Virology. The origin and control of pandemic influenza.
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Animals↗
Biomedical subjects
E Garman
Publications and source records attributed to E Garman.
A 1.8 A resolution structure of pig muscle 3-phosphoglycerate kinase with bound MgADP and 3-phosphoglycerate in open conformation: new insight into the role of the nucleotide in domain closure.
3-phosphoglycerate kinase (PGK) is a typical kinase with two structural domains. The domains each bind one of the two substrates, 3-phosphoglycerate (3-PG) and MgATP. For the phospho-transfer reaction to take place the substrates must be brought closer by a hinge-bending domain closure. Open and closed structures of the enzyme with different relative domain positions have been determined from different species, but a comprehensive description of this conformational transition is yet to be attained. Crystals of pig muscle PGK in complex with MgADP and 3-phosphoglycerate were grown under the conditions which have previously resulted in crystals of the closed, catalytically competent conformation of Trypanosoma brucei PGK. The X-ray structure of the pig muscle ternary complex was determined at 1.8 A and the model was refined to R=20.8% and Rfree=24.1%. Contrary to expectation, however, it represents an essentially open conformation compared to that of T. brucei PGK. In addition, the beta-phosphate group of ADP is mobile in the new structure, in contrast to its well-defined position in T. brucei PGK. An extensive comparison of the ternary complexes from these remote species has been carried out in order to establish general differences between the two conformations and is reported here. A second pair of the open and closed structures was also compared. These analyses have made it possible to define several characteristic changes which accompany the structural transition, in addition to those identified previously: (1) the operation of a hinge at beta-strand L in the inter-domain region which greatly affects the relative domain positions; (2) the rearrangement and movement of helix 8, regulated through the interactions with the nucleotide phosphate; and (3) the existence of another hinge between helix 14 and the rest of the C-terminal part of the chain, which allows fine adjustment of the N-domain position. The main hinge at beta-strand L acts in concert with the C-terminal hinge at helix 7 described previously. Simultaneous interactions of the nucleotide phosphate groups with the loop that precedes helix 8, beta-strand J and the N terminus of helix 13 are required for propagation of the nucleotide effect towards the beta-strand L molecular hinge. A detailed description of the role of nucleotide binding in the hinge operation is presented.
Adenosine Diphosphate↗
A DNA-porphyrin minor-groove complex at atomic resolution: the structural consequences of porphyrin ruffling.
The crystal structure of a B-type DNA hexanucleotide duplex complexed with the porphyrin molecule nickel-[tetra-N-methyl-pyridyl] porphyrin has been solved by multiwavelength anomalous diffraction phasing and refined to an R factor of 11.5% at a resolution of 0.9 A. The structure has been solved and refined as two crystallographically independent duplexes, stacked end to end. Contrary to expectation, the porphyrin molecule is not intercalated into the duplex but is stacked onto the ends of the two-duplex stack. The porphyrin molecule is highly buckled as a consequence of the nickel coordination, which produces large changes in local DNA structure. A second mode of porphyrin binding is apparent as a consequence of crystal packing, which places the ligand in the minor groove of an adjacent duplex. This structure thus provides, to our knowledge, the first atomic visualization of minor-groove binding for a porphyrin molecule. The geometry of groove binding provides a ready explanation for porphyrin-induced DNA strand cleavage at deoxyribose residues.
Base Pairing↗
Leaving no element of doubt: analysis of proteins using microPIXE.
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Calibration↗
Demystifying the synchrotron trip: a first time user's guide.
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Protein Conformation↗
Cool data: quantity AND quality.
The use of cryo-techniques in macromolecular crystallography has increased enormously over the last eight years and has become a vital part of modern X-ray data-collection methods. This paper presents some reasons for the rise in popularity of cryo-techniques and a brief outline of the basic methods, followed by a detailed discussion of factors to be considered when trying to optimize both the quantity and quality of the data collected. As more experimenters at synchrotrons observe significant radiation damage to crystals held near 100 K, the available options for further prolonging crystal lifetime and extending the techniques become worth investigating. Some possibilities and parameters to be considered are presented, although these must remain speculative until more experimental data are available.
