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.

Keywords: Small-angle scattering; X-ray crystallography; cryo-electron microscopy; ensembles; molecular recognition; nuclear magnetic resonance; protein flexibility; structural biology.

Figure 1

Visualizing molecular flexibility using structural ensembles. Ensemble refinement of macromolecular crystal structures: from a single, B factor-weighted static model to a superimposed “bouquet” of structural conformations, providing deeper understanding of local flexibility even inside the crystal lattice. The structural models (represented as sticks) and electron density maps (blue mesh, 2F_o–F_c maps contoured at 1.2 σ) for single- and ensemble-refined data were from PDB files 4CBN and 4CBO, respectively (Forneris et al., 2014). The structures are colored based on their isotropic atomic B factors, using the same scale from 10 (blue) to 100 (red) Ų. Figure prepared using PyMol (Schrödinger, LLC, 2010).

Figure 2

Trapping multiple conformations using modern cryo-EM. (A) Three different EM maps obtained from selective classification of the apo gamma secretase cryo micrographs show conformational changes in the transmembrane region of the enzyme complex. Shown are the experimental maps and the three-dimensional structures (obtained from EMDB maps 3238, 3239, 3240, and PDB IDs 5FN3, 5FN4, 5FN5, respectively, Bai et al., 2015b) with soluble nicastrin depicted in green, and the transmembrane region composed of Aph-1, PS1, and Pen-2 components in cyan. Transmembrane helices found in different conformations in the three different classes are shown in blue, red and orange. Arrows indicate the putative movements associated to the rearrangements of the transmembrane helices. (B) Three EM reconstructions relative to identification of multiple conformations in DNA-free and DNA-bound E. coli PolIIIα-clamp-exonuclease-τ_c micrographs (Fernandez-Leiro et al., 2015). PolIIIα is depicted in cyan, the clamp is shown in green, the exonuclease domain is in blue. DNA is colored in dark gray and is present only in classes 2 and 3. The moving regions, composed of the PolIIIα-tail and τ_c, are shown in orange and red, respectively (data from EMDB maps 3201, 3198, and 3202). The superposition shows the comparison between the structural models obtained from the DNA-free (class 1) and DNA-bound (class 2) states, shown as cartoon and colored in light and dark blue, respectively (PDB IDs 5FKU and 5FKV). DNA for the bound state is shown in gold. Figure prepared using Chimera (Pettersen et al., 2004).

Figure 3

Representative flowchart addressing modern experimental structural biology approaches for the understanding of molecular flexibility.