The application of the molecular replacement method to the de novo determination of protein structure

1991 ◽  
Vol 24 (4) ◽  
pp. 399-424 ◽  
Author(s):  
Michael C. Lawrence
Keyword(s):  
Protein Structure ◽  
De Novo ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Electron Density Map ◽  
Novel Protein

The determination of a novel protein structure by X-ray diffraction is seldom straightforward. Three hurdles present themselves (i) the protein must be purified in sufficient quantity to allow crystallization trials, (ii) crystals must be grown to adequate size and must diffract to a resolution that will allow atomic detail to be revealed, and (iii) phases must be determined for the diffracted X-ray beams in order that an initial electron-density map may be calculated.

scholarly journals Crystallization and X-ray structure determination of a thermoalkalophilic lipase fromGeobacillusSBS-4S

2013 ◽  
Vol 69 (4) ◽  
pp. 355-357
Author(s):  
Muhammad Tayyab ◽  
Naeem Rashid ◽  
Clement Angkawidjaja ◽  
Shigenori Kanaya ◽  
Muhammad Akhtar
Keyword(s):  
Diffusion Method ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Method ◽  
Wide Range ◽  
Molecular Replacement Method

A thermoalkalophilic lipase (LIPSBS) from the newly isolatedGeobacillusstrain SBS-4S which hydrolyzes a wide range of fatty acids has been characterized. In the present study, the crystallization of purified LIPSBSusing the sitting-drop vapour-diffusion method and its X-ray diffraction studies are described. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 55.13,b= 71.75,c= 126.26 Å. The structure was determined at 1.6 Å resolution by the molecular-replacement method using the lipase fromG. stearothermophilusL1 as a model.

scholarly journals Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF fromMicrocystis aeruginosa, a putative reductase participating in aeruginosin biosynthesis

2015 ◽  
Vol 71 (4) ◽  
pp. 466-470 ◽  
Author(s):  
Ruyi Ding ◽  
Cui Xu ◽  
Xu Chen ◽  
Mengyun Bao ◽  
Xiaoting Qiu
Keyword(s):  
Diffraction Analysis ◽  
Biosynthetic Pathway ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Antithrombotic Activity ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Replacement Method ◽  
Molecular Replacement Method

The 2-carboxy-6-hydroxyoctahydroindole moiety is an essential residue for the antithrombotic activity of aeruginosins, which are a class of cyanobacteria-derived bioactive linear tetrapeptides. The biosynthetic pathway of the 2-carboxy-6-hydroxyoctahydroindole moiety has not yet been resolved. AerF was indicated to be involved in the biosynthesis of the 2-carboxy-6-hydroxyoctahydroindole moiety. This study reports the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of AerF fromMicrocystis aeruginosawith a C-terminal His6tag. The crystal diffracted to a maximum resolution of 1.38 Å and belonged to the tetragonal space groupP4322, with unit-cell parametersa=b= 101.581,c= 116.094 Å. The calculated Matthews coefficient and solvent content of the crystal were 2.47 Å3 Da−1and 50.32%, respectively. The initial model of the structure was obtained by the molecular-replacement method and refinement of the structure is in progress.

scholarly journals Cloning, expression, purification, crystallization and X-ray crystallographic analysis of (S)-3-hydroxybutyryl-CoA dehydrogenase fromClostridium butyricum

2014 ◽  
Vol 70 (4) ◽  
pp. 485-488
Author(s):  
Eun-Jung Kim ◽  
Kyung-Jin Kim
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Crystal Volume

(S)-3-Hydroxybutyryl-CoA dehydrogenase fromClostridium butyricum(CbHBD) is an enzyme that catalyzes the second step in the biosynthesis ofn-butanol from acetyl-CoA by the reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. TheCbHBD protein was crystallized using the hanging-drop vapour-diffusion method in the presence of 2 Mammonium sulfate, 0.1 MCAPS pH 10.5, 0.2 Mlithium sulfate at 295 K. X-ray diffraction data were collected to a maximum resolution of 2.3 Å on a synchrotron beamline. The crystal belonged to space groupR3, with unit-cell parametersa=b= 148.5,c= 201.6 Å. With four molecules per asymmetric unit, the crystal volume per unit protein weight (VM) is 3.52 Å3 Da−1, which corresponds to a solvent content of approximately 65.04%. The structure was solved by the molecular-replacement method and refinement of the structure is in progress.

