Towards near-atomic resolution of non-crystalline biomolecular samples

Understanding how living cells work requires understanding the function of biomolecules. The structure of a biomolecule can provide direct clues about its function. Structures of macromolecular complexes reveal how individual proteins work in cohort to fulfil certain functions. Resolving the structures of macromolecular complexes in different conformational states provides fundamental mechanistic insights. Electron microscopy (EM) of macromolecules and complexes thereof is the sole technique that can yield three-dimensional structures of unlabelled, non-crystalline biomolecules. Our research group aims to answer biological questions of medical importance by studying macromolecules and macromolecular complexes using cryo-EM. Central in this proposal is the use of the single particle (SP) technique, in which images are recorded of individual molecules in different orientations. We typically arrive at 10.000 - 1.000.000 different projection images of a macromolecular complex. These images are computationally combined into one or a multitude of three dimensional structures. The ultimate aim is to obtain reconstructions at near-atomic resolution at which the direction of helices can be seen, beta-strands are well defined, and large side chains could be observed, allowing building de novo structures.