Structural and Functional Characterization of the Bacterial Type III Secretion Export Apparatus.

Bacterial type III protein secretion systems inject effector proteins into eukaryotic host cells in order to promote survival and colonization of Gram-negative pathogens and symbionts. Secretion across the bacterial cell envelope and injection into host cells is facilitated by a so-called injectisome. Its small hydrophobic export apparatus components SpaP and SpaR were shown to nucleate assembly of the needle complex and to form the central "cup" substructure of a Salmonella Typhimurium secretion system. However, the in vivo placement of these components in the needle complex and their function during the secretion process remained poorly defined. Here we present evidence that a SpaP pentamer forms a 15 Å wide pore and provide a detailed map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. We further refine the current view of export apparatus assembly, consolidate transmembrane topology models for SpaP and SpaR, and present intimate interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ, indicating how export apparatus and needle filament are connected to create a continuous conduit for substrate translocation.

Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement.

We describe a general approach for refining protein structure models on the basis of cryo-electron microscopy maps with near-atomic resolution. The method integrates Monte Carlo sampling with local density-guided optimization, Rosetta all-atom refinement and real-space B-factor fitting. In tests on experimental maps of three different systems with 4.5-Å resolution or better, the method consistently produced models with atomic-level accuracy largely independently of starting-model quality, and it outperformed the molecular dynamics-based MDFF method. Cross-validated model quality statistics correlated with model accuracy over the three test systems.

Topology and organization of the Salmonella typhimurium type III secretion needle complex components.

The correct organization of single subunits of multi-protein machines in a three dimensional context is critical for their functionality. Type III secretion systems (T3SS) are molecular machines with the capacity to deliver bacterial effector proteins into host cells and are fundamental for the biology of many pathogenic or symbiotic bacteria. A central component of T3SSs is the needle complex, a multiprotein structure that mediates the passage of effector proteins through the bacterial envelope. We have used cryo electron microscopy combined with bacterial genetics, site-specific labeling, mutational analysis, chemical derivatization and high-resolution mass spectrometry to generate an experimentally validated topographic map of a Salmonella typhimurium T3SS needle complex. This study provides insights into the organization of this evolutionary highly conserved nanomachinery and is the basis for further functional analysis.

Automated monitoring to reduce electron microscope downtime.

High-end transmission electron microscopes are complex and sensitive instruments. Failure of one of the external supplies, malfunction of the microscope hardware or maloperation are typical reasons for subsystems to fail. Especially if undiscovered for a longer period of time, this can cause unnecessary downtime, compromising user access and increasing operating costs. Utilizing the software introduced in this article ("MoniTEM"), we have succeeded to reduce downtime of an FEI Tecnai Polara by coupling constant monitoring of critical subsystems with automatic, remote feedback to the system supervisor, ensuring immediate problem solving. The software described here is freely available from http://www.imba.oeaw.ac.at/monitem/ and can be readily adapted for use with other FEI transmission electron microscopes.