Crystallization and preliminary X-ray diffraction studies of a Bowman–Birk inhibitor from Vigna unguiculata seeds

1999 ◽  
Vol 55 (11) ◽  
pp. 1920-1922 ◽  
Author(s):  
K. N. Rao ◽  
S. S. Hegde ◽  
R. J. Lewis ◽  
C. G. Suresh
Keyword(s):  
Vigna Unguiculata ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Citrate Phosphate

A Bowman–Birk type trypsin/chymotrypsin inhibitor isolated from Vigna unguiculata seeds has been crystallized. Crystals were grown using the vapour-diffusion method at pH 4.0 using citrate/phosphate as a buffer and 30% saturated ammonium sulfate as precipitant. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 32.4, b = 61.8, c = 32.9 Å, β = 114.5°. The Matthews coefficient calculated assuming two molecules in the asymmetric unit was 1.95 Å3 Da−1, which corresponds to a 37% solvent content. X-ray data were collected to 2.5 Å resolution from a flash-frozen crystal. The structure was solved using the molecular-replacement method using tracy soybean inhibitor structure (PDB entry 1pi2) as a model.

scholarly journals De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

2021 ◽  
Author(s):  
Guillaume Tetreau ◽  
Michael R. Sawaya ◽  
Elke De Zitter ◽  
Elena A. Andreeva ◽  
Anne-Sophie Banneville ◽  
...  
Keyword(s):  
De Novo ◽  
Mosquito Control ◽  
Crystal Packing ◽  
Molecular Replacement ◽  
Dispersion Method ◽  
X Ray ◽  
Naturally Occurring ◽  
Serial Femtosecond Crystallography

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

scholarly journals Sample preparation, crystallization and structure solution of HisC fromMycobacterium tuberculosis

2013 ◽  
Vol 69 (4) ◽  
pp. 445-448 ◽  
Author(s):  
Nazia Nasir ◽  
Rajan Vyas ◽  
Bichitra K. Biswal
Keyword(s):  
Gel Filtration ◽  
Nitrilotriacetic Acid ◽  
Monomethyl Ether ◽  
X Ray Diffraction ◽  
X Ray ◽  
Metal Affinity ◽  
Replacement Method ◽  
Diffusion Technique ◽  
Structure Solution ◽  
Molecular Replacement Method

Histidinolphosphate aminotransferase (HisC; Rv1600) fromMycobacterium tuberculosiswas overexpressed inM. smegmatisand purified to homogeneity using nickel–nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals suitable for X-ray analysis were grown by the hanging-drop vapour-diffusion technique using 30% polyethylene glycol monomethyl ether 2000 as the precipitant. The crystals belonged to the hexagonal space groupP3221, with an unusual high solvent content of 74.5%. X-ray diffraction data were recorded to 3.08 Å resolution from a single crystal using in-house Cu Kα radiation. The structure of HisC was solved by the molecular-replacement method using itsCorynebacterium glutamicumcounterpart as a search model. HisC is a dimer in the crystal as well as in solution.

Against the odds?De novostructure determination of a pilin with two cysteine residues by sulfur SAD

2015 ◽  
Vol 71 (5) ◽  
pp. 1095-1101 ◽  
Author(s):  
Manuela Gorgel ◽  
Andreas Bøggild ◽  
Jakob Jensen Ulstrup ◽  
Manfred S. Weiss ◽  
Uwe Müller ◽  
...  
Keyword(s):  
Protein Structure ◽  
Sodium Ion ◽  
Data Sets ◽  
Threshold Values ◽  
X Ray Diffraction ◽  
X Ray ◽  
Single Well ◽  
Anomalous Signal ◽  
Pilin Protein

Exploiting the anomalous signal of the intrinsic S atoms to phase a protein structure is advantageous, as ideally only a single well diffracting native crystal is required. However, sulfur is a weak anomalous scatterer at the typical wavelengths used for X-ray diffraction experiments, and therefore sulfur SAD data sets need to be recorded with a high multiplicity. In this study, the structure of a small pilin protein was determined by sulfur SAD despite several obstacles such as a low anomalous signal (a theoretical Bijvoet ratio of 0.9% at a wavelength of 1.8 Å), radiation damage-induced reduction of the cysteines and a multiplicity of only 5.5. The anomalous signal was improved by merging three data sets from different volumes of a single crystal, yielding a multiplicity of 17.5, and a sodium ion was added to the substructure of anomalous scatterers. In general, all data sets were balanced around the threshold values for a successful phasing strategy. In addition, a collection of statistics on structures from the PDB that were solved by sulfur SAD are presented and compared with the data. Looking at the quality indicatorRanom/Rp.i.m., an inconsistency in the documentation of the anomalousRfactor is noted and reported.

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