RIKEN aids international structural genomics efforts.
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Animals↗
Biomedical subjects
Thomas C Terwilliger
Publications and source records attributed to Thomas C Terwilliger.
Ligand identification using electron-density map correlations.
A procedure for the identification of ligands bound in crystal structures of macromolecules is described. Two characteristics of the density corresponding to a ligand are used in the identification procedure. One is the correlation of the ligand density with each of a set of test ligands after optimization of the fit of that ligand to the density. The other is the correlation of a fingerprint of the density with the fingerprint of model density for each possible ligand. The fingerprints consist of an ordered list of correlations of each the test ligands with the density. The two characteristics are scored using a Z-score approach in which the correlations are normalized to the mean and standard deviation of correlations found for a variety of mismatched ligand-density pairs, so that the Z scores are related to the probability of observing a particular value of the correlation by chance. The procedure was tested with a set of 200 of the most commonly found ligands in the Protein Data Bank, collectively representing 57% of all ligands in the Protein Data Bank. Using a combination of these two characteristics of ligand density, ranked lists of ligand identifications were made for representative (F(o) - F(c))exp(i(phi)c) difference density from entries in the Protein Data Bank. In 48% of the 200 cases, the correct ligand was at the top of the ranked list of ligands. This approach may be useful in identification of unknown ligands in new macromolecular structures as well as in the identification of which ligands in a mixture have bound to a macromolecule.
Algorithms↗
The structure and computational analysis of Mycobacterium tuberculosis protein CitE suggest a novel enzymatic function.
Fatty acid biosynthesis is essential for the survival of Mycobacterium tuberculosis and acetyl-coenzyme A (acetyl-CoA) is an essential precursor in this pathway. We have determined the 3-D crystal structure of M. tuberculosis citrate lyase beta-subunit (CitE), which as annotated should cleave protein bound citryl-CoA to oxaloacetate and a protein-bound CoA derivative. The CitE structure has the (beta/alpha)(8) TIM barrel fold with an additional alpha-helix, and is trimeric. We have determined the ternary complex bound with oxaloacetate and magnesium, revealing some of the conserved residues involved in catalysis. While the bacterial citrate lyase is a complex with three subunits, the M. tuberculosis genome does not contain the alpha and gamma subunits of this complex, implying that M. tuberculosis CitE acts differently from other bacterial CitE proteins. The analysis of gene clusters containing the CitE protein from 168 fully sequenced organisms has led us to identify a grouping of functionally related genes preserved in M. tuberculosis, Rattus norvegicus, Homo sapiens, and Mus musculus. We propose a novel enzymatic function for M. tuberculosis CitE in fatty acid biosynthesis that is analogous to bacterial citrate lyase but producing acetyl-CoA rather than a protein-bound CoA derivative.
Amino Acid Sequence↗
Structure of Mycobacterium tuberculosis RuvA, a protein involved in recombination.
The process of recombinational repair is crucial for maintaining genomic integrity and generating biological diversity. In association with RuvB and RuvC, RuvA plays a central role in processing and resolving Holliday junctions, which are a critical intermediate in homologous recombination. Here, the cloning, purification and structure determination of the RuvA protein from Mycobacterium tuberculosis (MtRuvA) are reported. Analysis of the structure and comparison with other known RuvA proteins reveal an octameric state with conserved subunit-subunit interaction surfaces, indicating the requirement of octamer formation for biological activity. A detailed analysis of plasticity in the RuvA molecules has led to insights into the invariant and variable regions, thus providing a framework for understanding regional flexibility in various aspects of RuvA function.
Bacterial Proteins↗
Automated ligand fitting by core-fragment fitting and extension into density.
A procedure for fitting of ligands to electron-density maps by first fitting a core fragment of the ligand to density and then extending the remainder of the ligand into density is presented. The approach was tested by fitting 9327 ligands over a wide range of resolutions (most are in the range 0.8-4.8 A) from the Protein Data Bank (PDB) into (Fo - Fc)exp(i phi(c)) difference density calculated using entries from the PDB without these ligands. The procedure was able to place 58% of these 9327 ligands within 2 A (r.m.s.d.) of the coordinates of the atoms in the original PDB entry for that ligand. The success of the fitting procedure was relatively insensitive to the size of the ligand in the range 10-100 non-H atoms and was only moderately sensitive to resolution, with the percentage of ligands placed near the coordinates of the original PDB entry for fits in the range 58-73% over all resolution ranges tested.
Algorithms↗
Functional and structural characterization of a thiol peroxidase from Mycobacterium tuberculosis.
