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At least 379 records · Page 21Linked to original sources
PubMed2006

Structure of 8Salpha globulin, the major seed storage protein of mung bean.

The 8S globulins of mung bean [Vigna radiata (L.) Wilczek] are vicilin-type seed storage globulins which consist of three isoforms: 8Salpha, 8Salpha' and 8Sbeta. The three isoforms have high sequence identities with each other (around 90%). The structure of 8Salpha globulin has been determined for the first time by X-ray crystallographic analysis and refined at 2.65 A resolution with a final R factor of 19.6% for 10-2.65 A resolution data. The refined 8Salpha globulin structure consisted of 366 of the 423 amino-acid residues (one subunit of the biological trimer). With the exception of several disordered regions, the overall 8Salpha globulin structure closely resembled those of other seed storage 7S globulins. The 8Salpha globulin exhibited the highest degree of sequence identity (68%) and structural similarity (a root-mean-square deviation of 0.6 A) with soybean beta-conglycinin beta (7S globulin). Their surface hydrophobicities are also similar to each other, although their solubilities differ under alkaline conditions at low ionic strength. This difference seems to be a consequence of charge-charge interactions and not hydrophobic interactions of the surfaces, based on a comparison of the electrostatic potentials of the molecular surfaces. The thermal stability of 8Salpha globulin is lower than that of soybean beta-conglycinin beta. This correlates with the cavity size derived from the crystal structure, although other structural features also have a small effect on the protein's thermal stability.

Amino Acid Sequence↗
PubMed2006

HKL-3000: the integration of data reduction and structure solution--from diffraction images to an initial model in minutes.

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.

Crystallography, X-Ray↗
PubMed1993

Crystallographic analyses of an active HIV-1 ribonuclease H domain show structural features that distinguish it from the inactive form.

. An active recombinant preparation of the carboxy-terminal ribonuclease H (RNase H) domain of HIV-I reverse transcriptase has produced crystals of several different forms, including a trigonal prism form (P3(1); a = b = 52.03, c = 113.9 A with two molecules per asymmetric unit) and a hexagonal tablet form (P6(2)22 or P6(4)22; a = b = 93.5, c = 74.1 A with one molecule per asymmetric unit). The former appears to be isomorphous with crystals of a similar, but inactive, version of the enzyme that was used for a prior crystal structure determination [Davies, Hostomska, Hostomsky, Jordan & Matthews (1991). Science, 252, 88-95]. We have also obtained a structure solution for this crystal form and have refined it with 2.8 A resolution data (R = 0.216). We report here details of our crystallization studies and some initial structural results that verify that the preparation of active HIV-1 RNase H yields a protein that is not just enzymatically, but also structurally, distinguishable from the inactive form. Evidence suggests that region 538-542, which may be involved in the catalytic site and which is disordered in both molecules in the prior structure determination, is ordered in the crystal structure of the active enzyme, although the ordering may include more than one conformation for this loop. It should also be noted that, in the crystal structure of the trigonal form, RNase H monomers associate to form noncrystallographic twofold-symmetric dimers by fusing five-stranded mixed beta sheets into a single ten-stranded dimerwide sheet, an assembly that was not remarked upon by previous investigators.

Journal Article↗
PubMed1994

Preliminary crystallographic analysis of glyceraldehyde 3-phosphate dehydrogenase from the extreme thermophile Thermus aquaticus.

Crystals have been obtained of glyceraldehyde 3-phosphate dehydrogenase from the extreme thermophile, Thermus aquaticus. This enzyme is stable and active at 363 K, thus its three-dimensional structure should add insight into the structural basis of protein thermostability. Large high-quality crystals were grown using isopropanol and polyethylene glycol at pH 8.4. They crystallize in the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 144.77 (6), b = 148.77 (5), c = 149.50 (7) A, and diffract to beyond 2.8 A. The volume of the unit cell and the packing observed in other GAPDH structures suggest that there are two tetramers per asymmetric unit. With 300 kDa/asymmetric unit expected in this form, its solution represents a challenging molecular replacement problem. A low-resolution data set has been recorded and used to carry out self-rotation, cross-rotation and Patterson-correlation refinement calculations. We found that the Q molecular axes of both tetramers are approximately coincident with the crystallographic a axis, and the non-crystallographic symmetry relating the two tetramers is approximately a rotation of 90 degrees about the a axis.

