The three-dimensional structures of macromolecules and their complexes are mainly elucidated by X-ray protein crystallography. A major limitation of this method is access to high-quality crystals, which is necessary to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields information of sufficiently high resolution with which to solve the crystal structure. The observation that crystals with reduced unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks1,2 suggests that crystallographic resolution for some macromolecules may be limited not by their heterogeneity, but by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern that is equal to the incoherent sum of diffraction from rigid individual molecular complexes aligned along several discrete crystallographic orientations and that, consequently, contains more information than Bragg peaks alone3. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins4—they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5-ångström limit of measurable Bragg peaks, which allows us to phase5 the pattern directly. Using the molecular envelope conventionally determined at 4.5 ångströms as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 ångströms. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography, using a method that exploits commonly encountered imperfect crystals and enables model-free phasing6,7.
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We acknowledge support of the Helmholtz Association through project-oriented funds and the Virtual Institute “Dynamic Pathways in Multidimensional Landscapes”; the DFG through the Gottfried Wilhelm Leibniz Program; the European Research Council under the European Union’s Seventh Framework Programme ERC Synergy Grant 609920 “AXSIS” and Marie Curie FP7-PEOPLE-2011-ITN Grant 317079 “Nanomem”; the BMBF through Project 05E13GU1; the Graduate College “GRK 1355” at the University of Hamburg, the International Max Planck Research School UFAST, the BioXFEL Science Technology Center (award 1231306); and the US National Institutes of Health (NIH), National Institute of General Medical Sciences grants R01 GM095583, U54 GM094599, and R01 GM097463. Parts of the sample injector used at the Linac Coherent Light Source (LCLS) for this research was funded by the NIH, P41GM103393, formerly P41RR001209. Use of the LCLS, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract number DE-AC02-76SF00515.
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The Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography instrument of the European XFEL: initial installation
Journal of Synchrotron Radiation (2019)