scholarly journals Development of a dose-limiting data collection strategy for serial synchrotron rotation crystallography

2017 ◽  
Vol 24 (1) ◽  
pp. 29-41 ◽  
Author(s):  
Kazuya Hasegawa ◽  
Keitaro Yamashita ◽  
Tomohiro Murai ◽  
Nipawan Nuemket ◽  
Kunio Hirata ◽  
...  
Keyword(s):  
Data Collection ◽  
Exposure Time ◽  
Signal To Noise Ratio ◽  
Single Shot ◽  
Resolution Limit ◽  
Sample Rotation ◽  
Data Collection Strategy ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Serial Femtosecond Crystallography

Serial crystallography, in which single-shot diffraction images are collected, has great potential for protein microcrystallography. Although serial femtosecond crystallography (SFX) has been successfully demonstrated, limited beam time prevents its routine use. Inspired by SFX, serial synchrotron crystallography (SSX) has been investigated at synchrotron macromolecular crystallography beamlines. Unlike SFX, the longer exposure time of milliseconds to seconds commonly used in SSX causes radiation damage. However, in SSX, crystals can be rotated during the exposure, which can achieve efficient coverage of the reciprocal space. In this study, mercury single-wavelength anomalous diffraction (Hg-SAD) phasing of the luciferin regenerating enzyme (LRE) was performed using serial synchrotron rotation crystallography. The advantages of rotation and influence of dose on the data collected were evaluated. The results showed that sample rotation was effective for accurate data collection, and the optimum helical rotation step depended on multiple factors such as multiplicity and partiality of reflections, exposure time per rotation angle and the contribution from background scattering. For the LRE microcrystals, 0.25° was the best rotation step for the achievable resolution limit, whereas a rotation step larger than or equal to 1° was favorable for Hg-SAD phasing. Although an accumulated dose beyond 1.1 MGy caused specific damage at the Hg site, increases in resolution and anomalous signal were observed up to 3.4 MGy because of a higher signal-to-noise ratio.

scholarly journals Data-collection strategy for challenging native SAD phasing

2016 ◽  
Vol 72 (3) ◽  
pp. 421-429 ◽  
Author(s):  
Vincent Olieric ◽  
Tobias Weinert ◽  
Aaron D. Finke ◽  
Carolin Anders ◽  
Dianfan Li ◽  
...  
Keyword(s):  
Data Collection ◽  
Data Sets ◽  
Yield Data ◽  
Guide Rna ◽  
Multiple Data ◽  
Data Collection Strategy ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Multiple Data Sets ◽  
Low X

Recent improvements in data-collection strategies have pushed the limits of native SAD (single-wavelength anomalous diffraction) phasing, a method that uses the weak anomalous signal of light elements naturally present in macromolecules. These involve the merging of multiple data sets from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose. Both approaches yield data of high multiplicity while minimizing radiation damage and systematic error, thus ensuring accurate measurements of the anomalous differences. Here, the combined use of these two strategies is described to solve cases of native SAD phasing that were particular challenges: the integral membrane diacylglycerol kinase (DgkA) with a low Bijvoet ratio of 1% and the large 200 kDa complex of the CRISPR-associated endonuclease (Cas9) bound to guide RNA and target DNA crystallized in the low-symmetry space groupC2. The optimal native SAD data-collection strategy based on systematic measurements performed on the 266 kDa multiprotein/multiligand tubulin complex is discussed.

A solution-free crystal-mounting platform for native SAD

2020 ◽  
Vol 76 (10) ◽  
pp. 938-945
Author(s):  
Jian Yu ◽  
Akira Shinoda ◽  
Koji Kato ◽  
Isao Tanaka ◽  
Min Yao
Keyword(s):  
Data Collection ◽  
Nucleic Acids ◽  
Protein Structures ◽  
Anomalous Scattering ◽  
X Rays ◽  
Long Wavelength ◽  
Light Atoms ◽  
Sad Phasing ◽  
Anomalous Signal

