACT

Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (Cas) proteins form the CRISPR/Cas system to defend against foreign nucleic acids of bacterial and archaeal origin. In the I-E subtype CRISPR/Cas system, eleven subunits from five Cas proteins (CasA1B2C6D1E1) assemble along a CRISPR RNA (crRNA) to form the Cascade complex. Here we report on the 3.05 Å crystal structure of the 405-kilodalton Escherichia coli Cascade complex that provides molecular details beyond those available from earlier lower-resolution cryo-electron microscopy structures. The bound 61-nucleotide crRNA spans the entire 11-protein subunit-containing complex, where it interacts with all six CasC subunits (named CasC1-6), with its 5' and 3' terminal repeats anchored by CasD and CasE, respectively. The crRNA spacer region is positioned along a continuous groove on the concave surface generated by the aligned CasC1-6 subunits. The five long ß-hairpins that project from individual CasC2-6 subunits extend across the crRNA, with each ß-hairpin inserting into the gap between the last stacked base and its adjacent splayed counterpart, and positioned within the groove of the preceding CasC subunit. Therefore, instead of continuously stacking, the crRNA spacer region is divided into five equal fragments, with each fragment containing five stacked bases flanked by one flipped-out base. Each of those crRNA spacer fragments interacts with CasC in a similar fashion. Furthermore, our structure explains why the seed sequence, with its outward-directed bases, has a critical role in target DNA recognition. In conclusion, our structure of the Cascade complex provides novel molecular details of protein-protein and protein-RNA alignments and interactions required for generation of a complex mediating RNA-guided immune surveillance.


Subject(s)
CRISPR-Associated Proteins/chemistry , Escherichia coli/chemistry , Escherichia coli/immunology , Immunologic Surveillance , Multiprotein Complexes/chemistry , RNA, Bacterial/genetics , RNA, Untranslated/genetics , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems/genetics , Crystallography, X-Ray , Escherichia coli/genetics , Models, Molecular , Multiprotein Complexes/metabolism , Protein Binding , Protein Subunits/chemistry , Protein Subunits/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Templates, Genetic
3.
Nat Methods ; 12(2): 127-30, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25532136

ABSTRACT

We describe a likelihood-based method for determining the substructure of anomalously scattering atoms in macromolecular crystals that allows successful structure determination by single-wavelength anomalous diffraction (SAD) X-ray analysis with weak anomalous signal. With the use of partial models and electron density maps in searches for anomalously scattering atoms, testing of alternative values of parameters and parallelized automated model-building, this method has the potential to extend the applicability of the SAD method in challenging cases.


Subject(s)
Crystallography, X-Ray/methods , Macromolecular Substances/chemistry , Software , Algorithms , Likelihood Functions , Models, Molecular , Signal-To-Noise Ratio
4.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 1): 144-54, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24419387

ABSTRACT

High-throughput drug-discovery and mechanistic studies often require the determination of multiple related crystal structures that only differ in the bound ligands, point mutations in the protein sequence and minor conformational changes. If performed manually, solution and refinement requires extensive repetition of the same tasks for each structure. To accelerate this process and minimize manual effort, a pipeline encompassing all stages of ligand building and refinement, starting from integrated and scaled diffraction intensities, has been implemented in Phenix. The resulting system is able to successfully solve and refine large collections of structures in parallel without extensive user intervention prior to the final stages of model completion and validation.


