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.

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 An overview of heavy-atom derivatization of protein crystals

2016 ◽  
Vol 72 (3) ◽  
pp. 303-318 ◽  
Author(s):  
Ashley C. W. Pike ◽  
Elspeth F. Garman ◽  
Tobias Krojer ◽  
Frank von Delft ◽  
Elisabeth P. Carpenter
Keyword(s):  
Heavy Atom ◽  
Protein Crystals ◽  
Data Sets ◽  
Low Resolution ◽  
Heavy Atoms ◽  
Density Maps ◽  
Resolution Electron ◽  
First Choice ◽  
First Time ◽  
Resolution Data

Heavy-atom derivatization is one of the oldest techniques for obtaining phase information for protein crystals and, although it is no longer the first choice, it remains a useful technique for obtaining phases for unknown structures and for low-resolution data sets. It is also valuable for confirming the chain trace in low-resolution electron-density maps. This overview provides a summary of the technique and is aimed at first-time users of the method. It includes guidelines on when to use it, which heavy atoms are most likely to work, how to prepare heavy-atom solutions, how to derivatize crystals and how to determine whether a crystal is in fact a derivative.

scholarly journals Volume and temperature transports through the main Arctic Gateways: A comparative study between an ocean reanalysis and mooring-derived data

2017 ◽  
Author(s):  
Marianne Pietschnig ◽  
Michael Mayer ◽  
Takamasa Tsubouchi ◽  
Andrea Storto ◽  
Sebastian Stichelberger ◽  
...  
Keyword(s):  
Cross Sections ◽  
Barents Sea ◽  
The Arctic ◽  
Data Sets ◽  
Ocean Reanalysis ◽  
Polar Climate ◽  
East Greenland Current ◽  
First Time ◽  
Climate Studies

Abstract. Oceanic transports through the Arctic gateways represent an integral part of the polar climate system, but comprehensive in-situ-based estimates of this quantity have been lacking in the past. New observation-based estimates of oceanic volume, temperature and freshwater transports have recently become available. Those estimates have been derived from moored observations in the four major gateways by applying mass and salinity constraints. We seize this opportunity to compare a recent ocean reanalysis release with those observation-based estimates. First, time series of integrated volume and temperature transports through each strait are considered. Good agreement is found for Davis Strait volume transports, but considerable disagreement of up to 1.1 Sv in Fram Strait and the Barents Sea Opening. The annual mean net volume export through the gateways is −0.03 ± 0.23 Sv in the reanalysis, weaker than the −0.15 ± 0.06 Sv derived from the observation-based estimate (uncertainties represent the monthly standard deviation). The net ocean heat transport to the Arctic Ocean is similar in the two datasets (observation-based: 153 ± 44 TW, reanalysis: 145 ± 35 TW). Discrepancies in the integrated transports are further investigated by studying cross-sections of velocity, temperature and temperature flux density. These reveal good qualitative agreement in all straits, but considerable differences in the strength of major features like the East Greenland Current and the West Spitzbergen Current. Examination of the instrumental coverage reveals that areas of discrepancy are often co-located with poorly observed regions. In conclusion, both types of data sets have their merits and are recommended to be used complementarily for climate studies in this data-sparse region. We hope that the results presented in this study can assist in planning future observational efforts and in the development of ocean reanalysis products.

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 MeshAndCollect: an automated multi-crystal data-collection workflow for synchrotron macromolecular crystallography beamlines

2015 ◽  
Vol 71 (11) ◽  
pp. 2328-2343 ◽  
Author(s):  
Ulrich Zander ◽  
Gleb Bourenkov ◽  
Alexander N. Popov ◽  
Daniele de Sanctis ◽  
Olof Svensson ◽  
...  
Keyword(s):  
Hierarchical Cluster ◽  
Data Sets ◽  
Macromolecular Crystallography ◽  
X Ray Diffraction ◽  
Data Set ◽  
X Ray ◽  
Partial Data ◽  
Final Data ◽  
Dispersion Technique

