scholarly journals Solution of the structure of tetrameric human glucose 6-phosphate dehydrogenase by molecular replacement

1999 ◽  
Vol 55 (4) ◽  
pp. 826-834 ◽  
Author(s):  
Shannon W. N. Au ◽  
Claire E. Naylor ◽  
Sheila Gover ◽  
Lucy Vandeputte-Rutten ◽  
Deborah A. Scopes ◽  
...  
Keyword(s):  
Crystal Form ◽  
Search Model ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Data Set ◽  
Space Groups ◽  
Successful Search ◽  
Using Data ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Resolution Data

Recombinant human glucose 6-phosphate dehydrogenase (G6PD) has been crystallized and its structure solved by molecular replacement. Crystals of the natural mutant R459L grow under similar conditions in space groups P212121 and C2221 with eight or four 515-residue molecules in the asymmetric unit, respectively. A non-crystallographic 222 tetramer was found in the C2221 crystal form using a 4 Å resolution data set and a dimer of the large β + α domains of the Leuconostoc mesenteroides enzyme as a search model. This tetramer was the only successful search model for the P212121 crystal form using data to 3 Å. Crystals of the deletion mutant ΔG6PD grow in space group F222 with a monomer in the asymmetric unit; 2.5 Å resolution data have been collected. Comparison of the packing of tetramers in the three space groups suggests that the N-terminal tail of the enzyme prevents crystallization with exact 222 molecular symmetry.

scholarly journals Expression, purification and crystallization of MnSOD fromArabidopsis thaliana

2014 ◽  
Vol 70 (5) ◽  
pp. 669-672 ◽  
Author(s):  
Alexandra T. Marques ◽  
Sandra P. Santos ◽  
Margarida G. Rosa ◽  
Mafalda A. A. Rodrigues ◽  
Isabel A. Abreu ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Superoxide Dismutase ◽  
Manganese Superoxide Dismutase ◽  
Search Model ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Plant Tolerance ◽  
Data Set ◽  
Manganese Superoxide ◽  
Primary Antioxidant

Manganese superoxide dismutase (MnSOD) is an essential primary antioxidant enzyme. MnSOD plays an important role in plant tolerance to abiotic stress and is a target candidate for increasing stress tolerance in crop plants. Although the structure and kinetic parameters of MnSODs from several organisms have been determined, this information is still lacking for plant MnSODs. Here, recombinant MnSOD fromArabidopsis thaliana(AtMnSOD) was expressed, purified and crystallized. A nearly complete data set could only be obtained when a total rotation range of 180° was imposed during data collection, despite the seemingly tetragonal metric of the AtMnSOD crystal diffraction. The data set extended to 1.95 Å resolution and the crystal belonged to space groupP1. Molecular-replacement calculations using an ensemble of homologous SOD structures as a search model gave a unique and unambiguous solution corresponding to eight molecules in the asymmetric unit. Structural and kinetic analysis of AtMnSOD is currently being undertaken.

The 1.1 Å resolution structure of a periplasmic phosphate-binding protein fromStenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank

2016 ◽  
Vol 72 (8) ◽  
pp. 933-943 ◽  
Author(s):  
Ronan Keegan ◽  
David G. Waterman ◽  
David J. Hopper ◽  
Leighton Coates ◽  
Graham Taylor ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Binding Protein ◽  
Data Bank ◽  
Crystal Form ◽  
Atomic Resolution ◽  
Search Model ◽  
Molecular Replacement ◽  
Phosphate Binding ◽  
Data Set ◽  
Phosphate Binding Protein

