Tag: serial crystallography

50th Anniversary of the SSRL synchrotron radiation & protein crystallography initiative

50th Anniversary of the SSRL synchrotron radiation & protein crystallography initiative

Synchrotron-based protein crystallography continues to accelerate, driven by new and upgraded high-brightness sources, improved optics, faster large-area detectors, robust automation and streamlined data handling. These advances are making increasingly challenging structural biology projects feasible and are reshaping how synchrotron experiments integrate with today’s wider structural biology methods. While AI models are now routinely used in  molecular replacement software for macromolecular crystal structure determination, synchrotron experimental methods remain vital for detailed model refinement, and even validating AI models. Also extracting key chemical information, with anomalous dispersion at tuneable beamlines still playing an important role especially in identifying metals and other such atoms in proteins.

This special issue in Journal of Synchrotron Radiation, edited by John R. Helliwell and Marian Szebenyi, and their Overview with Colin Nave, with a Perspective from Keith Hodgson, as well as articles from a majority of the facilities worldwide, explores the evolving landscape in depth. It also highlights the expanding impact of fragment screening and binding studies (from cryogenic up to body temperatures) and the rapidly developing frontiers of time-resolved and serial crystallography. In particular, the issue charts the synergy between XFEL-based serial femtosecond crystallography and serial synchrotron crystallography, culminating in recent demonstrations of microsecond time resolution at upgraded synchrotrons such as ESRF–EBS, pointing to a future where synchrotrons and X-ray lasers together enable ever more powerful studies of biological structure, dynamics and function.

Access the special issue here

Image Credit:

Phillips, J.C., Wlodawer, A., Yevitz, M.M. and Hodgson, K.O., 1976. Applications of synchrotron radiation to protein crystallography: preliminary results. Proceedings of the National Academy of Sciences, 73(1), pp.128-132. 

Rosenbaum, G., Holmes, K.C. and Witz, J., 1971. Synchrotron radiation as a source for X-ray diffraction. Nature, 230(5294), pp.434-437.

Room-temperature serial crystallography experiments with microsecond pulsed beams

Room-temperature serial crystallography experiments with microsecond pulsed beams

Scientists can now scan thousands of protein crystals at room temperature using X-ray microsecond pulses at the ESRF’s serial crystallography beamline, ID29. This capability is of utmost importance for time-resolved studies and drug discovery research at physiological conditions. The results are published in Communications Chemistry.

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.

Read more on ESRF website

Image: Daniele De Sanctis, scientist in charge of the ESRF, and Shibom Basu, from the EMBL, on the beamline.

Credit: S. Candé.

Acoustofluidic Crystallography: The next leap in serial crystallography at MAX IV

Acoustofluidic Crystallography: The next leap in serial crystallography at MAX IV

The innovative project “Acoustofluidic Crystallography” (AFX) is set to revolutionize the field of serial crystallography (SX) by introducing a novel sample delivery method that promises to enhance the efficiency and reliability of experiments conducted at synchrotrons and X-ray free-electron lasers (XFELs). This cutting-edge research is a collaborative effort involving KTH Royal Institute of Technology, DESY, and MAX IV, funded by the LEAPS-INNOV initiative (GA: 101004728).

The LEAPS-INNOV innovation pilot aims to develop the cutting-edge technology to support groundbreaking research and drive innovation, while strengthening the European scientific network and establishing long-lasting industry connections. As part of these efforts, a dedicated Work Package for Co-Creation, WP9, has been implemented. With an aim to establish new co-creation structures allowing for joint development of key technologies and novel access modes through seed funding for pilot implementation studies, a call for project proposals was processed. The result was a selection of 3 co-creation pilots, including the AFX project.

The essence of AFX

At the heart of AFX lies the integration of acoustofluidics with state-of-the-art injection nozzles, aimed at reducing sample consumption and preventing clogging—a common issue in current SX methods. The project leverages a piezoelectric actuator to generate a 2D ultrasonic standing wave, which focuses microcrystals into a single line within a silica capillary. This setup, combined with a flow cell or dual flow-focusing nozzle (DFFN), ensures precise and efficient sample delivery.

“We are combining acoustics with microfluidics to improve sample delivery of protein crystals,” explained Jonas Sellberg, coordinator of the AFX project. “Our goal is to reduce sample waste and make delivery more reliable, preventing crystals from getting trapped during the process.”

Read more at MAXIV website

Image: Varun Kumar Rajendran, post-doctoral fellow with the AFX project, installs a first prototype of the acoustofluidic device at MAX IV’s MicroMAX beamline during AFX beamtime in June 2024.

Credit: MAX IV