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Acknowledgements

We thank T. Nakane for assistance with real-time data analysis,

S. Boutet for advice on experimental design and P. Anfinrud for helpful

discussions about T-jump experiments. We acknowledge members

of the Engineering Team of RIKEN SPring-8 Center for technical

support. This work was supported by: grants to M.C.T. and J.S.F. from

the NSF BioXFEL Science and Technology Center (STC-1231306);

MEXT/JSPS KAKENHI Grants 19H05781 to E.N., 19H05784 to M.K., and

19H05776 to S.I.; the Platform Project for Supporting Drug Discovery

and Life Science Research (Basis for Supporting Innovative Drug

Discovery and Life Science Research) from Japan Agency for Medical

Research and Development under Grant JP21am0101070 to S.I.; and

the National Institutes of Health, grant GM117126 to N.K.S. The XFEL

experiments were performed at BL2 of SACLA with the approval of the

Japan Synchrotron Radiation Research Institute (JASRI) (proposal nos.

2017B8055 and 2018A8023).

Author contributions

M.C.T. and J.S.F. conceptualized the experiments. E.N., M.K., K.T., S.I.,

J.M.H. and N.K.S. contributed resources and methodology. A.M.W.,

E.N., I.D.Y., M.K., T.N., M.S., S.O., K.I., S.C., T.H., D.I., T.T., R.T., R.G.S., F.Y.

and M.C.T. conducted investigations. A.M.W., I.D.Y., A.S.B., B.A.B., A.B.,

L.J.O., N.K.S. and M.C.T. performed formal analysis of the data. A.M.W.

curated the data. J.S.F., M.C.T., N.K.S., E.N., M.K. and S.I. administered

the project, acquired funding, and supervised research. A.M.W., M.C.T.

and J.S.F. wrote the manuscript. M.C.T., A.M.W., J.S.F., E.N., I.D.Y., B.A.B.

and S.C. edited the manuscript.

Competing interests

The authors declare no competing interests.

Additional information

Extended data is available for this paper at

https://doi.org/10.1038/s41557-023-01329-4.

Supplementary information The online version

contains supplementary material available at

https://doi.org/10.1038/s41557-023-01329-4.

Correspondence and requests for materials should be addressed to

Eriko Nango or Michael C. Thompson.

Peer review information Nature Chemistry thanks the

anonymous reviewers for their contribution to the peer review

of this work.

Reprints and permissions information is available at

www.nature.com/reprints.

Article

Extended Data Fig. 1 | Qualitative and quantitative assessment of timeresolved difference electron density features. (a) Comparison of weighted

difference density maps for each pump-probe time delay (Flight – Fdark2) and

matched controls (Fdark1 – Fdark2) visualized at an absolute contour level of

± 0.04 e−/Å3 alongside initial refined models. Atoms with greater electron density,

such as the disulfide bridge between residues 76 and 94, display clear signals

Nature Chemistry

https://doi.org/10.1038/s41557-023-01329-4

across all experimental maps yet very little noise in matching controls.

(b) Pairwise correlation coefficients were calculated between all difference maps,

revealing varying levels of similarity between experimental maps and low noise

across controls. Labels correspond to time-delay (20ns, 20µs, 200µs) presence

of the inhibitor, chitobiose (CHI), whether a map was a matched control (CTRL),

or based on simulated (SIM) structure factors (see Methods for details).

Article

Extended Data Fig. 2 | Simulations of increased B-factors recapitulate signals

present at the 20 ns pump-probe time delay. The experimental 20ns difference

electron density map is visualized along with a simulated difference density map

Nature Chemistry

https://doi.org/10.1038/s41557-023-01329-4

created by linearly scaling the B-factors in the laser off structure by a factor of 1.2.

Negative peaks (yellow) are centered upon atoms in both maps, surrounded by

positive features (blue).

Article

Extended Data Fig. 3 | Normal mode analysis of the Apo laser off structure.

ProDy was used to generate an anisotropic network model based on the apo

ground state conformation. (a) The apo structure was then visualized as a ribbon

Nature Chemistry

https://doi.org/10.1038/s41557-023-01329-4

diagram (grey) along with the same model projected along the combined ANM

modes (green). (b) Per-residue RMSF values for the ANM model were plotted to

quantify local dynamics.

Article

Extended Data Fig. 4 | Effect of T-jump on average B-factor of refined apo

and chitobiose-bound structures. Models were refined against Laser Off,

Experiment (apo or chitobiose bound), or Control structure factors. Controls

exhibit similar B-factors across all time points, while B-factors for experimental

Nature Chemistry

https://doi.org/10.1038/s41557-023-01329-4

measurements increase following T-jump. Apo models reveal a decline in

B-factors at longer pump-probe time delays as complex motions develop,

while chitobiose-bound experimental models retain higher B-factors at 200 μs,

indicative of persistent, short-amplitude motions.

Article

https://doi.org/10.1038/s41557-023-01329-4

Extended Data Table 1 | Sample delivery and X-ray diffraction parameters for apo and chitobiose-bound data collection

Nature Chemistry

Article

Extended Data Table 2 | Crystallographic statistics for apo data collectiona

Statistics for the highest-resolution shell are shown in parentheses.

Nature Chemistry

https://doi.org/10.1038/s41557-023-01329-4

Article

Extended Data Table 3 | Refinement statistics for apo datasetsa

Statistics for the highest-resolution shell are shown in parentheses.

Nature Chemistry

https://doi.org/10.1038/s41557-023-01329-4

Article

https://doi.org/10.1038/s41557-023-01329-4

Extended Data Table 4 | Crystallographic statistics for chitobiose-bound data collectiona

Statistics for the highest-resolution shell are shown in parentheses.

Nature Chemistry

Article

Extended Data Table 5 | Refinement statistics for chitobiose-bound datasetsa

Statistics for the highest-resolution shell are shown in parentheses.

Nature Chemistry

https://doi.org/10.1038/s41557-023-01329-4

Last updated by author(s): Jul 21, 2023

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