Identifying and Visualizing Macromolecular Flexibility in Structural Biology

Abstract

Structural biology comprises a variety of tools to obtain atomic resolution data for the investigation of macromolecules. Conventional structural methodologies including crystallography, NMR and electron microscopy often do not provide sufficient details concerning flexibility and dynamics, even though these aspects are critical for the physiological functions of the systems under investigation. However, the increasing complexity of the molecules studied by structural biology (including large macromolecular assemblies, integral membrane proteins, intrinsically disordered systems, and folding intermediates) continuously demands in-depth analyses of the roles of flexibility and conformational specificity involved in interactions with ligands and inhibitors. The intrinsic difficulties in capturing often subtle but critical molecular motions in biological systems have restrained the investigation of flexible molecules into a small niche of structural biology. Introduction of massive technological developments over the recent years, which include time-resolved studies, solution X-ray scattering, and new detectors for cryo-electron microscopy, have pushed the limits of structural investigation of flexible systems far beyond traditional approaches of NMR analysis. By integrating these modern methods with powerful biophysical and computational approaches such as generation of ensembles of molecular models and selective particle picking in electron microscopy, more feasible investigations of dynamic systems are now possible. Using some prominent examples from recent literature, we review how current structural biology methods can contribute useful data to accurately visualize flexibility in macromolecular structures and understand its important roles in regulation of biological processes.

Challenges and advances in atomistic simulations of potassium and sodium ion channel gating and permeation

“…There is the potential for ion channel protein structures to undergo substantial modifications from physiological conditions due to the incorporation of buffer solutions, detergents, crystal packing and heavy atom staining (in X‐ray), and vitreous ice and radiation damage (in cryo‐EM) (Ubarretxena‐Belandia & Stokes, ; Palamini et al . ; Rawson et al . ).…”

Atomistic modeling towards predictive cardiotoxicity

“…For instance, liquid chromatography with mass spectrometry can elucidate pharmacokinetic information (12,13), mutagenesis studies and voltage patch-clamp techniques can tentatively identify specific residues involved in drugchannel interactions (14)(15)(16). Experimental techniques for determining high-resolution structures of drug-channel complexes at the atomic scale have their own limitations such as system size (NMR), bound state stability (X-ray) and resolution (cryogenic electron microscopy (cryo-EM) (17,18). The most important limitation for understanding drugprotein interactions, however, is the static nature of information provided by a single reported structure.…”

Expanding the structural biology toolbox with single-molecule holography

“…In electron microscopy, the signal-to-noise ratio is enhanced through averaging over hundreds of thousands of particles. Especially in crystallography and electron microscopy, this "averaged nature of the data" tends to blur the atomic fluctuations and conformational changes that underpin the complexity of the catalytic, signaling, and binding events of macromolecular processes (11,12). In this context, Longchamp et al (13) propose a highly innovative approach based on single-molecule holography, which is not significantly influenced by molecular sizes and bypasses the need for averaging because 3D electron densities are reconstructed directly from a single hologram (Fig.…”