A thiol peroxidase (Tpx) from Mycobacterium tuberculosis was functionally analyzed. The enzyme shows NADPH-linked peroxidase activity using a thioredoxin-thioredoxin reductase system as electron donor, and anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. It reduces H2O2, t-butyl hydroperoxide, and cumene hydroperoxide, and is inhibited by sulfhydryl reagents. Mutational studies revealed that the peroxidatic (Cys60) and resolving (Cys93) cysteine residues are critical amino acids for catalytic activity. The X-ray structure determined to a resolution of 1.75 A shows a thioredoxin fold similar to that of other peroxiredoxin family members. Superposition with structural homologues in oxidized and reduced forms indicates that the M. tuberculosis Tpx is a member of the atypical two-Cys peroxiredoxin family. In addition, the short distance that separates the Calpha atoms of Cys60 and Cys93 and the location of these cysteine residues in unstructured regions may indicate that the M. tuberculosis enzyme is oxidized, though the side-chain of Cys60 is poorly visible. It is solely in the reduced Streptococcus pneumoniae Tpx structure that both residues are part of two distinct helical segments. The M. tuberculosis Tpx is dimeric both in solution and in the crystal structure. Amino acid residues from both monomers delineate the active site pocket.
Amino Acid Sequence↗
Crystal structure of a putative pyridoxine 5'-phosphate oxidase (Rv2607) from Mycobacterium tuberculosis.
The three-dimensional structure of Rv2607, a putative pyridoxine 5'-phosphate oxidase (PNPOx) from Mycobacterium tuberculosis, has been determined by X-ray crystallography to 2.5 A resolution. Rv2607 has a core domain similar to known PNPOx structures with a flavin mononucleotide (FMN) cofactor. Electron density for two FMN at the dimer interface is weak despite the bright yellow color of the protein solution and crystal. The shape and size of the putative binding pocket is markedly different from that of members of the PNPOx family, which may indicate some significant changes in the FMN binding mode of this protein relative to members of the family.
Amino Acid Sequence↗
Is one solution good enough?
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Crystallography, X-Ray↗
Solution structure of the conserved hypothetical protein Rv2302 from Mycobacterium tuberculosis.
The Mycobacterium tuberculosis protein Rv2302 (80 residues; molecular mass of 8.6 kDa) has been characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. While the biochemical function of Rv2302 is still unknown, recent microarray analyses show that Rv2302 is upregulated in response to starvation and overexpression of heat shock proteins and, consequently, may play a role in the biochemical processes associated with these events. Rv2302 is a monomer in solution as shown by size exclusion chromatography and NMR spectroscopy. CD spectroscopy suggests that Rv2302 partially unfolds upon heating and that this unfolding is reversible. Using NMR-based methods, the solution structure of Rv2302 was determined. The protein contains a five-strand, antiparallel beta-sheet core with one C-terminal alpha-helix (A61 to A75) nestled against its side. Hydrophobic interactions between residues in the alpha-helix and beta-strands 3 and 4 hold the alpha-helix near the beta-sheet core. The electrostatic potential on the solvent-accessible surface is primarily negative with the exception of a positive arginine pocket composed of residues R18, R70, and R74. Steady-state {(1)H}-(15)N heteronuclear nuclear Overhauser effects indicate that the protein's core is rigid on the picosecond timescale. The absence of amide cross-peaks for residues G13 to H19 in the (1)H-(15)N heteronuclear single quantum correlation spectrum suggests that this region, a loop between beta-strands 1 and 2, undergoes motion on the millisecond to microsecond timescale. Dali searches using the structure closest to the average structure do not identify any high similarities to any other known protein structure, suggesting that the structure of Rv2302 may represent a novel protein fold.
Amino Acid Sequence↗
Engineering and characterization of a superfolder green fluorescent protein.
Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.
Bacterial Proteins↗
Structure of pyrR (Rv1379) from Mycobacterium tuberculosis: a persistence gene and protein drug target.
The Mycobacterium tuberculosis pyrR gene (Rv1379) encodes a protein that regulates the expression of pyrimidine-nucleotide biosynthesis (pyr) genes in a UMP-dependent manner. Because pyrimidine biosynthesis is an essential step in the progression of TB, the gene product pyrR is an attractive antitubercular drug target. The 1.9 A native structure of Mtb pyrR determined by the TB Structural Genomics Consortium facilities in trigonal space group P3(1)21 is reported, with unit-cell parameters a = 66.64, c = 154.72 A at 120 K and two molecules in the asymmetric unit. The three-dimensional structure and residual uracil phosphoribosyltransferase activity point to a common PRTase ancestor for pyrR. However, while PRPP- and UMP-binding sites have been retained in Mtb pyrR, a distinct dimer interaction among subunits creates a deep positively charged cleft capable of binding pyr mRNA. In silico screening of pyrimidine-nucleoside analogs has revealed a number of potential lead compounds that, if bound to Mtb pyrR, could facilitate transcriptional attenuation, particularly cyclopentenyl nucleosides.
Amino Acid Sequence↗
Recent advances in GFP folding reporter and split-GFP solubility reporter technologies. Application to improving the folding and solubility of recalcitrant proteins from Mycobacterium tuberculosis.
We have improved our green fluorescent protein (GFP) folding reporter technology [Waldo et al., (1999) Nat. Biotechnol. 17, 691-695] to evolve recalcitrant proteins from Mycobacterium tuberculosis. The target protein is inserted into the scaffolding of the GFP, eliminating false-positive artifacts caused by expression of truncated protein variants from internal cryptic ribosome binding sites in the target RNA. In parallel, we have developed a new quantitative fluorescent protein tagging and detection system based on micro-domains of GFP. This split-GFP system, which works both in vivo and in vitro, is amenable to high-throughput assays of protein expression and solubility [Cabantous et al., (2005) Nat. Biotechnol. 23, 102-107]. Together, the GFP folding reporter and split-GFP technologies offer a comprehensive system for manipulating and improving protein folding and solubility.