The native SAD phasing method uses the anomalous scattering signals from the S atoms contained in most proteins, the P atoms in nucleic acids or other light atoms derived from the solution used for crystallization. These signals are very weak and careful data collection is required, which makes this method very difficult. One way to enhance the anomalous signal is to use long-wavelength X-rays; however, these wavelengths are more strongly absorbed by the materials in the pathway. Therefore, a crystal-mounting platform for native SAD data collection that removes solution around the crystals has been developed. This platform includes a novel solution-free mounting tool and an automatic robot, which extracts the surrounding solution, flash-cools the crystal and inserts the loop into a UniPuck cassette for use in the synchrotron. Eight protein structures (including two new structures) have been successfully solved by the native SAD method from crystals prepared using this platform.

scholarly journals Native sulfur/chlorine SAD phasing for serial femtosecond crystallography

2015 ◽  
Vol 71 (12) ◽  
pp. 2519-2525 ◽  
Author(s):  
Takanori Nakane ◽  
Changyong Song ◽  
Mamoru Suzuki ◽  
Eriko Nango ◽  
Jun Kobayashi ◽  
...  
Keyword(s):  
Radiation Damage ◽  
Crystal Structures ◽  
Structure Determination ◽  
Anomalous Diffraction ◽  
Native Sulfur ◽  
Wide Range ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) allows structures to be determined with minimal radiation damage. However, phasing native crystals in SFX is not very common. Here, the structure determination of native lysozyme from single-wavelength anomalous diffraction (SAD) by utilizing the anomalous signal of sulfur and chlorine at a wavelength of 1.77 Å is successfully demonstrated. This sulfur SAD method can be applied to a wide range of proteins, which will improve the determination of native crystal structures.

scholarly journals Can I solve my structure by SAD phasing? Anomalous signal in SAD phasing

2016 ◽  
Vol 72 (3) ◽  
pp. 346-358 ◽  
Author(s):  
Thomas C. Terwilliger ◽  
Gábor Bunkóczi ◽  
Li-Wei Hung ◽  
Peter H. Zwart ◽  
Janet L. Smith ◽  
...  
Keyword(s):  
Signal To Noise Ratio ◽  
Atomic Displacement ◽  
Peak Height ◽  
High Ratio ◽  
Data Set ◽  
High Data ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Displacement Factor ◽  
Difference Fourier

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. A simple theoretical framework for describing measurements of anomalous differences and the resulting useful anomalous correlation and anomalous signal in a SAD experiment is presented. Here, the useful anomalous correlation is defined as the correlation of anomalous differences with ideal anomalous differences from the anomalous substructure. The useful anomalous correlation reflects the accuracy of the data and the absence of minor sites. The useful anomalous correlation also reflects the information available for estimating crystallographic phases once the substructure has been determined. In contrast, the anomalous signal (the peak height in a model-phased anomalous difference Fourier at the coordinates of atoms in the anomalous substructure) reflects the information available about each site in the substructure and is related to the ability to find the substructure. A theoretical analysis shows that the expected value of the anomalous signal is the product of the useful anomalous correlation, the square root of the ratio of the number of unique reflections in the data set to the number of sites in the substructure, and a function that decreases with increasing values of the atomic displacement factor for the atoms in the substructure. This means that the ability to find the substructure in a SAD experiment is increased by high data quality and by a high ratio of reflections to sites in the substructure, and is decreased by high atomic displacement factors for the substructure.

scholarly journals An optimal strategy for X-ray data collection on macromolecular crystals with position-sensitive detectors

1994 ◽  
Vol 27 (5) ◽  
pp. 791-793 ◽  
Author(s):  
I. Vicković ◽  
K. H. Kalk ◽  
J. Drenth ◽  
B. W. Dijkstra
Keyword(s):  
Data Collection ◽  
Exposure Time ◽  
Optimal Strategy ◽  
Actual Data ◽  
X Ray ◽  
Data Collection Strategy ◽  
Position Sensitive ◽  
Position Sensitive Detectors ◽  
Optimal Settings

X-ray data collection on macromolecular crystals is preferably done with minimum exposure time and high completeness. A Fortran procedure – DCS – has been written in the environment of the MADNES program to predict the completeness of data before the start of actual data collection. In addition, the program can check and refine the data-collection strategy and suggest optimal settings and rotation ranges for one or more crystals in different orientations that give highest completeness in minimum exposure time, thus extending the life of the crystal. The method has been tested on previously collected data as well as on new structures. Since the program has been in full use in this laboratory, the completeness of collected data has improved significantly.

