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 platform1,2,3. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time4. Here we use femtosecond X-ray free electron laser (XFEL) pulses5,6 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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions


  1. 1.

    , , , & Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria. Cell 113, 577–586 (2003)

  2. 2.

    et al. Genetic control by a metabolite binding mRNA. Chem. Biol. 9, 1043 (2002)

  3. 3.

    Riboswitches and the RNA world. Cold Spring Harb. Perspect. Biol. 4, a003566 (2012)

  4. 4.

    et al. Analyzing protein functions in four dimensions. Nat. Struct. Biol. 7, 1006–1012 (2000)

  5. 5.

    et al. High-resolution protein structure determination by serial femtosecond crystallography. Science 337, 362–364 (2012)

  6. 6.

    et al. Femtosecond X-ray protein nanocrystallography. Nature 470, 73–77 (2011)

  7. 7.

    et al. Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. Chem. Biol. 11, 1729–1741 (2004)

  8. 8.

    & Genetic control by metabolite-binding riboswitches. ChemBioChem 4, 1024–1032 (2003)

  9. 9.

    Structure and mechanism of purine-binding riboswitches. Q. Rev. Biophys. 45, 345–381 (2012)

  10. 10.

    , & Ligand-induced stabilization of the aptamer terminal helix in the add adenine riboswitch. RNA 19, 1517–1524 (2013)

  11. 11.

    & [The adenine riboswitch: a new gene regulation mechanism]. Med. Sci. (Paris) 22, 1053–1059 (2006)

  12. 12.

    , & Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine. Nature 432, 411–415 (2004)

  13. 13.

    & Dramatic improvement of crystals of large RNAs by cation replacement and dehydration. Structure 22, 1363–1371 (2014)

  14. 14.

    , , & Ligand-induced folding of the adenosine deaminase A-riboswitch and implications on riboswitch translational control. ChemBioChem 8, 896–902 (2007)

  15. 15.

    , , & Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain. J. Mol. Biol. 359, 754–768 (2006)

  16. 16.

    et al. Riboswitch structure: an internal residue mimicking the purine ligand. Nucleic Acids Res. 38, 2057–2068 (2010)

  17. 17.

    , , , & Comparison of a preQ1 riboswitch aptamer in metabolite-bound and free states with implications for gene regulation. J. Biol. Chem. 286, 24626–24637 (2011)

  18. 18.

    et al. Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein. Science 352, 725–729 (2016)

  19. 19.

    et al. Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein. Science 346, 1242–1246 (2014)

  20. 20.

    et al. Direct observation of ultrafast collective motions in CO myoglobin upon ligand dissociation. Science 350, 445–450 (2015)

  21. 21.

    et al. Millisecond X-ray diffraction and the first electron density map from Laue photographs of a protein crystal. Nature 329, 178–181 (1987)

  22. 22.

    , , & Double-focusing mixing jet for XFEL study of chemical kinetics. J. Synchrotron Radiat. 21, 1364–1366 (2014)

  23. 23.

    Mix and inject: reaction initiation by diffusion for time-resolved acromolecular crystallography. Adv. Condens. Matter Phys. 2013, 167276 (2013)

  24. 24.

    et al. Linkage between dynamics and catalysis in a thermophilic-mesophilic enzyme pair. Nat. Struct. Mol. Biol. 11, 945–949 (2004)

  25. 25.

    & RNA dynamics: it is about time. Curr. Opin. Struct. Biol. 18, 321–329 (2008)

  26. 26.

    et al. Calcium binding sites in tomato bushy stunt virus visualized by Laue crystallography. J. Mol. Biol. 214, 627–632 (1990)

  27. 27.

    , , & Modelling dynamics in protein crystal structures by ensemble refinement. eLife 1, e00311 (2012)

  28. 28.

    , , & Long-range pseudoknot interactions dictate the regulatory response in the tetrahydrofolate riboswitch. Proc. Natl Acad. Sci. USA 108, 14801–14806 (2011)

  29. 29.

    , , , & Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution. Proc. Natl Acad. Sci. USA 110, 4188–4193 (2013)

  30. 30.

    & Imaging enzyme kinetics at atomic resolution. IUCrJ 3, 228–229 (2016)

  31. 31.

    et al. Synthesis and applications of RNAs with position-selective labelling and mosaic composition. Nature 522, 368–372 (2015)

  32. 32.

    et al. A novel inert crystal delivery medium for serial femtosecond crystallography. IUCrJ 2, 421–430 (2015)

  33. 33.

