دورية أكاديمية
أعرض في EDS

Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals.

التفاصيل البيبلوغرافية
العنوان: Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals.
المؤلفون: Lahey-Rudolph JM; Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany.; Center for Free-Electron Laser Science (CFEL), Deutsches Elektronen Synchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany., Schönherr R; Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany.; Photon Science, Deutsches Elektronen Synchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany., Barthelmess M; Center for Free-Electron Laser Science (CFEL), Deutsches Elektronen Synchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany., Fischer P; Center for Free-Electron Laser Science (CFEL), Deutsches Elektronen Synchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany., Seuring C; Center for Free-Electron Laser Science (CFEL), Deutsches Elektronen Synchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany.; The Hamburg Center for Ultrafast Imaging, 22671 Hamburg, Germany., Wagner A; Diamond Light Source, Diamond House DH2-52, Chilton, Didcot OX11 0DE, United Kingdom., Meents A; Center for Free-Electron Laser Science (CFEL), Deutsches Elektronen Synchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany.; Photon Science, Deutsches Elektronen Synchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany., Redecke L; Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany.; Photon Science, Deutsches Elektronen Synchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany.
المصدر: IUCrJ [IUCrJ] 2021 Jun 18; Vol. 8 (Pt 4), pp. 665-677. Date of Electronic Publication: 2021 Jun 18 (Print Publication: 2021).
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: International Union of Crystallography Country of Publication: England NLM ID: 101623101 Publication Model: eCollection Cited Medium: Print ISSN: 2052-2525 (Print) Linking ISSN: 20522525 NLM ISO Abbreviation: IUCrJ Subsets: PubMed not MEDLINE
أسماء مطبوعة: Original Publication: Chester : International Union of Crystallography, [2014]-
مستخلص: 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.
(© J. Mia Lahey-Rudolph et al. 2021.)
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فهرسة مساهمة: Keywords: Roadrunner; fixed-target SFX; in cellulo crystallography; intracellular protein crystals; serial femtosecond crystallography; silicon chip
تواريخ الأحداث: Date Created: 20210714 Latest Revision: 20231107
رمز التحديث: 20240628
مُعرف محوري في PubMed: PMC8256716
DOI: 10.1107/S2052252521005297
PMID: 34258014
قاعدة البيانات: MEDLINE
الوصف
تدمد:2052-2525
DOI:10.1107/S2052252521005297