Here, an automated procedure is described to identify the positions of many cryocooled crystals mounted on the same sample holder, to rapidly predict and rank their relative diffraction strengths and to collect partial X-ray diffraction data sets from as many of the crystals as desired. Subsequent hierarchical cluster analysis then allows the best combination of partial data sets, optimizing the quality of the final data set obtained. The results of applying the method developed to various systems and scenarios including the compilation of a complete data set from tiny crystals of the membrane protein bacteriorhodopsin and the collection of data sets for successful structure determination using the single-wavelength anomalous dispersion technique are also presented.

scholarly journals Long-wavelength Mesh&Collect native SAD phasing from microcrystals

2019 ◽  
Vol 75 (2) ◽  
pp. 192-199 ◽  
Author(s):  
Michele Cianci ◽  
Max Nanao ◽  
Thomas R. Schneider
Keyword(s):  
Radiation Sensitivity ◽  
Time Frame ◽  
Data Sets ◽  
Data Set ◽  
Experimental Planning ◽  
Manual Intervention ◽  
Partial Data ◽  
Small Crystals ◽  
Anomalous Signal ◽  
Single Data

Harnessing the anomalous signal from macromolecular crystals with volumes of less than 10 000 µm3 for native phasing requires careful experimental planning. The type of anomalous scatterers that are naturally present in the sample, such as sulfur, phosphorus and calcium, will dictate the beam energy required and determine the level of radiation sensitivity, while the crystal size will dictate the beam size and the sample-mounting technique, in turn indicating the specifications of a suitable beamline. On the EMBL beamline P13 at PETRA III, Mesh&Collect data collection from concanavalin A microcrystals with linear dimensions of ∼20 µm or less using an accordingly sized microbeam at a wavelength of 1.892 Å (6.551 keV, close to the Mn edge at 6.549 keV) increases the expected Bijvoet ratio to 2.1% from an expected 0.7% at 12.6 keV (Se K edge), thus allowing experimental phase determination using the anomalous signal from naturally present Mn2+ and Ca2+ ions. Dozens of crystals were harvested and flash-cryocooled in micro-meshes, rapidly screened for diffraction (less than a minute per loop) and then used for serial Mesh&Collect collection of about 298 partial data sets (10° of crystal rotation per sample). The partial data sets were integrated and scaled. A genetic algorithm for combining partial data sets was used to select those to be merged into a single data set. This final data set showed high completeness, high multiplicity and sufficient anomalous signal to locate the anomalous scatterers, and provided phasing information which allowed complete auto-tracing of the polypeptide chain. To allow the complete experiment to run in less than 2 h, a practically acceptable time frame, the diffractometer and detector had to run together with limited manual intervention. The combination of several cutting-edge components allowed accurate anomalous signal to be measured from small crystals.

scholarly journals Making a difference in multi-data-set crystallography: simple and deterministic data-scaling/selection methods

2020 ◽  
Vol 76 (7) ◽  
pp. 636-652 ◽  
Author(s):  
Greta M. Assmann ◽  
Meitian Wang ◽  
Kay Diederichs
Keyword(s):  
Simulated Data ◽  
Test Case ◽  
Data Sets ◽  
Selection Methods ◽  
Data Set ◽  
Partial Data ◽  
Structure Solution ◽  
Making A Difference ◽  
Data Scaling ◽  
Anomalous Signal

Phasing by single-wavelength anomalous diffraction (SAD) from multiple crystallographic data sets can be particularly demanding because of the weak anomalous signal and possible non-isomorphism. The identification and exclusion of non-isomorphous data sets by suitable indicators is therefore indispensable. Here, simple and robust data-selection methods are described. A multi-dimensional scaling procedure is first used to identify data sets with large non-isomorphism relative to clusters of other data sets. Within each cluster that it identifies, further selection is based on the weighted ΔCC1/2, a quantity representing the influence of a set of reflections on the overall CC1/2 of the merged data. The anomalous signal is further improved by optimizing the scaling protocol. The success of iterating the selection and scaling steps was verified by substructure determination and subsequent structure solution. Three serial synchrotron crystallography (SSX) SAD test cases with hundreds of partial data sets and one test case with 62 complete data sets were analyzed. Structure solution was dramatically simplified with this procedure, and enabled solution of the structures after a few selection/scaling iterations. To explore the limits, the procedure was tested with much fewer data than originally required and could still solve the structure in several cases. In addition, an SSX data challenge, minimizing the number of (simulated) data sets necessary to solve the structure, was significantly underbid.

