scholarly journals Crystallographic molecular replacement using an in silico ‐generated search model of SARS‐CoV ‐2 ORF8

2021 ◽  
Author(s):  
Thomas G. Flower ◽  
James H. Hurley
Keyword(s):  
In Silico ◽  
Search Model ◽  
Molecular Replacement

Crystallization and preliminary X-ray diffraction studies of human catalase

1999 ◽  
Vol 55 (9) ◽  
pp. 1614-1615 ◽  
Author(s):  
R. A. P. Nagem ◽  
E. A. L. Martins ◽  
V. M. Gonçalves ◽  
R. Aparício ◽  
I. Polikarpov
Keyword(s):  
Search Model ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Beef Liver ◽  
X Ray ◽  
Diffusion Technique ◽  
Cell Dimensions ◽  
Synchrotron Radiation Diffraction ◽  
Replacement Solution ◽  
Unit Cell Dimensions

The enzyme catalase (H2O2–H2O2 oxidoreductase; E.C. 11.1.6) was purified from haemolysate of human placenta and crystallized using the vapour-diffusion technique. Synchrotron-radiation diffraction data have been collected to 1.76 Å resolution. The enzyme crystallized in the space group P212121, with unit-cell dimensions a = 83.6, b = 139.4, c = 227.5 Å. A molecular-replacement solution of the structure has been obtained using beef liver catalase (PDB code 4blc) as a search model.

scholarly journals X-ray crystal structure of a malonate-semialdehyde dehydrogenase fromPseudomonassp. strain AAC

2017 ◽  
Vol 73 (1) ◽  
pp. 24-28 ◽  
Author(s):  
Matthew Wilding ◽  
Colin Scott ◽  
Thomas S. Peat ◽  
Janet Newman
Keyword(s):  
Crystal Structure ◽  
Search Model ◽  
Molecular Replacement ◽  
X Rays ◽  
X Ray Diffraction ◽  
Acetyl Coa ◽  
Space Group ◽  
X Ray ◽  
Semialdehyde Dehydrogenase

The NAD-dependent malonate-semialdehyde dehydrogenase KES23460 fromPseudomonassp. strain AAC makes up half of a bicistronic operon responsible for β-alanine catabolism to produce acetyl-CoA. The KES23460 protein has been heterologously expressed, purified and used to generate crystals suitable for X-ray diffraction studies. The crystals belonged to space groupP212121and diffracted X-rays to beyond 3 Å resolution using the microfocus beamline of the Australian Synchrotron. The structure was solved using molecular replacement, with a monomer from PDB entry 4zz7 as the search model.

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 with multiple different models

2004 ◽  
Vol 37 (1) ◽  
pp. 159-161 ◽  
Author(s):  
Nicholas M. Glykos ◽  
Michael Kokkinidis
Keyword(s):  
Crystal Structure ◽  
Original Description ◽  
Natural Generalization ◽  
Divide And Conquer ◽  
Search Model ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Different Types ◽  
Replacement Problem ◽  
Special Case

Classical molecular replacement methods and the newer six-dimensional searches treat molecular replacement as a succession of sub-problems of reduced dimensionality. Due to their `divide-and-conquer' approach, these methods necessarily ignore (at least during their early stages) the very knowledge that a target crystal structure may comprise, for example, more than one copy of a search model, or several models of different types. An algorithm for a stochastic multi-dimensional molecular replacement search has been described previously and shown to locate solutions successfully, even in cases as complex as a 23-dimensional 4-body search. The original description of the method only dealt with a special case of molecular replacement, namely with the problem of placingncopies of only one search model in the asymmetric unit of a target crystal structure. Here a natural generalization of this algorithm is presented to deal with the full molecular replacement problem, that is, with the problem of determining the orientations and positions of a total ofncopies ofmdifferent models (withn≥m) which are assumed to be present in the asymmetric unit of a target crystal structure. The generality of this approach is illustrated through its successful application to a 17-dimensional 3-model problem involving one DNA and two protein molecules.

scholarly journals Expression, purification and crystallization of MnSOD fromArabidopsis thaliana

2014 ◽  
Vol 70 (5) ◽  
pp. 669-672 ◽  
Author(s):  
Alexandra T. Marques ◽  
Sandra P. Santos ◽  
Margarida G. Rosa ◽  
Mafalda A. A. Rodrigues ◽  
Isabel A. Abreu ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Superoxide Dismutase ◽  
Manganese Superoxide Dismutase ◽  
Search Model ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Plant Tolerance ◽  
Data Set ◽  
Manganese Superoxide ◽  
Primary Antioxidant

Manganese superoxide dismutase (MnSOD) is an essential primary antioxidant enzyme. MnSOD plays an important role in plant tolerance to abiotic stress and is a target candidate for increasing stress tolerance in crop plants. Although the structure and kinetic parameters of MnSODs from several organisms have been determined, this information is still lacking for plant MnSODs. Here, recombinant MnSOD fromArabidopsis thaliana(AtMnSOD) was expressed, purified and crystallized. A nearly complete data set could only be obtained when a total rotation range of 180° was imposed during data collection, despite the seemingly tetragonal metric of the AtMnSOD crystal diffraction. The data set extended to 1.95 Å resolution and the crystal belonged to space groupP1. Molecular-replacement calculations using an ensemble of homologous SOD structures as a search model gave a unique and unambiguous solution corresponding to eight molecules in the asymmetric unit. Structural and kinetic analysis of AtMnSOD is currently being undertaken.

