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

Tobias Weinert ,Florian Dworkowski,Dardan Gashi,Robert M Cooke,Steffen Brünle,Daniel James,Jörg Standfuss,Michael Hennig,Ezequiel Panepucci,D Ozerov ,Kathrin Jaeger,Petr Skopintsev,Mao Liang ,Gebhard F X Schertler ,Natacha Olieric,R.k Cheng ,Ulrich Ermler,Shibom Basu,Meitian Wang,A.e Prota ,Valérie Panneels ,A.s Dore ,Laura Vera,M Marsh ,Przemyslaw Nogly,Tao Geng ,Michel O Steinmetz

doi:10.1038/s41467-017-00630-4

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.

Highlights

Room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage
A promising solution is to adapt the methodology developed for macromolecular crystallography at X-ray free-electron lasers (XFELs) to synchrotron sources, where radiation damage cannot be outrun but where the radiation dose per crystal can be reduced by using many crystals
In our SMX setup (Fig. 1) we use nozzles between 50 and 75 μm, which is sufficient to avoid clogging by crystals, as larger crystals were usually crushed during mixing with a standard syringe coupler[23], without indications for loss of diffraction quality in our cases

Summary

Introduction

Room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. 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. The most direct approach is serial millisecond crystallography (SMX), which utilizes the same high-viscosity injectors successful at XFELs to distribute the radiation dose over thousands of crystals to determine room-temperature structures with minimum radiation-damage. We demonstrate how the combination of a high-viscosity injector[3] with the high frame rates of an EIGER 16 M detector[19] in a ‘crystal scanning’ approach allows SMX to be routinely applied to the three most common crystallographic techniques: high-resolution structure determination, ligand soaking and de novo phasing (Fig. 1).

Methods

Results

Conclusion