CDC2-CDC28 Kinases↗
A single sequence change destabilizes the influenza virus neuraminidase tetramer.
A single change (E119G) in the influenza A virus N9 neuraminidase (NA) results in resistance of the enzyme to the NA inhibitor 4-Guanidino-Neu5Ac2en (4-GuDANA). This change causes a salt link between Glu119, which sits in a pocket in the bottom of the active site of the enzyme, and the 4-guanidinium moiety of the inhibitor to be lost. NA "heads" of the resistant enzyme produced only a few small crystals under conditions in which the wild-type enzyme readily formed large crystals. These small crystals were of sufficient quality to yield X-ray crystallographic data which confirmed the E119G change and demonstrated the presence of electron density representing either a strong structural-water molecule or an anionic species in place of the glutamate carboxylate. NA heads of the resistant enzyme also have greatly reduced NA activity per milligram of total protein. We have now found that the mutant NA heads consist predominantly of monomers with a few dimers and tetramers, as determined by electron microscopic analysis of the protein. The low level of enzymatic activity as well as the small number of crystals obtained were probably from the few tetramers remaining intact in the preparation. The purified wild-type and 4-GuDANA-resistant enzymes were treated with the homobifunctional NHS-ester cross linker, DTSSP. SDS-PAGE analysis of the treated enzymes clearly revealed cross-linked dimers of the wild-type enzyme. In contrast, only a small proportion of the 4-GuDANA-resistant neuraminidase was cross-linked. An examination of the known X-ray crystallographic structure of the wild-type NA reveals a salt bridge between Glu119 and Arg156 of the same monomer. Arg156 is a conserved amino acid that is situated at the interface between monomers, and a salt link between this amino acid and Glu119 may contribute to the stability of enzyme tetramers. It is suggested that the E119G alteration in the 4-GuDANA-resistant NA leads to the abrogation of this interaction and thus to the instability of the NA tetramers.
Animals↗
Crystal structure of the DNA decamer d(CGCAATTGCG) complexed with the minor groove binding drug netropsin.
The crystal structure of netropsin bound to the decamer d(CGCAATTGCG) has been determined at 2.4 A resolution. This is the first example of a crystal structure of netropsin bound to decamer DNA. The central eight bases of each DNA single-strand base pair with a self-complementary strand to form an octamer B-DNA duplex. These duplexes lie end to end within the unit cell. The terminal 5'-C and G-3' bases are unpaired and interact with the neighboring duplexes via interactions within both the major and minor groove to form base triplet interactions of the type C(+)-G x C and G*(G x C), respectively. The triplet interaction of the type C(+)-G x C is known to exist within triplex DNA with the C+ base oriented parallel with the Watson-Crick guanine base to which it hydrogen bonds. The netropsin molecule lies within the minor groove of the octamer duplex and assumes a class I type position, with bifurcated hydrogen-bonding interactions from the amide groups of the netropsin to the A x T base pairs of the minor groove. The netropsin molecule fits within a five base pair long minor groove site by bending of the flexible amidinium group at one end of the drug.
Anti-Bacterial Agents↗
High resolution structures of HIV-1 RT from four RT-inhibitor complexes.
We have determined the structures of four complexes of HIV-1 reverse transcriptase with non-nucleoside inhibitors, three fully refined at high resolution. The highest resolution structure is of the RT-nevirapine complex which has an R-factor of 0.186 and a root-mean-square bond length deviation of 0.015 A for all data to 2.2 A. The structures reveal a common mode of binding for these chemically diverse compounds. The common features of binding are largely hydrophobic interactions and arise from induced shape complementarity achieved by conformational rearrangement of the enzyme and conformational/configurational rearrangement of the compounds.