    , , & A simple mechanical mixer for small viscous lipid-containing samples. Chem. Phys. Lipids 95, 11–21 (1998)

  34. 34.

    et al. Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography. Nat. Commun. 5, 3309 (2014)

  35. 35.

    et al. Cheetah: software for high-throughput reduction and analysis of serial femtosecond X-ray diffraction data. J. Appl. Crystallogr. 47, 1118–1131 (2014)

  36. 36.

    et al. CrystFEL: a software suite for snapshot serial crystallography. J. Appl. Crystallogr. 45, 335–341 (2012)

  37. 37.

    et al. Structure-factor analysis of femtosecond microdiffraction patterns from protein nanocrystals. Acta Crystallogr. A 67, 131–140 (2011)

  38. 38.

    et al. Crystallographic data processing for free-electron laser sources. Acta Crystallogr. D 69, 1231–1240 (2013)

  39. 39.

    & Linking crystallographic model and data quality. Science 336, 1030–1033 (2012)

  40. 40.

    & On the treatment of negative intensity observations. Acta Crystallogr. A 34, 517–525 (1978)

  41. 41.

    et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

  42. 42.

    et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

  43. 43.

    et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 (2011)

  44. 44.

    , , , & Correcting pervasive errors in RNA crystallography through enumerative structure prediction. Nat. Methods 10, 74–76 (2013)

  45. 45.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

  46. 46.

    The PyMOL Molecular Graphics System, Version Schrödinger, LLC

  47. 47.

    et al. A method for helical RNA global structure determination in solution using small-angle x-ray scattering and NMR measurements. J. Mol. Biol. 393, 717–734 (2009)

  48. 48.

    , & Reliable structural interpretation of small-angle scattering data from bio-molecules in solution—the importance of quality control and a standard reporting framework. BMC Struct. Biol. 12, 9 (2012)

  49. 49.

    et al. X-ray diffraction “fingerprinting” of DNA structure in solution for quantitative evaluation of molecular dynamics simulation. Proc. Natl Acad. Sci. USA 103, 3534–3539 (2006)

  50. 50.

    , & X-ray scattering combined with coordinate-based analyses for applications in natural and artificial photosynthesis. Photosynth. Res. 102, 267–279 (2009)

  51. 51.

    et al. Global molecular structure and interfaces: refining an RNA:RNA complex structure using solution X-ray scattering data. J. Am. Chem. Soc. 130, 3292–3293 (2008)

  52. 52.

    , , , & Standard atomic volumes in double-stranded DNA and packing in protein--DNA interfaces. Nucleic Acids Res. 29, 3362–3376 (2001)

  53. 53.

    & Calculation of standard atomic volumes for RNA and comparison with proteins: RNA is packed more tightly. J. Mol. Biol. 346, 477–492 (2005)

  54. 54.

    , , & The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 65–73 (2003)

  55. 55.

    et al. An unusual topological structure of the HIV-1 Rev response element. Cell 155, 594–605 (2013)

  56. 56.

    et al. Structure and dynamics of full-length HIV-1 capsid protein in solution. J. Am. Chem. Soc. 135, 16133–16147 (2013)

Download references


Portions of this research were carried out at the Linac Coherent Light Source, a National User Facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The CXI instrument was funded by the LCLS Ultrafast Science Instruments (LUSI) project funded by the US Department of Energy, Office of Basic Energy Sciences. Use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. We thank J. Strathern and M. Dunne for their support and S. Wakatsuki for discussions. This work is supported in part by the NSF-STC “BioXFEL” (NSF-1231306), the NIH Intramural Research Programs of NCI, CIT, NHLBI, and the US Department of Energy, Office of Biological and Environmental Research under Contract DE-AC02-06CH11357, the European Research Council, “Frontiers in Attosecond X-ray Science: Imaging and Spectroscopy (AXSIS)”, ERC-2013-SyG 609920, and the BMBF through project 05K16GU1.

Author information


  1. Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA

    • J. R. Stagno
    • , Y. Liu
    • , Y. R. Bhandari
    • , M. Swain
    • , D. R. Wendel
    • , R. A. Tuckey
    • , P. Yu
    • , M. Dyba
    • , S. G. Tarasov
    •  & Y.-X. Wang
  2. Department of Biochemistry, Arizona State University, Tempe, Arizona 85287, USA

    • C. E. Conrad
    • , J. D. Coe
    •  & P. Fromme
  3. Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, Arizona 85287, USA

    • C. E. Conrad
    • , J. D. Coe
    • , U. Weierstall
    • , P. Fromme
    • , N. A. Zatsepin
    •  & J. C. H. Spence
  4. Department of Biophysics, Johns Hopkins University, Baltimore, Maryland 21218, USA

    • S. Panja
    •  & S. A. Woodson
  5. Small Angle X-ray Scattering Core Facility, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA

