title: Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography.
abstract: Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of 'mix-and-inject' time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.
authors.affiliation: Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Stagno
authors.affiliation: Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: Y R
authors.initials: YR
authors.lastname: Bhandari
authors.affiliation: Department of Biochemistry, Arizona State University, Tempe, Arizona 85287, USA.|Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA.
authors.forename: C E
authors.initials: CE
authors.lastname: Conrad
authors.affiliation: Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 21218, USA.
authors.forename: S
authors.initials: S
authors.lastname: Panja
authors.affiliation: Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: M
authors.initials: M
authors.lastname: Swain
authors.affiliation: Small Angle X-ray Scattering Core Facility, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: L
authors.initials: L
authors.lastname: Fan
authors.affiliation: Department of Physics, Arizona State University, Tempe, Arizona 85287, USA.
authors.forename: G
authors.initials: G
authors.lastname: Nelson
authors.affiliation: Department of Physics, Arizona State University, Tempe, Arizona 85287, USA.
authors.forename: C
authors.initials: C
authors.lastname: Li
authors.affiliation: Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Wendel
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, 22607 Hamburg, Germany.
authors.forename: T A
authors.initials: TA
authors.lastname: White
authors.affiliation: Department of Biochemistry, Arizona State University, Tempe, Arizona 85287, USA.|Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Coe
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, 22607 Hamburg, Germany.|Department of Physics, University of Hamburg, Luruper Chaussee 149, 22607 Hamburg, Germany.
authors.forename: M O
authors.initials: MO
authors.lastname: Wiedorn
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, 22607 Hamburg, Germany.|Department of Physics, University of Hamburg, Luruper Chaussee 149, 22607 Hamburg, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Knoska
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, 22607 Hamburg, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Oberthuer
authors.affiliation: Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Tuckey
authors.affiliation: Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: P
authors.initials: P
authors.lastname: Yu
authors.affiliation: Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: M
authors.initials: M
authors.lastname: Dyba
authors.affiliation: Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: S G
authors.initials: SG
authors.lastname: Tarasov
authors.affiliation: Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA.|Department of Physics, Arizona State University, Tempe, Arizona 85287, USA.
authors.forename: U
authors.initials: U
authors.lastname: Weierstall
authors.affiliation: Hauptmann-Woodward Medical Research Institute, Buffalo, New York 14203, USA.
authors.forename: T D
authors.initials: TD
authors.lastname: Grant
authors.affiliation: Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892-5624, USA.
authors.forename: C D
authors.initials: CD
authors.lastname: Schwieters
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Laboratory of RNA Biophysics and Cellular Physiology, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: A R
authors.initials: AR
authors.lastname: Ferré-D'Amaré
authors.affiliation: Department of Biochemistry, Arizona State University, Tempe, Arizona 85287, USA.|Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA.
authors.forename: P
authors.initials: P
authors.lastname: Fromme
authors.affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Draper
authors.affiliation: Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA.
authors.forename: M
authors.initials: M
authors.lastname: Liang
authors.affiliation: Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA.
authors.forename: M S
authors.initials: MS
authors.lastname: Hunter
authors.affiliation: Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA.
authors.forename: S
authors.initials: S
authors.lastname: Boutet
authors.affiliation: Structural Biology Center, Biosciences Division, Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, USA.
authors.forename: K
authors.initials: K
authors.lastname: Tan
authors.affiliation: X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, USA.
authors.forename: X
authors.initials: X
authors.lastname: Zuo
authors.affiliation: Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: X
authors.initials: X
authors.lastname: Ji
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, 22607 Hamburg, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Barty
authors.affiliation: Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA.|Department of Physics, Arizona State University, Tempe, Arizona 85287, USA.
authors.forename: N A
authors.initials: NA
authors.lastname: Zatsepin
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, 22607 Hamburg, Germany.|Department of Physics, University of Hamburg, Luruper Chaussee 149, 22607 Hamburg, Germany.
authors.forename: H N
authors.initials: HN
authors.lastname: Chapman
authors.affiliation: Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA.|Department of Physics, Arizona State University, Tempe, Arizona 85287, USA.
authors.forename: J C H
authors.initials: JC
authors.lastname: Spence
authors.affiliation: Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 21218, USA.
authors.forename: S A
authors.initials: SA
authors.lastname: Woodson
authors.affiliation: Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA.
authors.forename: Y-X
authors.initials: YX
authors.lastname: Wang
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D052157:Aptamers, Nucleotide / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods*; D004058:Diffusion; D004583:Electrons; D007700:Kinetics; D007834:Lasers; D008024:Ligands; D008958:Models, Molecular; D036103:Nanotechnology / Q000379:methods*; D009690:Nucleic Acid Conformation; D059370:RNA Folding; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D058928:Riboswitch; D013997:Time Factors; D041261:Vibrio vulnificus / Q000235:genetics
chemical_list: D020121:5' Untranslated Regions; D052157:Aptamers, Nucleotide; D008024:Ligands; D012329:RNA, Bacterial; D058928:Riboswitch