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Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals

The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting th...

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Autores principales: Lahey-Rudolph, J. Mia, Schönherr, Robert, Barthelmess, Miriam, Fischer, Pontus, Seuring, Carolin, Wagner, Armin, Meents, Alke, Redecke, Lars
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Union of Crystallography 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8256716/
https://www.ncbi.nlm.nih.gov/pubmed/34258014
http://dx.doi.org/10.1107/S2052252521005297
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author Lahey-Rudolph, J. Mia
Schönherr, Robert
Barthelmess, Miriam
Fischer, Pontus
Seuring, Carolin
Wagner, Armin
Meents, Alke
Redecke, Lars
author_facet Lahey-Rudolph, J. Mia
Schönherr, Robert
Barthelmess, Miriam
Fischer, Pontus
Seuring, Carolin
Wagner, Armin
Meents, Alke
Redecke, Lars
author_sort Lahey-Rudolph, J. Mia
collection PubMed
description The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting the in cellulo crystals from environmental challenges. Serial femtosecond crystallography (SFX) at free-electron lasers (XFELs) allows the collection of detectable diffraction even from tiny protein crystals, but requires very fast sample exchange to utilize each XFEL pulse. Here, an efficient approach is presented for high-resolution structure elucidation using serial femtosecond in cellulo diffraction of micometre-sized crystals of the protein HEX-1 from the fungus Neurospora crassa on a fixed target. Employing the fast and highly accurate Roadrunner II translation-stage system allowed efficient raster scanning of the pores of micro-patterned, single-crystalline silicon chips loaded with living, crystal-containing insect cells. Compared with liquid-jet and LCP injection systems, the increased hit rates of up to 30% and reduced background scattering enabled elucidation of the HEX-1 structure. Using diffraction data from only a single chip collected within 12 min at the Linac Coherent Light Source, a 1.8 Å resolution structure was obtained with significantly reduced sample consumption compared with previous SFX experiments using liquid-jet injection. This HEX-1 structure is almost superimposable with that previously determined using synchrotron radiation from single HEX-1 crystals grown by sitting-drop vapour diffusion, validating the approach. This study demonstrates that fixed-target SFX using micro-patterned silicon chips is ideally suited for efficient in cellulo diffraction data collection using living, crystal-containing cells, and offers huge potential for the straightforward structure elucidation of proteins that form intracellular crystals at both XFELs and synchrotron sources.
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spelling pubmed-82567162021-07-12 Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals Lahey-Rudolph, J. Mia Schönherr, Robert Barthelmess, Miriam Fischer, Pontus Seuring, Carolin Wagner, Armin Meents, Alke Redecke, Lars IUCrJ Research Papers The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting the in cellulo crystals from environmental challenges. Serial femtosecond crystallography (SFX) at free-electron lasers (XFELs) allows the collection of detectable diffraction even from tiny protein crystals, but requires very fast sample exchange to utilize each XFEL pulse. Here, an efficient approach is presented for high-resolution structure elucidation using serial femtosecond in cellulo diffraction of micometre-sized crystals of the protein HEX-1 from the fungus Neurospora crassa on a fixed target. Employing the fast and highly accurate Roadrunner II translation-stage system allowed efficient raster scanning of the pores of micro-patterned, single-crystalline silicon chips loaded with living, crystal-containing insect cells. Compared with liquid-jet and LCP injection systems, the increased hit rates of up to 30% and reduced background scattering enabled elucidation of the HEX-1 structure. Using diffraction data from only a single chip collected within 12 min at the Linac Coherent Light Source, a 1.8 Å resolution structure was obtained with significantly reduced sample consumption compared with previous SFX experiments using liquid-jet injection. This HEX-1 structure is almost superimposable with that previously determined using synchrotron radiation from single HEX-1 crystals grown by sitting-drop vapour diffusion, validating the approach. This study demonstrates that fixed-target SFX using micro-patterned silicon chips is ideally suited for efficient in cellulo diffraction data collection using living, crystal-containing cells, and offers huge potential for the straightforward structure elucidation of proteins that form intracellular crystals at both XFELs and synchrotron sources. International Union of Crystallography 2021-06-18 /pmc/articles/PMC8256716/ /pubmed/34258014 http://dx.doi.org/10.1107/S2052252521005297 Text en © J. Mia Lahey-Rudolph et al. 2021 https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.
spellingShingle Research Papers
Lahey-Rudolph, J. Mia
Schönherr, Robert
Barthelmess, Miriam
Fischer, Pontus
Seuring, Carolin
Wagner, Armin
Meents, Alke
Redecke, Lars
Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals
title Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals
title_full Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals
title_fullStr Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals
title_full_unstemmed Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals
title_short Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals
title_sort fixed-target serial femtosecond crystallography using in cellulo grown microcrystals
topic Research Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8256716/
https://www.ncbi.nlm.nih.gov/pubmed/34258014
http://dx.doi.org/10.1107/S2052252521005297
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