scholarly journals Initiating heavy-atom-based phasing by multi-dimensional molecular replacement

2016 ◽  
Vol 72 (3) ◽  
pp. 440-445 ◽  
Author(s):  
Bjørn Panyella Pedersen ◽  
Pontus Gourdon ◽  
Xiangyu Liu ◽  
Jesper Lykkegaard Karlsen ◽  
Poul Nissen
Keyword(s):  
Conformational Changes ◽  
Wide Spectrum ◽  
Heavy Atom ◽  
Direct Methods ◽  
Data Sets ◽  
Molecular Replacement ◽  
Search Models ◽  
Experimental Phasing ◽  
Difference Fourier ◽  
P Type

To obtain an electron-density map from a macromolecular crystal the phase problem needs to be solved, which often involves the use of heavy-atom derivative crystals and concomitant heavy-atom substructure determination. This is typically performed by dual-space methods, direct methods or Patterson-based approaches, which however may fail when only poorly diffracting derivative crystals are available. This is often the case for, for example, membrane proteins. Here, an approach for heavy-atom site identification based on a molecular-replacement parameter matrix (MRPM) is presented. It involves ann-dimensional search to test a wide spectrum of molecular-replacement parameters, such as different data sets and search models with different conformations. Results are scored by the ability to identify heavy-atom positions from anomalous difference Fourier maps. The strategy was successfully applied in the determination of a membrane-protein structure, the copper-transporting P-type ATPase CopA, when other methods had failed to determine the heavy-atom substructure. MRPM is well suited to proteins undergoing large conformational changes where multiple search models should be considered, and it enables the identification of weak but correct molecular-replacement solutions with maximum contrast to prime experimental phasing efforts.

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.

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 High-throughput in situ experimental phasing

2020 ◽  
Vol 76 (8) ◽  
pp. 790-801 ◽  
Author(s):  
Joshua M. Lawrence ◽  
Julien Orlans ◽  
Gwyndaf Evans ◽  
Allen M. Orville ◽  
James Foadi ◽  
...  
Keyword(s):  
Heavy Atom ◽  
Data Sets ◽  
Human Intervention ◽  
Macromolecular Crystallography ◽  
New Approach ◽  
Partial Data ◽  
Experimental Phasing ◽  
Anomalous Signal ◽  
First Time

In this article, a new approach to experimental phasing for macromolecular crystallography (MX) at synchrotrons is introduced and described for the first time. It makes use of automated robotics applied to a multi-crystal framework in which human intervention is reduced to a minimum. Hundreds of samples are automatically soaked in heavy-atom solutions, using a Labcyte Inc. Echo 550 Liquid Handler, in a highly controlled and optimized fashion in order to generate derivatized and isomorphous crystals. Partial data sets obtained on MX beamlines using an in situ setup for data collection are processed with the aim of producing good-quality anomalous signal leading to successful experimental phasing.

Simplified heavy-atom derivatization of protein structures via co-crystallization with the MAD tetragon tetrabromoterephthalic acid

2021 ◽  
Vol 77 (5) ◽  
pp. 156-162
Author(s):  
Jia Q. Truong ◽  
Stephanie Nguyen ◽  
John B. Bruning ◽  
Keith E. Shearwin
Keyword(s):  
Heavy Atom ◽  
Protein Structures ◽  
Molecular Replacement ◽  
Hen Egg White Lysozyme ◽  
Phase Problem ◽  
Egg White Lysozyme ◽  
Mad Phasing ◽  
Experimental Phasing ◽  
Anomalous Signal ◽  
Hen Egg White

The phase problem is a persistent bottleneck that impedes the structure-determination pipeline and must be solved to obtain atomic resolution crystal structures of macromolecules. Although molecular replacement has become the predominant method of solving the phase problem, many scenarios still exist in which experimental phasing is needed. Here, a proof-of-concept study is presented that shows the efficacy of using tetrabromoterephthalic acid (B4C) as an experimental phasing compound. Incorporating B4C into the crystal lattice using co-crystallization, the crystal structure of hen egg-white lysozyme was solved using MAD phasing. The strong anomalous signal generated by its four Br atoms coupled with its compatibility with commonly used crystallization reagents render B4C an effective experimental phasing compound that can be used to overcome the phase problem.

