Correlated Motions in Structural Biology

Abstract

Correlated motions in proteins arising from the collective movements of residues have long been proposed to be fundamentally important to key properties of proteins, from allostery and catalysis to evolvability. Recent breakthroughs in structural biology have made it possible to capture proteins undergoing complex conformational changes, yet intrinsic correlated motions within a conformation remain one of the least understood facets of protein structure. For many decades, the analysis of total X-ray scattering held the promise of animating crystal structures with correlated motions. With recent advances in both X-ray detectors and data interpretation methods, this long-held promise can now be met. In this Perspective, we will introduce how correlated motions are captured in total scattering and provide guidelines for the collection, interpretation, and validation of data. As structural biology continues to push the boundaries, we see an opportunity to gain atomistic insight into correlated motions using total scattering as a bridge between theory and experiment.

Figure 1.

Combining theoretical and experimental approaches to determine how motions are correlated in proteins. Theoretical models include those based on sequence (represented by an MSA) and structure (represented by an ENM). Among experimental methods, total X-ray scattering from crystals stands out for its ability to measure high-resolution structure and correlated motions simultaneously. Shown on the right is a set of residues that was predicted to coevolve in DHFR (PDB: 1RX2) by statistical coupling analysis (shown in blue).

Figure 2.

Components of total scattering illustrated using the experimental X-ray structure of CAP (PDB: 1g6n) with dynamics added using ENM simulations of one unit cell. (A) The simulated diffraction image contains two signals: Bragg peaks (left) that depend on the average structure, and diffuse scattering (right) that arises from correlated atomic displacements (in this case, vibrations of the ENM). (B) B-factors refined to experimental Bragg data vary along the polypeptide chain (blue to red). (C) Normal modes of the ENM seem to explain regions of high experimental B-factor. The reality of such collective motions can be verified by diffuse scattering analysis.

Figure 3.

Total scattering analysis separates motion into lattice and internal components. (A) Three-dimensional map of diffuse scattering from triclinic lysozyme shown as intersecting central slices. A movie showing this volume from multiple perspectives is available online. The scattering includes an intense isotropic ring that may be subtracted to better visualize the halo and cloudy features. (B) Around Bragg peaks are three-dimensional halos (shown here as transparent contours, blue to yellow) attributed to thermally excited lattice vibrations. (C) Cloudy features due to short-ranged correlated motion are most visible in sections mid-way between Bragg planes. (D) Total motion and correlations are quantified using B-factor (top, PDB 6o2h) and diffuse Patterson maps (bottom), which report electron density fluctuations vs. inter-atomic vector, r. A lattice dynamics model fit to diffuse halos accounts for most of the B-factor for well-ordered atoms (total vs. lattice, top) and the correlated motions at large distances (total vs. lattice, bottom), but underestimates those at short distance (r < 10 Å, dashed circles). An ENM describing protein dynamics was fit to the residual B-factors (top right). The simulated diffuse Patterson of the protein dynamics model (bottom right) explains the remaining short-ranged correlations.

Figure 4.

Evolutionary and dynamic perspectives on residue-residue correlations. (Left) Evolutionary correlation according to SCA applied to an MSA of a hydrolase family that contains lysozyme (Pfam PF00062). (Right) Displacement correlations in lysozyme according to an ENM derived from total scattering analysis (Fig. 3). Establishing the connection between these two perspectives is necessary to fully understand protein function and allostery.