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Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography


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.

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Figure 1: Structure comparison of the ligand-bound and apo conformers.
Figure 2: Structures of the three-way junction in the absence of ligand.
Figure 3: Setup of mix-and-inject SFX and conversion of the structure and crystal lattice.
Figure 4: Visualizing the ligand-bound intermediate state.

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  1. Mandal, M., Boese, B., Barrick, J. E., Winkler, W. C. & Breaker, R. R. Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria. Cell 113, 577–586 (2003)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Winkler, W. C. & Breaker, R. R. Genetic control by metabolite-binding riboswitches. ChemBioChem 4, 1024–1032 (2003)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Di Palma, F., Colizzi, F. & Bussi, G. Ligand-induced stabilization of the aptamer terminal helix in the add adenine riboswitch. RNA 19, 1517–1524 (2013)

    Article  CAS  Google Scholar 

  11. Lemay, J. F. & Lafontaine, D. A. [The adenine riboswitch: a new gene regulation mechanism]. Med. Sci. (Paris) 22, 1053–1059 (2006)

    Article  Google Scholar 

  12. Batey, R. T., Gilbert, S. D. & Montange, R. K. Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine. Nature 432, 411–415 (2004)

    Article  ADS  CAS  Google Scholar 

  13. Zhang, J. & Ferré-D’Amaré, A. R. Dramatic improvement of crystals of large RNAs by cation replacement and dehydration. Structure 22, 1363–1371 (2014)

    Article  CAS  Google Scholar 

  14. Rieder, R., Lang, K., Graber, D. & Micura, R. Ligand-induced folding of the adenosine deaminase A-riboswitch and implications on riboswitch translational control. ChemBioChem 8, 896–902 (2007)

    Article  CAS  Google Scholar 

  15. Gilbert, S. D., Stoddard, C. D., Wise, S. J. & Batey, R. T. Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain. J. Mol. Biol. 359, 754–768 (2006)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Jenkins, J. L., Krucinska, J., McCarty, R. M., Bandarian, V. & Wedekind, J. E. Comparison of a preQ1 riboswitch aptamer in metabolite-bound and free states with implications for gene regulation. J. Biol. Chem. 286, 24626–24637 (2011)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  22. Wang, D., Weierstall, U., Pollack, L. & Spence, J. Double-focusing mixing jet for XFEL study of chemical kinetics. J. Synchrotron Radiat. 21, 1364–1366 (2014)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Al-Hashimi, H. M. & Walter, N. G. RNA dynamics: it is about time. Curr. Opin. Struct. Biol. 18, 321–329 (2008)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Burnley, B. T., Afonine, P. V., Adams, P. D. & Gros, P. Modelling dynamics in protein crystal structures by ensemble refinement. eLife 1, e00311 (2012)

    Article  Google Scholar 

  28. Huang, L., Ishibe-Murakami, S., Patel, D. J. & Serganov, A. Long-range pseudoknot interactions dictate the regulatory response in the tetrahydrofolate riboswitch. Proc. Natl Acad. Sci. USA 108, 14801–14806 (2011)

    Article  ADS  CAS  Google Scholar 

  29. Haller, A., Altman, R. B., Soulière, M. F., Blanchard, S. C. & Micura, R. Folding and ligand recognition of the TPP riboswitch aptamer at single-molecule resolution. Proc. Natl Acad. Sci. USA 110, 4188–4193 (2013)

    Article  ADS  CAS  Google Scholar 

  30. Spence, J. & Lattman, E. Imaging enzyme kinetics at atomic resolution. IUCrJ 3, 228–229 (2016)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  33. Cheng, A., Hummel, B., Qiu, H. & Caffrey, M. A simple mechanical mixer for small viscous lipid-containing samples. Chem. Phys. Lipids 95, 11–21 (1998)

    Article  CAS  Google Scholar 

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

    Article  ADS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Karplus, P. A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012)

    Article  ADS  CAS  Google Scholar 

  40. French, G. S. & Wilson, K. S. On the treatment of negative intensity observations. Acta Crystallogr. A 34, 517–525 (1978)

    Article  ADS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  44. Chou, F. C., Sripakdeevong, P., Dibrov, S. M., Hermann, T. & Das, R. Correcting pervasive errors in RNA crystallography through enumerative structure prediction. Nat. Methods 10, 74–76 (2013)

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  46. The PyMOL Molecular Graphics System, Version Schrödinger, LLC

  47. Wang, J. 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)

    Article  CAS  Google Scholar 

  48. Jacques, D. A., Guss, J. M. & Trewhella, J. 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)

    Article  Google Scholar 

  49. Zuo, X. 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)

    Article  ADS  CAS  Google Scholar 

  50. Tiede, D. M., Mardis, K. L. & Zuo, X. X-ray scattering combined with coordinate-based analyses for applications in natural and artificial photosynthesis. Photosynth. Res. 102, 267–279 (2009)

    Article  CAS  Google Scholar 

  51. Zuo, X. 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)

    Article  CAS  Google Scholar 

  52. Nadassy, K., Tomás-Oliveira, I., Alberts, I., Janin, J. & Wodak, S. J. Standard atomic volumes in double-stranded DNA and packing in protein--DNA interfaces. Nucleic Acids Res. 29, 3362–3376 (2001)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  54. Schwieters, C. D., Kuszewski, J. J., Tjandra, N. & Clore, G. M. The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 65–73 (2003)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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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.

