In-situ serial crystallography facilitates 96-well plate structuralanalysis at low symmetry

Abstract

The advent of serial crystallography has rejuvenated and popularised room temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g., large complexes, membrane proteins, and viruses) forming fragile crystals, are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallisation. Many medically relevant samples are expressed in mammalian cell-lines, producing a meagre quantity of protein that is incompatible for batch crystallisation. This can limit the scope of serial crystallography approaches. Direct in-situ data collection from a 96-well crystallisation plate enables not only the identification of the best diffracting crystallisation condition, but also the possibility for structure determination at ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallisation plates, mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility (ESRF). We applied our iSX approach on a challenging project, Autotaxin, a therapeutic target expressed in a stable human cell-line, to determine a structure in the lowest symmetry P1 space group at 3.0 [A] resolution. Our in situ data collection strategy provided a complete dataset for structure determination, while screening various crystallisation conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallisation plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons. SynopsisThe determination of a challenging structure in the P1 space group, the lowest symmetry possible, shows how our in-situ serial crystallography approach expands the application of crystallisation plates as a robust sample delivery method.