Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons

Abstract

Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000–10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.

Proton uptake mechanism in bacteriorhodopsin captured by serial synchrotron crystallography

“…Results are of comparable quality to our previous time-resolved studies at XFELs on the same crystals ( Figure S4), even though the resolution was limited by the available photon flux. However, the resolution is compensated by high levels of activation from the millisecond long light exposure, more accurate data due to the use of a photon counting detector and a more stable monochromatic beam as previously observed during de novo phasing (Weinert et al 2017). Due to the slow extrusion rate, less than 20 µl of prepared microcrystals were necessary to obtain these data instead of milliliters needed for the XFEL experiments.…”

“…Serial millisecond crystallography (SMX) approaches work well with high viscosity injectors (Weierstall et al 2014), as the injectors extrude crystals slowly and allow sufficient X-ray exposure to collect highresolution data with the photon flux available at synchrotrons (Grünbein & Nass Kovacs 2019). Our recent work has shown that modern high frame-rate and low-noise detectors make SMX a viable method for routine room-temperature structure determination (Weinert et al 2017). By extending the setup with a simple class 3R laser diode we enable time-resolved studies with millisecond time resolution (TR-SMX) and bring dynamic serial crystallography from the XFEL niche to the large community of synchrotron users worldwide.…”

“…The simple Class 3R diode laser we integrated into our SMX setup (Weinert et al 2017) is inexpensive, available at a range of wavelengths and requires significantly less strict safety protocols than the Class 3B and 4 lasers typically used for time-resolved experiments. The laser diode can be triggered at up to 10 kHz and provides sufficient light to "pump" each chromophore many times within a millisecond exposure to allow high excitation levels.…”

Poisson poisoning as the mechanism of action of the microtubule-targeting agent colchicine

“…The protein complex along with the nucleotides were all parametrized using the CHARMM-GUI interface (62). For our study, the tubulin structures were extracted from published tubulin-DARPin room-temperature serial millisecond crystallography (SMC) structures with a high resolution of 2.05 Å (PDB IDs: 5NM5, 5NQT) (63). These structures were chosen over the available tubulin-colchicine-Stathmin-like-domain (SLD) structures (55, 64) because we suspect the existence of the long chain of SLD might cause conformational changes that overshadows colchicine conformations, compared to DARPin-tubulin structure that has lower deviation from an equilibrated tubulin dimer in solution.…”

In situ crystallography as an emerging method for structure solution of membrane proteins: the case of CCR2A

“…The LCP jet provides a significant improvement in reducing sample consumption and extends the use to crystals grown in LCP [10]. The LCP jet today is predominately used for time-resolved femtosecond X-ray structure research [5,[11][12][13][14]. More recently, SX methods have been extended to using acoustic waves for crystal delivery.…”