S-SAD phasing of monoclinic histidine kinase fromBrucella abortuscombining data from multiple crystals and orientations: an example of data-collection strategy anda posteriorianalysis of different data combinations

2015 ◽  
Vol 71 (7) ◽  
pp. 1433-1443 ◽  
Author(s):  
Sebastián Klinke ◽  
Nicolas Foos ◽  
Jimena J. Rinaldi ◽  
Gastón Paris ◽  
Fernando A. Goldbaum ◽  
...  
Keyword(s):  
Data Collection ◽  
Structure Determination ◽  
Histidine Kinase ◽  
Structural Information ◽  
Data Sets ◽  
X Rays ◽  
X Ray Crystallography ◽  
Data Collection Strategy ◽  
Sad Phasing ◽  
The Impact

The histidine kinase (HK) domain belonging to the light–oxygen–voltage histidine kinase (LOV-HK) fromBrucella abortusis a member of the HWE family, for which no structural information is available, and has low sequence identity (20%) to the closest HK present in the PDB. The `off-edge' S-SAD method in macromolecular X-ray crystallography was used to solve the structure of the HK domain from LOV-HK at low resolution from crystals in a low-symmetry space group (P21) and with four copies in the asymmetric unit (∼108 kDa). Data were collected both from multiple crystals (diffraction limit varying from 2.90 to 3.25 Å) and from multiple orientations of the same crystal, using the κ-geometry goniostat on SOLEIL beamline PROXIMA 1, to obtain `true redundancy'. Data from three different crystals were combined for structure determination. An optimized HK construct bearing a shorter cloning artifact yielded crystals that diffracted X-rays to 2.51 Å resolution and that were used for final refinement of the model. Moreover, a thorougha posteriorianalysis using several different combinations of data sets allowed us to investigate the impact of the data-collection strategy on the success of the structure determination.

scholarly journals Data collection with a tailored X-ray beam size at 2.69 Å wavelength (4.6 keV): sulfur SAD phasing of Cdc23Nterm

2016 ◽  
Vol 72 (3) ◽  
pp. 403-412 ◽  
Author(s):  
Michele Cianci ◽  
Matthew R. Groves ◽  
David Barford ◽  
Thomas R. Schneider
Keyword(s):  
Data Collection ◽  
Anaphase Promoting Complex ◽  
X Ray ◽  
Cell Parameters ◽  
Structure Solution ◽  
Sad Phasing ◽  
A Subunit ◽  
Anomalous Signal ◽  
Crystal Twinning ◽  
Methionine Residues

The capability to reach wavelengths of up to 3.1 Å at the newly established EMBL P13 beamline at PETRA III, the new third-generation synchrotron at DESY in Hamburg, provides the opportunity to explore very long wavelengths to harness the sulfur anomalous signal for phase determination. Data collection at λ = 2.69 Å (4.6 keV) allowed the crystal structure determination by sulfur SAD phasing of Cdc23Nterm, a subunit of the multimeric anaphase-promoting complex (APC/C). At this energy, Cdc23Ntermhas an expected Bijvoet ratio 〈|Fanom|〉/〈F〉 of 2.2%, with 282 residues, including six cysteines and five methionine residues, and two molecules in the asymmetric unit (65.4 kDa; 12 Cys and ten Met residues). Selectively illuminating two separate portions of the same crystal with an X-ray beam of 50 µm in diameter allowed crystal twinning to be overcome. The crystals diffracted to 3.1 Å resolution, with unit-cell parametersa=b= 61.2,c = 151.5 Å, and belonged to space groupP43. The refined structure to 3.1 Å resolution has anRfactor of 18.7% and anRfreeof 25.9%. This paper reports the structure solution, related methods and a discussion of the instrumentation.

