Mycobacterial type VII secretion systems

Mycobacteria, such as the pathogen M. tuberculosis, utilize up to five paralogous type VII secretion systems to transport proteins across their cell envelope. Since these proteins associate in pairs that depend on each other for transport to a different extent, the secretion pathway to the bacterial surface remained challenging to address. Structural characterization of the inner-membrane embedded secretion machineries along with recent advances on the substrates' co-dependencies for transport allow for the first time more detailed and testable models for secretion.Biophysical Structural Chemistr

Haruspex: A Neural Network for the Automatic Identification of Oligonucleotides and Protein Secondary Structure in Cryo‐Electron Microscopy Maps

In recent years, three‐dimensional density maps reconstructed from single particle images obtained by electron cryo‐microscopy (cryo‐EM) have reached unprecedented resolution. However, map interpretation can be challenging, in particular if the constituting structures require de‐novo model building or are very mobile. Herein, we demonstrate the potential of convolutional neural networks for the annotation of cryo‐EM maps: our network Haruspex has been trained on a carefully curated set of 293 experimentally derived reconstruction maps to automatically annotate RNA/DNA as well as protein secondary structure elements. It can be straightforwardly applied to newly reconstructed maps in order to support domain placement or as a starting point for main‐chain placement. Due to its high recall and precision rates of 95.1 % and 80.3 %, respectively, on an independent test set of 122 maps, it can also be used for validation during model building. The trained network will be available as part of the CCP‐EM suite

Dealing with incompleteness in automata-based model checking

A software specification is often the result of an iterative process that transforms an initial incomplete model through refinement decisions. A model is incomplete because the implementation of certain functionalities is postponed to a later development step or is delegated to third parties. An unspecified functionality may be later replaced by alternative solutions, which may be evaluated to analyze tradeoffs. Model checking has been proposed as a technique to verify that a model of the system under development is compliant with a formal specification of its requirements. However, most classical model checking approaches assume that a complete model of the system is given: they do not support incompleteness. A verification-driven design process would instead benefit from the ability to apply formal verification at any stage, hence also to incomplete models. After any change, it is desirable that only the portion affected by the change, called replacement, is analyzed. To achieve this goal, this paper extends the classical automata-based model checking procedure to deal with incompleteness. The proposed model checking approach is able not only to evaluate whether a property definitely holds, possibly holds or does not hold in an incomplete model but, when the satisfaction of the specification depends on the incomplete parts, to compute the constraints that must be satisfied by their future replacements. Constraints are properties on the unspecified components that, if satisfied by the replacement, guarantee the satisfaction of the original specification in the refined model. Each constraint is verified in isolation on the corresponding replacement

Structure and dynamics of a mycobacterial type VII secretion system

Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year(1). Specialized protein transport systems—known as type VII secretion systems (T7SSs)—are central to the virulence of this pathogen, and are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope(2,3). Here we present the structure of an intact T7SS inner-membrane complex of M. tuberculosis. We show how the 2.32-MDa ESX-5 assembly, which contains 165 transmembrane helices, is restructured and stabilized as a trimer of dimers by the MycP(5) protease. A trimer of MycP(5) caps a central periplasmic dome-like chamber that is formed by three EccB(5) dimers, with the proteolytic sites of MycP(5) facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP(5) show disruption of the EccB(5) periplasmic assembly and increased flexibility, which highlights the importance of MycP(5) for complex integrity. Beneath the EccB(5)–MycP(5) chamber, dimers of the EccC(5) ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC(5) domains adopt two distinctive conformations that probably reflect different secretion states. Our work suggests a previously undescribed mechanism of protein transport and provides a structural scaffold to aid in the development of drugs against this major human pathogen