Serial crystallography

Serial crystallography is a method for obtaining structural information on an atomic level of a protein, without the need for large protein crystals. Instead, small diffraction datasets are collected on many small protein crystals, which are usually easier to obtain than large ones. Serial crystallography is an ideal method for collecting diffraction data of proteins at room temperature, where the onset of radiation damage from the X-ray beam is rapid.

Serial Crystallography: Experiment setup at G3

Serial crystallography experiment set up at Beamline ID7B2, MacCHESS

One major bottleneck in the protein crystallography experiment is collection of a complete dataset prior to the onset of radiation damage. In certain cases, such as with microcrystalline samples, or data collection at room temperature, complete data sets are most efficiently obtained by collecting partial datasets from many crystals. With improved microbeam performance, novel techniques in sample delivery, and advances in post-processing of partial data sets, MacCHESS offers users an advanced serial microcrystallography experience.

There are many cases in macromolecular crystallography where crystals cannot be grown to a size sufficient to collect a full dataset for structural analysis, due in part to rapid onset of radiation damage. In such cases, where microcrystal formation dominates the crystallization landscape, much effort has been undertaken to use the microcrystals that are available to their fullest extent. To that end, the technique of serial crystallography- the collection of a series of small datasets of partial completeness from many crystals- was developed. In this technique, a small dataset is collected from each crystal, and the collection of partial data is evaluated and merged together to generate a complete dataset. Collecting crystal datasets serially is preferable in cases where the small size of the crystals is not the limiting factor, e.g. where diffraction data can be more accurately measured from multiple crystals than from a single crystal, due to reduced radiation damage to each crystal and increased data multiplicity. We are developing methodologies for streamlining serial crystallography, so that the technique can be made available for any user.

Room temperature serial oscillation crystallography

Schematic of major beamline components
Schematic of major beamline components. Shown is the arrangement with oscillation about the Z axis. The beam path is shown in yellow. A, beam-defining slit; B, CRL box with aperture; C, CRL positioning motors; D, air-bearing goniometer; E, three-axis piezo translation stage; F, chip holder; G, EIGER detector.

What did the Scientists Discover?
Our team has developed a new serial crystallography method for rapid serial data collection with oscillation, lowering the total number of crystals needed. Crystals are deposited onto a silicon chip with thousands of microwells in precise locations. Using a piezoelectric translation stage, each crystal-containing microwell can be positioned in front of the X-ray beam rapidly and precisely. With oscillation, the total amount of data per crystal that can be obtained is improved, without effects from radiation damage. Optimizing data collection by minimizing the effects of background X-ray scatter by air was also paramount.

Broader Impacts of this work?
This work should enable structural biologists to more easily access serial crystallographic techniques, and importantly, simplify room temperature data collection. This in turn should have a dramatic effect on our understanding of protein folding and dynamics under physiological conditions.

Why is this important?
Serial crystallography has much untapped potential within the broader structural biology community, but access to simple and readily available techniques is lacking. Further, economical use of protein crystals- by maximizing the amount of data that can be obtained from each crystal- will make the technique more attractive to users as a whole.

Why did this research need CHESS
MacCHESS has the flexibility needed to design beamlines tailored to user needs. At beamline ID7B2- FlexX, we designed a serial crystallography diffractometer tailor-made for this experiment, and deployed it in a couple of days. This level of rapidity is unique to MacCHESS among other beamlines in the country. We are equally capable of running users with unique needs as well as those who require routine data collection.

Collaborators:
Jennifer L. Wierman, MacCHESS, Cornell University
Olivier Paré-Labrosse, Departments of Chemistry & Physics, University of Toronto and Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany
Antoine Sarracini, Departments of Chemistry & Physics, University of Toronto
Jessica Besaw,  Departments of Chemistry & Physics, University of Toronto
Michael J. Cook, MacCHESS, Cornell University
Saeed Oghbaey,  Departments of Chemistry & Physics, University of Toronto
Hazem Daoud,  Departments of Chemistry & Physics, University of Toronto
Pedram Mehrabi, Max Planck Institute for the Structure and Dynamics of Matter, Hamburg, Germany
Irina Kriksunov, MacCHESS, Cornell University
Anling Kuo, Departments of Biochemistry and Molecular Genetics, University of Toronto
David J. Schuller, MacCHESS, Cornell University
Scott Smith, MacCHESS, Cornell University
Oliver P. Ernst, Departments of Biochemistry and Molecular Genetics, University of Toronto
Doletha M. E. Szebenyi, MacCHESS, Cornell University
Sol M. Gruner, MacCHESS, Department of Physics and Kavli Institute for Nanoscale Science, Cornell University
R. J. Dwayne Miller,  Departments of Chemistry & Physics, University of Toronto
Aaron D. Finke, MacCHESS, Cornell University

Protein crystals on chips enable high throughput

The standard X-ray protein crystallography experiment requires a single protein crystal specimen that is large enough to collect a “complete” data set, that is, to collect all the available diffraction peaks to a given resolution.

But what if large crystals are not available? A team of scientists at MacCHESS and the University of Toronto is pushing what is possible for small protein crystals at storage ring sources.

While structural biologists have expanded their purview to increasingly large and complex biological systems, the necessity for reliable, atomic resolution structural data for those systems has not changed. However, it is simply not possible to grow sufficiently large crystals for many systems. The necessity of large crystals in protein crystallography stems primarily from two factors. First, all other things being equal, microcrystals diffract more weakly than large ones, because the crystal volume, and thus number of protein molecules diffracting the X-rays, is lessened. Second, and more insidiously, protein microcrystals succumb more quickly to radiation damage – a loss of diffraction intensity resulting from X-ray induced, stochastic ionization and bond cleavage. These factors result in apparently contradictory solutions: increase the beam intensity to induce more diffraction, but at the expense of crystal lifetime; or lower the beam intensity, but collect weak data.

One solution to this problem is serial microcrystallography– collecting small data sets from several microcrystals and merging them together. This technique, which originated at XFELs, has found utility in storage ring sources as well. Because standard techniques are increasingly unsuitable for smaller and smaller crystals, delivering thousands to millions of small crystals to the X-ray beam presents its own brand of challenges.

Scientists from MacCHESS and the University of Toronto have teamed together to create an X-ray diffraction beamline specifically optimized for serial microcrystallography. Using the G3 beamline at CHESS, MacCHESS postdoc Jeney Wierman, engineer Mike Cook, and staff scientist Aaron Finke have built a diffractometer station with a new focusing element, the compound refractive lens (CRL), to deliver a beam that is less than 20 microns in diameter, well suited to collecting diffraction data from small crystals. The CRL is a series of beryllium lenses that focuses the X-ray source beam to an intense microbeam. Scientists from Dwayne Miller’s group from the University of Toronto have developed a sample loading chip system that allows for high throughput loading of up to over 100,000 microcrystals on a single chip, loaded onto a fast and highly accurate piezoelectric stage, which efficiently delivers each crystal to the X-ray beam. In combination with the DECTRIS EIGER 1M detector, data can be collected hundreds of times per second, so microcrystals diffraction data is collected as quickly as possible to avoid radiation damage.

The combination of a serial microcrystallography diffractometer setup with chip loading technology leads to a highly efficient, modular, and scalable sample delivery system for serial microcrystallography. The method is potentially suitable for any microcrystal system.

The Serial Microcrystallography Team
The Serial Microcrystallography Team. Front from left: Mike Cook, Jessica Besaw, Jeney Wierman, Antoine Sarrachini. Back from left: Hazem Daoud, John Feng, Pedram Mehrabi, Aaron Finke.