scholarly journals Protein structure determination by single-wavelength anomalous diffraction phasing of X-ray free-electron laser data

IUCrJ ◽  
2016 ◽  
Vol 3 (3) ◽  
pp. 180-191 ◽  
Author(s):  
Karol Nass ◽  
Anton Meinhart ◽  
Thomas R. M. Barends ◽  
Lutz Foucar ◽  
Alexander Gorel ◽  
...  
Keyword(s):  
Structure Determination ◽  
Free Electron ◽  
De Novo ◽  
Protein Structure Determination ◽  
Anomalous Scattering ◽  
X Ray ◽  
Sad Phasing ◽  
Anomalous Signal ◽  
Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) offers unprecedented possibilities for macromolecular structure determination of systems that are prone to radiation damage. However, phasing XFEL datade novois complicated by the inherent inaccuracy of SFX data, and only a few successful examples, mostly based on exceedingly strong anomalous or isomorphous difference signals, have been reported. Here, it is shown that SFX data from thaumatin microcrystals can be successfully phased using only the weak anomalous scattering from the endogenous S atoms. Moreover, a step-by-step investigation is presented of the particular problems of SAD phasing of SFX data, analysing data from a derivative with a strong anomalous signal as well as the weak signal from endogenous S atoms.

scholarly journals Can I solve my structure by SAD phasing? Planning an experiment, scaling data and evaluating the useful anomalous correlation and anomalous signal

2016 ◽  
Vol 72 (3) ◽  
pp. 359-374 ◽  
Author(s):  
Thomas C. Terwilliger ◽  
Gábor Bunkóczi ◽  
Li-Wei Hung ◽  
Peter H. Zwart ◽  
Janet L. Smith ◽  
...  
Keyword(s):  
Figure Of Merit ◽  
Signal To Noise Ratio ◽  
Theoretical Framework ◽  
Data Sets ◽  
Signal To Noise ◽  
Acta Cryst ◽  
Data Set ◽  
Local Scaling ◽  
Sad Phasing ◽  
Anomalous Signal

A key challenge in the SAD phasing method is solving a structure when the anomalous signal-to-noise ratio is low. Here, algorithms and tools for evaluating and optimizing the useful anomalous correlation and the anomalous signal in a SAD experiment are described. A simple theoretical framework [Terwilligeret al.(2016),Acta Cryst.D72, 346–358] is used to develop methods for planning a SAD experiment, scaling SAD data sets and estimating the useful anomalous correlation and anomalous signal in a SAD data set. Thephenix.plan_sad_experimenttool uses a database of solved and unsolved SAD data sets and the expected characteristics of a SAD data set to estimate the probability that the anomalous substructure will be found in the SAD experiment and the expected map quality that would be obtained if the substructure were found. Thephenix.scale_and_mergetool scales unmerged SAD data from one or more crystals using local scaling and optimizes the anomalous signal by identifying the systematic differences among data sets, and thephenix.anomalous_signaltool estimates the useful anomalous correlation and anomalous signal after collecting SAD data and estimates the probability that the data set can be solved and the likely figure of merit of phasing.

Online, Asynchronous Data Collection in Qualitative Research

2019 ◽  
pp. 442-467
Author(s):  
Tracy Spencer ◽  
Linnea Rademaker ◽  
Peter Williams ◽  
Cynthia Loubier
Keyword(s):  
Qualitative Research ◽  
Data Collection ◽  
Face To Face ◽  
Research Outcomes ◽  
Data Collection Strategy ◽  
Time Distance ◽  
Privacy Issues

The authors discuss the use of online, asynchronous data collection in qualitative research. Online interviews can be a valuable way to increase access to marginalized participants, including those with time, distance, or privacy issues that prevent them from participating in face-to-face interviews. The resulting greater participant pool can increase the rigor and validity of research outcomes. The authors also address issues with conducting in-depth asynchronous interviews such as are needed in phenomenology. Advice from the field is provided for rigorous implementation of this data collection strategy. The authors include extensive excerpts from two studies using online, asynchronous data collection.

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