Reflections on the many facets of protein microcrystallography

The use of X-ray crystallography for the structure determination of biological macromolecules has experienced a steady expansion over the last 20 years with the Protein Data Bank growing from 100000 in 2014. The large number of structures determined each year not only reflects the impact of X-ray crystallography on many disciplines in the biological and medical fields but also its accessibility to non-expert laboratories. Thus protein crystallography is now largely a mainstream research technique and is routinely integrated in high-throughput pipelines such as structural genomics projects and structure-based drug design. Yet, significant frontiers remain that continuously require methodological developments. In particular, membrane proteins, large assemblies, and proteins from scarce natural sources still represent challenging targets for which obtaining the large diffracting crystals required for classical crystallography is often difficult. These limitations have fostered the emergence of microcrystallography, novel approaches in structural biology that collectively aim at determining structures from the smallest crystals. Here, we review the state of the art of macromolecular microcrystallography and recent progress achieved in this field.