Journal Article↗
PubMed1995

On the application of phase relationships to complex structures. XXXV. Some experiments with 2-Zn insulin.

The direct-methods program SAYTAN has been applied to the known structure of 2-Zn insulin with 806 atoms, excluding solvent, in the asymmetric unit. Useful sets of phases can be obtained and selected by figures of merit for data resolutions between 1.5 and 2.25 A and these can be extended by SAYTAN to give mean phase errors of 68 degrees for more than 2000 reflections. A feature of the phases so found is that the phase errors decrease with increasing resolution - which is the opposite of the situation when phases are found by isomorphous-replacement techniques.

Journal Article↗
PubMed1996

A challenging case for protein crystal structure determination: the mating pheromone Er-1 from Euplotes raikovi.

Four different phasing methods have been applied to the determination of the crystal structure of the 40 amino-acid mating pheromone of the unicellular ciliated protozoan Euplotes raikovi. The difficulties, failures and successes in attempts to solve the structure by: (1) molecular replacement, (2) direct phasing using the 'Shake and Bake' algorithm, (3) isomorphous replacement, and (4) multiple-wavelength anomalous dispersion are described. The structure was first solved by molecular replacement, and then was the first successful structure determination by 'Shake and Bake' without the direct involvement of its authors. A description of the current status of the high-resolution refinement of the structure is also given. The model is refined against 1 A resolution data to an R factor of 12.9%, and includes H atoms and discretely disordered side chains.

Journal Article↗
PubMed1998

Structure and proposed amino-acid sequence of a pepsin from atlantic cod (Gadus morhua).

The crystal structure of a pepsin from the gastric mucosa of Atlantic cod has been determined to 2.16 A resolution. Data were collected on orthorhombic crystals with cell dimensions a = 35.98, b = 75.40 and c = 108.10 A, on a FAST area-detector system. The phase problem was solved by the molecular-replacement method using porcine pepsin (PDB entry 5PEP) as a search model. The structure has been refined to a crystallographic R factor of 20.8% using all reflections between 8.0 and 2.16 A, without prior knowledge of the primary sequence. The resulting crystal structure is very similar to the porcine enzyme, consisting of two domains with predominantly beta-sheet structure in the same sequential positions as the enzyme from pig. In the course of the model building, 122 residues were substituted and two residues deleted from the starting model to give a polypeptide chain of 324 amino acids and a sequence identity of 57.7% with the pig pepsin. No carbohydrate residues were located. Sequence alignment with available aspartic proteinases, indicates that the fish enzyme seems to be more related to mammalian gastric pepsins than to the mammalian gastricsins and chymosins, lysosomal cathepsin D's and a pepsin from tuna fish. The amino-acid composition of the cod enzyme, however, is more in accordance with the cathepsin D's.

Amino Acid Sequence↗
PubMed1998

Miscellaneous algorithms for density modification.

Various algorithms are described, developed for the dm density modification package, which have not been described elsewhere. Methods are described for the following problems: determination of the absolute scale and overall temperature factor of a data set, by a method which is less dependent on data resolution than Wilson statistics; an efficient interpolation algorithm for averaging and its application to refinement of averaging operators; a method for the automatic determination of averaging masks.

Algorithms↗
PubMed1998

Purification, crystallization and preliminary X-ray analysis of the Escherichia coli phytase.

A recombinant form of Escherichia coli phytase, which hydrolyzes phytic acid into phosphate and myo-inositol, has been expressed, purified and crystallized. Crystals have been obtained by the method of bulk crystallization in 10 mM sodium acetate buffer (pH 4.5) without using a conventional precipitant. The enzyme crystallized in space group P21, with unit-cell dimensions a = 74.9, b = 72.2, c = 82.4 A, and beta = 92.0 degrees. Crystals diffract to at least 2.2 A at a rotating-anode X-ray source and a 2.3 A resolution data set has been collected, giving completeness of 98.0% and an Rsym of 0.072. Assuming there are two phytase molecules in the asymmetric unit, the solvent content is calculated to be 42.1%. A self-rotation function shows a clear twofold non-crystallographic symmetry relating two molecules of E. coli phytase in the asymmetric unit.

