title: Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser.
abstract: To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense femtosecond-duration pulses from X-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm3in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm3in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.
authors.forename: Cornelius
authors.initials: C
authors.lastname: Gati
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Oberthuer
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.
authors.forename: Oleksandr
authors.initials: O
authors.lastname: Yefanov
authors.affiliation: School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand.
authors.forename: Richard D
authors.identifier: 0000-0001-8866-5290
authors.initials: RD
authors.lastname: Bunker
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.
authors.forename: Francesco
authors.initials: F
authors.lastname: Stellato
authors.affiliation: School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand.
authors.forename: Elaine
authors.initials: E
authors.lastname: Chiu
authors.affiliation: School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand.
authors.forename: Shin-Mei
authors.initials: SM
authors.lastname: Yeh
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.|European XFEL GmbH, Hamburg 22761, Germany.
authors.forename: Andrew
authors.initials: A
authors.lastname: Aquila
authors.affiliation: School of Molecular Sciences, Arizona State University, Tempe, AZ 85287-1604.|Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001.
authors.forename: Shibom
authors.initials: S
authors.lastname: Basu
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.|European XFEL GmbH, Hamburg 22761, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Bean
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Beyerlein
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Heidelberg 69120, Germany.
authors.forename: Sabine
authors.initials: S
authors.lastname: Botha
authors.affiliation: Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, CA 94025.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Boutet
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.|SLAC National Accelerator Laboratory, Menlo Park, CA 94025.
authors.forename: Daniel P
authors.initials: DP
authors.lastname: DePonte
authors.affiliation: Department of Physics, Arizona State University, Tempe, AZ 85287.
authors.forename: R Bruce
authors.initials: RB
authors.lastname: Doak
authors.affiliation: School of Molecular Sciences, Arizona State University, Tempe, AZ 85287-1604.|Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001.
authors.forename: Raimund
authors.initials: R
authors.lastname: Fromme
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Galli
authors.affiliation: School of Molecular Sciences, Arizona State University, Tempe, AZ 85287-1604.
authors.forename: Ingo
authors.initials: I
authors.lastname: Grotjohann
authors.affiliation: Department of Physics, Arizona State University, Tempe, AZ 85287.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: James
authors.affiliation: School of Molecular Sciences, Arizona State University, Tempe, AZ 85287-1604.|Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001.
authors.forename: Christopher
authors.initials: C
authors.lastname: Kupitz
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Heidelberg 69120, Germany.
authors.forename: Lukas
authors.initials: L
authors.lastname: Lomb
authors.affiliation: Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, CA 94025.
authors.forename: Marc
authors.initials: M
authors.lastname: Messerschmidt
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.
authors.forename: Karol
authors.initials: K
authors.lastname: Nass
authors.affiliation: School of Molecular Sciences, Arizona State University, Tempe, AZ 85287-1604.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Rendek
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Heidelberg 69120, Germany.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Shoeman
authors.affiliation: Department of Physics, Arizona State University, Tempe, AZ 85287.
authors.forename: Dingjie
authors.initials: D
authors.lastname: Wang
authors.affiliation: Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001.|Department of Physics, Arizona State University, Tempe, AZ 85287.
authors.forename: Uwe
authors.initials: U
authors.lastname: Weierstall
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: White
authors.affiliation: Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, CA 94025.
authors.forename: Garth J
authors.initials: GJ
authors.lastname: Williams
authors.affiliation: Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001.|Department of Physics, Arizona State University, Tempe, AZ 85287.
authors.forename: Nadia A
authors.initials: NA
authors.lastname: Zatsepin
authors.affiliation: School of Molecular Sciences, Arizona State University, Tempe, AZ 85287-1604.|Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001.
authors.forename: Petra
authors.initials: P
authors.lastname: Fromme
authors.affiliation: Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ 85287-5001.|Department of Physics, Arizona State University, Tempe, AZ 85287.
authors.forename: John C H
authors.initials: JC
authors.lastname: Spence
authors.affiliation: Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University Basel, Basel CH-4058, Switzerland.
authors.forename: Kenneth N
authors.initials: KN
authors.lastname: Goldie
authors.affiliation: Institute for Biological Control, Julius Kuehn Institute (JKI), 64287 Darmstadt, Germany.
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Jehle
authors.affiliation: School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand; henry.chapman@desy.de peter.metcalf@auckland.ac.nz.
authors.forename: Peter
authors.initials: P
authors.lastname: Metcalf
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.
authors.forename: Anton
authors.initials: A
authors.lastname: Barty
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany; henry.chapman@desy.de peter.metcalf@auckland.ac.nz.|Department of Physics, University of Hamburg, Hamburg 20355, Germany.|Center for Ultrafast Imaging, University of Hamburg, Hamburg 20355, Germany.
authors.forename: Henry N
authors.identifier: 0000-0002-4655-1743
authors.initials: HN
authors.lastname: Chapman
mesh_terms: D003461:Crystallography / Q000295:instrumentation / Q000379:methods*; D004583:Electrons; D025722:Granulovirus / Q000737:chemistry / Q000648:ultrastructure*; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry*; D007834:Lasers; D008958:Models, Molecular; D000077153:Progranulins; D017433:Protein Structure, Secondary; D017356:Synchrotrons
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D000077153:Progranulins
keywords: SFX; XFEL; bioimaging; nanocrystals; structural biology