Co-flow injection for serial crystallography at X-ray free-electron lasers

2022 ◽  
Vol 55 (1) ◽  
Author(s):  
Diandra Doppler ◽  
Mohammad T. Rabbani ◽  
Romain Letrun ◽  
Jorvani Cruz Villarreal ◽  
Dai Hyun Kim ◽  
...  

Serial femtosecond crystallography (SFX) is a powerful technique that exploits X-ray free-electron lasers to determine the structure of macromolecules at room temperature. Despite the impressive exposition of structural details with this novel crystallographic approach, the methods currently available to introduce crystals into the path of the X-ray beam sometimes exhibit serious drawbacks. Samples requiring liquid injection of crystal slurries consume large quantities of crystals (at times up to a gram of protein per data set), may not be compatible with vacuum configurations on beamlines or provide a high background due to additional sheathing liquids present during the injection. Proposed and characterized here is the use of an immiscible inert oil phase to supplement the flow of sample in a hybrid microfluidic 3D-printed co-flow device. Co-flow generation is reported with sample and oil phases flowing in parallel, resulting in stable injection conditions for two different resin materials experimentally. A numerical model is presented that adequately predicts these flow-rate conditions. The co-flow generating devices reduce crystal clogging effects, have the potential to conserve protein crystal samples up to 95% and will allow degradation-free light-induced time-resolved SFX.

2019 ◽  
Vol 20 (6) ◽  
pp. 1401 ◽  
Author(s):  
Marius Schmidt

The focus of structural biology is shifting from the determination of static structures to the investigation of dynamical aspects of macromolecular function. With time-resolved macromolecular crystallography (TRX), intermediates that form and decay during the macromolecular reaction can be investigated, as well as their reaction dynamics. Time-resolved crystallographic methods were initially developed at synchrotrons. However, about a decade ago, extremely brilliant, femtosecond-pulsed X-ray sources, the free electron lasers for hard X-rays, became available to a wider community. TRX is now possible with femtosecond temporal resolution. This review provides an overview of methodological aspects of TRX, and at the same time, aims to outline the frontiers of this method at modern pulsed X-ray sources.


2011 ◽  
Vol 98 (18) ◽  
pp. 182504 ◽  
Author(s):  
N. Pontius ◽  
T. Kachel ◽  
C. Schüßler-Langeheine ◽  
W. F. Schlotter ◽  
M. Beye ◽  
...  

2014 ◽  
Vol 5 (1) ◽  
Author(s):  
C. Behrens ◽  
F.-J. Decker ◽  
Y. Ding ◽  
V. A. Dolgashev ◽  
J. Frisch ◽  
...  

2016 ◽  
Vol 3 (5) ◽  
pp. 054301 ◽  
Author(s):  
George D. Calvey ◽  
Andrea M. Katz ◽  
Chris B. Schaffer ◽  
Lois Pollack

2022 ◽  
Vol 4 (1) ◽  
Author(s):  
Philipp Dijkstal ◽  
Alexander Malyzhenkov ◽  
Paolo Craievich ◽  
Eugenio Ferrari ◽  
Romain Ganter ◽  
...  

2020 ◽  
Vol 10 (10) ◽  
pp. 3642 ◽  
Author(s):  
Grant Mills ◽  
Richard Bean ◽  
Adrian P. Mancuso

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.


Crystals ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 628
Author(s):  
Suraj Pandey ◽  
Ishwor Poudyal ◽  
Tek Narsingh Malla

With time-resolved crystallography (TRX), it is possible to follow the reaction dynamics in biological macromolecules by investigating the structure of transient states along the reaction coordinate. X-ray free electron lasers (XFELs) have enabled TRX experiments on previously uncharted femtosecond timescales. Here, we review the recent developments, opportunities, and challenges of pump-probe TRX at XFELs.


Author(s):  
Christopher Jones ◽  
Brandon Tran ◽  
Chelsie Conrad ◽  
Jason Stagno ◽  
Robert Trachman ◽  
...  

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.


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