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Authors and Affiliations



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.

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Correspondence to Y.-X. Wang.

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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 figures and tables

Extended Data Figure 1 Characterization of RNA crystals.

Microcrystals of apo-rA71 were grown using batch crystallization as described in Methods. A SONICC Imager (Formulatrix) was used to image each sample (0.5–1.0 μl) of crystals using three different methods: visible light (a); UV-TPEF (b); and second-order nonlinear imaging of chiral crystals (SONICC) (c). d, Crystal samples were observed using a stereomicroscope (Zeiss) under cross-polarized light. e, Without cross-polarization, crystals were barely observable. f, The relative quality of the crystalline samples was measured by powder X-ray diffraction (APS beamline 19-ID), with a maximum observable resolution of approximately 6 Å. The resolution ring (red) corresponds to 6.8 Å.

Extended Data Figure 2 Characterization of rA71 in solution by small angle X-ray scattering.

a, Comparison of the Kratky plots of the solution X-ray scattering curves of rA71 in the apo (black) and bound (red) states. b, Plot of back-calculated small angle X-ray scattering (SAXS) profiles of apo1 and apo2 conformers along with solution X-ray scattering curves of rA71 in the apo (red) states. c, Experimental SAXS curve shown in red with red error bars and superimposed with the SAXS curves that were back-calculated from 128 structures using the two-member ensemble calculation. The ratio of the two conformers (apo2:apo1) is approximately 0.9:0.1 to give the best fit to the experiment data with χ2 values ranging approximately from 0.1 to 0.3.

Extended Data Figure 3 Three-way junction undergoes notable structural rearrangements to accommodate ligand, compressing the major groove.

a, The three-way junction, depicted in three orientations, as observed in the apo1 (blue), apo2 (cyan,), and ligand-bound (magenta, PDB code 4TZX) structures. Virtually all residues in the three-way junction undergo considerable conformational changes upon ligand binding. Most notable are the ‘swinging’ residues in the hinge (U22, A23) and latch (U48, U49, U51) regions, the atomic positions of which differ by as much as 17 Å in the apo conformers relative to the ligand-bound conformer. b, In the absence of ligand, concerted movement of the hinge (depicted as white surface and stick model) and latch regions results in considerable narrowing of the major groove formed between helices P1 and P3, which measures 9.4 Å, 10.0 Å and 16.6 Å for apo1, apo2 and ligand-bound conformers, respectively. Major groove distances were measured between the phosphorous atoms of U71 and A19 (or U20 in the case of apo1 owing to a difference in register).

Extended Data Figure 4 Multistage ligand binding kinetics to the rA71 riboswitch.