During efforts to crystallize the enzyme 2,4-dihydroxyacetophenone dioxygenase (DAD) fromAlcaligenessp. 4HAP, a small number of strongly diffracting protein crystals were obtained after two years of crystal growth in one condition. The crystals diffracted synchrotron radiation to almost 1.0 Å resolution and were, until recently, assumed to be formed by the DAD protein. However, when another crystal form of this enzyme was eventually solved at lower resolution, molecular replacement using this new structure as the search model did not give a convincing solution with the original atomic resolution data set. Hence, it was considered that these crystals might have arisen from a protein impurity, although molecular replacement using the structures of common crystallization contaminants as search models again failed. A script to perform molecular replacement usingMOLREPin which the first chain of every structure in the PDB was used as a search model was run on a multi-core cluster. This identified a number of prokaryotic phosphate-binding proteins as scoring highly in theMOLREPpeak lists. Calculation of an electron-density map at 1.1 Å resolution based on the solution obtained with PDB entry 2q9t allowed most of the amino acids to be identified visually and built into the model. ABLASTsearch then indicated that the molecule was most probably a phosphate-binding protein fromStenotrophomonas maltophilia(UniProt ID B4SL31; gene ID Smal_2208), and fitting of the corresponding sequence to the atomic resolution map fully corroborated this. Proteins in this family have been linked to the virulence of antibiotic-resistant strains of pathogenic bacteria and with biofilm formation. The structure of theS. maltophiliaprotein has been refined to anRfactor of 10.15% and anRfreeof 12.46% at 1.1 Å resolution. The molecule adopts the type II periplasmic binding protein (PBP) fold with a number of extensively elaborated loop regions. A fully dehydrated phosphate anion is bound tightly between the two domains of the protein and interacts with conserved residues and a number of helix dipoles.

Atomic resolution structure of a chimeric DNA–RNA Z-type duplex in complex with Ba2+ions: a case of complicated multi-domain twinning

2016 ◽  
Vol 72 (2) ◽  
pp. 211-223 ◽  
Author(s):  
Miroslaw Gilski ◽  
Pawel Drozdzal ◽  
Ryszard Kierzek ◽  
Mariusz Jaskolski
Keyword(s):  
Molecular Replacement ◽  
Higher Symmetry ◽  
Barium Ions ◽  
Space Groups ◽  
Left Handed ◽  
Crystal Twinning ◽  
Using Data ◽  
High Degree ◽  
Packing Analysis ◽  
Resolution Structure

The self-complementary dCrGdCrGdCrG hexanucleotide, in which not only the pyrimidine/purine bases but also the ribo/deoxy sugars alternate along the sequence, was crystallized in the presence of barium cations in the form of a left-handed Z-type duplex. The asymmetric unit of theP21crystal with a pseudohexagonal lattice contains four chimeric duplexes and 16 partial Ba2+sites. The chimeric (DNA–RNA)2duplexes have novel patterns of hydration and exhibit a high degree of discrete conformational disorder of their sugar-phosphate backbones, which can at least partly be correlated with the fractional occupancies of the barium ions. The crystals of the DNA–RNA chimeric duplex in complex with Ba2+ions and also with Sr2+ions exhibit complicated twinning, which in combination with structural pseudosymmetry made structure determination difficult. The structure could be successfully solved by molecular replacement in space groupsP1 andP21but not in orthorhombic or higher symmetry and, after scrupulous twinning and packing analysis, was refined in space groupP21to anRandRfreeof 11.36 and 16.91%, respectively, using data extending to 1.09 Å resolution. With the crystal structure having monoclinic symmetry, the sixfold crystal twinning is a combination of threefold and twofold rotations. The paper describes the practical aspects of dealing with cases of complicated twinning and pseudosymmetry, and compares the available software tools for the refinement and analysis of such cases.