Subject(s)
Crystallography, X-Ray/methods , Proteins/chemistry , Animals , Drug Design , Factor Xa/chemistry , Factor Xa/metabolism , HIV Protease/chemistry , HIV Protease/metabolism , HIV-1/enzymology , Humans , Ligands , Models, Molecular , Protein Binding , Proteins/metabolism , Thrombin/chemistry , Thrombin/metabolism
5.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 11): 2209-15, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24189232

ABSTRACT

The estimate of the root-mean-square deviation (r.m.s.d.) in coordinates between the model and the target is an essential parameter for calibrating likelihood functions for molecular replacement (MR). Good estimates of the r.m.s.d. lead to good estimates of the variance term in the likelihood functions, which increases signal to noise and hence success rates in the MR search. Phaser has hitherto used an estimate of the r.m.s.d. that only depends on the sequence identity between the model and target and which was not optimized for the MR likelihood functions. Variance-refinement functionality was added to Phaser to enable determination of the effective r.m.s.d. that optimized the log-likelihood gain (LLG) for a correct MR solution. Variance refinement was subsequently performed on a database of over 21,000 MR problems that sampled a range of sequence identities, protein sizes and protein fold classes. Success was monitored using the translation-function Z-score (TFZ), where a TFZ of 8 or over for the top peak was found to be a reliable indicator that MR had succeeded for these cases with one molecule in the asymmetric unit. Good estimates of the r.m.s.d. are correlated with the sequence identity and the protein size. A new estimate of the r.m.s.d. that uses these two parameters in a function optimized to fit the mean of the refined variance is implemented in Phaser and improves MR outcomes. Perturbing the initial estimate of the r.m.s.d. from the mean of the distribution in steps of standard deviations of the distribution further increases MR success rates.


Subject(s)
Amino Acid Sequence , Amino Acid Substitution , Databases, Protein/trends , Signal-To-Noise Ratio , Amino Acid Sequence/genetics , Amino Acid Substitution/genetics , Crystallography, X-Ray/instrumentation , Crystallography, X-Ray/methods , Databases, Protein/classification , Likelihood Functions , Models, Molecular , Mutation , Protein Folding , Sequence Alignment , Software , X-Ray Diffraction
6.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 11): 2276-86, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24189240

ABSTRACT

Phaser.MRage is a molecular-replacement automation framework that implements a full model-generation workflow and provides several layers of model exploration to the user. It is designed to handle a large number of models and can distribute calculations efficiently onto parallel hardware. In addition, phaser.MRage can identify correct solutions and use this information to accelerate the search. Firstly, it can quickly score all alternative models of a component once a correct solution has been found. Secondly, it can perform extensive analysis of identified solutions to find protein assemblies and can employ assembled models for subsequent searches. Thirdly, it is able to use a priori assembly information (derived from, for example, homologues) to speculatively place and score molecules, thereby customizing the search procedure to a certain class of protein molecule (for example, antibodies) and incorporating additional biological information into molecular replacement.


Subject(s)
Amino Acid Substitution , Computational Biology/methods , Databases, Protein , Software , Artificial Intelligence , Crystallography, X-Ray/methods , Crystallography, X-Ray/trends , Databases, Protein/standards , Models, Molecular , Protein Multimerization , Protein Structure, Tertiary
7.
J Struct Funct Genomics ; 13(2): 81-90, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22418934

ABSTRACT

The combination of algorithms from the structure-modeling field with those of crystallographic structure determination can broaden the range of templates that are useful for structure determination by the method of molecular replacement. Automated tools in phenix.mr_rosetta simplify the application of these combined approaches by integrating Phenix crystallographic algorithms and Rosetta structure-modeling algorithms and by systematically generating and evaluating models with a combination of these methods. The phenix.mr_rosetta algorithms can be used to automatically determine challenging structures. The approaches used in phenix.mr_rosetta are described along with examples that show roles that structure-modeling can play in molecular replacement.


