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.
Highlights
Most known biological processes require precise and often highly regulated interactions among macromolecules to exert macroscopic events including signal transduction, metabolism, tissue homeostasis, immune responses, and development
A valuable example is provided by the molecular recognition displayed in numerous epigenetic regulators of post-translationally modified histone tails, frequently resulting in opposite gene expression states depending on the readout of the specific histone tail reader or modifier involved (Bowman and Poirier, 2015; DesJarlais and Tummino, 2016; McGinty and Tan, 2016)
Over the course of this review we have described how several cutting-edge structural biology techniques may provide a broad toolbox to explore molecular flexibility, with emphasis on the possible outcome of the investigation and on the methodological approaches to employ
Summary
Most known biological processes require precise and often highly regulated interactions among macromolecules to exert macroscopic events including signal transduction, metabolism, tissue homeostasis, immune responses, and development. When considered altogether, structural ensembles capture the multiple conformations displayed by various regions of the crystallized macromolecules, poorly represented and hard to understand by B factor analysis (Lang et al, 2014; Woldeyes et al, 2014) Even though these methods are not recent, their diffusion has so far been very limited, mostly due to the heavy computational resources that are needed to generate reliable ensembles (Burnley and Gros, 2013; van den Bedem and Fraser, 2015). Using time-averaged ER on high resolution data collected from protease complement factor D (FD) crystals, it was possible to highlight dramatic conformational dynamics in regions where the electron density was poorly defined after conventional refinement In this case, the ER analysis revealed an unprecedented aspect of FD biology, showing that this protease undergoes a highly flexible intermediate state during recognition and interaction with its macromolecular substrate. Such an idea has been exploited recently (Keedy et al, 2015), providing the first example of a conformational ensemble from XFEL data and suggesting exciting developments for the detection of concerted conformational changes upon ultrafast temperature changes, offering an opportunity to study correlated motions inside macromolecular crystals using ensembles
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