Identifying dynamic, partially occupied residues using anomalous scattering.

Serena Rocchio,Loïc Salmon,Armin Wagner,Scott Horowitz,Ramona Duman,Christian Orr,Vitaliy Mykhaylyk,Zhen Yan,Kamel El Omari,James C A Bardwell

doi:10.1107/s2059798319014475

Abstract

Although often presented as taking single `snapshots' of the conformation of a protein, X-ray crystallography provides an averaged structure over time and space within the crystal. 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 electron and anomalous 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 occupancies as low as ∼12% is demonstrated. The number and positions of these anomalous scatterers are consistent with previous biophysical, kinetic and structural data that suggest that the protein Im7 binds to the chaperone Spy in multiple partially occupied conformations. Finally, READ selections demonstrate that re-measured data using the new protocols are consistent with the previously characterized structural ensemble of the chaperone Spy with its client Im7. 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 disordered sections of crystal structures.

Highlights

In crystallography, anomalous scattering is commonly used to help solve the phase problem (Hendrickson, 2014)
We recently introduced a method called residual electron and anomalous density (READ) that uses anomalous maps to allow the reconstruction of highly heterogeneous conformational ensembles in regions of the protein that are not sufficiently well ordered for traditional model building (Salmon et al, 2018; Horowitz et al, 2016)
The Super Spy chaperone mutant Spy H96L (Quan et al, 2014) was used here in co-crystallization experiments, as we found that it formed crystals more readily than the wild-type Spy protein (Horowitz et al, 2016)

Summary

Introduction

Anomalous scattering is commonly used to help solve the phase problem (Hendrickson, 2014). We recently introduced a method called residual electron and anomalous density (READ) that uses anomalous maps to allow the reconstruction of highly heterogeneous conformational ensembles in regions of the protein that are not sufficiently well ordered for traditional model building (Salmon et al, 2018; Horowitz et al, 2016). This method uses selective anomalous labeling with iodophenylalanine (pI-Phe) to generate multiple partially occupied iodine anomalous signals. The Im7 ensembles generated by READ were highly consistent with orthogonal experimental approaches that were used to characterize Im7 binding and folding with Spy, including NMR spectroscopy and thermodynamic and kinetic analyses (Salmon et al, 2018; Horowitz et al, 2016; Koldewey et al, 2016; Stull et al, 2016; He & Hiller, 2018; He et al, 2016)

Methods

Results

Conclusion