A quantitative analysis of single protein-ligand complex separation with the atomic force microscope

Force measurements on and within single macromolecular complexes utilizing techniques such as atomic force microscopy, optical trapping, flexible glass fibers, and magnetic beads provide a rich source of quantitative data on biomolecular processes. Stochastic thermal fluctuations, an undesirable source of noise in macroscopic biochemical experiments, are an essential element of these sensitive and novel experiments. With the proper analysis, a great deal of information can be gleaned from measurements of these fluctuations. A quantitative framework for analyzing such measurements, based on Kramers' theory of molecular dissociation, is developed. The analysis reveals the kinetic origin and stochastic nature of the measurements. This framework is presented in the context of protein-ligand separation with the atomic force microscope.     

Normal mode based flexible fitting of high-resolution structure into low-resolution experimental data from cryo-EM

A new method for the flexible fitting of high-resolution structures into low-resolution maps of macromolecular complexes from electron microscopy has been recently described in applications to simulated electron density maps. This method uses a linear combination of low-frequency normal modes in an iterative manner to deform the structure optimally to conform to the low-resolution electron density map. Gradient-following techniques in the coordinate space of collective normal modes are used to optimize the overall correlation coefficient between computed and measured electron densities. With this approach, multi-scale flexible fitting can be performed using all-atoms or Calpha atoms. In this paper, illustrative studies of normal mode based flexible fitting to experimental cryo-EM maps are presented for three different systems. Large, functionally relevant conformational changes for elongation factor G bound to the ribosome, Escherichia coli RNA polymerase and cowpea chlorotic mottle virus are elucidated as the result of the application of NMFF from high-resolution structures to cryo-electron microscopy maps.     

Assembly of ribonucleoparticle complexes

RNA-proteins interactions are involved in numerous cellular functions. These interactions are found in most cases within complex macromolecular assemblies. The recent development of tools and techniques to study RNA-protein complexes has significantly increased our knowledge in the nature of these specific interactions. The aim of this article is to present the different techniques used to study RNA-protein complexes, as well as recent data concerning the application of RNA as therapeutic molecules.     

Distance-restrained docking of rifampicin and rifamycin SV to RNA polymerase using systematic FRET measurements: developing benchmarks of model quality and reliability

We are developing distance-restrained docking strategies for modeling macromolecular complexes that combine available high-resolution structures of the components and intercomponent distance restraints derived from systematic fluorescence resonance energy transfer (FRET) measurements. In this article, we consider the problem of docking small-molecule ligands within macromolecular complexes. Using simulated FRET data, we have generated a series of benchmarks that permit estimation of model accuracy based on the quantity and quality of FRET-derived distance restraints, including the number, random error, systematic error, distance distribution, and radial distribution of FRET-derived distance restraints. We find that expected model accuracy is 10 A or better for models based on: i), > or =20 restraints with up to 15% random error and no systematic error, or ii), > or =20 restraints with up to 15% random error, up to 10% systematic error, and a symmetric radial distribution of restraints. Model accuracies can be improved to 5 A or better by increasing the number of restraints to > or =40 and/or by optimizing the distance distribution of restraints. Using experimental FRET data, we have defined the positions of the binding sites within bacterial RNA polymerase of the small-molecule inhibitors rifampicin (Rif) and rifamycin SV (Rif SV). The inferred binding sites for Rif and Rif SV were located with accuracies of, respectively, 7 and 10 A relative to the crystallographically defined binding site for Rif. These accuracies agree with expectations from the benchmark simulations and suffice to indicate that the binding sites for Rif and Rif SV are located within the RNA polymerase active-center cleft, overlapping the binding site for the RNA-DNA hybrid.