A tool for visualizing protein motions in time-resolved crystallography

Cecilia Wickstrand,Joerg Standfuss,Gergely Katona,Eriko Nango,Takanori Nakane,Richard Neutze,Przemyslaw Nogly

doi:10.1063/1.5126921

Abstract

Time-resolved serial femtosecond crystallography (TR-SFX) at an x-ray free electron laser enables protein structural changes to be imaged on time-scales from femtoseconds to seconds. It can, however, be difficult to grasp the nature and timescale of global protein motions when structural changes are not isolated near a single active site. New tools are, therefore, needed to represent the global nature of electron density changes and their correlation with modeled protein structural changes. Here, we use TR-SFX data from bacteriorhodopsin to develop and validate a method for quantifying time-dependent electron density changes and correlating them throughout the protein. We define a spherical volume of difference electron density about selected atoms, average separately the positive and negative electron difference densities within each volume, and walk this spherical volume through all atoms within the protein. By correlating the resulting difference electron density amplitudes with time, our approach facilitates an initial assessment of the number and timescale of structural intermediates and highlights quake-like motions on the sub-picosecond timescale. This tool also allows structural models to be compared with experimental data using theoretical difference electron density changes calculated from refined resting and photo-activated structures.

Highlights

Structural dynamics are as intrinsic to the protein function as the protein structure
The growth of electron density changes along the extracellular side of helix C44,45 is at first associated with Thr89 and Asp85 and only at later times do structural changes spread to Arg82
This is important for characterizing ultrafast motions18,19,25,35 and protein motions stimulated by electric fields and THz radiation29,30 since protein motions recovered under these conditions are likely to be spread throughout the protein and are not understood by looking at a few difference electron density features alone

Summary

Introduction

Structural dynamics are as intrinsic to the protein function as the protein structure. We illustrate the power of this representation using difference Fourier electron density maps and the results of structural refinement from two recent TR-SFX studies of bacteriorhodopsin.24,25 This reanalysis of time-dependent electron density changes does not touch upon an important but separate scientific question concerning the possible impact of multi-photon excitation of the retinal chromophore on the protein’s observed trajectory.39 We first demonstrate this method using TR-SFX data recorded for thirteen time-points from Dt 1⁄4 16 ns to 1.7 ms following retinal photo-isomerization.24 We further illustrate how these tools can be used to analyze a sequence of refined crystallographic structures and correlate these with the experimental electron density changes.

Results

Conclusion