Warming up to room-temperature crystallography

By growing proteins into a crystal and probing them with X-rays, structural biologists can determine the shape of the biomolecule, including the twists and turns in the amino acid chain. In the early days of X-ray crystallography, scientists would irradiate the crystals at room temperature but found that the beam often destroyed the crystal before capturing enough structural data. Crystallographers found a solution to the problem: cryopreserve the crystals to withstand the damage caused by the X-ray beam.

Mike Hough, the Principal Beamline Scientist at Diamond’s VMXi beamline, said;

Crystal cooling expanded the limits of X-ray crystallography, increasing the number of structures that researchers could uncover; however, cryopreservation hampered structural biology in a different regard. Most proteins are flexible and take on a variety of conformations, but at low temperatures they tend to adopt the state with the lowest energy, like a spring with its tension released. In some cases, proteins may even adopt an artefactual “frozen out” state. Hough continued;

The team at Diamond’s VMXi beamline reverted to room-temperature crystallography to avoid potential artefacts that come with cryopreservation. To overcome issues with X-ray damage, they don’t limit themselves to studying single crystals. Hough said;

This enables users to capture highly mobile proteins in various subtly different shapes and study dynamic changes in a protein as it interacts with other chemicals, like drugs. Their technique also expands the scope of crystallography by allowing scientists to study fragile crystals that break when cryo-cooled, like ones containing large protein complexes or proteins from cell membranes.With this, Hough said;

The beamline uses a microfocus X-ray beam, which allows crystals as small as a hundredth of a millimetre to be used. Users have harnessed this beam to successfully study peroxidase enzymes that carry out chemical reactions related to biofuel production despite the hurdle that these proteins carry iron-laden heme groups that are sensitive to X-ray damage. Hough noted;

The VMXi team can work with crystals that come in many sizes, ranging from 7 to 200 μm, which can help them study smaller crystals like those of lysozyme prepared in a viscous liquid called in lipidic cubic phase. Researchers use this medium because it’s suitable for preparing crystals of membrane proteins. However, Hough said;

Despite the fragility of these crystals, the VMXi team managed to resolve structures from 21 crystals, demonstrating that a disulfide bond in lysozyme, which is usually weak and liable to breaking, remained intact in their irradiated samples.

Other users from Macquarie University in Australia came to VMXi to explore a cohort of proteins that marine cyanobacteria use for nutrient uptake. The team produced crystal hits for eight proteins, most of which had never been resolved before. For one of the structures, where the team achieved a high resolution of 1.8 Ångströms (Å), X-ray crystallography under cryo-conditions had previously been used to capture its 3D nature. Both the room-temperature and cryopreserved crystals revealed a phosphate group in a ligand binding site, suggesting the techniques can produce similar, complementary results.

Besides offering a different strategy to probe protein structure, room-temperature crystallography can streamline the synthesis of optimal crystals. When scientists prepare samples under cryogenic conditions, they often judge the quality of the crystal based on their visual appearance under a microscope before committing to resolving the protein’s structure, but this approach produces unreliable forecasts. Taking an alternative approach, users routinely run VMXi’s swift technique to screen crystals because room-temperature crystallography can provide insight into the sample’s properties, such as how efficiently the proteins pack together. It allows researchers to compare arrays of crystals and rapidly pinpoint optimal varieties that they can subsequently cryopreserve and study. For example, users from the University of Oxford studied an unusually long antibody produced in cattle, and the VMXi beamline helped them quickly compare several crystallisation hits to work out the optimal conditions to produce and harvest crystals for this protein.

As VMXi team prepare for Diamond-II upgrades, they aim to make their technique even more versatile. Hough said they plan to capture structures from even smaller crystals moving forward, thereby opening up the technique to even more challenging proteins.

To find out more about VMXi or discuss potential applications, please contact principal beamline scientist Mike Hough: [email protected].