Amino Acid Sequence↗
Crystal structure of Vibrio cholerae neuraminidase reveals dual lectin-like domains in addition to the catalytic domain.
BACKGROUND: Vibrio cholerae neuraminidase is part of a mucinase complex which may function in pathogenesis by degrading the mucin layer of the gastrointestinal tract. The neuraminidase, which has been the target of extensive inhibitor studies, plays a subtle role in the pathology of the bacterium, by processing higher order gangliosides to GM1, the receptor for cholera toxin. RESULTS: We report here the X-ray crystal structure of V. cholerae neuraminidase at 2.3 A resolution. The 83 kDa enzyme folds into three distinct domains. The central catalytic domain has the canonical neuraminidase beta-propeller fold, and is flanked by two domains which possess identical legume lectin-like topologies but without the usual metal-binding loops. The active site has many features in common with other viral and bacterial neuraminidases but, uniquely, has an essential Ca2+ ion which plays a crucial structural role. CONCLUSIONS: The environment of the small intestine requires V. cholerae to secrete several adhesins, and it is known that its neuraminidase can bind to cell surfaces, and remain active. The unexpected lectin-like domains possibly mediate this attachment. These bacterial lectin folds represent additional members of a growing lectin superfamily.
Bacterial Adhesion↗
Crystallization and preliminary X-ray studies of influenza A virus neuraminidase of subtypes N5, N6, N8 and N9.
Neuraminidase heads from the following subtypes of influenza A virus have been crystallized: N5, N6, N8 and whale virus N9. The last three yielded crystals of X-ray diffraction quality; the N5 crystals obtained so far are not suitable for high resolution studies. The cell dimensions and space groups of crystals grown from the four subtypes have been determined.
Crystallography↗
X-ray crystallographic structure of a papain-leupeptin complex.
The three-dimensional structure of the papain-leupeptin complex has been determined by X-ray crystallography to a resolution of 2.1 A (overall R-factor = 19.8%). The structure indicates that: (i) leupeptin contacts the S subsites of the papain active site and not the S' subsites; (ii) the 'carbonyl' carbon atom of the inhibitor is covalently bound by the Cys-25 sulphur atom of papain and is tetrahedrally coordinated; (iii) the 'carbonyl' oxygen atom of the inhibitor faces the oxyanion hole and makes hydrogen bond contacts with Gln-19 and Cys-25.
Amino Acid Sequence↗
Purification, crystallization and preliminary crystallographic study of neuraminidase from Vibrio cholerae and Salmonella typhimurium LT2.
The nanH genes of Vibrio cholerae and Salmonella typhimurium LT2 coding neuraminidase were cloned separately in Escherichia coli, and the expression products purified. Single crystals of the V. cholerae neuraminidase were obtained using the hanging drop vapour diffusion method with polyethylene glycol as precipitant at pH 7.2. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 71.9 A, b = 79.0 A, c = 165.7 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 2.5 A. Single crystals of the S. typhimurium neuraminidase were obtained by hanging drop with potassium phosphate as precipitant at pH 7.2. The crystals also belong to the orthorhombic space group P2(1)2(1)2(1), with unit cell dimensions a = 47.4 A, b = 82.8 A, c = 92.4 A, and with one molecule in the asymmetric unit. Diffraction extends to at least 1.8 A.
Amino Acid Sequence↗
Crystal structure of a berenil-d(CGCAAATTTGCG) complex. An example of drug-DNA recognition based on sequence-dependent structural features.
The AT-selective drug berenil has been co-crystallized with the dodecanucleotide sequence d(CGCAAATTTGCG)2. The crystal structure has been solved to a resolution of 2.0 A and an R factor of 18.3%, with the location of 65 water molecules. The drug is symmetrically bound in the 5'-AATT region of the minor groove, with its amidinium groups hydrogen-bonding to O-2 atoms of the thymine base at each end of the binding site. This arrangement is distinct from that previously found for berenil with the sequence d(CGCGAATTCGCG)2, which has the drug bound to the sequencing 5'-ATT via hydrogen bonds to adenine N-3 atoms with the involvement of a bridging water molecule at one end of the binding site. The reasons for these differences are discussed in terms of changes in helical parameters; in particular propeller twist and base-pair roll are considered to be important. The conformational and base-pair geometry of the dodecanucleotide in the structure reported here, is closely similar to that for the native structure, suggesting that the 5'-AAATTT sequence does not significantly alter during drug binding, either because of its inflexibility or because its geometry is nearly ideal for berenil binding.
