Advanced Crystallographic Data Collection Protocols for Experimental Phasing

2016 ◽  
pp. 175-191 ◽  
Author(s):  
Aaron D. Finke ◽  
Ezequiel Panepucci ◽  
Clemens Vonrhein ◽  
Meitian Wang ◽  
Gérard Bricogne ◽  
...  
Keyword(s):  
Data Collection ◽  
Crystallographic Data ◽  
Experimental Phasing

scholarly journals PRIGo: a new multi-axis goniometer for macromolecular crystallography

2015 ◽  
Vol 22 (4) ◽  
pp. 895-900 ◽  
Author(s):  
Sandro Waltersperger ◽  
Vincent Olieric ◽  
Claude Pradervand ◽  
Wayne Glettig ◽  
Marco Salathe ◽  
...  
Keyword(s):  
Data Collection ◽  
High Precision ◽  
Optimal Strategies ◽  
Crystallographic Data ◽  
Air Bearing ◽  
Sample Holder ◽  
Macromolecular Crystallography ◽  
Parallel Kinematics ◽  
Experimental Phasing ◽  
Parallel Robotics

The Parallel Robotics Inspired Goniometer (PRIGo) is a novel compact and high-precision goniometer providing an alternative to (mini-)kappa, traditional three-circle goniometers and Eulerian cradles used for sample reorientation in macromolecular crystallography. Based on a combination of serial and parallel kinematics, PRIGo emulates an arc. It is mounted on an air-bearing stage for rotation around ω and consists of four linear positioners working synchronously to achievex, y, ztranslations and χ rotation (0–90°), followed by a φ stage (0–360°) for rotation around the sample holder axis. Owing to the use of piezo linear positioners and active correction, PRIGo features spheres of confusion of <1 µm, <7 µm and <10 µm for ω, χ and φ, respectively, and is therefore very well suited for micro-crystallography. PRIGo enables optimal strategies for both native and experimental phasing crystallographic data collection. Herein, PRIGo hardware and software, its calibration, as well as applications in macromolecular crystallography are described.

scholarly journals Current status of protein micro-crystallography at SPring-8

2014 ◽  
Vol 70 (a1) ◽  
pp. C333-C333 ◽  
Author(s):  
Kunio Hirata ◽  
Yoshiaki Kawano ◽  
Keitaro Yamashita ◽  
Go Ueno ◽  
Takaaki Hikima ◽  
...  
Keyword(s):  
Data Collection ◽  
Current Status ◽  
Photon Flux ◽  
Protein Structure Analysis ◽  
Experimental Conditions ◽  
Automatic Data ◽  
Sports Science ◽  
Experimental Phasing ◽  
Raster Scan ◽  
Data Collections

Protein micro-crystallography is one of the most advanced technologies for protein structure analysis. In order to realize this, an undulator beamline, named BL32XU, was constructed at SPring-8. The beamline can provide beam with size of 0.9 x 0.9 µm and photon flux of 6E10 photons/s. The beam size can be easily changed by users from 1 to 10 µm square with the same flux density. Through three years user operation, we have established several key systems for efficient protein micro-crystallography. One of them is the software for precise positioning of micro-crystals in `raster scan'. SHIKA is a program with GUI which searches diffraction spots in a plenty of low dose diffraction images obtained in raster scan. Finally, it generates 2D map of crystal positions based on the number of spots or spot intensities. Parameters and thresholds in peak search have been empirically optimized for LCP crystals and it provides robust results. Another system is for the data collection strategy. Almost all successful data collections were conducted via `helical data collection' on BL32XU using the line-focused beam. The GUI software, named KUMA, enables estimation of an accumulated dose and suggests suitable experimental conditions for helical data collection. The system is proven to be useful for experimental phasing using tiny LCP crystals of membrane proteins[1-3]. Based on them, the rapid and automatic data collection system using protein micro-crystals is under development. The new CCD detector, Rayonix MX225HS, was installed for faster data acquisition in 10 Hz with the pixel size of 78 µm square. The new SHIKA using GPUs is under development for faster and more accurate crystal alignment. Following this step, KUMA system can suggest experimental conditions for each crystal found on the loop. We also report about the effects of higher dose rate in protein crystallography up to the order of 100 MGy/s. This work was supported by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

The crystal structure of peroxymyoglobin generated through cryoradiolytic reduction of myoglobin compound III during data collection

