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

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.

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

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319003103 ◽

2019 ◽

Vol 75 (3) ◽

pp. 262-271 ◽

Cited By ~ 5

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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Facilitating best practices in collecting anomalous scattering data forde novostructure solution at the ESRF Structural Biology Beamlines

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798316001042 ◽

2016 ◽

Vol 72 (3) ◽

pp. 413-420 ◽

Cited By ~ 6

Author(s):

Daniele de Sanctis ◽

Marcus Oscarsson ◽

Alexander Popov ◽

Olof Svensson ◽

Gordon Leonard

Keyword(s):

Structural Biology ◽

De Novo ◽

Heavy Atom ◽

Scattering Data ◽

Anomalous Scattering ◽

Automatic Data ◽

Structure Solution ◽

Experimental Phasing ◽

Wide Range ◽

Collection Practice

The constant evolution of synchrotron structural biology beamlines, the viability of screening protein crystals for a wide range of heavy-atom derivatives, the advent of efficient protein labelling and the availability of automatic data-processing and structure-solution pipelines have combined to makede novostructure solution in macromolecular crystallography a less arduous task. Nevertheless, the collection of diffraction data of sufficient quality for experimental phasing is still a difficult and crucial step. Here, some examples of good data-collection practice for projects requiring experimental phasing are presented and recent developments at the ESRF Structural Biology beamlines that have facilitated these are illustrated.

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Current status of protein micro-crystallography at SPring-8

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314096661 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C333-C333 ◽

Cited By ~ 1

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.

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Synchrotron microcrystal native-SAD phasing at a low energy

IUCrJ ◽

10.1107/s2052252519004536 ◽

2019 ◽

Vol 6 (4) ◽

pp. 532-542 ◽

Cited By ~ 5

Author(s):

Gongrui Guo ◽

Ping Zhu ◽

Martin R. Fuchs ◽

Wuxian Shi ◽

Babak Andi ◽

...

Keyword(s):

Data Analysis ◽

Data Collection ◽

Diffraction Data ◽

De Novo ◽

Low Energy ◽

Anomalous Scattering ◽

Free Electron Lasers ◽

X Ray ◽

Structural Evaluation ◽

Sad Phasing

De novo structural evaluation of native biomolecules from single-wavelength anomalous diffraction (SAD) is a challenge because of the weakness of the anomalous scattering. The anomalous scattering from relevant native elements – primarily sulfur in proteins and phosphorus in nucleic acids – increases as the X-ray energy decreases toward their K-edge transitions. Thus, measurements at a lowered X-ray energy are promising for making native SAD routine and robust. For microcrystals with sizes less than 10 µm, native-SAD phasing at synchrotron microdiffraction beamlines is even more challenging because of difficulties in sample manipulation, diffraction data collection and data analysis. Native-SAD analysis from microcrystals by using X-ray free-electron lasers has been demonstrated but has required use of thousands of thousands of microcrystals to achieve the necessary accuracy. Here it is shown that by exploitation of anomalous microdiffraction signals obtained at 5 keV, by the use of polyimide wellmounts, and by an iterative crystal and frame-rejection method, microcrystal native-SAD phasing is possible from as few as about 1 200 crystals. Our results show the utility of low-energy native-SAD phasing with microcrystals at synchrotron microdiffraction beamlines.

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ZOO: an automatic data-collection system for high-throughput structure analysis in protein microcrystallography

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798318017795 ◽

2019 ◽

Vol 75 (2) ◽

pp. 138-150 ◽

Cited By ~ 42

Author(s):

Kunio Hirata ◽

Keitaro Yamashita ◽

Go Ueno ◽

Yoshiaki Kawano ◽

Kazuya Hasegawa ◽

...

