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.

Time-Resolved Serial Femtosecond Crystallography at the European X-ray Free Electron Laser

2019 ◽  
Author(s):  
Marius Schmidt ◽  
Suraj Pandey ◽  
Adrian Mancuso ◽  
Richard Bean
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
Crystallographic Data ◽  
Time Resolved ◽  
X Ray ◽  
Serial Femtosecond Crystallography

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

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 Two mirror X-ray pulse split and delay instrument for femtosecond time resolved investigations at the LCLS free electron laser facility

2016 ◽  
Vol 24 (11) ◽  
pp. 11768 ◽  
Author(s):  
Nora Berrah ◽  
Li Fang ◽  
Brendan F Murphy ◽  
Edwin Kukk ◽  
Timur Y. Osipov ◽  
...  
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
Time Resolved ◽  
X Ray ◽  
Laser Facility

scholarly journals Direct autocorrelation of soft-x-ray free-electron-laser pulses by time-resolved two-photon double ionization of He

2009 ◽  
Vol 80 (2) ◽  
Author(s):  
R. Mitzner ◽  
A. A. Sorokin ◽  
B. Siemer ◽  
S. Roling ◽  
M. Rutkowski ◽  
...  
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
Laser Pulses ◽  
Double Ionization ◽  
Time Resolved ◽  
X Ray ◽  
Two Photon

scholarly journals Co-crystal structure of the iMango-III fluorescent RNA aptamer using an X-ray free-electron laser

2019 ◽  
Vol 75 (8) ◽  
pp. 547-551 ◽  
Author(s):  
Robert J. Trachman ◽  
Jason R. Stagno ◽  
Chelsie Conrad ◽  
Christopher P. Jones ◽  
Pontus Fischer ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Single Crystal ◽  
Free Electron ◽  
Free Electron Laser ◽  
Structural Model ◽  
Data Sets ◽  
Data Set ◽  
X Ray ◽  
Turn On

Turn-on aptamers are in vitro-selected RNAs that bind to conditionally fluorescent small molecules and enhance their fluorescence. Upon binding TO1-biotin, the iMango-III aptamer achieves the largest fluorescence enhancement reported for turn-on aptamers (over 5000-fold). This aptamer was generated by structure-guided engineering and functional reselection of the parental aptamer Mango-III. Structures of both Mango-III and iMango-III have previously been determined by conventional cryocrystallography using synchrotron X-radiation. Using an X-ray free-electron laser (XFEL), the room-temperature iMango-III–TO1-biotin co-crystal structure has now been determined at 3.0 Å resolution. This structural model, which was refined against a data set of ∼1300 diffraction images (each from a single crystal), is largely consistent with the structures determined from single-crystal data sets collected at 100 K. This constitutes a technical benchmark on the way to XFEL pump–probe experiments on fluorescent RNA–small molecule complexes.

Determination of x-ray free electron laser power using a room-temperature calorimeter

Metrologia ◽  
2016 ◽  
Vol 53 (1) ◽  
pp. 98-102 ◽  
Author(s):  
T Tanaka ◽  
M Kato ◽  
N Saito ◽  
K Tono ◽  
M Yabashi ◽  
...  
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
Laser Power ◽  
Room Temperature ◽  
X Ray

scholarly journals Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser

2015 ◽  
Vol 71 (7) ◽  
pp. 856-860 ◽  
Author(s):  
Wenting Wu ◽  
Przemyslaw Nogly ◽  
Jan Rheinberger ◽  
Leonhard M. Kick ◽  
Cornelius Gati ◽  
...  
Keyword(s):  
Free Electron ◽  
Free Electron Laser ◽  
Second Harmonic ◽  
Night Vision ◽  
Salt Crystallization ◽  
Time Resolved ◽  
Batch Crystallization ◽  
X Ray ◽  
Vapour Diffusion ◽  
Serial Crystallography

Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup.

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