6-Phytase↗
PubMed1998

Crystallographic characterization of Pap1-DNA complex.

Pap1 is a fission yeast transcription factor that activates genes related with resistance against staurosporine, a potent inhibitor of protein kinase C, and has been shown to be involved in cell growth, cell cycle, carcinogenesis and differentiation. Pap1 has the bZIP DNA-binding domain but binds to non-consensus DNA sequences for the bZIP motif. Highly ordered crystals of the DNA-binding domain complexed with a DNA fragment that has an ATF/CREB-like non-consensus sequence have been obtained. The crystals grew by the vapor-diffusion technique with polyethylene glycol 6000 and belong to space group R3 with a = b = 240.78, c = 43.85 A. A 2.0 A resolution data set was collected with a cryo-crystallographic technique.

Basic-Leucine Zipper Transcription Factors↗
PubMed1999

Crystallization of L-aspartate oxidase, the first enzyme in the bacterial de novo biosynthesis of NAD.

The flavoenzyme L-aspartate oxidase from Escherichia coli was crystallized using the hanging-drop vapour-diffusion technique with PEG 4000 as precipitant. The crystals belong to space group P3121 (or P3221) with unit-cell parameters a = b = 84.9, c = 159.9 A. A solvent content of 42% corresponds to a monomer (60 kDa) in the asymmetric unit. A complete 2.8 A resolution data set was collected using a rotating-anode X-ray generator.

Amino Acid Oxidoreductases↗
PubMed1999

Crystallization and preliminary x-ray crystallographic analysis of the B800-820 light-harvesting complex from Rhodopseudomonas acidophila strain 7050.

The B800-820 peripheral light-harvesting complex, an integral membrane protein from Rhodopseudomonas acidophila strain 7050, has been crystallized in a form suitable for X-ray diffraction analysis. The crystals belong to space group R32 with hexagonal cell dimensions a = 117.20, c = 295.14 A (at 100 K). A complete 2.8 A resolution data set has been collected and a structure solution obtained using molecular-replacement methods.

Amino Acid Sequence↗
PubMed1999

Ab initio structure determination of a small protein, rubredoxin, by direct methods.

The direct-methods program SAYTAN has been applied successfully to a known protein, rubredoxin, which contains 52 amino-acid residues including an FeS4 unit, a sulfate ion and 102 solvent water molecules. Starting with initially random phases, useful sets can be obtained from multiple trials and selected by figures of merit at different resolutions. Phase extension followed by weighted Fourier recycling reveals a recognizable structure of rubredoxin. The model is refined against 1 A resolution data to an R factor of 14.5% using the program SHELXL93.

Bacterial Proteins↗
PubMed1999

A two-dimensional histogram-matching method for protein phase refinement and extension.

A new method has been developed for protein phase improvement using the joint distribution of the electron density and its gradient (two-dimensional histogram) as a constraint in a density-modification procedure. Matching the two-dimensional (2D) histogram of a given map to that of an ideal 2D histogram was achieved through alternating applications of one-dimensional (1D) histogram matching on electron density and on density gradient. The 2D histogram-matching method was compared with the 1D density histogram-matching method for phase refinement and extension starting from either medium-resolution or high-resolution data on three different types of phases. These included phase refinement and extension using MIR phases of T(6) insulin and phases with randomly generated errors. The test results demonstrated significant improvement of the phases and the overall map quality using the 2D histogram-matching method compared with the 1D density histogram-matching method in each of the three test cases.

Algorithms↗
PubMed2000

Structure of a new neurotoxin from the scorpion Buthus martensii Karsch at 1.76 A.