Molecular replacement with multiple different models

2004 ◽  
Vol 37 (1) ◽  
pp. 159-161 ◽  
Author(s):  
Nicholas M. Glykos ◽  
Michael Kokkinidis
Keyword(s):  
Crystal Structure ◽  
Original Description ◽  
Natural Generalization ◽  
Divide And Conquer ◽  
Search Model ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Different Types ◽  
Replacement Problem ◽  
Special Case

Classical molecular replacement methods and the newer six-dimensional searches treat molecular replacement as a succession of sub-problems of reduced dimensionality. Due to their `divide-and-conquer' approach, these methods necessarily ignore (at least during their early stages) the very knowledge that a target crystal structure may comprise, for example, more than one copy of a search model, or several models of different types. An algorithm for a stochastic multi-dimensional molecular replacement search has been described previously and shown to locate solutions successfully, even in cases as complex as a 23-dimensional 4-body search. The original description of the method only dealt with a special case of molecular replacement, namely with the problem of placingncopies of only one search model in the asymmetric unit of a target crystal structure. Here a natural generalization of this algorithm is presented to deal with the full molecular replacement problem, that is, with the problem of determining the orientations and positions of a total ofncopies ofmdifferent models (withn≥m) which are assumed to be present in the asymmetric unit of a target crystal structure. The generality of this approach is illustrated through its successful application to a 17-dimensional 3-model problem involving one DNA and two protein molecules.

scholarly journals Crystallization and preliminary X-ray studies of the C-terminal domain ofMycobacterium tuberculosisLexA

2010 ◽  
Vol 66 (9) ◽  
pp. 1093-1095 ◽  
Author(s):  
Anu V. Chandran ◽  
J. Rajan Prabu ◽  
G. P. Manjunath ◽  
K. Neelakanteshwar Patil ◽  
K. Muniyappa ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
X Ray ◽  
Space Groups ◽  
Terminal Domain

The C-terminal domain ofMycobacterium tuberculosisLexA has been crystallized in two different forms. The form 1 and form 2 crystals belonged to space groupsP3121 andP31, respectively. Form 1 contains one domain in the asymmetric unit, while form 2 contains six crystallographically independent domains. The structures have been solved by molecular replacement.

Application of the molecular replacement method to a heavy-atom problem

1999 ◽  
Vol 32 (3) ◽  
pp. 472-474
Author(s):  
John G. Arnez
Keyword(s):  
Heavy Atom ◽  
Crystal Form ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Atom Model ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
The Absolute

The molecular replacement method has been applied to the heavy-atom problem in a case for which a heavy-atom model existed for one crystal form, and both native and derivative data were available for another crystal form of the same molecule; the absolute values of difference amplitudes for the second form were used in the calculation. The key requirement for obtaining a solution is that the contents of the asymmetric unit are the same and the same heavy-atom compound is used in both cases.

scholarly journals Crystal structure of a phosphoribosyl anthranilate isomerase from the hyperthermophilic archaeonThermococcus kodakaraensis

2016 ◽  
Vol 72 (11) ◽  
pp. 804-812 ◽  
Author(s):  
Sumera Perveen ◽  
Naeem Rashid ◽  
Anastassios C. Papageorgiou
Keyword(s):  
Pyrococcus Furiosus ◽  
Helical Structure ◽  
Structural Similarity ◽  
Molecular Replacement ◽  
Salt Bridges ◽  
Structural Comparison ◽  
Space Groups ◽  
Tim Barrel ◽  
Potential Factors ◽  
Using Data

A phosphoribosyl anthranilate isomerase,TkTrpF, fromThermococcus kodakaraensiswas expressed inEscherichia coliand purified to homogeneity.TkTrpF was crystallized and its structure was determined by molecular replacement in two different space groups (C2 andP1) using data to 1.85 and 1.75 Å resolution, respectively.TkTrpF belongs to the class of TIM-barrel proteins. Structural comparison with other phosphoribosyl anthranilate isomerases (TrpFs) showed the highest structural similarity toPyrococcus furiosusTrpF. Similarly toP. furiosusTrpF,TkTrpF is a monomer in solution, in contrast to other thermophilic enzymes, which exist as functional dimers. Although in space groupP1TkTrpF crystallizes with two molecules in the asymmetric unit, the interface is highly improbable in solution. Potential factors for the thermostability ofTkTrpF were attributed to an increase in helical structure, an increased number of charged residues and an increase in the number of salt bridges.