Keyword(s):

Data Collection ◽

Structural Information ◽

Protein Crystal ◽

Quality Data ◽

Collection System ◽

Automated Data Collection ◽

Automatic Data ◽

Data Collection System ◽

Automatic Data Collection ◽

Processing Techniques

Owing to the development of brilliant microfocus beamlines, rapid-readout detectors and sample changers, protein microcrystallography is rapidly becoming a popular technique for accessing structural information from complex biological samples. However, the method is time-consuming and labor-intensive and requires technical expertise to obtain high-resolution protein crystal structures. At SPring-8, an automated data-collection system named ZOO has been developed. This system enables faster data collection, facilitates advanced data-collection and data-processing techniques, and permits the collection of higher quality data. In this paper, the key features of the functionality put in place on the SPring-8 microbeam beamline BL32XU are described and the major advantages of this system are outlined. The ZOO system will be a major driving force in the evolution of the macromolecular crystallography beamlines at SPring-8.

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Data collection tools for challenging samples at the MX beamlines at Diamond

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314096703 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C329-C329

Author(s):

Ralf Flaig

Keyword(s):

Data Collection ◽

Radiation Damage ◽

Special Focus ◽

Data Set ◽

Automatic Data ◽

Standard Data ◽

Experimental Phasing ◽

The Difference ◽

The Uk ◽

Collection Strategies

Diamond Light Source [1] is the UK third generation synchrotron facility located south of Oxford and it started with the user programme in early 2007. Currently, there are five operational macromolecular crystallography (MX) beamlines that provide state of the art facilities to the user community and eventually there will be seven beamlines dedicated to MX [2]. All MX beamlines provide tools for standard data collection but given the increasing complexity and associated challenges with bigger macromolecular complexes, membrane proteins, smaller crystals, and radiation damage, different approaches are often required to get the best possible data out of these samples. Tools for sample location and characterization are a first step. Often, because of radiation damage and sample deterioration, multiple crystals are needed in order to obtain a complete data set and a number of tools and different experiment setups that help to address this problem will be described, including use of suitable software tools to get the best data set, fast data collection, crystal humidity control, in situ screening and use of a mini kappa goniometer. These tools enable new data collection strategies which can make the difference towards a successful structure determination. A special focus will be on the use and potential of multi-axis goniometers. Given the limited amount of beam time and ever faster data acquisition rates, quick decision making during the beam time becomes more important. Therefore, data collection strategies and crystal and diffraction image characterization are provided automatically. Very shortly after the data collection has finished the results from our automatic data processing routines are available and we also provide difference electron density map, molecular replacement and experimental phasing pipelines.

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De novo protein structure determination by heavy-atom soaking in lipidic cubic phase and SIRAS phasing using serial synchrotron crystallography

IUCrJ ◽

10.1107/s2052252518009223 ◽

2018 ◽

Vol 5 (5) ◽

pp. 524-530 ◽

Cited By ~ 4

Author(s):

S. Botha ◽

D. Baitan ◽

K. E. J. Jungnickel ◽

D. Oberthür ◽

C. Schmidt ◽

...

Keyword(s):

Synchrotron Radiation ◽

Data Collection ◽

Diffraction Data ◽

Heavy Atom ◽

Proteinase K ◽

Cubic Phase ◽

Anomalous Scattering ◽

Experimental Phasing ◽

Lipidic Cubic Phase ◽

Serial Crystallography

During the past few years, serial crystallography methods have undergone continuous development and serial data collection has become well established at high-intensity synchrotron-radiation beamlines and XFEL radiation sources. However, the application of experimental phasing to serial crystallography data has remained a challenging task owing to the inherent inaccuracy of the diffraction data. Here, a particularly gentle method for incorporating heavy atoms into micrometre-sized crystals utilizing lipidic cubic phase (LCP) as a carrier medium is reported. Soaking in LCP prior to data collection offers a new, efficient and gentle approach for preparing heavy-atom-derivative crystals directly before diffraction data collection using serial crystallography methods. This approach supports effective phasing by utilizing a reasonably low number of diffraction patterns. Using synchrotron radiation and exploiting the anomalous scattering signal of mercury for single isomorphous replacement with anomalous scattering (SIRAS) phasing resulted in high-quality electron-density maps that were sufficient for building a complete structural model of proteinase K at 1.9 Å resolution using automatic model-building tools.

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