Identifying dynamic, partially occupied residues using anomalous scattering

Abstract

X-ray crystallography is generally used to take single snapshots of a protein’s conformation. The important but difficult task of characterizing structural ensembles in crystals is typically limited to small conformational changes, such as multiple side-chain conformations. A crystallographic method was recently introduced that utilizes Residual Anomalous and Electron Density (READ) to characterize structural ensembles encompassing large-scale structural changes. Key to this method is an ability to accurately measure anomalous signals and distinguish them from noise or other anomalous scatterers. This report presents an optimized data collection and analysis strategy for partially occupied iodine anomalous signals. Using the long wavelength-optimized beamline I23 at Diamond Light Source, the ability to accurately distinguish the positions of anomalous scatterers with as low as ~12% occupancy is demonstrated. The number and position of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. This study shows that a long-wavelength beamline results in easily validated anomalous signals that are strong enough to be used to detect and characterize highly dynamic sections of crystal structures.