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Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals

DOI: 10.1107/S2052252521005297 DOI Help

Authors: J. Mia Lahey-Rudolph (University of Lübeck; Center for Free-Electron Laser Science (CFEL), Deutsches Elektronen Synchrotron (DESY)) , Robert Schönherr (University of Lübeck; Deutsches Elektronen Synchrotron (DESY)) , Miriam Barthelmess (Center for Free-Electron Laser Science (CFEL), Deutsches Elektronen Synchrotron (DESY)) , Pontus Fischer (Center for Free-Electron Laser Science (CFEL), Deutsches Elektronen Synchrotron (DESY)) , Carolin Seuring (Center for Free-Electron Laser Science (CFEL), Deutsches Elektronen Synchrotron (DESY); The Hamburg Center for Ultrafast Imaging) , Armin Wagner (Diamond Light Source) , Alke Meents (Center for Free-Electron Laser Science (CFEL), Deutsches Elektronen Synchrotron (DESY)) , Lars Redecke (University of Lübeck; Deutsches Elektronen Synchrotron (DESY))
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Iucrj , VOL 8 , PAGES 665 - 677

State: Published (Approved)
Published: July 2021

Open Access Open Access

Abstract: 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.

Journal Keywords: fixed-target SFX; serial femtosecond crystallography; in cellulo crystallography; intracellular protein crystals; silicon chip; Roadrunner

Subject Areas: Technique Development, Biology and Bio-materials

Facility: MFX at LCLS

Added On: 19/07/2021 09:55


Discipline Tags:

Technique Development - Life Sciences & Biotech Structural biology Life Sciences & Biotech

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