Well-based crystallization of lipidic cubic phase microcrystals for serial X-ray crystallography experiments

Abstract

Serial crystallography is having an increasing impact on structural biology. This emerging technique opens up new possibilities for studying protein structures at room temperature and investigating structural dynamics using time-resolved X-ray diffraction. A limitation of the method is the intrinsic need for large quantities of well ordered micrometre-sized crystals. Here, a method is presented to screen for conditions that produce microcrystals of membrane proteins in the lipidic cubic phase using a well-based crystallization approach. A key advantage over earlier approaches is that the progress of crystal formation can be easily monitored without interrupting the crystallization process. In addition, the protocol can be scaled up to efficiently produce large quantities of crystals for serial crystallography experiments. Using the well-based crystallization methodology, novel conditions for the growth of showers of microcrystals of three different membrane proteins have been developed. Diffraction data are also presented from the first user serial crystallography experiment performed at MAX IV Laboratory.

BioMAX – the first macromolecular crystallography beamline at MAX IV Laboratory

“…BioMAX has been used for a number of serial crystallography experiments including both the HVE injector and fixed target scanning (Andersson et al, 2019;Shilova et al, 2020). While the oscillation data collections in most cases uses a defocused beam and a flux-reducing aperture to set the beam size, the serial crystallography experiments take full advantage of the full flux of the fully focused beam.…”

“…In order to establish and use new experimental techniques relevant for the MicroMAX beamline, we started a collaboration with Bruce Doak from the Max Planck Institute for Medical Research, Heidelberg, Germany, to develop a high-viscosity extrusion (HVE) injector for SSX applications at BioMAX (Shilova et al, 2020;Andersson et al, 2019). This device can be easily mounted on the MD3 omega axis (instead of the single axis or mini-kappa) and uses the MD3 alignment translations and sample environment including the OAV.…”

Lipidic cubic phase serial femtosecond crystallography structure of a photosynthetic reaction centre

“…1b), but due to their being very thin in one direction only the largest crystals gave diffraction and the hit rate was extremely low. Screening around these conditions in crystallization plates (Andersson et al, 2019) instead of syringes yielded crystals with a rhomboidlike topology with an acceptable crystal thickness in all directions (Fig. 1c).…”

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

“…However, as LCP is viscous, increasing the crystal density by centrifugation would not be feasible and the crystallization conditions need to be optimized to yield high density of microcrystals. Optimization during scale up in Hamilton syringes, however, may not be very efficient and an alternative well-based approach using normal crystallization trays has been proposed [77]. This also facilitates easier tracking of crystallization progress with microscopic imaging.…”