Advantages of Serial Femtosecond Crystallography for RNA
Structure Determination
J.R. Stagno1, Y. R. Bhandari1, C.E.
Conrad2,3, Y. Liu1, M. Swain1, Lixin Fan4,
G. Nelson5, C. Li5, D.R. Wendel1, T.A. White6,
A. Barty6, R.A. Tuckey1, P. Yu1, U.
Weierstall5, N.A. Zatsepin5, T.D. Grant7, C.D.
Schwieters8, J. Zhang9, A. Ferré-D’Amaré10, P.
Fromme2, D.E. Draper11, K. Tan12, X. Zuo13,
X. Ji14, J.C.H. Spence5 & Y.-X. Wang1
1Structural Biophysics
Laboratory, National Cancer Institute, Frederick, MD 21702; 2Department
of Biochemistry, Arizona State University, Tempe, AZ 85287, USA; 3Center
for Applied Structural Discovery, The Biodesign Institute, Arizona State
University, Tempe, AZ, 85287, USA; 4The Small Angle X-ray
Scattering Core Facility, Center for Cancer Research, National Cancer
Institute, Frederick, MD 21702, USA; 5Department of Physics, Arizona
State University, Tempe, AZ 85287, USA; 6Center for Free-Electron
Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, 22607
Hamburg, Germany; 7Hauptmann-Woodward Medical Research Institute,
Buffalo, NY 14203, USA; 8Center for Information Technology, National
Institutes of Health, Bethesda, MD 20892-5624, USA. 9Laboratory of
Molecular Biology, National Institute of Diabetes and Digestive and Kidney
Diseases, 10Laboratory of RNA Biophysics and Cellular Physiology,
National Heart Lung and Blood Institute, National Institutes of Health,
Bethesda, MD 20892, USA. 11Department of Chemistry, Johns Hopkins
University, Baltimore, Maryland 21218, USA. 12Structural Biology
Center, Biosciences, 13X-ray Science Division, Advanced Photon
Source, Argonne National Laboratory, Argonne, IL 60439, USA. 14Macromolecular
Crystallography Laboratory, National Cancer Institute, Frederick, MD 21702,
USA.
Discovery of
the important and diverse biological roles of RNA molecules is ever increasing.
Similar to proteins, knowledge of the three-dimensional structures of RNAs is
critical to understanding their functions. However, structure elucidation of
RNAs using conventional methods has been extremely
hampered by technical challenges, and is reflected in the overwhelmingly few
RNA structures in the protein data bank. In addition to molecular size
limitations faced by nuclear magnetic resonance (NMR), the intrinsically similar
chemical signatures of nucleotides result in severe peak overlap in NMR
spectra. RNAs are often difficult to crystallize, and when available, RNA
crystals often exhibit high mosaicity, high solvent content, and high
susceptibility to radiation damage. In addition, RNAs tend to be very dynamic,
and low-temperature data collection on a single crystal may not provide the
most accurate depiction of its structure. Clearly, there is an acute need for
advanced methods for RNA structure determination. Serial femtosecond
crystallography (SFX) using an X-ray free electron laser (XFEL) has the
potential to revolutionize RNA crystallography by overcoming many of these
technical challenges. Its advantages include the use of nano/micro-sized
crystals, room-temperature data collection, the ability to outrun radiation
damage, and a high-throughput oversampling of crystal data. We have used SFX to
determine the structure of the adenine riboswitch RNA apatamer domain in the
ligand-free state. For the first time, these results provide a structural basis
for the ligand-induced conformational switch required for the regulation of
gene expression.
This material is based upon work
supported by funds from the NIH Intramural Research Program.