Subject(s)
Algorithms , Computational Biology/methods , Models, Molecular , Protein Conformation , Software , Crystallography, X-Ray/methods , Databases, Protein , Internet , Macromolecular Substances/chemistry , Magnetic Resonance Spectroscopy , Proteins/analysis , Proteins/chemistry , Proteomics/methods
8.
Methods ; 55(1): 94-106, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21821126

ABSTRACT

X-ray crystallography is a critical tool in the study of biological systems. It is able to provide information that has been a prerequisite to understanding the fundamentals of life. It is also a method that is central to the development of new therapeutics for human disease. Significant time and effort are required to determine and optimize many macromolecular structures because of the need for manual interpretation of complex numerical data, often using many different software packages, and the repeated use of interactive three-dimensional graphics. The Phenix software package has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on automation. This has required the development of new algorithms that minimize or eliminate subjective input in favor of built-in expert-systems knowledge, the automation of procedures that are traditionally performed by hand, and the development of a computational framework that allows a tight integration between the algorithms. The application of automated methods is particularly appropriate in the field of structural proteomics, where high throughput is desired. Features in Phenix for the automation of experimental phasing with subsequent model building, molecular replacement, structure refinement and validation are described and examples given of running Phenix from both the command line and graphical user interface.


Subject(s)
Automation, Laboratory/methods , Crystallography, X-Ray , Data Collection/methods , Proteins/analysis , Proteomics/methods , Software , Algorithms , Automation, Laboratory/instrumentation , Crystallography, X-Ray/instrumentation , Crystallography, X-Ray/methods , High-Throughput Screening Assays , Molecular Structure , Proteins/chemistry
9.
Acta Crystallogr D Biol Crystallogr ; 67(Pt 4): 303-12, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21460448

ABSTRACT

In molecular replacement, the quality of models can be improved by transferring information contained in sequence alignment to the template structure. A family of algorithms has been developed that make use of the sequence-similarity score calculated from residue-substitution scores smoothed over nearby residues to delete or downweight parts of the model that are unreliable. These algorithms have been implemented in the program Sculptor, together with well established methods that are in common use for model improvement. An analysis of the new algorithms has been performed by studying the effect of algorithm parameters on the quality of models. Benchmarking against existing techniques shows that models from Sculptor compare favourably, especially if the alignment is unreliable. Carrying out multiple trials using alternative models created from the same structure but using different algorithm parameters can significantly improve the success rate.


Subject(s)
Algorithms , Software Design , Models, Molecular , Surface Properties
10.
Structure ; 17(1): 128-38, 2009 Jan 14.
Article in English | MEDLINE | ID: mdl-19141289

ABSTRACT

About 10% of all protein kinases are predicted to be enzymatically inactive pseudokinases, but the structural details of kinase inactivation have remained unclear. We present the first structure of a pseudokinase, VRK3, and that of its closest active relative, VRK2. Profound changes to the active site region underlie the loss of catalytic activity, and VRK3 cannot bind ATP because of residue substitutions in the binding pocket. However, VRK3 still shares striking structural similarity with VRK2, and appears to be locked in a pseudoactive conformation. VRK3 also conserves residue interactions that are surprising in the absence of enzymatic function; these appear to play important architectural roles required for the residual functions of VRK3. Remarkably, VRK3 has an "inverted" pattern of sequence conservation: although the active site is poorly conserved, portions of the molecular surface show very high conservation, suggesting that they form key interactions that explain the evolutionary retention of VRK3.


Subject(s)
Phosphotransferases/chemistry , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Catalytic Domain , Models, Molecular , Molecular Sequence Data , Phosphotransferases/metabolism , Protein Conformation , Protein Folding
11.
Acta Crystallogr D Biol Crystallogr ; 66(Pt 2): 213-21, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20124702

ABSTRACT

Macromolecular X-ray crystallography is routinely applied to understand biological processes at a molecular level. However, significant time and effort are still required to solve and complete many of these structures because of the need for manual interpretation of complex numerical data using many software packages and the repeated use of interactive three-dimensional graphics. PHENIX has been developed to provide a comprehensive system for macromolecular crystallographic structure solution with an emphasis on the automation of all procedures. This has relied on the development of algorithms that minimize or eliminate subjective input, the development of algorithms that automate procedures that are traditionally performed by hand and, finally, the development of a framework that allows a tight integration between the algorithms.