Bacterial Proteins↗
Structural and functional features of an NDP kinase from the hyperthermophile crenarchaeon Pyrobaculum aerophilum.
Nucleoside diphosphate (NDP) kinases are ubiquitous enzymes that transfer gamma-phosphates from nucleoside triphosphates to nucleoside diphosphates via a ping-pong mechanism. The important role of this large family of enzymes in controlling cellular functions and developmental processes along with their crystallizability has made them good candidates for structural studies. We recently determined the structure of an evolved version of an NDP kinase from Pyrobaculum aerophilum, an extreme thermophile. This NDP kinase has similarity to the 42 other NDP kinases deposited in the Protein Data Bank (PDB) but differs significantly in sequence, structure, and biophysical properties. The P. aerophilum NDP kinase sequence contains two unique segments not present in other NDP kinases, comprising residues 66-100 and 156-165. We show that deletion mutants of the P. aerophilum NDP kinase lacking either or both of these inserts have an altered substrate specificity, allowing dGTP as the phosphate donor. A structural analysis of the evolved NDP kinase in conjunction with mutagenesis experiments suggests that the substrate specificity of the P. aerophilum NDP kinase is related to the presence of these two inserts.
Archaeal Proteins↗
Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein.
Existing protein tagging and detection methods are powerful but have drawbacks. Split protein tags can perturb protein solubility or may not work in living cells. Green fluorescent protein (GFP) fusions can misfold or exhibit altered processing. Fluorogenic biarsenical FLaSH or ReASH substrates overcome many of these limitations but require a polycysteine tag motif, a reducing environment and cell transfection or permeabilization. An ideal protein tag would be genetically encoded, would work both in vivo and in vitro, would provide a sensitive analytical signal and would not require external chemical reagents or substrates. One way to accomplish this might be with a split GFP, but the GFP fragments reported thus far are large and fold poorly, require chemical ligation or fused interacting partners to force their association, or require coexpression or co-refolding to produce detectable folded and fluorescent GFP. We have engineered soluble, self-associating fragments of GFP that can be used to tag and detect either soluble or insoluble proteins in living cells or cell lysates. The split GFP system is simple and does not change fusion protein solubility.
Amino Acid Motifs↗
Using prime-and-switch phasing to reduce model bias in molecular replacement.
Atomic models are commonly used to calculate phases in macromolecular crystallography. When combined with measured amplitudes, model-based phases yield electron-density maps with features of the correct structure but with a significant bias towards features of the model. The present contribution shows applications of the technique of prime-and-switch phasing to reduce this bias. An atomic model is used to generate phases that are close to the correct set but that may be biased. An unbiased source of phase information, an estimate of the probability that the electron-density map corresponds to a macromolecule, is then used to select a set of phases that are near the biased set, without further reference to the biased phases. The probability that the electron-density map corresponds to a macromolecule is based on agreement of the map with expectations such as a flat solvent region. Prime-and-switch phasing can be useful even for crystals with low solvent content and may reduce errors in interpretation of electron density in a wide range of applications of macromolecular crystallography, including molecular replacement, model building, ligand-binding and conformation-change studies, refinement and structure validation.
Bias↗
Mycobacterium tuberculosis RmlC epimerase (Rv3465): a promising drug-target structure in the rhamnose pathway.
The Mycobacterium tuberculosis rmlC gene encodes dTDP-4-keto-6-deoxyglucose epimerase, the third enzyme in the M. tuberculosis dTDP-L-rhamnose pathway which is essential for mycobacterial cell-wall synthesis. Because it is structurally unique, highly substrate-specific and does not require a cofactor, RmlC is considered to be the most promising drug target in the pathway, and the M. tuberculosis rmlC gene was selected in the initial round of TB Structural Genomics Consortium targets for structure determination. The 1.7 A native structure determined by the consortium facilities is reported and implications for in silico screening of ligands for structure-guided drug design are discussed.
Amino Acid Sequence↗
Structures and technology for biologists.
Structural genomics efforts are already producing a quarter of all 'new' macromolecular structures (<30% sequence identity to previously solved structures) and are stimulating development of systematic and automated approaches to structure determination. The thousands of new structures likely to be determined and the technologies and infrastructure likely to be developed over the next decade will benefit all biologists.
Automation↗
Recent developments in the PHENIX software for automated crystallographic structure determination.
A new software system called PHENIX (Python-based Hierarchical ENvironment for Integrated Xtallography) is being developed for the automation of crystallographic structure solution. This will provide the necessary algorithms to proceed from reduced intensity data to a refined molecular model, and facilitate structure solution for both the novice and expert crystallographer. Here, the features of PHENIXare reviewed and the recent advances in infrastructure and algorithms are briefly described.
Algorithms↗