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 ◽  
...  

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.

IUCrJ ◽  
2020 ◽  
Vol 7 (6) ◽  
pp. 965-975 ◽  
Author(s):  
Karol Nass ◽  
Robert Cheng ◽  
Laura Vera ◽  
Aldo Mozzanica ◽  
Sophie Redford ◽  
...  

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.


Nature ◽  
2013 ◽  
Vol 505 (7482) ◽  
pp. 244-247 ◽  
Author(s):  
Thomas R. M. Barends ◽  
Lutz Foucar ◽  
Sabine Botha ◽  
R. Bruce Doak ◽  
Robert L. Shoeman ◽  
...  

IUCrJ ◽  
2016 ◽  
Vol 3 (3) ◽  
pp. 180-191 ◽  
Author(s):  
Karol Nass ◽  
Anton Meinhart ◽  
Thomas R. M. Barends ◽  
Lutz Foucar ◽  
Alexander Gorel ◽  
...  

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.


Author(s):  
Marius Schmidt ◽  
Suraj Pandey ◽  
Adrian Mancuso ◽  
Richard Bean

Abstract This protocol introduces step by step into the collection of time resolved crystallographic data and their analysis at the European Free Electron Laser.


2014 ◽  
Vol 369 (1647) ◽  
pp. 20130337 ◽  
Author(s):  
Uwe Weierstall

X-ray free-electron lasers overcome the problem of radiation damage in protein crystallography and allow structure determination from micro- and nanocrystals at room temperature. To ensure that consecutive X-ray pulses do not probe previously exposed crystals, the sample needs to be replaced with the X-ray repetition rate, which ranges from 120 Hz at warm linac-based free-electron lasers to 1 MHz at superconducting linacs. Liquid injectors are therefore an essential part of a serial femtosecond crystallography experiment at an X-ray free-electron laser. Here, we compare different techniques of injecting microcrystals in solution into the pulsed X-ray beam in vacuum. Sample waste due to mismatch of the liquid flow rate to the X-ray repetition rate can be addressed through various techniques.


2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Mark S. Hunter ◽  
Chun Hong Yoon ◽  
Hasan DeMirci ◽  
Raymond G. Sierra ◽  
E. Han Dao ◽  
...  

IUCrJ ◽  
2015 ◽  
Vol 2 (4) ◽  
pp. 409-420 ◽  
Author(s):  
Maike Bublitz ◽  
Karol Nass ◽  
Nikolaj D. Drachmann ◽  
Anders J. Markvardsen ◽  
Matthias J. Gutmann ◽  
...  

Membrane proteins are key players in biological systems, mediating signalling events and the specific transport ofe.g.ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with functionally or medically important ligands. Structure determination, however, is hampered by the difficulty of obtaining well diffracting, macroscopic crystals. Here, the feasibility of X-ray free-electron-laser-based serial femtosecond crystallography (SFX) for the structure determination of membrane protein–ligand complexes using microcrystals of various native-source and recombinant P-type ATPase complexes is demonstrated. The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate. By analyzing the resolution dependence of ligand densities and overall model qualities, SFX data quality metrics as well as suitable refinement procedures are discussed. Even at relatively low resolution and multiplicity, the identification of ligands can be demonstrated. This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.


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