Subject(s)
Crystallography, X-Ray/methods , Software Design , Algorithms , Models, Molecular
12.
Drug Discov Today ; 25(3): 485-490, 2020 03.
Article in English | MEDLINE | ID: mdl-31877353

ABSTRACT

Recent advances in electron cryo-microscopy (cryo-EM) structure determination have pushed the resolutions obtainable by the method into the range widely considered to be of utility for drug discovery. Here, we review the use of cryo-EM in fragment-based drug discovery (FBDD) based on in-house method development. We demonstrate not only that cryo-EM can reveal details of the molecular interactions between fragments and a protein, but also that the current reproducibility, quality, and throughput are compatible with FBDD. We exemplify this using the test system ß-galactosidase (Bgal) and the oncology target pyruvate kinase 2 (PKM2).


Subject(s)
Cryoelectron Microscopy/methods , Drug Discovery/methods , Carrier Proteins/chemistry , Carrier Proteins/metabolism , High-Throughput Screening Assays , Humans , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Reproducibility of Results , Thyroid Hormones/chemistry , Thyroid Hormones/metabolism , beta-Galactosidase/chemistry , beta-Galactosidase/metabolism , Thyroid Hormone-Binding Proteins
13.
Structure ; 15(10): 1215-26, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17937911

ABSTRACT

Apoptosis signal-regulating kinase 1 (ASK1) plays an essential role in stress and immune response and has been linked to the development of several diseases. Here, we present the structure of the human ASK1 catalytic domain in complex with staurosporine. Analytical ultracentrifugation (AUC) and crystallographic analysis showed that ASK1 forms a tight dimer (K(d) approximately 0.2 microM) interacting in a head-to-tail fashion. We found that the ASK1 phosphorylation motifs differ from known ASK1 phosphorylation sites but correspond well to autophosphorylation sites identified by mass spectrometry. Reporter gene assays showed that all three identified in vitro autophosphorylation sites (Thr813, Thr838, Thr842) regulate ASK1 signaling, but site-directed mutants showed catalytic activities similar to wild-type ASK1, suggesting a regulatory mechanism independent of ASK1 kinase activity. The determined high-resolution structure of ASK1 and identified ATP mimetic inhibitors will provide a first starting point for the further development of selective inhibitors.


Subject(s)
MAP Kinase Kinase Kinase 5/chemistry , Amino Acid Sequence , Binding Sites , Catalysis , Dimerization , Enzyme Activation , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , MAP Kinase Kinase Kinase 5/metabolism , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Conformation , Protein Structure, Tertiary , Staurosporine/chemistry , Staurosporine/metabolism , Structure-Activity Relationship , Substrate Specificity
14.
Acta Crystallogr D Struct Biol ; 75(Pt 10): 861-877, 2019 Oct 01.
Article in English | MEDLINE | ID: mdl-31588918

ABSTRACT

Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.


Subject(s)
Automation/methods , Macromolecular Substances/chemistry , Software Design , Software Validation , Cryoelectron Microscopy/methods , Crystallography, X-Ray/methods , Models, Molecular , Molecular Conformation
15.
Chem Biol ; 14(11): 1243-53, 2007 Nov.
Article in English | MEDLINE | ID: mdl-18022563

ABSTRACT

Mammals utilize a single phosphopantetheinyl transferase for the posttranslational modification of at least three different apoproteins: the carrier protein components of cytosolic and mitochondrial fatty acid synthases and the aminoadipate semialdehyde reductase involved in lysine degradation. We determined the crystal structure of the human phosphopantetheinyl transferase, a eukaryotic phosphopantetheinyl transferase characterized, complexed with CoA and Mg(2+), and in ternary complex with CoA and ACP. The involvement of key residues in ligand binding and catalysis was confirmed by mutagenesis and kinetic analysis. Human phosphopantetheinyl transferase exhibits an alpha/beta fold and 2-fold pseudosymmetry similar to the Sfp phosphopantetheinyl transferase from Bacillus subtilis. Although the bound ACP exhibits a typical four-helix structure, its binding is unusual in that it is facilitated predominantly by hydrophobic interactions. A detailed mechanism is proposed describing the substrate binding and catalytic process.