scholarly journals With phases: how two wrongs can sometimes make a right

2010 ◽  
Vol 66 (4) ◽  
pp. 420-425 ◽  
Author(s):  
Pietro Roversi ◽  
Steven Johnson ◽  
Susan M. Lea
Keyword(s):  
Crystal Structures ◽  
Case Studies ◽  
Electron Density ◽  
Heavy Atom ◽  
Direct Methods ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
Partial Model ◽  
Density Maps

In isolation, both weak isomorphous/anomalous difference signals from heavy-atom derivatization and phases from partial molecular-replacement solutions for a subset of the asymmetric unit often fall short of producing interpretable electron-density maps. Phases generated from very partial molecular-replacement models (if generated carefully) can be used to reliably locate heavy-atom sites, even if the signal is not sufficiently strong to allow robust finding of the sites using Patterson interpretation or direct methods. Additional advantages are that using molecular-replacement phases to define the heavy-atom substructure avoids the need for subsequent hand determination and/or origin-choice reconciliation and that the partial model can be used to aid the mask determination during solvent flattening. Two case studies are presented in which it was only by combining experimental and molecular-replacement phasing approaches that the crystal structures could be determined.

scholarly journals An introduction to experimental phasing of macromolecules illustrated bySHELX; new autotracing features

2018 ◽  
Vol 74 (2) ◽  
pp. 106-116 ◽  
Author(s):  
Isabel Usón ◽  
George M. Sheldrick
Keyword(s):  
Heavy Atom ◽  
Direct Methods ◽  
Anomalous Scattering ◽  
Structure Factors ◽  
Structure Solution ◽  
Experimental Phasing ◽  
Simplifying Assumptions ◽  
Efficient Route ◽  
Tracing Algorithm

For the purpose of this article, experimental phasing is understood to mean the determination of macromolecular structures by exploiting small intensity differences of Friedel opposites and possibly of reflections measured at different wavelengths or for heavy-atom derivatives, without the use of specific structural models. TheSHELXprograms provide a robust and efficient route for routine structure solution by the SAD, MAD and related methods, but involve a number of simplifying assumptions that may limit their applicability in borderline cases. The substructure atoms (i.e.those with significant anomalous scattering) are first located by direct methods, and the experimental data are then used to estimate phase shifts that are added to the substructure phases to obtain starting phases for the native reflections. These are then improved by density modification and, if the resolution of the data and the type of structure permit, polyalanine tracing. A number of extensions to the tracing algorithm are discussed; these are designed to improve its performance at low resolution. Given native data to 2.5 Å resolution or better, a correlation coefficient greater than 25% between the structure factors calculated from such a trace and the native data is usually a good indication that the structure has been solved.

scholarly journals Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing

Crystals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 273
Author(s):  
Yoshita Srivastava ◽  
Rachel Bonn-Breach ◽  
Sai Shashank Chavali ◽  
Geoffrey M. Lippa ◽  
Jermaine L. Jenkins ◽  
...  
Keyword(s):  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Molecular Replacement ◽  
Hydrophobic Core ◽  
Anomalous Diffraction ◽  
Recognition Motif ◽  
Determination Process ◽  
Experimental Phasing ◽  
Crystal Contacts ◽  
Apical Loop

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (KD of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ1-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (KD of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.

Crystallization and preliminary X-ray studies of sialidase L from the leech Macrobdella decora

1998 ◽  
Vol 54 (1) ◽  
pp. 111-113 ◽  
Author(s):  
Yu Luo ◽  
Min-yuan Chou ◽  
Su-chen Li ◽  
Yu-teh Li ◽  
Ming Luo
Keyword(s):  
Escherichia Coli ◽  
Unit Cell ◽  
Data Sets ◽  
Molecular Replacement ◽  
Unit Cell Parameters ◽  
Solvent Content ◽  
X Ray ◽  
Cell Parameters ◽  
Mercury Derivative ◽  
Macrobdella Decora

Functional monomeric 83 kDa sialidase L, a NeuAcα2→3Gal-specific sialidase from Macrobdella leech, was expressed in Escherichia coli and readily crystallized by a macroseeding technique. The crystal belongs to space group P1 with unit-cell parameters a = 46.4, b = 69.3, c = 72.5 Å, α = 113.5, β = 95.4 and γ = 107.3°. There is one molecule per unit cell, giving a Vm = 2.4 Å3 Da−1 and a solvent content of 40%. Native and mercury-derivative data sets were collected to 2.0 Å resolution. Threading and molecular-replacement calculations confirmed the existence of a bacterial sialidase-like domain.