Ligand binding to the rA71 riboswitch was monitored by replacing U48 with the fluorescent base analogue 2-aminopurine (2AP; termed rA71-U482AP). The 2AP fluorescence emission intensity increases upon ligand-induced reorganization of the binding pocket. The 2AP substitution does not change the secondary structure of the apo rA71 riboswitch, as judged by partial RNase digestion. a, Equilibrium titration of rA71-U482AP with adenine yields Kd = 5 μM (black symbols). A similar Kd value was obtained from the endpoints of the kinetics progress curves (red symbols) and from in-line probing experiments. This value is about tenfold higher than adenine binding to the unmodified riboswitch, possibly because 2AP forms more stable base-stacking interactions in the apo structure. Error bars show the standard deviation from the average of two or more independent trials. b, The ligand-binding kinetics is consistent with a four-state mechanism. Binding of 0.5–1,600 μM adenine to 0.5 μM rA71-U482AP was measured by stopped-flow fluorescence (1.8 ms deadtime), as described in the Methods. The apparent rate constants for adenine association, λfast and λslow, were obtained from fits of individual trajectories to a biphasic rate equation, ∆F = Afast(1 − exp(−λfastt)) + Aslow(1 − exp(−λslowt)), where A denotes for adenine concentrations. Error bars as in a. The nonlinear increase in λfast (filled symbols) with adenine concentration over the full range of ligand concentrations indicates the presence of one or intermediates in the binding mechanism. The ligand-independent phase, λslow (open symbols), results in biphasic trajectories above 50 μM adenine and is explained by slow exchange between binding competent and binding incompetent forms of the riboswitch. c, The apparent bimolecular rate constant for adenine association is slower than diffusion and was obtained from λfast versus [adenine], under pseudo-first order conditions (0.5–25 μM adenine) in which ligand binding to the competent (open) riboswitch is rate-limiting. In 10 mM MgCl2 (filled symbols), kon = 1.9 × 105 M−1 s−1 and koff = 1.7 s−1. In 1.25 mM MgCl2 (open symbols), kon = 5.2 × 104 M−1 s−1 and koff = 2.1 s−1. The error bars are as in a with n = 3 over three independent trials. d, e, The same set of reduced experimental data in Fig. 2c was globally fit to simpler three-state kinetic mechanisms as described in the Supplementary Discussion. These models were not able to describe the solution binding kinetics over the full-range of ligand concentrations tested. Therefore, the four-state model in equation (1) is the simplest mechanism capable of describing the data. We do not exclude the possibility that the riboswitch samples additional apo states and intermediate complexes that may contribute to the robustness of the switch mechanism. d, Three-state mechanism with only one apo state. The parameters obtained from the fitting are: kon = 0.28 μM−1 s−1, koff = 37 s−1, kf = 103 s−1, kr = 5.1 s−1, sc = 2.03. Err(k, sc) = 0.053, where sc is scaling value and rate constants (k) by minimizing a Chi-squared error function Err(k, sc) that describes the discrepancy between calculated curves and experimental data sets (Supplementary Discussion). e, Three-state mechanism with two apo states and no binding intermediate. Parameters: kop = 2.5 s−1, kcl = 0.58 s−1, kon = 0.16 μM−1 s−1, koff = 0.79 s−1, sc = 2.44. Err(k,sc) = 0.056.

Extended Data Figure 5 Time-course simulation of the IB concentration in the crystal and comparison of the unit cell dimensions of the apo, IB and bound structures.

a, Simulated time courses of the IB buildup and changes in concentrations of ligand, apo2 and bound (B) states in the crystal. See also Methods. b, c, Space group and unit-cell dimensions of the crystals of apo, IB and bound states. The structure was converted in the crystal from the apo to the bound state after at least 10 min of mixing with adenine ligand. The crystal lattice remains unchanged after 10 s of mixing with ligand.

Extended Data Figure 6 Determining the structure of the IB conformer.

a, To first verify whether there were changes in the IB state relative to apo2, the apo2 structure was refined against the 10-s-mix data; 2 Fo − Fc (1σ, blue) and Fo − Fc (3σ, green) electron density maps are shown. Both maps indicated alternative positions for both U48 and A21. b, The occupancies of U48 and A21 of the apo2 state were set to 0.5 and refined in the same manner. The Fo − Fc map (3σ, green) clearly indicated the alternative (IB) conformation of U48 with a blob of density in the original U48 position corresponding to the adenine ligand. The alternative conformation of A21 was much less pronounced and is partially disordered along with the adjacent hinge residues (U22 and A23). c, Keeping the occupancy of A21 at 0.5, the structure was refined with U48 omitted. The Fo − Fc map (3σ, green) again supports the alternative configuration of U48, A21, and density for the adenine ligand. d, The final refined structure of the IB state with the adenine ligand (red), and alternative conformations for both U48 and A21 modelled at 0.5 occupancy, shown with 2 Fo − Fc electron density map (1σ, blue).

Extended Data Figure 7 Fo − Fc and 2 Fo − Fc electron density maps, and ensemble refinement model for the 10-s-mix data.

a, Weighted Fo − Fc difference electron density maps in green (3σ) and red (−3σ), computed using the apo structure (apo2, cyan; apo1, blue) and the structure factors from the 10-s-mix data. The isolated peaks, most likely corresponding to backbone phosphates, indicate a mixture of conformational states, and are predominantly in and around the three-way junction of the apo2 structure. b, 2 Fo − Fc electron density map (1σ, left) and time-averaged molecular dynamics ensemble refinement model (right) for the ‘apo1-like’ molecule of the 10-s-mix structure. c, Superimposition of apo1 (blue) and the apo1-like molecule of the 10-s-mix structure (orange), indicating no structural changes to apo1 after 10 s of mixing with ligand.

Extended Data Table 1 Crystal data and refinement statistics
Extended Data Table 2 R.m.s.d. values of all atoms among apo and ligand-bound conformers
Extended Data Table 3 Average B-factors (Å2)

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Stagno, J., Liu, Y., Bhandari, Y. et al. Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography. Nature 541, 242–246 (2017).

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