scholarly journals Experimental phasing: best practice and pitfalls

2010 ◽  
Vol 66 (4) ◽  
pp. 458-469 ◽  
Author(s):  
Airlie J. McCoy ◽  
Randy J. Read
Keyword(s):  
Best Practice ◽  
Model Building ◽  
Protein Crystal ◽  
Protein Crystal Structure ◽  
Experimental Phasing ◽  
Log Likelihood ◽  
Map Generation ◽  
Sad Phasing ◽  
Supplementary Material ◽  
Crystal Twinning

Developments in protein crystal structure determination by experimental phasing are reviewed, emphasizing the theoretical continuum between experimental phasing, density modification, model building and refinement. Traditional notions of the composition of the substructure and the best coefficients for map generation are discussed. Pitfalls such as determining the enantiomorph, identifying centrosymmetry (or pseudo-symmetry) in the substructure and crystal twinning are discussed in detail. An appendix introduces combined real–imaginary log-likelihood gradient map coefficients for SAD phasing and their use for substructure completion as implemented in the softwarePhaser. Supplementary material includes animated probabilistic Harker diagrams showing how maximum-likelihood-based phasing methods can be used to refine parameters in the case of SIR and MIR; it is hoped that these will be useful for those teaching best practice in experimental phasing methods.

scholarly journals Making routine native SAD a reality: lessons from beamline X06DA at the Swiss Light Source

2019 ◽  
Vol 75 (3) ◽  
pp. 262-271 ◽  
Author(s):  
Shibom Basu ◽  
Aaron Finke ◽  
Laura Vera ◽  
Meitian Wang ◽  
Vincent Olieric
Keyword(s):  
Data Collection ◽  
Light Source ◽  
Low Dose ◽  
Model Building ◽  
De Novo ◽  
Anomalous Scattering ◽  
Orientation Data ◽  
Swiss Light Source ◽  
Experimental Phasing ◽  
Sad Phasing

Native single-wavelength anomalous dispersion (SAD) is the most attractive de novo phasing method in macromolecular crystallography, as it directly utilizes intrinsic anomalous scattering from native crystals. However, the success of such an experiment depends on accurate measurements of the reflection intensities and therefore on careful data-collection protocols. Here, the low-dose, multiple-orientation data-collection protocol for native SAD phasing developed at beamline X06DA (PXIII) at the Swiss Light Source is reviewed, and its usage over the last four years on conventional crystals (>50 µm) is reported. Being experimentally very simple and fast, this method has gained popularity and has delivered 45 de novo structures to date (13 of which have been published). Native SAD is currently the primary choice for experimental phasing among X06DA users. The method can address challenging cases: here, native SAD phasing performed on a streptavidin–biotin crystal with P21 symmetry and a low Bijvoet ratio of 0.6% is highlighted. The use of intrinsic anomalous signals as sequence markers for model building and the assignment of ions is also briefly described.

scholarly journals Protein-complex structure completion usingIPCAS(Iterative Protein Crystal structure Automatic Solution)

2015 ◽  
Vol 71 (7) ◽  
pp. 1487-1492 ◽  
Author(s):  
Weizhe Zhang ◽  
Hongmin Zhang ◽  
Tao Zhang ◽  
Haifu Fan ◽  
Quan Hao
Keyword(s):  
Crystal Structure ◽  
Protein Complex ◽  
Model Building ◽  
Protein Complexes ◽  
Direct Method ◽  
Complex Structure ◽  
Protein Crystal ◽  
Molecular Replacement ◽  
Protein Crystal Structure ◽  
Building Program

Protein complexes are essential components in many cellular processes. In this study, a procedure to determine the protein-complex structure from a partial molecular-replacement (MR) solution is demonstrated using a direct-method-aided dual-space iterative phasing and model-building program suite,IPCAS(Iterative Protein Crystal structure Automatic Solution). TheIPCASiteration procedure involves (i) real-space model building and refinement, (ii) direct-method-aided reciprocal-space phase refinement and (iii) phase improvement through density modification. The procedure has been tested with four protein complexes, including two previously unknown structures. It was possible to useIPCASto build the whole complex structure from one or less than one subunit once the molecular-replacement method was able to give a partial solution. In the most challenging case,IPCASwas able to extend to the full length starting from less than 30% of the complex structure, while conventional model-building procedures were unsuccessful.

