The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.

Australian Research Council Centre of Excellence in Advanced Molecular Imaging Department of Chemistry and Physics La Trobe Institute for Molecular Sciences La Trobe University Bundoora VIC 3086 Australia

Biological Research Centre Hungarian Academy of Sciences Temesvári krt 62 Szeged 6726 Hungary

Biomedical and 10 Ray Physics Department of Applied Physics AlbaNova University Center KTH Royal Institute of Technology Stockholm 106 91 Sweden

Center for Free Electron Laser Science Deutsches Elektronen Synchrotron DESY Notkestrasse 85 22607 Hamburg Germany

Condensed Matter Physics Department of Physics Chalmers University of Technology Gothenburg 412 96 Sweden

Depart Ingeniería Aeroespacial y Mecánica de Fluidos ETSI Universidad de Sevilla 41092 Sevilla Spain

Department of Cellular and Molecular Biophysics Max Planck Institute of Biochemistry 82152 Martinsried Germany

Department of Chemistry Universität Hamburg Martin Luther King Platz 6 20146 Hamburg Germany

Department of Chemistry University at Buffalo 359 Natural Sciences Complex Buffalo NY 14260 USA

Department of Physics Arizona State University Tempe AZ 85287 USA

Department of Physics Universität Hamburg Luruper Chaussee 149 22761 Hamburg Germany

Deutsches Elektronen Synchrotron DESY Notkestrasse 85 22607 Hamburg Germany

Diamond Light Source Research Complex at Harwell and University of Oxford Diamond House Harwell Science and Innovation Campus Didcot Oxfordshire OX11 0DE UK

Division of Structural Biology Headington Oxford OX3 7BN UK

ELI Beamlines Institute of Physics of the Czech Academy of Sciences Na Slovance 2 182 21 Prague Czech Republic

Engineering and the Environment University of Southampton SO17 1BJ Southampton UK

European XFEL GmbH Holzkoppel 4 22869 Schenefeld Germany

Hamburg University of Technology Vision Systems E 2 Harburger Schloßstr 20 21079 Hamburg Germany

Institute for Biochemistry and Molecular Biology Laboratory for Structural Biology of Infection and Inflammation Universität Hamburg Notkestrasse 85 22607 Hamburg Germany

Institute for Glycomics Griffith University Southport QLD 4222 Australia

Institute of Biochemistry Center for Structural and Cell Biology in Medicine University of Lübeck Ratzeburger Allee 160 23562 Lübeck Germany

Institute of Medical Microbiology Virology and Hygiene University Medical Center Hamburg Eppendorf 20246 Hamburg Germany

Institute of Molecular Biology SAS Dubravska cesta 21 845 51 Bratislava Slovakia

Institute of Nanostructure and Solid State Physics Department of Physics Universität Hamburg Luruper Chaussee 149 22761 Hamburg Germany

Integrated Biology Infrastructure Life Science Facility at the European XFEL Holzkoppel 4 22869 Schenefeld Germany

Laboratory of Molecular Biophysics Department of Cell and Molecular Biology Uppsala University Uppsala 751 24 Sweden

Lawrence Livermore National Laboratory 7000 East Avenue Livermore CA 94550 USA

Linac Coherent Light Source SLAC National Accelerator Laboratory Menlo Park 94025 CA USA

Max Planck Institute for Medical Research Jahnstr 29 69120 Heidelberg Germany

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

Mid Sweden University Holmgatan 10 85170 Sundsvall Sweden

NERSC Lawrence Berkeley National Laboratory Berkeley 94720 CA USA

Paul Scherrer Institut Forschungsstrasse 111 5232 Villigen Switzerland

Physics Department Rutgers University Newark Newark NJ 07102 USA

Physics Department University of Wisconsin Milwaukee 3135 N Maryland Ave Milwaukee WI 53211 USA

School of Chemistry and Molecular Biosciences Institute for Molecular Bioscience and Australian Infectious Diseases Research Centre University of Queensland Brisbane QLD 4072 Australia

School of Molecular Sciences and Biodesign Center for Applied Structural Discovery Arizona State University Tempe AZ 85287 1604 USA

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

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