Subject(s)
Fatty Acid Synthases/metabolism , Amino Acid Sequence , Catalysis , Coenzyme A/metabolism , Crystallography, X-Ray , Fatty Acid Synthases/chemistry , Fatty Acid Synthases/genetics , Humans , Kinetics , Magnesium/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Sequence Homology, Amino Acid , Substrate Specificity
16.
Biochem J ; 402(3): 419-27, 2007 Mar 15.
Article in English | MEDLINE | ID: mdl-17067289

ABSTRACT

To this day, a significant proportion of the human genome remains devoid of functional characterization. In this study, we present evidence that the previously functionally uncharacterized product of the human DHRS10 gene is endowed with 17beta-HSD (17beta-hydroxysteroid dehydrogenase) activity. 17beta-HSD enzymes are primarily involved in the metabolism of steroids at the C-17 position and also of other substrates such as fatty acids, prostaglandins and xenobiotics. In vitro, DHRS10 converts NAD+ into NADH in the presence of oestradiol, testosterone and 5-androstene-3beta,17beta-diol. Furthermore, the product of oestradiol oxidation, oestrone, was identified in intact cells transfected with a construct plasmid encoding the DHRS10 protein. In situ fluorescence hybridization studies have revealed the cytoplasmic localization of DHRS10. Along with tissue expression data, this suggests a role for DHRS10 in the local inactivation of steroids in the central nervous system and placenta. The crystal structure of the DHRS10 apoenzyme exhibits secondary structure of the SDR (short-chain dehydrogenase/reductase) family: a Rossmann-fold with variable loops surrounding the active site. It also reveals a broad and deep active site cleft into which NAD+ and oestradiol can be docked in a catalytically competent orientation.


Subject(s)
17-Hydroxysteroid Dehydrogenases/chemistry , 17-Hydroxysteroid Dehydrogenases/metabolism , Cytosol/enzymology , 17-Hydroxysteroid Dehydrogenases/genetics , 17-Hydroxysteroid Dehydrogenases/isolation & purification , Amino Acid Sequence , Binding Sites , Cell Line , Crystallography, X-Ray , Gene Expression , Humans , Kinetics , Ligands , Models, Molecular , Molecular Sequence Data , NAD/metabolism , Organ Specificity , Oxidation-Reduction , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Alignment , Structural Homology, Protein
17.
Nat Commun ; 8: 15786, 2017 06 08.
Article in English | MEDLINE | ID: mdl-28593992

ABSTRACT

Hunter syndrome is a rare but devastating childhood disease caused by mutations in the IDS gene encoding iduronate-2-sulfatase, a crucial enzyme in the lysosomal degradation pathway of dermatan sulfate and heparan sulfate. These complex glycosaminoglycans have important roles in cell adhesion, growth, proliferation and repair, and their degradation and recycling in the lysosome is essential for cellular maintenance. A variety of disease-causing mutations have been identified throughout the IDS gene. However, understanding the molecular basis of the disease has been impaired by the lack of structural data. Here, we present the crystal structure of human IDS with a covalently bound sulfate ion in the active site. This structure provides essential insight into multiple mechanisms by which pathogenic mutations interfere with enzyme function, and a compelling explanation for severe Hunter syndrome phenotypes. Understanding the structural consequences of disease-associated mutations will facilitate the identification of patients that may benefit from specific tailored therapies.