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 CO2 adsorption in Y zeolite: a structural and dynamic view by a novel principal-component-analysis-assisted in situ single-crystal X-ray diffraction experiment

2019 ◽  
Vol 75 (2) ◽  
pp. 214-222 ◽  
Author(s):  
Eleonora Conterosito ◽  
Luca Palin ◽  
Rocco Caliandro ◽  
Wouter van Beek ◽  
Dmitry Chernyshov ◽  
...  
Keyword(s):  
Principal Component Analysis ◽  
Single Crystal ◽  
Principal Component ◽  
Active Species ◽  
Data Sets ◽  
X Ray Diffraction ◽  
Y Zeolite ◽  
X Ray ◽  
First Time

The increasing efficiency of detectors and brightness of X-rays in both laboratory and large-scale facilities allow the collection of full single-crystal X-ray data sets within minutes. The analysis of these `crystallographic big data' requires new tools and approaches. To answer these needs, the use of principal component analysis (PCA) is proposed to improve the efficiency and speed of the analysis. Potentialities and limitations of PCA were investigated using single-crystal X-ray diffraction (XRD) data collected in situ on Y zeolite, in which CO2, acting as an active species, is thermally adsorbed while cooling from 300 to 200 K. For the first time, thanks to the high sensitivity of single-crystal XRD, it was possible to determine the sites where CO2 is adsorbed, the increase in their occupancy while the temperature is decreased, and the correlated motion of active species, i.e. CO2, H2O and Na+. PCA allowed identification and elimination of problematic data sets, and better understanding of the trends of the occupancies of CO2, Na+ and water. The quality of the data allowed for the first time calculation of the enthalpy (ΔH) and entropy (ΔS) of the CO2 adsorption by applying the van 't Hoff equation to in situ single-crystal data. The calculation of thermodynamic values was carried out by both traditional and PCA-based approaches, producing comparable results. The obtained ΔH value is significant and involves systems (CO2 and Y zeolite) with no toxicity, superb stability and chemical inertness. Such features, coupled with the absence of carbonate formation and framework inertness upon adsorption, were demonstrated for the bulk crystal by the single-crystal experiment, and suggest that the phenomenon can be easily reversed for a large number of cycles, with CO2 released on demand. The main advantages of PCA-assisted analysis reside in its speed and in the possibility of it being applied directly to raw data, possibly as an `online' data-quality test during data collection, without any a priori knowledge of the crystal structure.

scholarly journals Physical Barriers, Cultural Connections: Prehistoric Metallurgy across the Alpine Region

2015 ◽  
Vol 18 (4) ◽  
pp. 599-632 ◽  
Author(s):  
Laura Perucchetti ◽  
Peter Bray ◽  
Andrea Dolfini ◽  
A. Mark Pollard
Keyword(s):  
Bronze Age ◽  
Metal Flow ◽  
Western Alps ◽  
Alpine Region ◽  
Data Sets ◽  
New Approach ◽  
Physical Barriers ◽  
Flow Through ◽  
Cultural Connections ◽  
First Time

This paper considers the early copper and copper-alloy metallurgy of the entire Alpine region. It introduces a new approach to the interpretation of chemical composition data sets, which has been applied to a comprehensive regional database for the first time. The Alpine Chalcolithic and Early Bronze Age each have distinctive patterns of metal use, which can be interpreted through changes in mining, social choice, and major landscape features such as watersheds and river systems. Interestingly, the Alpine range does not act as a north-south barrier, as major differences in composition tend to appear on an east-west axis. Central among these is the prevalence of tin-bronze in the western Alps compared to the east. This ‘tin-line’ is discussed in terms of metal flow through the region and evidence for a deeply rooted geographical division that runs through much of Alpine prehistory.

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