In situ protein crystal diffraction screening

The generation of high quality diffracting crystals remains an area that requires considerable person-power. Not only do crystals need to be produced and/or optimized, but such samples also have to be analyzed for their diffraction properties. While crystal shape and size are critical parameters controlling diffraction strength, diffraction screening remains the optimal manner to guide crystal growth optimization protocols. Mounting of the crystals to an appropriate sample support often requires transfer into a buffer significantly different from that the crystal is grown in, often resulting in a negative impact upon crystal morphology and diffraction properties. Modern synchrotron sources are increasingly using in situ exposure to X-rays to characterize protein crystals, with a growing number of structures being solved directly in situ, without recourse to manual handling of delicate samples. An automated approach requires crystal identification, positioning and collection of X-ray diffraction data for analysis. However, limitations are imposed by the variation in crystal size, morphology and space group. Here we review the availability and limitations of both commercial and synchrotron based infrastructures for in situ diffraction screening. We review the status of methods to establish the basic geometric features of crystals, the limitations currently inherent with in situ screening methods and we also describe our and other researchers efforts to overcome these limitations.