scholarly journals Native phasing of x-ray free-electron laser data for a G protein–coupled receptor

2016 ◽  
Vol 2 (9) ◽  
pp. e1600292 ◽  
Author(s):  
Alexander Batyuk ◽  
Lorenzo Galli ◽  
Andrii Ishchenko ◽  
Gye Won Han ◽  
Cornelius Gati ◽  
...  
Keyword(s):  
Free Electron ◽  
Room Temperature ◽  
De Novo ◽  
Ultrashort Pulses ◽  
Temperature Structure ◽  
X Ray ◽  
Anomalous Signal ◽  
G Protein Coupled ◽  
Serial Femtosecond Crystallography ◽  
General Method

Serial femtosecond crystallography (SFX) takes advantage of extremely bright and ultrashort pulses produced by x-ray free-electron lasers (XFELs), allowing for the collection of high-resolution diffraction intensities from micrometer-sized crystals at room temperature with minimal radiation damage, using the principle of “diffraction-before-destruction.” However, de novo structure factor phase determination using XFELs has been difficult so far. We demonstrate the ability to solve the crystallographic phase problem for SFX data collected with an XFEL using the anomalous signal from native sulfur atoms, leading to a bias-free room temperature structure of the human A2A adenosine receptor at 1.9 Å resolution. The advancement was made possible by recent improvements in SFX data analysis and the design of injectors and delivery media for streaming hydrated microcrystals. This general method should accelerate structural studies of novel difficult-to-crystallize macromolecules and their complexes.

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 Liquid sample delivery techniques for serial femtosecond crystallography

2014 ◽  
Vol 369 (1647) ◽  
pp. 20130337 ◽  
Author(s):  
Uwe Weierstall
Keyword(s):  
Flow Rate ◽  
Free Electron ◽  
Repetition Rate ◽  
Free Electron Laser ◽  
Room Temperature ◽  
Liquid Flow Rate ◽  
Free Electron Lasers ◽  
Liquid Sample ◽  
X Ray ◽  
Serial Femtosecond Crystallography

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.

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 Double-flow focused liquid injector for efficient serial femtosecond crystallography

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Dominik Oberthuer ◽  
Juraj Knoška ◽  
Max O. Wiedorn ◽  
Kenneth R. Beyerlein ◽  
David A. Bushnell ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Room Temperature ◽  
Temperature Structure ◽  
Flow Focusing ◽  
X Ray ◽  
Jet Stability ◽  
Efficient Delivery ◽  
Structural Details ◽  
Liquid Injector ◽  
Serial Femtosecond Crystallography

Abstract Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.

scholarly journals Lipidic cubic phase serial millisecond crystallography using synchrotron radiation

IUCrJ ◽  
2015 ◽  
Vol 2 (2) ◽  
pp. 168-176 ◽  
Author(s):  
Przemyslaw Nogly ◽  
Daniel James ◽  
Dingjie Wang ◽  
Thomas A. White ◽  
Nadia Zatsepin ◽  
...  
Keyword(s):  
Room Temperature ◽  
Cubic Phase ◽  
Temperature Structure ◽  
Free Electron Lasers ◽  
X Ray ◽  
Cubic Phases ◽  
Lipidic Cubic Phase ◽  
Effective Delivery ◽  
Serial Femtosecond Crystallography ◽  
Delivery Medium

Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.

scholarly journals Co-crystal structure of the Fusobacterium ulcerans ZTP riboswitch using an X-ray free-electron laser

2019 ◽  
Vol 75 (7) ◽  
pp. 496-500
Author(s):  
Christopher Jones ◽  
Brandon Tran ◽  
Chelsie Conrad ◽  
Jason Stagno ◽  
Robert Trachman ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Small Molecules ◽  
Free Electron ◽  
Free Electron Laser ◽  
Room Temperature ◽  
Temperature Structure ◽  
Data Set ◽  
Time Resolved ◽  
Regulate Gene Expression ◽  
X Ray

Riboswitches are conformationally dynamic RNAs that regulate gene expression by binding specific small molecules. ZTP riboswitches bind the purine-biosynthetic intermediate 5-aminoimidazole-4-carboxamide riboside 5′-monophosphate (ZMP) and its triphosphorylated form (ZTP). Ligand binding to this riboswitch ultimately upregulates genes involved in folate and purine metabolism. Using an X-ray free-electron laser (XFEL), the room-temperature structure of the Fusobacterium ulcerans ZTP riboswitch bound to ZMP has now been determined at 4.1 Å resolution. This model, which was refined against a data set from ∼750 diffraction images (each from a single crystal), was found to be consistent with that previously obtained from data collected at 100 K using conventional synchrotron X-radiation. These experiments demonstrate the feasibility of time-resolved XFEL experiments to understand how the ZTP riboswitch accommodates cognate ligand binding.

Single-Crystal X-Ray Scattering Study of Superstructures and Phase Transitions in Graphite-Hno3 and Graphite-Br2 Intercalation Compounds

1982 ◽  
Vol 20 ◽  
Author(s):  
R. Moret ◽  
R. Comes ◽  
G. Furdin ◽  
H. Fuzellier ◽  
F. Rousseaux
Keyword(s):  
Phase Transitions ◽  
Low Temperature ◽  
Room Temperature ◽  
Temperature Phase ◽  
Temperature Structure ◽  
X Ray ◽  
X Ray Scattering ◽  
Scattering Study ◽  
Temperature Liquid ◽  
Low Temperature Phase

ABSTRACTIn α-C5n-HNO3 the condensation of the room-temperature liquid-like diffuse ring associated with the disorder-order transition around 250 K is studied and the low-temperature. superstructure is examined.It is found that β-C8n-HNO3 exhibits an in-plane incommensurate order at room temperature.Two types of graphite-Br2 are found. Low-temperature phase transitions in C8Br are observed at T1 ≍ 277 K and T2 ≍ 297 K. The room-temperature structure of C14Br is reexamined. Special attention is given to diffuse scattering and incommensurability.

scholarly journals Soft x-ray absorption spectroscopy of metalloproteins and high-valent metal-complexes at room temperature using free-electron lasers

2017 ◽  
Vol 4 (5) ◽  
pp. 054307 ◽  
Author(s):  
Markus Kubin ◽  
Jan Kern ◽  
Sheraz Gul ◽  
Thomas Kroll ◽  
Ruchira Chatterjee ◽  
...  
Keyword(s):  
Metal Complexes ◽  
Absorption Spectroscopy ◽  
Free Electron ◽  
Room Temperature ◽  
Free Electron Lasers ◽  
X Ray ◽  
High Valent ◽  
X Ray Absorption
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