Crystallization and preliminary X-ray studies of hORF6, a novel human antioxidant enzyme

1998 ◽  
Vol 54 (3) ◽  
pp. 436-436 ◽  
Author(s):  
Hee-Jeong Choi ◽  
Sang Won Kang ◽  
Chul-Hak Yang ◽  
Sue Goo Rhee ◽  
Seong-Eon Ryu
Keyword(s):  
Antioxidant Enzyme ◽  
Unit Cell ◽  
X Rays ◽  
Asymmetric Unit ◽  
Data Set ◽  
X Ray ◽  
Space Groups ◽  
Cell Dimensions ◽  
Hanging Drop Method ◽  
Unit Cell Dimensions

HORF6 is a member of the novel antioxidant enzyme family found in humans. A recombinant form of hORF6 expressed and purified from E. coli has been crystallized by the hanging-drop method using various PEG's as precipitating agents. HORF6 crystallizes in two different monoclinic space groups, P21 and C2. The P21 crystals have unit-cell dimensions of a = 47.85, b = 75.17, c = 63.30 Å and β = 110.21° and contain two monomers per asymmetric unit, while the C2 crystals have unit-cell dimensions of a = 165.27, b = 95.44, c = 166.44 Å and β = 128.97° and contain more than six monomers per asymmetric unit. The P21 crystals with the smaller unit cell diffract X-rays better and behave well for the X-ray analysis. A native data set from a single crystal of the P21 space group gas been collected to 2.0 Å resolution.

Dehydration leads to a phase transition in monoclinic factor XIII crystals

1999 ◽  
Vol 55 (11) ◽  
pp. 1858-1862 ◽  
Author(s):  
Manfred S. Weiss ◽  
Rolf Hilgenfeld
Keyword(s):  
Phase Transition ◽  
Rigid Body ◽  
Cell Volume ◽  
Factor Xiii ◽  
Unit Cell Volume ◽  
Crystal Form ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Body Rotation ◽  
Peg 6000

Monoclinic factor XIII crystals have been transferred to a solution containing increasing amounts of the precipitant PEG 6000. At a concentration of about 36%(w/v) PEG 6000, a phase transition was observed. The space group of the crystals was preserved on the transition, but half of the 21 screw axes were lost, which meant that the unit-cell volume and the content of the asymmetric unit were doubled. The structure of factor XIII in the new crystal form was solved by molecular replacement. About 80% of the changes accompanying the transition can be explained by a rigid-body rotation of half of the factor XIII dimers in the lattice by about 5°. The remaining changes are mostly small interdomain movements of the four domains which constitute one factor XIII monomer.

scholarly journals Feasibility of one-shot-per-crystal structure determination using Laue diffraction

2009 ◽  
Vol 66 (1) ◽  
pp. 2-11 ◽  
Author(s):  
Sterling Cornaby ◽  
Doletha M. E. Szebenyi ◽  
Detlef-M. Smilgies ◽  
David J. Schuller ◽  
Richard Gillilan ◽  
...  
Keyword(s):  
Structure Determination ◽  
Crystal Size ◽  
Simultaneous Recording ◽  
Protein Crystal ◽  
Single Shot ◽  
Molecular Replacement ◽  
Data Set ◽  
Diffraction Patterns ◽  
Using Data ◽  
Lysozyme Crystals

Crystal size is an important factor in determining the number of diffraction patterns which may be obtained from a protein crystal before severe radiation damage sets in. As crystal dimensions decrease this number is reduced, eventually falling to one, at which point a complete data set must be assembled using data from multiple crystals. When only a single exposure is to be collected from each crystal, the polychromatic Laue technique may be preferable to monochromatic methods owing to its simultaneous recording of a large number of fully recorded reflections per image. To assess the feasibility of solving structures using single Laue images from multiple crystals, data were collected using a `pink' beam at the CHESS D1 station from groups of lysozyme crystals with dimensions of the order of 20–30 µm mounted on MicroMesh grids. Single-shot Laue data were used for structure determination by molecular replacement and correct solutions were obtained even when as few as five crystals were used.

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