scholarly journals The copper(II)-binding tripeptide GHK, a valuable crystallization and phasing tag for macromolecular crystallography

2020 ◽  
Vol 76 (12) ◽  
pp. 1222-1232
Author(s):  
Alexander Mehr ◽  
Fabian Henneberg ◽  
Ashwin Chari ◽  
Dirk Görlich ◽  
Trevor Huyton
Keyword(s):  
Absorption Edge ◽  
Current Trend ◽  
Macromolecular Crystallography ◽  
Native Sulfur ◽  
High Affinity ◽  
Redundant Data ◽  
Experimental Phasing ◽  
Log Likelihood ◽  
N Terminus ◽  
Sad Phasing

The growth of diffraction-quality crystals and experimental phasing remain two of the main bottlenecks in protein crystallography. Here, the high-affinity copper(II)-binding tripeptide GHK was fused to the N-terminus of a GFP variant and an MBP-FG peptide fusion. The GHK tag promoted crystallization, with various residues (His, Asp, His/Pro) from symmetry molecules completing the copper(II) square-pyramidal coordination sphere. Rapid structure determination by copper SAD phasing could be achieved, even at a very low Bijvoet ratio or after significant radiation damage. When collecting highly redundant data at a wavelength close to the copper absorption edge, residual S-atom positions could also be located in log-likelihood-gradient maps and used to improve the phases. The GHK copper SAD method provides a convenient way of both crystallizing and phasing macromolecular structures, and will complement the current trend towards native sulfur SAD and MR-SAD phasing.

scholarly journals The magic triangle goes MAD: experimental phasing with a bromine derivative

2010 ◽  
Vol 66 (4) ◽  
pp. 374-380 ◽  
Author(s):  
Tobias Beck ◽  
Tim Gruene ◽  
George M. Sheldrick
Keyword(s):  
Functional Groups ◽  
Heavy Atom ◽  
Anomalous Dispersion ◽  
Proteinase K ◽  
Nonspecific Binding ◽  
Heavy Atoms ◽  
Carboxylate Groups ◽  
Experimental Phasing ◽  
Sad Phasing ◽  
Bromine Derivative

Experimental phasing is an essential technique for the solution of macromolecular structures. Since many heavy-atom ion soaks suffer from nonspecific binding, a novel class of compounds has been developed that combines heavy atoms with functional groups for binding to proteins. The phasing tool 5-amino-2,4,6-tribromoisophthalic acid (B3C) contains three functional groups (two carboxylate groups and one amino group) that interact with proteinsviahydrogen bonds. Three Br atoms suitable for anomalous dispersion phasing are arranged in an equilateral triangle and are thus readily identified in the heavy-atom substructure. B3C was incorporated into proteinase K and a multiwavelength anomalous dispersion (MAD) experiment at the Br Kedge was successfully carried out. Radiation damage to the bromine–carbon bond was investigated. A comparison with the phasing tool I3C that contains three I atoms for single-wavelength anomalous dispersion (SAD) phasing was also carried out.

scholarly journals Escherichia coliMltA: MAD phasing and refinement of a tetartohedrally twinned protein crystal structure. Addendum and erratum

2005 ◽  
Vol 61 (8) ◽  
pp. 1172-1172
Author(s):  
Thomas R. M. Barends ◽  
René M. de Jong ◽  
Karin E. van Straaten ◽  
Andy-Mark W. H. Thunnissen ◽  
Bauke W. Dijkstra
Keyword(s):  
Crystal Structure ◽  
Protein Crystal ◽  
Mad Phasing ◽  
Protein Crystal Structure

scholarly journals S-SAD phasing on SOLEIL Beamline PROXIMA 1

2014 ◽  
Vol 70 (a1) ◽  
pp. C609-C609
Author(s):  
Patrick Gourhant ◽  
Beatriz Guimaraes ◽  
Tatiana Isabet ◽  
Sebastian Klinke ◽  
Pierre Legrand ◽  
...  
Keyword(s):  
Model Building ◽  
De Novo ◽  
Signal To Noise Ratio ◽  
Beam Line ◽  
Synchrotron Source ◽  
Combining Data ◽  
Low Energies ◽  
Sad Phasing ◽  
Multiple Sample ◽  
Data Collections