Determination of the site of incorporation of cobalt in CoZnPO-CZP by multiple-wavelength anomalous-dispersion crystallography

1999 ◽  
Vol 55 (3) ◽  
pp. 327-332 ◽  
Author(s):  
M. Helliwell ◽  
J. R. Helliwell ◽  
V. Kaucic ◽  
N. Zabukovec Logar ◽  
L. Barba ◽  
...  
Keyword(s):  
Metal Atom ◽  
Peak Height ◽  
Data Sets ◽  
White Line ◽  
Data Set ◽  
Small Peak ◽  
Multiple Wavelength ◽  
Subsequent Calculation ◽  
Difference Fourier ◽  
Two Peaks

Data were collected from a crystal of CoZnPO-CZP {sodium cobalt–zinc phosphate hydrate, Na6[Co0.2Zn0.8PO4]6.6H2O} using synchrotron radiation at ELETTRA at the inflection point and `white line' for both the cobalt and zinc K edges, and at 1.45 Å, a wavelength remote from the K edges of both metals. The data were processed using the programs DENZO and SCALEPACK. The CCP4 program suite was used for the scaling of data sets and the subsequent calculation of dispersive difference Fourier maps. Optimal scaling was achieved by using a subset of reflections with little or no contribution from the metal atoms (i.e. which were essentially wavelength independent in their intensities) and using weights based on the σ's to obtain an overall scale factor in each case. Phases were calculated with SHELXL97 based on the refined structure using a much higher resolution and complete Cu Kα data set. An occupancy of 100% by zinc at the two metal-atom sites was assumed. The dispersive difference Fourier map calculated for zinc gave two peaks above the background of similar heights at the expected metal-atom sites. The peak height at the Zn1 site was a little higher than at the Zn2 site. The dispersive difference Fourier map calculated for cobalt gave just one peak above the background, at the Zn1 site, and only a small peak at the Zn2 site, thus indicating that incorporation of cobalt takes place mainly at one site. Refinement of the zinc occupancies using MLPHARE reinforces this conclusion. The chemical environment of each site is discussed.

scholarly journals Purification, crystallization and preliminary X-ray crystallographic studies of FMN-bound and FMN-free forms of aromatic acid decarboxylase (CpsUbiX) from the psychrophilic bacteriumColwellia psychrerythraea34H

2014 ◽  
Vol 70 (2) ◽  
pp. 215-220 ◽  
Author(s):  
Hackwon Do ◽  
Chang Woo Lee ◽  
Se Jong Han ◽  
Sung Gu Lee ◽  
Hak Jun Kim ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Protein Interactions ◽  
Aromatic Acid ◽  
Diffusion Method ◽  
Flavin Mononucleotide ◽  
Acid Decarboxylase ◽  
Data Sets ◽  
Single Mutation ◽  
Homologous Proteins ◽  
Space Group

TheubiXgene (UniProtKB code Q489U8) ofColwellia psychrerythraeastrain 34H has been annotated as a putative flavin mononucleotide (FMN)-dependent aromatic acid decarboxylase. Based on previous studies of homologous proteins, CpsUbiX is thought to catalyze the decarboxylation of 3-octaprenyl-4-hydroxybenzoate to produce 2-polyprenylphenol in the ubiquinone-biosynthesis pathway using a noncovalently bound FMN molecule as a cofactor. However, the detailed mechanisms of this important enzyme are not yet clear and need to be further elucidated. In this study, it was found that the V47S single mutation resulted in a loss of FMN binding, resulting in the production of FMN-free CpsUbiX protein. This mutation is likely to destabilize FMN–protein interactions without affecting the overall structural folding. To fully characterize the conformational changes upon FMN binding and the enzymatic mechanism at the molecular level, the wild-type (FMN-bound) and V47S mutant (FMN-free) CpsUbiX proteins were purified and crystallized using the sitting-drop vapour-diffusion method. Furthermore, complete diffraction data sets for FMN-bound (space groupC2221) and FMN-free (space groupP23) forms were obtained to 2.0 and 1.76 Å resolution, respectively.

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