Subject(s)
Glycoproteins/chemistry , Glycoproteins/metabolism , Mucopolysaccharidosis II/enzymology , Catalytic Domain , Crystallography, X-Ray , Glycoproteins/genetics , Humans , Models, Molecular , Mucopolysaccharidosis II/etiology , Mutation , Protein Conformation , Protein Processing, Post-Translational , Sulfates/metabolism
18.
Acta Crystallogr D Struct Biol ; 72(Pt 3): 346-58, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26960122

ABSTRACT

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. A simple theoretical framework for describing measurements of anomalous differences and the resulting useful anomalous correlation and anomalous signal in a SAD experiment is presented. Here, the useful anomalous correlation is defined as the correlation of anomalous differences with ideal anomalous differences from the anomalous substructure. The useful anomalous correlation reflects the accuracy of the data and the absence of minor sites. The useful anomalous correlation also reflects the information available for estimating crystallographic phases once the substructure has been determined. In contrast, the anomalous signal (the peak height in a model-phased anomalous difference Fourier at the coordinates of atoms in the anomalous substructure) reflects the information available about each site in the substructure and is related to the ability to find the substructure. A theoretical analysis shows that the expected value of the anomalous signal is the product of the useful anomalous correlation, the square root of the ratio of the number of unique reflections in the data set to the number of sites in the substructure, and a function that decreases with increasing values of the atomic displacement factor for the atoms in the substructure. This means that the ability to find the substructure in a SAD experiment is increased by high data quality and by a high ratio of reflections to sites in the substructure, and is decreased by high atomic displacement factors for the substructure.


Subject(s)
Crystallography, X-Ray/methods , Proteins/chemistry , Algorithms , Models, Molecular , Protein Conformation , Signal-To-Noise Ratio , Software
19.
Acta Crystallogr D Struct Biol ; 72(Pt 3): 359-74, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26960123

ABSTRACT

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. Here, algorithms and tools for evaluating and optimizing the useful anomalous correlation and the anomalous signal in a SAD experiment are described. A simple theoretical framework [Terwilliger et al. (2016), Acta Cryst. D72, 346-358] is used to develop methods for planning a SAD experiment, scaling SAD data sets and estimating the useful anomalous correlation and anomalous signal in a SAD data set. The phenix.plan_sad_experiment tool uses a database of solved and unsolved SAD data sets and the expected characteristics of a SAD data set to estimate the probability that the anomalous substructure will be found in the SAD experiment and the expected map quality that would be obtained if the substructure were found. The phenix.scale_and_merge tool scales unmerged SAD data from one or more crystals using local scaling and optimizes the anomalous signal by identifying the systematic differences among data sets, and the phenix.anomalous_signal tool estimates the useful anomalous correlation and anomalous signal after collecting SAD data and estimates the probability that the data set can be solved and the likely figure of merit of phasing.


Subject(s)
Crystallography, X-Ray/methods , Proteins/chemistry , Algorithms , Probability , Protein Conformation , Signal-To-Noise Ratio
20.
J Mol Biol ; 318(3): 723-32, 2002 May 03.
Article in English | MEDLINE | ID: mdl-12054818

ABSTRACT

The vancomycin-related antibiotics balhimycin and degluco-balhimycin have been crystallized in complexes with di-, tri- and pentapeptides that emulate bacterial cell-wall precursors, and four structures determined at atomic resolution (<1 A). In addition to the features expected from previous structural and spectroscopic studies, two new motifs were observed that may prove important in the design of antibiotics modified to overcome bacterial resistance. A changed binding mode was found in two dipeptide complexes, and a new type of face-to-face oligomerization (in addition to the well-established back-to-back dimerization) was seen when the model peptide reaches a critical fraction of the size of the cell-wall precursor pentapeptide. The extensive interactions involving both antibiotic and peptide molecules in this interface should appreciably enhance the kinetic and thermodynamic stability of the complexes. In the pentapeptide complex, the relative positions of the peptides are close to those required for d-Ala elimination, so this structure may provide a realistic model for the prevention of the enzyme-catalyzed cell-wall crosslinking by antibiotic binding.


Subject(s)
Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Cell Wall/chemistry , Vancomycin/analogs & derivatives , Vancomycin/chemistry , Binding Sites , Crystallography, X-Ray , Dimerization , Drug Resistance, Bacterial , Ligands , Models, Molecular , Molecular Structure , Protein Conformation , Protein Precursors/chemistry
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