Base Sequence↗
Crystallization and preliminary X-ray study of a lipase from Pseudomonas glumae.
Lipase from Pseudomonas glumae has been purified and crystallized in two forms, using the hanging drop method of vapour diffusion at 4 degrees C and 15 degrees C. Both forms grew at pH 9.0 from 0.1 M-Tris buffer in the presence of 10% (v/v) acetone. Form 1 was crystallized from 27 to 29% polyethylene glycol in the presence of less than 0.5% (v/v) n-dodecyl-beta-D-glucopyranoside. Form 2 was grown from 17 to 19% ammonium sulphate in the presence of 1% n-octyl-beta-D-glucopyranoside. Form 1 is orthorhombic with space group P2(1)2(1)2(1), and cell dimensions of a = 158.1 A, b = 158.6 A, c = 63.4 A, Form 2 is tetragonal with space group P4(1)2(1)2 (or P4(3)2(1)2) and cell dimensions of a = 89.3 A, c = 180.4 A. Form 1 probably has four molecules per asymmetric unit and diffracts to at least 2.5 A. Form 2 has two molecules per asymmetric unit and diffracts to at least 3.0 A.
Crystallization↗
Crystal and solution structures of the oligonucleotide d(ATGCGCAT)2: a combined X-ray and NMR study.
A combined crystal-structure determination and NMR analysis of the octanucleotide d(ATGCGCAT)2 is reported. The X-ray analysis shows that the structure is A-form duplex in crystal state. The NMR study shows that in solution this sequence is B-type. The conformational results from each technique are presented in detail. The implications of these findings in terms of conformational flexibility and ligand binding are discussed.
Base Sequence↗
Crystal structure of a berenil-dodecanucleotide complex: the role of water in sequence-specific ligand binding.
The three-dimensional structure of a complex between the dodecanucleotide d(CGCGAATTCGCG) and the anti-trypanocidal drug berenil, has been determined to a resolution of 2.5 A. The structure has been solved by molecular replacement and refined to an R factor of 0.177. A total of 49 water molecules have been located. The drug is bound at the 5'-AAT-3' region of the oligonucleotide. At one end of the drug the amidinium group is in hydrogen-bonded contact with N3 of the adenine base complementary to the thymine of the AAT. The other amidinium group does not make direct interactions with the DNA. Instead, a water molecule mediates between them. This is in hydrogen-bonded contact with an amidinium nitrogen atom, N3 of the 5' end adenine base and the ring oxygen atom of an adjacent deoxyribose. Molecular mechanics calculations have been performed on this complex, with the drug at various positions along the sequence. These show that the observed position is only 0.8 kcal/mol higher in energy than the best position. It is suggested that there is a broad energy well in the AATT region for this drug, and that water molecules as well as the neighbouring sequence, will determine precise positioning. More general aspects of minor groove binding are discussed.
Amidines↗
Preliminary crystallographic data for NAD(P)H quinone reductase isolated from the Walker 256 rat carcinoma cell line.
An NAD(P)H quinone reductase isolated from Walker rat 256 carcinoma cells has been crystallized in a form suitable for high-resolution structural analysis. The crystals belong to orthorhombic space group P2(1)2(1)2(1) with cell parameters a = 168.15 A, b = 105.09 A and c = 67.38 A and contain four monomeric or two dimeric enzyme molecules per asymmetric unit. Diffraction extends beyond 2.3 A resolution.
Animals↗