Although often presented as taking single 'snapshots' of the conformation ofa protein, X-ray crystallography provides an averaged structure over time andspace within the crystal. The important but difficult task of characterizingstructural ensembles in crystals is typically limited to small conformationalchanges, such as multiple side-chain conformations. A crystallographic methodwas recently introduced that utilizes residual electron and anomalous density(READ) to characterize structural ensembles encompassing large-scalestructural changes. Key to this method is an ability to accurately measureanomalous signals and distinguish them from noise or other anomalousscatterers. This report presents an optimized data-collection and analysisstrategy for partially occupied iodine anomalous signals. Using the long-wavelength-optimized beamline I23 at Diamond Light Source, the ability toaccurately distinguish the positions of anomalous scatterers with occupancies aslow as approx12% is demonstrated. The number and positions of these anomalousscatterers are consistent with previous biophysical, kinetic and structural datathat suggest that the protein Im7 binds to the chaperone Spy in multiple partiallyoccupied conformations. Finally, READ selections demonstrate that re-measured data using the new protocols are consistent with the previouslycharacterized structural ensemble of the chaperone Spy with its client Im7. Thisstudy shows that a long-wavelength beamline results in easily validatedanomalous signals that are strong enough to be used to detect and characterizehighly disordered sections of crystal structures.
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