We introduce a liquid application method for time-resolved analyses (LAMA), an in situ mixing approach for serial crystallography. Picoliter-sized droplets are shot onto chip-mounted protein crystals, achieving near-full ligand occupancy within theoretical diffusion times. We demonstrate proof-of-principle binding of GlcNac to lysozyme, and resolve glucose binding and subsequent ring opening in a time-resolved study of xylose isomerase.
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The authors thank D. Oberthuer (CFEL, DESY Hamburg) for the provision of the GlcNAc3 substrate immediately before data collection. The LAMA TR-SSX data were collected at beamline P14 and P14-2 (T-REXX) operated by EMBL Hamburg at the PETRA III storage ring (DESY, Hamburg, Germany). Construction of T-REXX was funded by the BMBF (Verbund Forschungsprojekt 05K16GU1). The authors are grateful to R. Dousza, L. Bultema and J. Besaw for their technical assistance during data collection. T-REXX beamtime was awarded as part of the EMBL BAG MX-660. The authors gratefully acknowledge the support provided by the Max Planck Society and the excellence cluster ‘The Hamburg Centre for Ultrafast Imaging—Structure, Dynamics and Control of Matter at the Atomic Scale’ of the Deutsche Forschungsgemeinschaft EXC 1074 project ID 194651731 (R.J.D.M.). P.M. was supported by the Alexander von Humboldt-Stiftung for postdoctoral researchers.
The authors declare no competing interests.
Peer review information Allison Doerr was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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Integrated supplementary information
a) Overview CAD render of the LAMA setup. The humidifier is connected to the translation stage with a hose represented in orange. The diffractometer, chip holder and translation stages were previously described in detail1. b) Closeup view of a) displays the injector nozzle (beige) mounted at a fixed point above the chip (black). The landing point of the droplets intersects with the X-ray beam at the position of each individual protein microcrystal inside the chip features as the chip is rastered across the X-ray beam. The humidified air nozzle is 3D-printed to provide a homogenous humidity stream over chip, preventing crystal dehydration. c) A ~150 pl droplet generated by a single pulse waveform of the piezo transducer (microdrop Technologies). d) A ~75 pl droplet generated by a triple pulse waveform of the piezo transducer (microdrop Technologies).
Supplementary Figure 2 Absolute value electron density maps for the different Lysozyme and Xylose isomerase time-points.
(a–c) GlcNac3 is shown in a ball-and-stick representation. Absolute-value maps contoured at 0.5 e− Å−3 from 0.05 s—1 s display a gradual increase of the electron density as a function of time. (d–i) Coloring and representation as in Fig. 2. All absolute value electron density maps are shown with the density contoured at 0.7 e− Å−3.
The plot shows the on/off times of the various components of the LAMA setup. Initially the stage moves into position and there is a pre-exposure time of 1 ms during which the detector is triggered, and a t0 image is taken. Subsequently a TTL pulse is sent to the droplet generator; the droplet has a time-of-flight of ~0.5 ms which is encompassed in the post-exposure time (4 ms). After droplet injection additional images are recorded at different feature positions. Finally, the sequence is repeated without any further droplet injection. The total time delay corresponds to a HARE number1.
a) The XI active site of the 0 s structure is clearly empty and shows only strong density for the metal binding sites. Metals were modelled as cobalt (green sphere) and magnesium (pink sphere) since these matched the 2Fo-Fc electron density best. The POLDER-OMIT map for the metals is shown at 3σ. b) Superimposition of XI with a previously determined neutron structure of XI in complex with glucose in closed ring conformation (cyan, 3KCL). c) Superimposition of XI with a previously determined neutron structure of XI in complex with glucose in open ring conformation (cyan, 3KBN).
a) Cartoon representation of the 7 XI-monomer structures superimposed with an overall r.m.s.d of 0.09 Å2. indicating that differences between all the XI structures are within error. b) The active site of all the superimposed XI structures showing that the side chains as well as the structural waters within a 7 Å radius around the ligand also show near identical conformations from one time point to the next.
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Mehrabi, P., Schulz, E.C., Agthe, M. et al. Liquid application method for time-resolved analyses by serial synchrotron crystallography. Nat Methods 16, 979–982 (2019). https://doi.org/10.1038/s41592-019-0553-1
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