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.

scholarly journals Expression, purification, crystallization and preliminary X-ray analysis of glucose-1-phosphate uridylyltransferase (GalU) fromErwinia amylovora

2014 ◽  
Vol 70 (9) ◽  
pp. 1249-1251 ◽  
Author(s):  
Mirco Toccafondi ◽  
Michele Cianci ◽  
Stefano Benini
Keyword(s):  
Erwinia Amylovora ◽  
Search Model ◽  
Data Sets ◽  
Molecular Replacement ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
E Coli ◽  
Maximum Resolution ◽  
His Tag

Glucose-1-phosphate uridylyltransferase fromErwinia amylovoraCFPB1430 was expressed as a His-tag fusion protein inEscherichia coli. After tag removal, the purified protein was crystallized from 100 mMTris pH 8.5, 2 Mammonium sulfate, 5% ethylene glycol. Diffraction data sets were collected to a maximum resolution of 2.46 Å using synchrotron radiation. The crystals belonged to the hexagonal space groupP62, with unit-cell parametersa= 80.67,b= 80.67,c = 169.18. The structure was solved by molecular replacement using the structure of theE. colienzyme as a search model.

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

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 solution wide-angle X-ray scattering and crystallography: determination of molecular envelope and heavy-atom sites

2009 ◽  
Vol 42 (2) ◽  
pp. 259-264 ◽  
Author(s):  
Xinguo Hong ◽  
Quan Hao
Keyword(s):  
Structure Determination ◽  
Heavy Atom ◽  
Unit Cell ◽  
Wide Angle ◽  
X Ray ◽  
X Ray Scattering ◽  
Molecular Envelope ◽  
Waxs Data ◽  
Ray Scattering

Solving the phase problem remains central to crystallographic structure determination. A six-dimensional search method of molecular replacement (FSEARCH) can be used to locate a low-resolution molecular envelope determined from small-angle X-ray scattering (SAXS) within the crystallographic unit cell. This method has now been applied using the higher-resolution envelope provided by combining SAXS and WAXS (wide-angle X-ray scattering) data. The method was tested on horse hemoglobin, using the most probable model selected from a set of a dozen bead models constructed from SAXS/WAXS data using the programGASBORat 5 Å resolution (qmax= 1.25 Å−1) to phase a set of single-crystal diffraction data. It was found that inclusion of WAXS data is essential for correctly locating the molecular envelope in the crystal unit cell, as well as for locating heavy-atom sites. An anomalous difference map was calculated using phases out to 8 Å resolution from the correctly positioned envelope; four distinct peaks at the 3.2σ level were identified, which agree well with the four iron sites of the known structure (Protein Data Bank code 1ns9). In contrast, no peaks could be found close to the iron sites if the molecular envelope was constructed using the data from SAXS alone (qmax= 0.25 Å−1). The initial phases can be used as a starting point for a variety of phase-extension techniques, successful application of which will result in complete phasing of a crystallographic data set and determination of the internal structure of a macromolecule to atomic resolution. It is anticipated that the combination ofFSEARCHand WAXS techniques will facilitate the initial structure determination of proteins and provide a good foundation for further structure refinement.

scholarly journals Exploiting subtle structural differences in heavy-atom derivatives for experimental phasing

2014 ◽  
Vol 70 (7) ◽  
pp. 1873-1883 ◽  
Author(s):  
Jimin Wang ◽  
Yue Li ◽  
Yorgo Modis
Keyword(s):  
Structure Determination ◽  
Heavy Atom ◽  
Data Sets ◽  
Diarrhea Virus ◽  
Isomorphous Replacement ◽  
Experimental Phasing ◽  
Structural Differences ◽  
Structure Determinations ◽  
Weak Derivatives ◽  
Viral Diarrhea Virus

Structure determination using the single isomorphous replacement (SIR) or single-wavelength anomalous diffraction (SAD) methods with weak derivatives remains very challenging. In a recent structure determination of glycoprotein E2 from bovine viral diarrhea virus, three isomorphous uranium-derivative data sets were merged to obtain partially interpretable initial experimental maps. Small differences between them were then exploited by treating them as three independent SAD data sets plus three circular pairwise SIR data sets to improve the experimental maps. Here, how such subtle structural differences were exploited for experimental phasing is described in detail. The basis for why this approach works is also provided: the effective resolution of isomorphous signals between highly isomorphous derivatives is often much higher than the effective resolution of the anomalous signals of individual derivative data sets. Hence, the new phasing approaches outlined here will be generally applicable to structure determinations involving weak derivatives.

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

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

STRUCTURE DETERMINATION OF THE Ge(111)-(3×1)Ag SURFACE RECONSTRUCTION

1999 ◽  
Vol 06 (06) ◽  
pp. 1061-1065 ◽  
Author(s):  
D. GROZEA ◽  
E. BENGU ◽  
C. COLLAZO-DAVILA ◽  
L. D. MARKS
Keyword(s):  
Electron Diffraction ◽  
Surface Reconstruction ◽  
Diffraction Data ◽  
Structure Determination ◽  
Heavy Atom ◽  
Direct Methods ◽  
Electron Diffraction Data ◽  
Transmission Electron ◽  
First Time

For the first time, during the investigation of the Ag submonolayer on the Ge(111) system, large, independent domains of the Ge (111)-(3×1) Ag phase were imaged and investigated. Previous studies have reported it only as small insets between Ge (111)-(4×4) Ag and Ge (111)- c (2×8) domains. The transmission electron diffraction data were analyzed using a Direct Methods approach and "heavy-atom holography," with the result of an atomic model of the structure similar to that of Ge (111)-(3×1) Ag .

The use of Mo Kα radiation in the assignment of the absolute configuration of light-atom molecules; the importance of high-resolution data

2014 ◽  
Vol 70 (4) ◽  
pp. 660-668 ◽  
Author(s):  
Eduardo C. Escudero-Adán ◽  
Jordi Benet-Buchholz ◽  
Pablo Ballester
Keyword(s):  
High Resolution ◽  
Crystal Structures ◽  
Structure Determination ◽  
Great Majority ◽  
Data Sets ◽  
Light Atom ◽  
High Resolution Data ◽  
The Absolute ◽  
Absolute Structure

Recent studies have confirmed the usefulness of the Hooft and Parsons methodologies for determination of the absolute crystal structures of enantiopure light-atom compounds using CuKα radiation. While many single-crystal diffractometers used for small-molecule structure determination are equipped with molybdenum anodes, use of data from such instruments for the absolute structure determination of light-atom crystal structures is rarely documented and has often been found to be unsuccessful. The Hooft and Parsons methodologies have been applied to 44 data sets obtained from single crystals containing light-atom molecules of known chirality using Mo Kαradiation. Several factors influencing the calculation of accurate and precise values for the Hooft and Parsons parameters obtained from these data sets have been identified, the inclusion of high-resolution diffraction data being particularly important. The correct absolute structure was obtained in all cases, with the standard uncertainties of the final absolute structure parameters below 0.1 for the great majority.

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