The idea of using ultrashort X-ray pulses to obtain images of single proteins frozen in time has fascinated and inspired many. It was one of the arguments for building X-ray free-electron lasers. According to theory, the extremely intense pulses provide sufficient signal to dispense with using crystals as an amplifier, and the ultrashort pulse duration permits capturing the diffraction data before the sample inevitably explodes. This was first demonstrated on biological samples a decade ago on the giant mimivirus. Since then, a large collaboration has been pushing the limit of the smallest sample that can be imaged. The ability to capture snapshots on the timescale of atomic vibrations, while keeping the sample at room temperature, may allow probing the entire conformational phase space of macromolecules. Here we show the first observation of an X-ray diffraction pattern from a single protein, that of Escherichia coli GroEL which at 14 nm in diameter is the smallest biological sample ever imaged by X-rays, and demonstrate that the concept of diffraction before destruction extends to single proteins. From the pattern, it is possible to determine the approximate orientation of the protein. Our experiment demonstrates the feasibility of ultrafast imaging of single proteins, opening the way to single-molecule time-resolved studies on the femtosecond timescale.

Biomedical and 10 Ray Physics Department of Applied Physics AlbaNova University Center KTH Royal Institute of Technology SE 10691 Stockholm Sweden

Center for Free Electron Laser Science DESY 22607 Hamburg Germany

Center for Free Electron Laser Science Luruper Chaussee 149 22761 Hamburg Germany

Department of Chemistry and Physics La Trobe Institute for Molecular Science La Trobe University Melbourne VIC 3086 Australia

Department of Chemistry Universität Hamburg 20146 Hamburg Germany

Department of Physics and Astronomy Uppsala University Box 516 SE 75120 Uppsala Sweden

Department of Physics Universität Hamburg Luruper Chaussee 149 22761 Hamburg Germany

Deutsches Electronen Synchrotron DESY Notkestrasse 85 22607 Hamburg Germany

Diamond Light Source Harwell Science and Innovation Campus Didcot OX11 0DE UK

Dipartimento di Fisica Aldo Pontremoli Università degli Studi di Milano via Celoria 16 20133 Milano Italy

Division of Scientific Computing Science for Life Laboratory Department of Information Technology Uppsala University Box 337 SE 75105 Uppsala Sweden

ELI Beamlines IoP Institute of Physics AS CR v v i Na Slovance 2 182 21 Prague 8 Czech Republic

European Molecular Biology Laboratory c o DESY Notkestrasse 85 22607 Hamburg Germany

European XFEL Holzkoppel 4 22869 Schenefeld Germany

Laboratory of Molecular Biophysics Department of Cell and Molecular Biology Uppsala University Husargatan 3 SE 75124 Uppsala Sweden

Leibniz Institute for Experimental Virology Centre for Structural Systems Biology Notkestraße 85 22607 Hamburg Germany

Max Planck Institute for the Structure and Dynamics of Matter Luruper Chaussee 149 22761 Hamburg Germany

Multi User CryoEM Facility Centre for Structural Systems Biology Notkestr 85 22607 Hamburg Germany

NERSC Lawrence Berkeley National Laboratory Berkeley CA 94720 USA

Plant Biology Section School of Integrative Plant Science Cornell University Ithaca NY 14853 USA

PNSensor GmbH Otto Hahn Ring 6 D 81739 München Germany

The Hamburg Center for Ultrafast Imaging Universität Hamburg Luruper Chaussee 149 22761 Hamburg Germany

University Medical Center Hamburg Eppendorf Martinistrasse 52 20246 Hamburg Germany

University of Stavanger Centre Organelle Research Richard Johnsensgate 4 4021 Stavanger Norway

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