Comparison of DLS measurements in thin capillaries and a quartz cuvette.

<p>A) It shows an overlay plot of the radius distribution of glucose isomerase in capillaries and a quartz cuvette; B) It shows an overlay of the auto correlation function from which the radius distribution were derived; C) Picture showing the focus of the laser inside a 0.1 mm capillary and the scattering associated to the capillary-walls, which does not affect the measurements.</p

Pictures of the size distribution (X-axis) as a function of time (Y-axis) obtained from DLS measurements in a single capillary.

<p>The number on the bottom right corner of each picture denotes the distance of the measurement in millimetres from the open end of the capillary. Time is displayed in arbitrary units (a.u.), where each unit corresponds to 1/10 of a day. The picture at the bottom shows an overview of the capillary and the position of the measurements (*) in the GCB-D.</p

Graphical representation of a typical counter-diffusion experiment set-up in a GCB-Domino (Triana S&T).

<p>The dimension of the GCB-Domino is approximately 70 mm high × 17.2 mm wide × 7.0 mm thick. The open end of the capillary allows the precipitant (in blue) to diffuse against the much slower diffusive protein solution, thereby inducing the precipitation of the latter. The capillaries are kept in place by an agarose plug sitting at the top of the precipitant solution. Each mark (*) shows a DLS measurement position, at 0.7, 2.2, 5.1, 9.9, 11.8, 18.7 and 24.5 mm from the open end.</p

Additional file 1: of Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography

Figure S1. Schematics of the short-time-point mixing injector. Figure S2. Selected views of the CEF binding site in the BlaC shard crystals including simulated annealing omit maps. Figure S3. Structural details, and simulated annealing omit maps, shard crystal form, subunit B (stereo representation, from 30 ms to 2 s). Figure S4. Structural details and simulated annealing omit maps, shard crystal form, subunit D (stereo representation, from 30 ms to 2 s). Figure S5. Structural details, and simulated annealing omit maps, needle crystal form (stereo representation, from 30 ms to 2 s). Figure S6. Backside view of the catalytic cleft of BlaC in the shard crystal form, structural details and simulated annealing omit maps (stereo representation, selected time points). Figure S7. 2mFo-DFc electron density in the catalytic clefts of BlaC in the shard crystal form (stereo representation, from 30 ms to 2 s). Figure S8. 2mFo-DFc electron density and structural details in the catalytic clefts of BlaC in the needle crystal form (stereo representation from 30 ms to 2 s). Figure S9. Details in the catalytic cleft of subunit B in the shard crystal form at 500 ms including the stacked CEF, 2FoFc maps, and simulated annealing omit maps (stereo representation). Figure S10. The catalytic cleft of BlaC, further details, including a difference map between the 500 ms and 100 ms time points. Figure S11. Crystal packing in shards and needles. Figure S12. Dynamic light scattering results. Table S1. B-factors for CEF species observed in the shard crystals at different time delays. (PDF 1646 kb

Megahertz pulse trains enable multi-hit serial femtosecond crystallography experiments at X-ray free electron lasers

The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX