Fixed-target Serial Crystallography Research Articles

Fixed-target serial crystallography at the Structural Biology Center.

Serial synchrotron crystallography enables the study of protein structures under physiological temperature and reduced radiation damage by collection of data from thousands of crystals. The Structural Biology Center at Sector 19 of the Advanced Photon Source has implemented a fixed-target approach with a new 3D-printed mesh-holder optimized for sample handling. The holder immobilizes a crystal suspension or droplet emulsion on a nylon mesh, trapping and sealing a near-monolayer of crystals in its mother liquor between two thin Mylar films. Data can be rapidly collected in scan mode and analyzed in near real-time using piezoelectric linear stages assembled in an XYZ arrangement, controlled with a graphical user interface and analyzed using a high-performance computing pipeline. Here, the system was applied to two β-lactamases: a class D serine β-lactamase from Chitinophaga pinensis DSM 2588 and L1 metallo-β-lactamase from Stenotrophomonas maltophilia K279a.

Room-temperature serial synchrotron crystallography of Drosophila cryptochrome.

Fixed-target serial crystallography allows the high-throughput collection of diffraction data from small crystals at room temperature. This methodology is particularly useful for difficult samples that have sensitivity to radiation damage or intolerance to cryoprotection measures; fixed-target methods also have the added benefit of low sample consumption. Here, this method is applied to the structure determination of the circadian photoreceptor cryptochrome (CRY), previous structures of which have been determined at cryogenic temperature. In determining the structure, several data-filtering strategies were tested for combining observations from the hundreds of crystals that contributed to the final data set. Removing data sets based on the average correlation coefficient among equivalent reflection intensities between a given data set and all others was most effective at improving the data quality and maintaining overall completeness. CRYs are light sensors that undergo conformational photoactivation. Comparisons between the cryogenic and room-temperature CRY structures reveal regions of enhanced mobility at room temperature in loops that have functional importance within the CRY family of proteins. The B factors of the room-temperature structure correlate well with those predicted from molecular-dynamics simulations.

A simple vapor-diffusion method enables protein crystallization inside the HARE serial crystallography chip.

Fixed-target serial crystallography has become an important method for the study of protein structure and dynamics at synchrotrons and X-ray free-electron lasers. However, sample homogeneity, consumption and the physical stress on samples remain major challenges for these high-throughput experiments, which depend on high-quality protein microcrystals. The batch crystallization procedures that are typically applied require time- and sample-intensive screening and optimization. Here, a simple protein crystallization method inside the features of the HARE serial crystallography chips is reported that circumvents batch crystallization and allows the direct transfer of canonical vapor-diffusion conditions to in-chip crystallization. Based on conventional hanging-drop vapor-diffusion experiments, the crystallization solution is distributed into the wells of the HARE chip and equilibrated against a reservoir with mother liquor. Using this simple method, high-quality microcrystals were generated with sufficient density for the structure determination of four different proteins. A new protein variant was crystallized using the protein concentrations encountered during canonical crystallization experiments, enabling structure determination from ∼55 µg of protein. Additionally, structure determination from intracellular crystals grown in insect cells cultured directly in the features of the HARE chips is demonstrated. In cellulo crystallization represents a comparatively unexplored space in crystallization, especially for proteins that are resistant to crystallization using conventional techniques, and eliminates any need for laborious protein purification. This in-chip technique avoids harvesting the sensitive crystals or any further physical handling of the crystal-containing cells. These proof-of-principle experiments indicate the potential of this method to become a simple alternative to batch crystallization approaches and also as a convenient extension to canonical crystallization screens.

Fixed-target serial crystallography at SACLA

Fixed-target serial crystallography at SACLA