Joint Laboratory for Structural Biology of Infection and Inflammation, Institute of Biochemistry and Molecular Biology, University of Hamburg, and Institute of Biochemistry, University of Lübeck, Notkestrasse 85, 22607 Hamburg, Germany
Dirk Rehders
Joint Laboratory for Structural Biology of Infection and Inflammation, Institute of Biochemistry and Molecular Biology, University of Hamburg, and Institute of Biochemistry, University of Lübeck, Notkestrasse 85, 22607 Hamburg, Germany
Francesco Stellato
Center for Free-Electron Laser Science (CFEL), Deutsches Elektronensynchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany
Dominik Oberthür
Center for Free-Electron Laser Science (CFEL), Deutsches Elektronensynchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany
Oleksandr Yefanov
Center for Free-Electron Laser Science (CFEL), Deutsches Elektronensynchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany
Benjamin P. Sommer
Institute of Biochemistry and Molecular Biology, University of Hamburg, Notkestrasse 85, 22607 Hamburg, Germany
Stefan Mogk
Interfaculty Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Strasse 4, 72076 Tübingen, Germany
Michael Duszenko
Interfaculty Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Strasse 4, 72076 Tübingen, Germany
Christian Betzel
Institute of Biochemistry and Molecular Biology, University of Hamburg, Notkestrasse 85, 22607 Hamburg, Germany
Center for Free-Electron Laser Science (CFEL), Deutsches Elektronensynchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany
Lars Redecke
Joint Laboratory for Structural Biology of Infection and Inflammation, Institute of Biochemistry and Molecular Biology, University of Hamburg, and Institute of Biochemistry, University of Lübeck, Notkestrasse 85, 22607 Hamburg, Germany
Crystal structure determinations of biological macromolecules are limited by the availability of sufficiently sized crystals and by the fact that crystal quality deteriorates during data collection owing to radiation damage. Exploiting a micrometre-sized X-ray beam, high-precision diffractometry and shutterless data acquisition with a pixel-array detector, a strategy for collecting data from many micrometre-sized crystals presented to an X-ray beam in a vitrified suspension is demonstrated. By combining diffraction data from 80 Trypanosoma brucei procathepsin B crystals with an average volume of 9 µm3, a complete data set to 3.0 Å resolution has been assembled. The data allowed the refinement of a structural model that is consistent with that previously obtained using free-electron laser radiation, providing mutual validation. Further improvements of the serial synchrotron crystallography technique and its combination with serial femtosecond crystallography are discussed that may allow the determination of high-resolution structures of micrometre-sized crystals.