High-throughput counter-diffusion capillary crystallization and in situ diffraction using high-pressure freezing in protein crystallography

Post-crystallization treatments such as manual fishing of crystals and soaking in cryoprotectant solutions, especially of large macromolecular complexes and membrane proteins, are cumbersome and often lead to crystal damage and reduced diffraction data quality. Here, a capillary crystallization plate is presented that simultaneously allows counter-diffusion crystallization at the nanolitre scale in a high-throughput screening mode, low-temperature in situ diffraction data collection from crystals after cryoprotection and low-temperature in situ data collection of crystals without the addition of any cryoprotectant after high-pressure (HP) freezing. The development of this plate and plunge cooling of crystals in the capillaries is a major step towards implementing automated in situ high-throughput crystal diffraction data collection at a synchrotron beamline. In combination with HP freezing this offers a new opportunity to obtain structural information from fragile crystals of supramolecular complexes that might otherwise not be feasible.