scholarly journals Molecular replacement in the `twilight zone': structure determination of the non-haem iron oxygenase NovR fromStreptomyces spheroidesthrough repeated density modification of a poor molecular-replacement solution

2006 ◽  
Vol 62 (12) ◽  
pp. 1564-1570 ◽  
Author(s):  
Sascha Keller ◽  
Florence Pojer ◽  
Lutz Heide ◽  
David M. Lawson
Keyword(s):  
Structure Determination ◽  
Twilight Zone ◽  
Molecular Replacement ◽  
Zone Structure ◽  
Replacement Solution ◽  
Haem Iron

scholarly journals Structure determination of Murine Norovirus NS6 proteases with C-terminal extensions designed to probe protease-substrate interactions

2014 ◽  
Author(s):  
Humberto Fernandes ◽  
Eoin N Leen ◽  
Hamlet Cromwell Jnr ◽  
Marc-Philipp Pfeil ◽  
Stephen Curry
Keyword(s):  
Structure Determination ◽  
Crystal Form ◽  
Molecular Replacement ◽  
Murine Norovirus ◽  
Substrate Interactions ◽  
C Terminus ◽  
Peptide Binding Site ◽  
Specific Protease ◽  
Protease Substrate

Noroviruses are positive-sense single-stranded RNA viruses. They encode an NS6 protease that cleaves a viral polyprotein at specific sites to produce mature viral proteins. In an earlier study we obtained crystals of murine norovirus (MNV) NS6 protease in which crystal contacts were mediated by specific insertion of the C-terminus of one protein (which contains residues P5-P1 of the NS6-7 cleavage junction) into the peptide binding site of an adjacent molecule, forming an adventitious protease-product complex. We sought to reproduce this crystal form to investigate protease-substrate complexes by extending the C-terminus of NS6 construct to include residues on the C-terminal (P´) side of the cleavage junction. We report the crystallization and crystal structure determination of inactive mutants of murine norovirus NS6 protease with C-terminal extensions of one, two and four residues from the N-terminus of the adjacent NS7 protein (NS6 1´, NS6 2´, NS6 4´). We also determined the structure of a chimeric extended NS6 protease in which the P4 P4′ sequence of the NS6-7 cleavage site was replaced with the corresponding sequence from the NS2-3 cleavage junction (NS6 4´ 2|3). The constructs NS6 1′ and NS6 2′ yielded crystals that diffracted anisotropically. We found that, although the uncorrected data could be phased by molecular replacement, refinement of the structures stalled unless the data were ellipsoidally truncated and corrected with anisotropic B-factors. These corrections significantly improved phasing by molecular replacement and subsequent refinement. The refined structures of all four extended NS6 proteases are very similar in structure to the mature MNV NS6 — and in one case reveal additional details of a surface loop. Although the packing arrangement observed showed some similarities to those observed in the adventitious protease-product crystals reported previously, in no case were specific protease-substrate interactions observed.

scholarly journals Combining experimental data for structure determination of flexible multimeric macromolecules by molecular replacement

2006 ◽  
Vol 62 (5) ◽  
pp. 467-475 ◽  
Author(s):  
Stefano Trapani ◽  
Chantal Abergel ◽  
Irina Gutsche ◽  
Cristina Horcajada ◽  
Ignacio Fita ◽  
...  
Keyword(s):  
Experimental Data ◽  
Structure Determination ◽  
Molecular Replacement

Structure determination using poorly diffracting membrane-protein crystals: the H+-ATPase and Na+,K+-ATPase case history

2010 ◽  
Vol 66 (3) ◽  
pp. 309-313 ◽  
Author(s):  
Bjørn P. Pedersen ◽  
J. Preben Morth ◽  
Poul Nissen
Keyword(s):  
Structure Determination ◽  
Heavy Atom ◽  
Real Space ◽  
Search Model ◽  
Data Sets ◽  
Molecular Replacement ◽  
Maximum Resolution ◽  
Phase Combination ◽  
Site Identification

An approach is presented for the structure determination of membrane proteins on the basis of poorly diffracting crystals which exploits molecular replacement for heavy-atom site identification at 6–9 Å maximum resolution and improvement of the heavy-atom-derived phases by multi-crystal averaging using quasi-isomorphous data sets. The multi-crystal averaging procedure allows real-space density averaging followed by phase combination between non-isomorphous native data sets to exploit crystal-to-crystal nonisomorphism despite the crystals belonging to the same space group. This approach has been used in the structure determination of H+-ATPase and Na+,K+-ATPase using Ca2+-ATPase models and its successful application to the Mhp1 symporter using LeuT as a search model is demonstrated.

