FitEM2EM—Tools for Low Resolution Study of Macromolecular Assembly and Dynamics

Studies of the structure and dynamics of macromolecular assemblies often involve comparison of low resolution models obtained using different techniques such as electron microscopy or atomic force microscopy. We present new computational tools for comparing (matching) and docking of low resolution structures, based on shape complementarity. The matched or docked objects are represented by three dimensional grids where the value of each grid point depends on its position with regard to the interior, surface or exterior of the object. The grids are correlated using fast Fourier transformations producing either matches of related objects or docking models depending on the details of the grid representations. The procedures incorporate thickening and smoothing of the surfaces of the objects which effectively compensates for differences in the resolution of the matched/docked objects, circumventing the need for resolution modification. The presented matching tool FitEM2EMin successfully fitted electron microscopy structures obtained at different resolutions, different conformers of the same structure and partial structures, ranking correct matches at the top in every case. The differences between the grid representations of the matched objects can be used to study conformation differences or to characterize the size and shape of substructures. The presented low-to-low docking tool FitEM2EMout ranked the expected models at the top

Molecular Architecture of the Human Mediator–RNA Polymerase II–TFIIF Assembly

The authors perform a cryo-EM study of the 1.9 MDa human Mediator-RNA polymerase II-TFIIF assembly, which reveals the structural organization of the human transcription initiation apparatus

Synchrotron Small-Angle X-Ray Scattering on Biological Macromolecules in Solution

Small-angle X-ray scattering (SAXS) is a powerful method for the structural analysis of macromolecular solutions, allowing one to study the structure of native particles and complexes and to rapidly analyze structural changes in response to variations in external conditions. On synchrotrons, SAXS benefits enormously from the high brilliance of the radiation, providing a considerable advantage for the timescale of measurements (allowing also for time-resolved experiments) and the small amounts of material required. Emerging automation of the scattering experiment, data processing, and interpretation make synchrotron solution SAXS a streamline tool for large-scale structural studies in molecular biology. In the present chapter, a brief account will be given of the basic principles of SAXS by macromolecular solutions and of the synchrotron SAXS instrumentation. The main concepts of SAXS data analysis from monodisperse solutions will be considered and the methods for computation of the overall structural parameters and ab initio low-resolution shape reconstructions will be presented. Further, approaches combining SAXS with other structural, biophysical, and biochemical techniques including validation of predicted or experimentally obtained high-resolution models in solution and identification of biologically active oligomers will be considered. Modeling methods of the quaternary structure of macromolecular complexes in terms of rigid body movements/rotations of individual subunits or domains will be reviewed. The approaches will also be considered to study oligomeric mixtures and to quantitatively characterize flexible macromolecular systems, including intrinsically unfolded proteins. The new methodological developments in SAXS will be illustrated by examples of practical applications