Single particle Cryo-EM studies of the structure of the T4SS core complex from Xanthomonas citri

Abstract

One of the main objectives of this postdoctoral project is to obtain structural information regarding the Xac Type IV Secretion System core complex, which forms the pore through both the inner and outer membrane layers of the bacterial envelope. This complex is made up of 14 copies each of VirB7, VirB9, and VirB10 subunits. Due to the core complex's large size (~1.2 MDa) and association with the bacterial outer membrane, the method of choice for structural analysis is the rapidly evolving technique of Cryo-electron microscopy (Cryo-EM).So far, we have made significant progress in our work on the Xanthomonas T4SS core complex. Beginning with heterologous expression in E. coli, we have been able to purify the membrane-bound Xanthomonas core complex using mild detergents in combination with affinity and size-exclusion chromatography. Initial characterization of the particles using negative staining electron microscopy indicate that they are toroidal in shape with a symmetry similar or equal to the C14 symmetry observed for the complexes from the conjugative plasmids pKM1011 and R3882 described by the group of Dr. Gabriel Waksman at Birkbeck College, London. Furthermore, we have recently been successful in preparing Cryo-EM grids for analysis. These preliminary results, obtained in collaboration with the group of Rodrigo Portugal at the Laboratório Nacional de Nanotecnologia (LNNano-CNPEM) in Campinas are extremely promising and suggest that we are very close to being ready to collect definitive NS-EM and Cryo-EM datasets to build 3D models of the Xac T4SS core complex. These models will give us the first structural insights into the organization of the unique Xanthomonadaceae T4SS and will provide significant advances in our understanding of the how this machine carries out its function at the molecular level. To achieve this goal, we have established a collaboration with a world-class laboratory with both the expertise and necessary infrastructure to carry out high resolution single particle electron microscopy analysis as well as experience in structural studies of Type IV secretion systems. This proposal for a BEPE scholarship describes our plans for a 6-month visit to the laboratory of Dr. Gabriel Waksman at Birkbeck College in London. During this visit, Dr. Germán Sgro will have access to a state-of-the-art Cryo-EM infrastructure and will be able to interact intensively with a highly competent group that specializes in EM studies of bacterial secretion systems.