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Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers

Knowledge of protein structure provides essential insight into function, enhancing our understanding of diseases and enabling new treatment development. X-ray crystallography has been used to solve the structures of more than 100 000 proteins; however, the vast majority represent long-lived states t...

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Autores principales: Calvey, George D., Katz, Andrea M., Schaffer, Chris B., Pollack, Lois
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Crystallographic Association 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5010557/
https://www.ncbi.nlm.nih.gov/pubmed/27679802
http://dx.doi.org/10.1063/1.4961971
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author Calvey, George D.
Katz, Andrea M.
Schaffer, Chris B.
Pollack, Lois
author_facet Calvey, George D.
Katz, Andrea M.
Schaffer, Chris B.
Pollack, Lois
author_sort Calvey, George D.
collection PubMed
description Knowledge of protein structure provides essential insight into function, enhancing our understanding of diseases and enabling new treatment development. X-ray crystallography has been used to solve the structures of more than 100 000 proteins; however, the vast majority represent long-lived states that do not capture the functional motions of these molecular machines. Reactions triggered by the addition of a ligand can be the most challenging to detect with crystallography because of the difficulty of synchronizing reactions to create detectable quantities of transient states. The development of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) enables new approaches for solving protein structures following the rapid diffusion of ligands into micron sized protein crystals. Conformational changes occurring on millisecond timescales can be detected and time-resolved. Here, we describe a new XFEL injector which incorporates a microfluidic mixer to rapidly combine reactant and sample milliseconds before the sample reaches the X-ray beam. The mixing injector consists of bonded, concentric glass capillaries. The fabrication process, employing custom laser cut centering spacers and UV curable epoxy, ensures precise alignment of capillaries for repeatable, centered sample flow and dependable mixing. Crystal delivery capillaries are 50 or 75 μm in diameter and can contain an integrated filter depending on the demands of the experiment. Reaction times can be varied from submillisecond to several hundred milliseconds. The injector features rapid and uniform mixing, low sample dilution, and high hit rates. It is fully compatible with existing SFX beamlines.
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spelling pubmed-50105572016-09-27 Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers Calvey, George D. Katz, Andrea M. Schaffer, Chris B. Pollack, Lois Struct Dyn ARTICLES Knowledge of protein structure provides essential insight into function, enhancing our understanding of diseases and enabling new treatment development. X-ray crystallography has been used to solve the structures of more than 100 000 proteins; however, the vast majority represent long-lived states that do not capture the functional motions of these molecular machines. Reactions triggered by the addition of a ligand can be the most challenging to detect with crystallography because of the difficulty of synchronizing reactions to create detectable quantities of transient states. The development of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) enables new approaches for solving protein structures following the rapid diffusion of ligands into micron sized protein crystals. Conformational changes occurring on millisecond timescales can be detected and time-resolved. Here, we describe a new XFEL injector which incorporates a microfluidic mixer to rapidly combine reactant and sample milliseconds before the sample reaches the X-ray beam. The mixing injector consists of bonded, concentric glass capillaries. The fabrication process, employing custom laser cut centering spacers and UV curable epoxy, ensures precise alignment of capillaries for repeatable, centered sample flow and dependable mixing. Crystal delivery capillaries are 50 or 75 μm in diameter and can contain an integrated filter depending on the demands of the experiment. Reaction times can be varied from submillisecond to several hundred milliseconds. The injector features rapid and uniform mixing, low sample dilution, and high hit rates. It is fully compatible with existing SFX beamlines. American Crystallographic Association 2016-08-29 /pmc/articles/PMC5010557/ /pubmed/27679802 http://dx.doi.org/10.1063/1.4961971 Text en © 2016 Author(s). 2329-7778/2016/3(5)/054301/19 All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle ARTICLES
Calvey, George D.
Katz, Andrea M.
Schaffer, Chris B.
Pollack, Lois
Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers
title Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers
title_full Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers
title_fullStr Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers
title_full_unstemmed Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers
title_short Mixing injector enables time-resolved crystallography with high hit rate at X-ray free electron lasers
title_sort mixing injector enables time-resolved crystallography with high hit rate at x-ray free electron lasers
topic ARTICLES
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5010557/
https://www.ncbi.nlm.nih.gov/pubmed/27679802
http://dx.doi.org/10.1063/1.4961971
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