Microcrystallography

Microcrystallography is a field of X-ray crystallography that deals with determining molecular structures from crystals too small for conventional data collection, typically in the micrometer to tens of micrometers range. Crystals of this size are often obtained from challenging proteins, membrane proteins, or complexes that resist growth to larger forms. Because of their small volume, microcrystals yield weaker diffraction and are more sensitive to radiation damage.

Data collection is enabled by microfocus X-ray beams on synchrotron beamlines and by X-ray free-electron lasers. In microcrystallography, diffraction data may be collected from single microcrystals by rotating the crystal in the beam (serial or conventional) or from many microcrystals in serial fashion. Serial crystallography uses repetitive, short exposures from many crystals, either on a fixed target or in a liquid jet, and can be performed at room temperature or cryogenic conditions. XFELs enable single-shot data collection with diffraction-before-destruction, reducing radiation damage, while modern synchrotrons provide high-throughput microbeam capabilities and high redundancy.

Phasing can be done by molecular replacement, experimental phasing using anomalous or isomorphous differences, or single-wavelength anomalous dispersion, with data often merged from many crystals to improve signal-to-noise.

Data processing involves indexing, integration, scaling, and merging of many partial reflections, followed by structure solution and refinement. The use of microcrystals has expanded the range of structures accessible, including flexible domains and membrane proteins, and supports time-resolved studies to capture reaction intermediates.

Sample delivery approaches include fixed targets, microfluidic chips, and viscous or aerosol jets. The field continues to advance with improved detectors, better software, and integrated pipelines for rapid screening and structure determination from microcrystals.