    • L. Fan
  6. Department of Physics, Arizona State University, Tempe, Arizona 85287, USA

    • G. Nelson
    • , C. Li
    • , U. Weierstall
    • , N. A. Zatsepin
    •  & J. C. H. Spence
  7. Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, 22607 Hamburg, Germany

    • T. A. White
    • , M. O. Wiedorn
    • , J. Knoska
    • , D. Oberthuer
    • , A. Barty
    •  & H. N. Chapman
  8. Department of Physics, University of Hamburg, Luruper Chaussee 149, 22607 Hamburg, Germany

    • M. O. Wiedorn
    • , J. Knoska
    •  & H. N. Chapman
  9. Hauptmann-Woodward Medical Research Institute, Buffalo, New York 14203, USA

    • T. D. Grant
  10. Center for Information Technology, National Institutes of Health, Bethesda, Maryland 20892-5624, USA

    • C. D. Schwieters
  11. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

    • J. Zhang
  12. Laboratory of RNA Biophysics and Cellular Physiology, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

    • A. R. Ferré-D’Amaré
  13. Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, USA

    • D. E. Draper
  14. Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California 94025, USA

    • M. Liang
    • , M. S. Hunter
    •  & S. Boutet
  15. Structural Biology Center, Biosciences Division, Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, USA

    • K. Tan
  16. X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, USA

    • X. Zuo
  17. Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA

    • X. Ji


  1. Search for J. R. Stagno in:

  2. Search for Y. Liu in:

  3. Search for Y. R. Bhandari in:

  4. Search for C. E. Conrad in:

  5. Search for S. Panja in:

  6. Search for M. Swain in:

  7. Search for L. Fan in:

  8. Search for G. Nelson in:

  9. Search for C. Li in:

  10. Search for D. R. Wendel in:

  11. Search for T. A. White in:

  12. Search for J. D. Coe in:

  13. Search for M. O. Wiedorn in:

  14. Search for J. Knoska in:

  15. Search for D. Oberthuer in:

  16. Search for R. A. Tuckey in:

  17. Search for P. Yu in:

  18. Search for M. Dyba in:

  19. Search for S. G. Tarasov in:

  20. Search for U. Weierstall in:

  21. Search for T. D. Grant in:

  22. Search for C. D. Schwieters in:

  23. Search for J. Zhang in:

  24. Search for A. R. Ferré-D’Amaré in:

  25. Search for P. Fromme in:

  26. Search for D. E. Draper in:

  27. Search for M. Liang in:

  28. Search for M. S. Hunter in:

  29. Search for S. Boutet in:

  30. Search for K. Tan in:

  31. Search for X. Zuo in:

  32. Search for X. Ji in:

  33. Search for A. Barty in:

  34. Search for N. A. Zatsepin in:

  35. Search for H. N. Chapman in:

  36. Search for J. C. H. Spence in:

  37. Search for S. A. Woodson in:

  38. Search for Y.-X. Wang in:


J.R.S. and Y.-X.W. designed experiments; Y.L. and P.Y. provided the RNA samples; J.R.S. and Y.L. crystallized the RNA; J.R.S., Y.L., Y.R.B., D.R.W., C.E.C., J.D.C., G.N., C.L., N.A.Z., M.O.W., D.O., J.K., T.D.G., M.S.H., S.B., M.L. and Y.-X.W. collected the SFX data; M.O.W., D.O., J.K. and H.N.C. designed the mixing setup; J.R.S., Y.R.B., D.R.W., T.A.W., A.B., R.A.T., N.A.Z., X.J. and T.D.G. processed and analysed the SFX data; J.R.S., Y.R.B., M.S., X.J. and Y.-X.W. interpreted the SFX data; N.A.Z., A.B., M.S.H., S.B., M.L., U.W., P.F., H.N.C. and J.C.H.S. contributed the XFEL expertise and support; Y.R.B., L.F., X.Z., C.D.S. and Y.-X.W. collected, analysed and interpreted SAXS data; J.R.S., C.E.C., K.T. and Y.-X.W. characterized samples; M.D. and Y.L. performed mass spectroscopy; S.G.T. and Y.L. performed ITC and fluorescence titration; S.P. and Y.L. collected and analysed binding data; X.Z. and S.A.W. modelled ligand binding kinetics; X.Z., S.P., S.A.W. and Y.-X.W. interpreted kinetic data; D.E.D., A.F.D. and J.Z. contributed discussions; J.R.S. and Y.-X.W. drafted the manuscript and all authors contributed to the revision.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Y.-X. Wang.

Reviewer Information Nature thanks J. Hajdu, R. Micura and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Discussion

    This file contains a Supplementary Discussion and Supplementary References.

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.