Structural characterization of macromolecular assemblies at the atomic level

Developing cures for diseases requires an understanding of how they work. We know, for example, that bacteria must penetrate the membranes of cells to cause infection, but we do not know much about the underlying mechanisms. Which part of the membrane serves as the entry point? Why are some membranes vulnerable while others are not? By answering these kinds of questions, we can take the first steps towards designing drugs capable of blocking these processes. Structural biology aids this project by providing pictures of parts of cells. This is done through the use of experimental techniques such as cryogenic electron microscopy, a method whereby cell components are frozen and then bombarded with electrons, yielding an image of the sample. Though such techniques have led to great discoveries, there are significant limitations to what they can accomplish. The pictures they provide are usually only partial, leaving us with significant gaps in our knowledge. My research aims to overcome this gap. Working with the electron microscopy group at Birkbeck college, I develop computational methods to complement experimental techniques. The project focuses on three diseases-pneumonia, herpes, and Alzheimer‘s-and we use computer modelling to pull together information from a variety of experimental sources to construct clearer and more complete images of the crucial cellular components and processes at work in these diseases.

One of the central challenges in biology is to gather structural information at the molecular level. Experimental techniques such as X-ray crystallography, NMR spectroscopy, and cryo-electron microscopy (cryoEM) have so far provided a wealth of structural data on proteins, DNA, and RNA, as well as on their complexes. Despite these successes, many important biological questions remain unanswered, due in part to the limitations of these experimental methods. My research goal is to overcome these limitations and provide more accurate characterizations of macromolecular assemblies at the atomic level through the development a novel hybrid computational method that integrates information from experiments, physical theories, and bioinformatics.
There has been a good deal of important work done that combines low- and intermediate-resolution cryoEM density maps of whole assemblies with atomic-resolution structures of individual components to provide atomic models of the assemblies. My aim in this project is to take the next step forward, building and refining atomic models of assemblies that integrate cryoEM, crystallography, NMR, low-resolution experimental data (chemical cross-linking, site-directed spin labelling, etc.), and protein structure prediction, with the aid of methods from physical and statistical theories. This integrative model will not only provide a superior picture of assemblies, but it will also enable us to observe and simulate dynamic processes so that we can gain a better understanding of how proteins assemble, how their components interact, and what kind of structural rearrangements they undergo.
The specific methodological aims of the project are:
Aim 1: Develop a method of combining computational protein-protein docking, cryoEM density fitting, and other experimental restraints.
Aim 2: Develop a method for modelling new structural folds by direct analysis of subnanometer-resolution cryoEM maps.
Aim 3: Develop a real-space refinement method for modelling conformational changes in atomic structures of assembly components.
Aim 4: Implement and integrate an automated protocol for building atomic models of whole assemblies using cryoEM maps.
Working in collaboration with experimentalists in the School of Crystallography at Birkbeck College (Image Processing Group), the research will focus initially on protein assemblies involved in three biologically and medically important cellular processes, each of which presents significant methodological challenges:
(1) Bacterial Infection (S. pneumoniae toxin pneumolysin)
(2) Viral infection (The connector of Bacillus subtilis bacteriophage SPP1)
(3) Chaperonin-mediated protein folding (E. coli chaperonin folding machine GroEL-GroES).