Unique beamline achieves room-temperature serial crystallography experiments with microsecond pulsed beams

Studying macromolecular complexes at room temperature has always been challenging because of X-ray damage to the biological samples. Usually this is mitigated by collecting diffraction data at cryogenic conditions, but under these conditions functional dynamics are hindered.

Serial crystallography can provide an alternative way to collect data at physiological conditions with limited X-ray damage andto visualise functional dynamics that become untrapped. Serial femtosecond crystallography at X-ray free electron lasers (XFELs) allow scientists to decode macromolecular structures by acquiring data of tiny protein crystals at room temperature, outrunning the damage thanks to the extremely short pulses on the femtosecond. The transfer of the same technology to 3rd generation synchrotrons has been often limited to longer exposure time, flux and spatial resolution.

At the ESRF, thanks to the Extremely Brilliant Source, the ID29 beamline today has a flux density of ( > 1014 ph/s/µm2), three times higher than 3rd generation synchrotron sources. With this, scientists can deliver X-rays in very short pulses, on the microsecond time resolution, and at a very high repetition rate for macromolecular structure determination at room temperature.

Combined with a slightly polychromatic beam, this allows to measure complete reflections and ultimately accurate structure factor from thousands of microcrystals, even from low redundant datasets. This combination minimizes the sample consumption down to only a few microliters of crystal slurry, in contrast to larger amounts that are frequently needed for serial experiments, and allows complete data to be collected in the fraction of the time.

“Our beamline is the first in the world at a high energy 4th generation synchrotron which is designed to use the high flux density to study macromolecules at room temperature, with a microsecond time resolution”, explains Daniele de Sanctis, scientist in charge of ID29 together with Shibom Basu, EMBL scientist. “The technique, called serial microsecond crystallography (SµX), allows researchers to use less sample to achieve comprehensive structural detail of proteins under physiological conditions and also to visualise molecular movies in action on this time domain. Our work initiates a new future of time-resolved serial microsecond crystallography experiments at 4th generation storage rings, that will ultimately complement X-ray free electron laser (XFEL) experiments.”

A versatile sample environment

One specificity of serial crystallography is the set-up. How do scientists deliver a slurry of hundreds to thousands of microcrystals to the beam? This is a constantly evolving field and ID29 can accommodate different kinds of sample delivery methods with its flexible setup. The researchers applied the unique beam of ID29 to different sample delivery methods: fixed target (foils and chips) and  three different types of high viscosity extruders demonstrating how structures obtained do not present any evident sign of radiation damage. The data quality obtained allows to unambiguously identify the electron density map of ligated molecules.

Pharmaceutical applications

One of the experiments reported in the paper shows how Istradefylline, a drug used to treat Parkinson disease, binds to a membrane protein receptor. “There is a clear application of this technique for pharmaceutical research”, explains de Sanctis. “We are convinced that the easy operation and fast data acquisition of our unique set-up will attract industrial clients to perform large ligand screening campaigns, with optimal sample consumption at near physiological conditions, in the future”, he adds.

Leading the way for future beamlines

The concept design of ID29, together with the technological advances, is a revolutionary step in macromolecular crystallography.

The publication proves that the new ID29 bridges the standard structural biology beamlines and XFELs and establishes serial microsecond crystallography. “The results lead the way for the construction and commissioning of similar beamlines at other synchrotrons, currently undergoing or planning the upgrade of their accelerators”, concludes de Sanctis. 

The beamline is run jointly with the EMBL, as part of the long-standing collaboration that the ESRF and EMBL have set up on the EPN science campus in Grenoble.

Reference:

Orlans, J., Rose, S.L., Ferguson, G. et al. Advancing macromolecular structure determination with microsecond X-ray pulses at a 4th generation synchrotron. Commun Chem 8, 6 (2025). https://doi.org/10.1038/s42004-024-01404-y

Text Montserrat Capellas Espuny

Top image: Daniele De Sanctis, scientist in charge of the ESRF, and Shibom Basu, from the EMBL, on the beamline. Credits: S, Candé.