An approach to multi-copy search in molecular replacement

Vagin, A.A.; Teplyakov, A.

doi:10.1107/s0907444900013780

Cited by 692 publications

(544 citation statements)

References 8 publications

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“…These crystals belong to the same space group (P1), but the unit cell dimensions differ from those obtained with the enzyme-FAD complex crystals, giving four molecules per unit cell. The structure was determined by molecular replacement using the enzyme-FAD complex as a search model (26). A composite omit map showed residual electron density for FAD and methimazole only.…”

Section: Methodsmentioning

confidence: 99%

Mechanism of action of a flavin-containing monooxygenase

Eswaramoorthy

Bonanno

Burley³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

164

170

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Elimination of nonnutritional and insoluble compounds is a critical task for any living organism. Flavin-containing monooxygenases (FMOs) attach an oxygen atom to the insoluble nucleophilic compounds to increase solubility and thereby increase excretion. Here we analyze the functional mechanism of FMO from Schizosaccharomyces pombe using the crystal structures of the wild type and protein–cofactor and protein–substrate complexes. The structure of the wild-type FMO revealed that the prosthetic group FAD is an integral part of the protein. FMO needs NADPH as a cofactor in addition to the prosthetic group for its catalytic activity. Structures of the protein–cofactor and protein–substrate complexes provide insights into mechanism of action. We propose that FMOs exist in the cell as a complex with a reduced form of the prosthetic group and NADPH cofactor, readying them to act on substrates. The 4α-hydroperoxyflavin form of the prosthetic group represents a transient intermediate of the monooxygenation process. The oxygenated and reduced forms of the prosthetic group help stabilize interactions with cofactor and substrate alternately to permit continuous enzyme turnover.

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Section: Methodsmentioning

confidence: 99%

Mechanism of action of a flavin-containing monooxygenase

Eswaramoorthy

Bonanno

Burley³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

164

170

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“…29 The final model was then used to search for a molecular replacement solution using the higher resolution 1.30 Å data from the YscU N263A/P264A crystal and the 1.53 Å resolution data collected from the YscU N263A crystal with the program MOLREP from the CCP4 suite. 30 The model was Atomic Resolution Structure of YscU refined using REFMAC5 31 and manually corrected using COOT.…”

Section: Protein Crystallizationmentioning

confidence: 99%

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion

et al. 2009

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Crystal structures of cleaved and uncleaved forms of the YscU cytoplasmic domain, an essential component of the type III secretion system (T3SS) in Yersinia pestis, have been solved by single-wavelength anomolous dispersion and refined with X-ray diffraction data extending up to atomic resolution (1.13 Å ). These crystallographic studies provide structural insights into the conformational changes induced upon auto-cleavage of the cytoplasmic domain of YscU. The structures indicate that the cleaved fragments remain bound to each other. The conserved NPTH sequence that contains the site of the N263-P264 peptide bond cleavage is found on a b-turn which, upon cleavage, undergoes a major reorientation of the loop away from the catalytic N263, resulting in altered electrostatic surface features at the site of cleavage. Additionally, a significant conformational change was observed in the N-terminal linker regions of the cleaved and noncleaved forms of YscU which may correspond to the molecular switch that influences substrate specificity. The YscU structures determined here also are in good agreement with the auto-cleavage mechanism described for the flagellar homolog FlhB and E. coli EscU.

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“…The model from the monoclinic crystal form was then used to solve the triclinic crystal form, again by molecular replacement using MOL-REP (19). The MOLREP search for six monomers worked with default settings, probably because of the high quality of the model from the monoclinic crystal form and because of the presence of pseudotranslation vectors relating several monomers in identical or near identical orientations.…”

Section: Methodsmentioning

confidence: 99%

“…Two copies of the partial model for subunit A were subsequently located in the monoclinic crystal form using MOLREP (19) and used to calculate starting phases for an ARP/WARP-run at 1.4-Å resolution, which yielded near complete models and a confident sequence assignment for both monomers in this crystal form.…”

Section: Methodsmentioning

confidence: 99%

Peptidoglycan Amidase MepA Is a LAS Metallopeptidase

Marcyjaniak

Odintsov

Sabala

et al. 2004

Journal of Biological Chemistry

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LAS enzymes are a group of metallopeptidases that share an active site architecture and a core folding motif and have been named according to the group members lysostaphin, D-Ala-D-Ala carboxypeptidase and sonic hedgehog. Escherichia coli MepA is a periplasmic, penicillin-insensitive murein endopeptidase that cleaves the D-alanyl-meso-2,6-diamino-pimelyl amide bond in E. coli peptidoglycan. The enzyme lacks sequence similarity with other peptidases, and is currently classified as a peptidase of unknown fold and catalytic class in all major data bases. Here, we build on our observation that two motifs, characteristic of the newly described LAS group of metallopeptidases, are conserved in MepA-type sequences. We demonstrate that recombinant E. coli MepA is sensitive to metal chelators and that mutations in the predicted Zn 2؉ ligands His-113, Asp-120, and His-211 inactivate the enzyme. Moreover, we present the crystal structure of MepA. The active site of the enzyme is most similar to the active sites of lysostaphin and D-Ala-D-Ala carboxypeptidase, and the fold is most closely related to the N-domain of sonic hedgehog. We conclude that MepAtype peptidases are LAS enzymes.

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An approach to multi-copy search in molecular replacement

Cited by 692 publications

References 8 publications

Mechanism of action of a flavin-containing monooxygenase

Mechanism of action of a flavin-containing monooxygenase

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion

Peptidoglycan Amidase MepA Is a LAS Metallopeptidase

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