‘Towards high resolution structures of biological machines at work: computational tools to push the limits of three-dimensional electron microscopy’

Alberto Domingo Pascual Montano, Javier Angel Velázquez Muriel, José Jesús Fernández Rodríguez, José María Carazo García, José Román Bilbao Castro, Mª Carmen San Martín Pastrana, Mónica Chagoyen Quiles, Roberto Marabini Ruiz, Sjors Scheres.

Many protein structures have been solved using electron microscopy (EM). In contrast to X-ray crystallography and nuclear magnetic resonance (NMR), EM can deal with a wide range of specimen sizes (from single proteins to whole organelles) and does not require sample crystallization. One of the main reasons that limit the resolution attained in three-dimensional EM studies is structural heterogeneity in the sample.

The first goal of this project is therefore to develop computational approaches to tackle sample heterogeneity in 3D image processing. In particular, we aim at (i) detecting and locating structural heterogeneity, (ii) modeling the conformational freedom and (iii) producing a set of 3D reconstructions showing (or sampling) the different conformations.Resolutions in the range of 10-20 Å are not high enough to provide a direct visualization of the secondary structure, but they contain a fair amount of information that cannot be directly interpreted. As a second goal of this project, we propose to develop a methodology that, in the absence of known fragments, will be able to detect and place folds obtained from a generic fold data base, such as CATH, in the 3D reconstructions provided by EM. Biological studies have become more challenging and computational complex, and speed is of the essence. However, management of the large amounts of data involved in the 3D reconstruction is not a minor item. As its third goal, this project undertakes the task of parallelizing the more expensive routines using grid technologies.The tools developed in this project will be applied to the study of Simian Virus 40 large T antigen (T-ag) in complex with the viral origin of replication SV40 ori. The results obtained will allow a better understanding of the initiation of DNA replication, while the tools developed will be of further applicability to other relevant biological systems.