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

Author(s):  
Wenting Wu ◽  
Przemyslaw Nogly ◽  
Jan Rheinberger ◽  
Leonhard M. Kick ◽  
Cornelius Gati ◽  
...  

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.

2019 ◽  
Vol 52 (6) ◽  
pp. 1385-1396 ◽  
Author(s):  
John H. Beale ◽  
Rachel Bolton ◽  
Stephen A. Marshall ◽  
Emma V. Beale ◽  
Stephen B. Carr ◽  
...  

Serial crystallography, at both synchrotron and X-ray free-electron laser light sources, is becoming increasingly popular. However, the tools in the majority of crystallization laboratories are focused on producing large single crystals by vapour diffusion that fit the cryo-cooled paradigm of modern synchrotron crystallography. This paper presents several case studies and some ideas and strategies on how to perform the conversion from a single crystal grown by vapour diffusion to the many thousands of micro-crystals required for modern serial crystallography grown by batch crystallization. These case studies aim to show (i) how vapour diffusion conditions can be converted into batch by optimizing the length of time crystals take to appear; (ii) how an understanding of the crystallization phase diagram can act as a guide when designing batch crystallization protocols; and (iii) an accessible methodology when attempting to scale batch conditions to larger volumes. These methods are needed to minimize the sample preparation gap between standard rotation crystallography and dedicated serial laboratories, ultimately making serial crystallography more accessible to all crystallographers.


Crystals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 655
Author(s):  
Ki-Hyun Nam

Serial crystallography (SX) is an emerging X-ray crystallographic method for determining macromolecule structures. It can address concerns regarding the limitations of data collected by conventional crystallography techniques, which require cryogenic-temperature environments and allow crystals to accumulate radiation damage. Time-resolved SX studies using the pump-probe methodology provide useful information for understanding macromolecular mechanisms and structure fluctuation dynamics. This Special Issue deals with the serial crystallography approach using an X-ray free electron laser (XFEL) and synchrotron X-ray source, and reviews recent SX research involving synchrotron use. These reports provide insights into future serial crystallography research trends and approaches.


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.


2016 ◽  
Vol 24 (11) ◽  
pp. 11768 ◽  
Author(s):  
Nora Berrah ◽  
Li Fang ◽  
Brendan F Murphy ◽  
Edwin Kukk ◽  
Timur Y. Osipov ◽  
...  

2009 ◽  
Vol 80 (2) ◽  
Author(s):  
R. Mitzner ◽  
A. A. Sorokin ◽  
B. Siemer ◽  
S. Roling ◽  
M. Rutkowski ◽  
...  

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