The 1.1 Å resolution structure of a periplasmic phosphate-binding protein fromStenotrophomonas maltophilia: a crystallization contaminant identified by molecular replacement using the entire Protein Data Bank

2016 ◽  
Vol 72 (8) ◽  
pp. 933-943 ◽  
Author(s):  
Ronan Keegan ◽  
David G. Waterman ◽  
David J. Hopper ◽  
Leighton Coates ◽  
Graham Taylor ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Binding Protein ◽  
Data Bank ◽  
Crystal Form ◽  
Atomic Resolution ◽  
Search Model ◽  
Molecular Replacement ◽  
Phosphate Binding ◽  
Data Set ◽  
Phosphate Binding Protein

During efforts to crystallize the enzyme 2,4-dihydroxyacetophenone dioxygenase (DAD) fromAlcaligenessp. 4HAP, a small number of strongly diffracting protein crystals were obtained after two years of crystal growth in one condition. The crystals diffracted synchrotron radiation to almost 1.0 Å resolution and were, until recently, assumed to be formed by the DAD protein. However, when another crystal form of this enzyme was eventually solved at lower resolution, molecular replacement using this new structure as the search model did not give a convincing solution with the original atomic resolution data set. Hence, it was considered that these crystals might have arisen from a protein impurity, although molecular replacement using the structures of common crystallization contaminants as search models again failed. A script to perform molecular replacement usingMOLREPin which the first chain of every structure in the PDB was used as a search model was run on a multi-core cluster. This identified a number of prokaryotic phosphate-binding proteins as scoring highly in theMOLREPpeak lists. Calculation of an electron-density map at 1.1 Å resolution based on the solution obtained with PDB entry 2q9t allowed most of the amino acids to be identified visually and built into the model. ABLASTsearch then indicated that the molecule was most probably a phosphate-binding protein fromStenotrophomonas maltophilia(UniProt ID B4SL31; gene ID Smal_2208), and fitting of the corresponding sequence to the atomic resolution map fully corroborated this. Proteins in this family have been linked to the virulence of antibiotic-resistant strains of pathogenic bacteria and with biofilm formation. The structure of theS. maltophiliaprotein has been refined to anRfactor of 10.15% and anRfreeof 12.46% at 1.1 Å resolution. The molecule adopts the type II periplasmic binding protein (PBP) fold with a number of extensively elaborated loop regions. A fully dehydrated phosphate anion is bound tightly between the two domains of the protein and interacts with conserved residues and a number of helix dipoles.

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.

scholarly journals Molecular replacement then and now

2013 ◽  
Vol 69 (11) ◽  
pp. 2266-2275 ◽  
Author(s):  
Giovanna Scapin
Keyword(s):  
New York ◽  
Structural Similarity ◽  
Correct Choice ◽  
Search Model ◽  
Molecular Replacement ◽  
Acta Cryst ◽  
Replacement Method ◽  
Molecular Replacement Method ◽  
Selection Of

The `phase problem' in crystallography results from the inability to directly measure the phases of individual diffracted X-ray waves. While intensities are directly measured during data collection, phases must be obtained by other means. Several phasing methods are available (MIR, SAR, MAD, SAD and MR) and they all rely on the premise that phase information can be obtained if the positions of marker atoms in the unknown crystal structure are known. This paper is dedicated to the most popular phasing method, molecular replacement (MR), and represents a personal overview of the development, use and requirements of the methodology. The first description of noncrystallographic symmetry as a tool for structure determination was explained by Rossmann and Blow [Rossmann & Blow (1962),Acta Cryst.15, 24–31]. The term `molecular replacement' was introduced as the name of a book in which the early papers were collected and briefly reviewed [Rossmann (1972),The Molecular Replacement Method.New York: Gordon & Breach]. Several programs have evolved from the original concept to allow faster and more sophisticated searches, including six-dimensional searches and brute-force approaches. While careful selection of the resolution range for the search and the quality of the data will greatly influence the outcome, the correct choice of the search model is probably still the main criterion to guarantee success in solving a structure using MR. Two of the main parameters used to define the `best' search model are sequence identity (25% or more) and structural similarity. Another parameter that may often be undervalued is the quality of the probe: there is clearly a relationship between the quality and the correctness of the chosen probe and its usefulness as a search model. Efforts should be made by all structural biologists to ensure that their deposited structures, which are potential search probes for future systems, are of the best possible quality.

scholarly journals Crystallographic molecular replacement using an in silico-generated search model of SARS-CoV-2 ORF8

2021 ◽  
Author(s):  
Thomas G. Flower ◽  
James H. Hurley
Keyword(s):  
Ab Initio ◽  
Structural Parameters ◽  
Search Model ◽  
Problem Solution ◽  
Molecular Replacement ◽  
Phase Problem ◽  
Search Models ◽  
Sad Phasing ◽  
Predicted Model ◽  
Β Sheet

AbstractThe majority of crystal structures are determined by the method of molecular replacement (MR). The range of application of MR is limited mainly by the need for an accurate search model. In most cases, pre-existing experimentally determined structures are used as search models. In favorable cases, ab initio predicted structures have yielded search models adequate for molecular replacement. The ORF8 protein of SARS-CoV-2 represents a challenging case for MR using an ab initio prediction because ORF8 has an all β-sheet fold and few orthologs. We previously determined experimentally the structure of ORF8 using the single anomalous dispersion (SAD) phasing method, having been unable to find an MR solution to the crystallographic phase problem. Following a report of an accurate prediction of the ORF8 structure, we assessed whether the predicted model would have succeeded as an MR search model. A phase problem solution was found, and the resulting structure was refined, yielding structural parameters equivalent to the original experimental solution.

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