Flexible-body refinement for Cryogenic Electron Microscopy Applications

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Scientists are interested in the atomic structure of biological molecules, in other words what the molecules look like. Knowing in detail what a molecule looks like provides important clues to how it might work. If we can go further and capture molecules in the process of interacting with other biological molecules, or artificial compounds such as drugs, we get a clearer picture of how they work.Most of our knowledge of the structure of biological molecules comes from X-ray crystallography. However over the past decade a new technique, electron microscopy (EM) has become popular. Individual molecules held in a thin film of liquid solvent are frozen and placed in an electron microscope, which captures images of the molecules. Many individual views can be combined to construct a model of the structure of the molecule in 3 dimensions. Electron microscopy has developed rapidly over the past decades due to new kinds of electron detector and new software methods, leading to a 'resolution revolution' enabling a much greater understanding of the molecules.In the most common cases, images of molecules are 'fuzzy' enough that we can't see individual atoms. The EM user therefore needs to have some knowledge of the structure of the molecule, or at least parts of it, in advance. This prior knowledge may come from other techniques, such as X-ray crystallography or computational modelling. The prior models can then be fitted into the EM image to give an indication of the whole structure, and allowed large molecular machines such as the Ribosome to be understood.The prior model is generally only a poor match for the true structure, either because it came from a different species, or because it was distorted by crystallisation, or because of limitations in the computational modelling process. The model must therefore be adjusted in order to fit into the observed EM images. This is performed using both automated software such as Flex-EM which breaks the structure into successively smaller fragments and adjusts their positions to fit the density, and by time consuming manual modelling using 3D graphics.The aim of this project is to take an existing method called 'shift field refinement' for distorting one 3D image to better fit another, and apply it to several problems in the determination of molecular structures from EM images. The method was developed by Professor Cowtan for problems in X-ray crystallography, but is sufficiently general to apply to other problems. The first problem we will address is fitting a known molecular structure into a 3D EM image. Rather than breaking the model up into smaller fragments which are each fitted separately, shift field refinement can very rapidly determine smooth deformations of the model which improve its fit to the image.We will also look at the problem of improving EM images of flexible molecules. In this case, the 3D EM image is blurred because it is combined from 2D images of thousands of particles, with each particle being slightly different. We will improve the 3D particle image by averaging together smaller clusters of more similar particles, and then using shift field refinement to adjust for the differences between the clusters before averaging them together to produce final 3D images.The project involves adding new steps to existing computer software for these problems and implementing new methods in a way which can be easily integrated with the existing software. We are working with existing software tools, including Flex-EM, rather than developing a suite of software from scratch to reduce the cost of the project, improve its chances of success, and to exploit the best features of the new and existing methods.All of the software produced by the project will be distributed freely to academic users through existing software suites for electron microscopy. The source code for software will also be distributed so that other developers can learn from it or modify it.