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

PeerJ ◽  
2015 ◽  
Vol 3 ◽  
pp. e798 ◽  
Author(s):  
Humberto Fernandes ◽  
Eoin N. Leen ◽  
Hamlet Cromwell Jr ◽  
Marc-Philipp Pfeil ◽  
Stephen Curry
Keyword(s):  
Structure Determination ◽  
Murine Norovirus ◽  
Substrate Interactions ◽  
Protease Substrate

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 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.

Ab initio structure determination of cytochrome c6 by combined reciprocal space-real space approach

2003 ◽  
Vol 218 (1) ◽  
Author(s):  
K. Chowdhury ◽  
S. Ghosh ◽  
M. Mukherjee
Keyword(s):  
Cytochrome C ◽  
Ab Initio ◽  
Structure Determination ◽  
Direct Method ◽  
Real Space ◽  
Reciprocal Space ◽  
Cytochrome C6 ◽  
Ab Initio Structure ◽  
Space Approach

AbstractThe direct method program SAYTAN has been applied successfully to redetermine the structure of cytochrome c

STRUCTURE DETERMINATION OF 5 MEMBERED SILANE RINGS USING MICROWAVE SPECTROSCOPY

2018 ◽  
Author(s):  
Frank Marshall ◽  
G. Grubbs II ◽  
Gamil Guirgis ◽  
Theodore Carrigan-Broda ◽  
Grier Jones ◽  
...  
Keyword(s):  
Structure Determination ◽  
Microwave Spectroscopy
Sign in / Sign up

Export Citation Format

Share Document