Molecular-replacement structure determination of two different antibody:antigen complexes

1990 ◽  
Vol 46 (3) ◽  
pp. 418-425 ◽  
Author(s):  
S. Sheriff ◽  
E. A. Padlan ◽  
G. H. Cohen ◽  
D. R. Davies
Keyword(s):  
Structure Determination ◽  
Molecular Replacement

Implementation of a six-dimensional search using theAMoRetranslation function for difficult molecular-replacement problems

1999 ◽  
Vol 32 (1) ◽  
pp. 98-101 ◽  
Author(s):  
Steven Sheriff ◽  
Herbert E. Klei ◽  
Malcolm E. Davis
Keyword(s):  
Structure Determination ◽  
Molecular Replacement ◽  
Replacement Procedure ◽  
Translation Function ◽  
Conventional Manner ◽  
Potential Complex

A six-dimensional molecular-replacement procedure has been implemented using Perl scripts and theCCP4 version of the translation function of the molecular-replacement programAMoRe. These tools have allowed the structure determination of CTLA-4 monomer from NMR-derived coordinates, and of a potential complex of MurD with a substrate. In both cases other molecular-replacement programs,X-PLORandAMoRe, when used in a conventional manner, either failed or did not yield an obvious solution.

scholarly journals A practical overview of molecular replacement: Clostridioides difficile PilA1, a difficult case study

2020 ◽  
Vol 76 (3) ◽  
pp. 261-271
Author(s):  
Adam D. Crawshaw ◽  
Arnaud Baslé ◽  
Paula S. Salgado
Keyword(s):  
Structure Determination ◽  
Three Dimensional ◽  
Difficult Case ◽  
Molecular Replacement ◽  
Type Iv ◽  
Clostridioides Difficile ◽  
Α Helix ◽  
Key Aspects

Many biologists are now routinely seeking to determine the three-dimensional structures of their proteins of choice, illustrating the importance of this knowledge, but also of the simplification and streamlining of structure-determination processes. Despite the fact that most software packages offer simple pipelines, for the non-expert navigating the outputs and understanding the key aspects can be daunting. Here, the structure determination of the type IV pili (TFP) protein PilA1 from Clostridioides difficile is used to illustrate the different steps involved, the key decision criteria and important considerations when using the most common pipelines and software. Molecular-replacement pipelines within CCP4i2 are presented to illustrate the more commonly used processes. Previous knowledge of the biology and structure of TFP pilins, particularly the presence of a long, N-terminal α-helix required for pilus formation, allowed informed decisions to be made during the structure-determination strategy. The PilA1 structure was finally successfully determined using ARCIMBOLDO and the ab initio MR strategy used is described.

scholarly journals Structure determination of an 11-subunit exosome in complex with RNA by molecular replacement

2013 ◽  
Vol 69 (11) ◽  
pp. 2226-2235 ◽  
Author(s):  
Debora Lika Makino ◽  
Elena Conti
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structure Determination ◽  
Rna Degradation ◽  
Molecular Replacement ◽  
Rna Transcripts ◽  
Hydrolytic Reaction ◽  
Evolutionarily Conserved ◽  
Rna Exosome ◽  
Exosome Complex

The RNA exosome is an evolutionarily conserved multi-protein complex involved in the 3′ degradation of a variety of RNA transcripts. In the nucleus, the exosome participates in the maturation of structured RNAs, in the surveillance of pre-mRNAs and in the decay of a variety of noncoding transcripts. In the cytoplasm, the exosome degrades mRNAs in constitutive and regulated turnover pathways. Several structures of subcomplexes of eukaryotic exosomes or related prokaryotic exosome-like complexes are known, but how the complete assembly is organized to fulfil processive RNA degradation has been unclear. An atomic snapshot of aSaccharomyces cerevisiae420 kDa exosome complex bound to an RNA substrate in the pre-cleavage state of a hydrolytic reaction has been determined. Here, the crystallographic steps towards the structural elucidation, which was carried out by molecular replacement, are presented.

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