scholarly journals Advances in long-wavelength native phasing at X-ray free-electron lasers

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 965-975 ◽  
Author(s):  
Karol Nass ◽  
Robert Cheng ◽  
Laura Vera ◽  
Aldo Mozzanica ◽  
Sophie Redford ◽  
...  
Keyword(s):  
Data Quality ◽  
Structure Determination ◽  
Free Electron ◽  
De Novo ◽  
Geometry Optimization ◽  
Protein Structure Determination ◽  
Time Resolved ◽  
X Ray ◽  
Long Wavelength ◽  
Order Of Magnitude

Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized de novo structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies.

scholarly journals Pink-beam serial femtosecond crystallography for accurate structure-factor determination at an X-ray free-electron laser

IUCrJ ◽  
2021 ◽  
Vol 8 (6) ◽  
Author(s):  
Karol Nass ◽  
Camila Bacellar ◽  
Claudio Cirelli ◽  
Florian Dworkowski ◽  
Yaroslav Gevorkov ◽  
...  
Keyword(s):  
Data Quality ◽  
Structure Determination ◽  
Structure Factor ◽  
Free Electron ◽  
Free Electron Laser ◽  
De Novo ◽  
Similar Data ◽  
X Ray ◽  
Redundant Data ◽  
Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables essentially radiation-damage-free macromolecular structure determination using microcrystals that are too small for synchrotron studies. However, SFX experiments often require large amounts of sample in order to collect highly redundant data where some of the many stochastic errors can be averaged out to determine accurate structure-factor amplitudes. In this work, the capability of the Swiss X-ray free-electron laser (SwissFEL) was used to generate large-bandwidth X-ray pulses [Δλ/λ = 2.2% full width at half-maximum (FWHM)], which were applied in SFX with the aim of improving the partiality of Bragg spots and thus decreasing sample consumption while maintaining the data quality. Sensitive data-quality indicators such as anomalous signal from native thaumatin micro-crystals and de novo phasing results were used to quantify the benefits of using pink X-ray pulses to obtain accurate structure-factor amplitudes. Compared with data measured using the same setup but using X-ray pulses with typical quasi-monochromatic XFEL bandwidth (Δλ/λ = 0.17% FWHM), up to fourfold reduction in the number of indexed diffraction patterns required to obtain similar data quality was achieved. This novel approach, pink-beam SFX, facilitates the yet underutilized de novo structure determination of challenging proteins at XFELs, thereby opening the door to more scientific breakthroughs.

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 Experimental phase determination with selenomethionine or mercury-derivatization in serial femtosecond crystallography

IUCrJ ◽  
2017 ◽  
Vol 4 (5) ◽  
pp. 639-647 ◽  
Author(s):  
Keitaro Yamashita ◽  
Naoyuki Kuwabara ◽  
Takanori Nakane ◽  
Tomohiro Murai ◽  
Eiichi Mizohata ◽  
...  
Keyword(s):  
Structure Determination ◽  
Free Electron ◽  
De Novo ◽  
Heavy Atom ◽  
Protein Structure Determination ◽  
High Energy ◽  
X Rays ◽  
Replacement Method ◽  
Serial Femtosecond Crystallography

Serial femtosecond crystallography (SFX) using X-ray free-electron lasers (XFELs) holds enormous potential for the structure determination of proteins for which it is difficult to produce large and high-quality crystals. SFX has been applied to various systems, but rarely to proteins that have previously unknown structures. Consequently, the majority of previously obtained SFX structures have been solved by the molecular replacement method. To facilitate protein structure determination by SFX, it is essential to establish phasing methods that work efficiently for SFX. Here, selenomethionine derivatization and mercury soaking have been investigated for SFX experiments using the high-energy XFEL at the SPring-8 Angstrom Compact Free-Electron Laser (SACLA), Hyogo, Japan. Three successful cases are reported of single-wavelength anomalous diffraction (SAD) phasing using X-rays of less than 1 Å wavelength with reasonable numbers of diffraction patterns (13 000, 60 000 and 11 000). It is demonstrated that the combination of high-energy X-rays from an XFEL and commonly used heavy-atom incorporation techniques will enable routinede novostructural determination of biomacromolecules.

scholarly journals First Experiments in Structural Biology at the European X-ray Free-Electron Laser

2020 ◽  
Vol 10 (10) ◽  
pp. 3642 ◽  
Author(s):  
Grant Mills ◽  
Richard Bean ◽  
Adrian P. Mancuso
Keyword(s):  
Structural Biology ◽  
Structure Determination ◽  
Free Electron ◽  
Free Electron Laser ◽  
Pulse Frequency ◽  
Free Electron Lasers ◽  
Time Resolved ◽  
New Era ◽  
X Ray ◽  
Rapid Pulse

Ultrabright pulses produced in X-ray free-electron lasers (XFELs) offer new possibilities for industry and research, particularly for biochemistry and pharmaceuticals. The unprecedented brilliance of these next-generation sources enables structure determination from sub-micron crystals as well as radiation-sensitive proteins. The European X-Ray Free-Electron Laser (EuXFEL), with its first light in 2017, ushered in a new era for ultrabright X-ray sources by providing an unparalleled megahertz-pulse repetition rate, with orders of magnitude more pulses per second than previous XFEL sources. This rapid pulse frequency has significant implications for structure determination; not only will data collection be faster (resulting in more structures per unit time), but experiments requiring large quantities of data, such as time-resolved structures, become feasible in a reasonable amount of experimental time. Early experiments at the SPB/SFX instrument of the EuXFEL demonstrate how such closely-spaced pulses can be successfully implemented in otherwise challenging experiments, such as time-resolved studies.

De novo protein crystal structure determination from X-ray free-electron laser data

Nature ◽  
2013 ◽  
Vol 505 (7482) ◽  
pp. 244-247 ◽  
Author(s):  
Thomas R. M. Barends ◽  
Lutz Foucar ◽  
Sabine Botha ◽  
R. Bruce Doak ◽  
Robert L. Shoeman ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structure Determination ◽  
Free Electron ◽  
Free Electron Laser ◽  
De Novo ◽  
Crystal Structure Determination ◽  
Protein Crystal ◽  
X Ray ◽  
Protein Crystal Structure ◽  
Laser Data

Beyond X-rays: an overview of emerging structural biology methods

2021 ◽  
Author(s):  
Jason E. Schaffer ◽  
Vandna Kukshal ◽  
Justin J. Miller ◽  
Vivian Kitainda ◽  
Joseph M. Jez
Keyword(s):  
Structure Determination ◽  
De Novo ◽  
Three Dimensional ◽  
Atomic Scale ◽  
X Rays ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
New Methods ◽  
Main Technique

Structural biologists rely on X-ray crystallography as the main technique for determining the three-dimensional structures of macromolecules; however, in recent years, new methods that go beyond X-ray-based technologies are broadening the selection of tools to understand molecular structure and function. Simultaneously, national facilities are developing programming tools and maintaining personnel to aid novice structural biologists in de novo structure determination. The combination of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) now enable time-resolved structure determination that allows for capture of dynamic processes, such as reaction mechanism and conformational flexibility. XFEL and SFX, along with microcrystal electron diffraction (MicroED), help side-step the need for large crystals for structural studies. Moreover, advances in cryogenic electron microscopy (cryo-EM) as a tool for structure determination is revolutionizing how difficult to crystallize macromolecules and/or complexes can be visualized at the atomic scale. This review aims to provide a broad overview of these new methods and to guide readers to more in-depth literature of these methods.

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