2008 ◽  
Vol 412 (2) ◽  
pp. 257-264 ◽  
Author(s):  
Hans-Petter Hersleth ◽  
Ya-Wen Hsiao ◽  
Ulf Ryde ◽  
Carl Henrik Görbitz ◽  
K. Kristoffer Andersson
Keyword(s):  
Crystal Structure ◽  
Hydrogen Peroxide ◽  
Data Collection ◽  
Intermediate Compound ◽  
Crystallographic Data ◽  
X Rays ◽  
Biologically Relevant ◽  
Short Annealing ◽  
High Valent ◽  
Compound Ii

Myoglobin has the ability to react with hydrogen peroxide, generating high-valent complexes similar to peroxidases (compounds I and II), and in the presence of excess hydrogen peroxide a third intermediate, compound III, with an oxymyoglobin-type structure is generated from compound II. The compound III is, however, easily one-electron reduced to peroxymyoglobin by synchrotron radiation during crystallographic data collection. We have generated and solved the 1.30 Å (1 Å=0.1 nm) resolution crystal structure of the peroxymyoglobin intermediate, which is isoelectric to compound 0 and has a Fe–O distance of 1.8 Å and O–O bond of 1.3 Å in accordance with a FeII–O–O− (or FeIII–O–O2−) structure. The generation of the peroxy intermediate through reduction of compound III by X-rays shows the importance of using single-crystal microspectrophotometry when doing crystallography on metalloproteins. After having collected crystallographic data on a peroxy-generated myoglobin crystal, we were able (by a short annealing) to break the O–O bond leading to formation of compound II. These results indicate that the cryoradiolytic-generated peroxymyoglobin is biologically relevant through its conversion into compound II upon heating. Additionally, we have observed that the Xe1 site is occupied by a water molecule, which might be the leaving group in the compound II to compound III reaction.

RSPACE – a reciprocal-space modelling tool

1989 ◽  
Vol 22 (6) ◽  
pp. 624-627
Author(s):  
M. R. Harris ◽  
M. Fitzgibbon ◽  
F. Hage
Keyword(s):  
Computer Graphics ◽  
Data Collection ◽  
Reciprocal Space ◽  
Crystallographic Data ◽  
Complex Data ◽  
Educational Tool ◽  
Interactive Computer Graphics ◽  
Area Detector ◽  
The Relationship ◽  
Collection Strategies

RSPACE is an interactive computer-graphics program that models the relationship between crystallographic data-collection hardware and reciprocal space. It is designed to help crystallographers plan efficient complex data-collection strategies for area detector systems, and as an educational tool. Because RSPACE models the interaction of crystal, detector and goniostat geometry in a general way, it is of particular interest to users of area detectors interfaced with multi-axis goniostats.

High-throughput crystallographic data collection at synchrotrons

2007 ◽  
pp. 173-190 ◽  
Author(s):  
Stephen R. Wasserman ◽  
David W. Smith ◽  
Kevin L. D'Amico ◽  
John W. Koss ◽  
Laura L. Morisco ◽  
...  
Keyword(s):  
Data Collection ◽  
High Throughput ◽  
Crystallographic Data

scholarly journals Guidelines for de novo phasing using multiple small-wedge data collection

2021 ◽  
Vol 28 (5) ◽  
pp. 1284-1295 ◽  
Author(s):  
Seiki Baba ◽  
Hiroaki Matsuura ◽  
Takashi Kawamura ◽  
Naoki Sakai ◽  
Yuki Nakamura ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Data Collection ◽  
De Novo ◽  
Optimal Dose ◽  
Dose Dependence ◽  
Systematic Investigation ◽  
Quality Data ◽  
Anomalous Scattering ◽  
Automatic Data ◽  
Experimental Phasing

Intense micro-focus X-ray beamlines available at synchrotron facilities have achieved high-quality data collection even from the microcrystals of membrane proteins. The automatic data collection system developed at SPring-8, named ZOO, has contributed to many structure determinations of membrane proteins using small-wedge synchrotron crystallography (SWSX) datasets. The `small-wedge' (5–20°) datasets are collected from multiple crystals and then merged to obtain the final structure factors. To our knowledge, no systematic investigation on the dose dependence of data accuracy has so far been reported for SWSX, which is between `serial crystallography' and `rotation crystallography'. Thus, herein, we investigated the optimal dose conditions for experimental phasing with SWSX. Phase determination using anomalous scattering signals was found to be more difficult at higher doses. Furthermore, merging more homogeneous datasets grouped by hierarchical clustering with controlled doses mildly reduced the negative factors in data collection, such as `lack of signal' and `radiation damage'. In turn, as more datasets were merged, more probable phases could be obtained across a wider range of doses. Therefore, our findings show that it is essential to choose a lower dose than 10 MGy for de novo structure determination by SWSX. In particular, data collection using a dose of 5 MGy proved to be optimal in balancing the amount of signal available while reducing the amount of damage as much as possible.

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.

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