Snapshots of Internal Protein Crystal Architecture at the Nanoscale.

Macromolecular crystallography has historically inferred models of internal crystal architecture from reciprocal-space measurements of Bragg reflections. Nevertheless, direct real-space visualization of crystallographic disorder remains elusive, particularly at the nanoscale. Using a 15-nanometer probe, here we apply both ambient-temperature and cryogenic four-dimensional scanning transmission electron microscopy (4D-STEM) to map the topography of coherently diffracting domains (CDDs) in lysozyme and myoglobin microcrystals at length scales 100 × finer than conventional X-ray and electron beams. Virtual dark-field images show that each Bragg peak arises from spatially distinct subvolumes representing smooth and continuous variations in local lattice orientation. Under sustained irradiation, protein CDDs undergo rearrangements spanning several micrometers of internal movement. Furthermore, pinpoint high-dose "impact crater" experiments reveal delocalized radiolytic damage propagating hundreds of nanometers from primary irradiation sites, behavior similar to small-molecule crystals but amplified in both rate and magnitude. Together, these results establish macromolecular microcrystals as dynamic assemblies whose internal architecture continuously reorganizes during irradiation, laying the foundation for realistic models of mosaicity directly informed by both real-space and reciprocal-space observations.