"PROXIMA 1, a beamline for macro-molecular crystallography at the 3rd generation synchrotron source SOLEIL, is equipped with a multi-circle goniometer (alpha 50 degrees) as well as a PILATUS 6M detector. These features, along with the extended energy range of the beam line towards the low energies (down to 5.5 keV) and the possibility to adapt the source size to the sample in order to optimize signal to noise ratio, have made the beam line very attractive for S-SAD phasing with more than seven examples of successful de novo phasing achieved over the last two years. The use of low energies has also proved a significant aid in assisting with MODEL building. The technical capabilities of the beam line for low energy data collections will be presented, along with a number of examples of the successful use of low wavelengths on the beam line. The importance of combining data from multiple sample orientations in order to achieve ""true multiplicity"" will be highlighted, as well as the importance of combining data from multiple crystals in order to achieve high multiplicity."

scholarly journals Softer but stronger? Sulfur SAD with low energy X-rays of 2.7 Å

2014 ◽  
Vol 70 (a1) ◽  
pp. C604-C604
Author(s):  
Dorothee Liebschner ◽  
Naohiro Matsugaki ◽  
Miki Senda ◽  
Yusuke Yamada ◽  
Toshiya Senda
Keyword(s):  
Absorption Edge ◽  
Protein Crystal ◽  
X Rays ◽  
Long Wavelength ◽  
Heavy Atoms ◽  
Statistical Validity ◽  
Experimental Phasing ◽  
Recent Developments ◽  
Long Time ◽  
Anomalous Signal

Single wavelength anomalous diffraction (SAD) is a powerful experimental phasing technique used in macromolecular crystallography (MX). SAD is based on the absorption of X-rays by heavy atoms, which can be either incorporated into the protein (crystal) or naturally present in the structure, such as sulfur or metal ions. In particular, sulfur seems to be an attractive candidate for phasing, because most proteins contain a considerable number of S atoms. However, the K-absorption edge of sulfur is around 5.1 Å wavelength (2.4 keV), which is far from the optimal wavelength of most MX-beamlines at synchrotrons. Therefore, phasing experiments have to be performed further away from the absorption edge, which results in weaker anomalous signal. This explains why S-SAD was not commonly used for a long time, although its feasibility was illustrated by the ground-breaking study by Hendrickson and Teeter [1]. Recent developments in instrumentation, software and methodology made it possible to measure intensities more accurately, and, as a consequence, S-SAD has lately obtained more and more attention [2]. The beamline BL-1A at Photon factory (KEK, Japan) is designed to take full advantage of a long wavelength X-ray beam at around 3 Å to further enhance anomalous signals. We performed S-SAD experiments at BL-1A using two different wavelengths (1.9 Å and 2.7 Å) and compared their phasing capabilities. This methodological study was performed with ferredoxin reductase crystals of various sizes. In order to guarantee statistical validity and to exclude the influence of a particular sample, we repeated the comparison with several crystals. The novelty in the approach consists in using very long wavelengths (2.7 Å), not fully exploited in the literature so far. According to our study, the 2.7 Å wavelength shows - despite strong absorption effects of the diffracted X-rays - more successful phasing results than at 1.9 Å.

scholarly journals Microfluidic Approaches for Protein Crystal Structure Analysis

2016 ◽  
Vol 32 (1) ◽  
pp. 3-9 ◽  
Author(s):  
Masatoshi MAEKI ◽  
Hiroshi YAMAGUCHI ◽  
Manabu TOKESHI ◽  
Masaya MIYAZAKI
Keyword(s):  
Crystal Structure ◽  
Structure Analysis ◽  
Crystal Structure Analysis ◽  
Protein Crystal ◽  
Protein Crystal Structure
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