A new neurotoxin BmK M2, toxic to both mammals and insects, with the strongest toxicity in the BmK toxin series, has been purified from the Chinese scorpion Buthus martensii Karsch and crystallized with MPD at pH 7.5. The crystals are orthorhombic, belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 36.64, b = 36.95, c = 37.23 A. The structure was solved by molecular replacement and refined to R = 0.186 for all reflections to a resolution of 1.76 A. The whole sequence (64 residues) of BmK M2 was determined by crystallographic analysis based on high-resolution data and the homologous model of BmK M8. The refined BmK M2 structure shows a non-proline cis peptide bond between Pro9 and His10 which enables the C-terminal segment to adopt a conformation different to that of the weak toxin BmK M8. Recently, a mutation analysis had suggested that both the tenth residue and the C-terminus play key roles in receptor binding. Therefore, these features may be related to the binding selectivity of the group III alpha-like toxins. The charge changes of residues 8, 10, 18, 28, 55 and 59 from neutral or negative to positive or neutral, which leads to a positive electrostatic potential surface, may be responsible for the high toxicity of BmK M2.

Amino Acid Sequence↗
PubMed2000

Structure of human erythrocyte catalase.

Catalase (E.C. 1.11.1.6) was purified from human erythrocytes and crystallized in three different forms: orthorhombic, hexagonal and tetragonal. The structure of the orthorhombic crystal form of human erythrocyte catalase (HEC), with space group P2(1)2(1)2(1) and unit-cell parameters a = 84.9, b = 141.7, c = 232.5 A, was determined and refined with 2.75 A resolution data. Non-crystallographic symmetry restraints were employed and the resulting R value and R(free) were 0.206 and 0.272, respectively. The overall structure and arrangement of HEC molecules in the orthorhombic unit cell were very similar to those of bovine liver catalase (BLC). However, no NADPH was observed in the HEC crystal and a water was bound to the active-site residue His75. Conserved lattice interactions suggested a common growth mechanism for the orthorhombic crystals of HEC and BLC.

Animals↗
PubMed2005

Crystallization and preliminary X-ray crystallographic analysis of Sulfolobus solfataricus thioredoxin reductase.

A thermostable thioredoxin reductase isolated from Sulfolobus solfataricus (SsTrxR) has been successfully crystallized in the absence and in the presence of NADP. Two different crystal forms have been obtained. Crystals of the form that yields higher resolution data (1.8 A) belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 76.77, b = 120.68, c = 126.85 A. The structure of the enzyme has been solved by MAD methods using the anomalous signal from the Se atoms of selenomethionine-labelled SsTrxR.

Adenosine Diphosphate↗
PubMed2002

Transmembrane helix predictions revisited.

Methods that predict membrane helices have become increasingly useful in the context of analyzing entire proteomes, as well as in everyday sequence analysis. Here, we analyzed 27 advanced and simple methods in detail. To resolve contradictions in previous works and to reevaluate transmembrane helix prediction algorithms, we introduced an analysis that distinguished between performance on redundancy-reduced high- and low-resolution data sets, established thresholds for significant differences in performance, and implemented both per-segment and per-residue analysis of membrane helix predictions. Although some of the advanced methods performed better than others, we showed in a thorough bootstrapping experiment based on various measures of accuracy that no method performed consistently best. In contrast, most simple hydrophobicity scale-based methods were significantly less accurate than any advanced method as they overpredicted membrane helices and confused membrane helices with hydrophobic regions outside of membranes. In contrast, the advanced methods usually distinguished correctly between membrane-helical and other proteins. Nonetheless, few methods reliably distinguished between signal peptides and membrane helices. We could not verify a significant difference in performance between eukaryotic and prokaryotic proteins. Surprisingly, we found that proteins with more than five helices were predicted at a significantly lower accuracy than proteins with five or fewer. The important implication is that structurally unsolved multispanning membrane proteins, which are often important drug targets, will remain problematic for transmembrane helix prediction algorithms. Overall, by establishing a standardized methodology for transmembrane helix prediction evaluation, we have resolved differences among previous works and presented novel trends that may impact the analysis of entire proteomes.

Algorithms↗