bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.10.28.762456

Cryo-EM structures of the YsaN ATPase reveal dodecameric and tetradecameric assemblies in the Yersinia enterocolitica T3SS

Bhattacharyya, B.·Chakraborty, B.·Nandy, S.·Datta, S.·Patra, D.
View original source ↗Export metadata ↓

Abstract

Bacterial pathogens deploy sophisticated nanomachines such as the Type III Secretion System (T3SS) to inject effector proteins directly into host cells, thereby subverting cellular defenses and promoting infection. Central to this machinery is a conserved homo-oligomeric ATPase that fuels substrate unfolding and translocation. Despite extensive structural and biochemical studies, the precise mechanism of T3SS ATPase oligomer assembly and regulation remains incompletely understood, events crucial for the overall functionality of the T3SS. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of the Yersinia enterocolitica T3SS ATPase YsaN, captured in two previously uncharacterized oligomeric states, a dodecamer (12-mer) at 3.51 [A] and a tetradecamer (14-mer) at 3.80 [A] resolution. Both assemblies exhibit helical-like symmetry and conformational heterogeneity. A conserved longitudinal fissure interrupts the helical lattice and appears to mediate interconversion between the two states, while an N-terminal interface stabilized by inter-tier disulfide bonds and an extensive non-covalent network locks the assembly together. Molecular dynamics simulations reveal that ATP hydrolysis drives a global, correlated compaction upon transition to the ADP-bound state. Together, these structural, biochemical, and MD simulation data establish T3SS ATPases as dynamic, oligomerically plastic molecular motors and provide a mechanistic framework for chaperone-effector dissociation and substrate translocation.

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Bhattacharyya, B., Chakraborty, B., Nandy, S., Datta, S., Patra, D.. 2026-10-30. Cryo-EM structures of the YsaN ATPase reveal dodecameric and tetradecameric assemblies in the Yersinia enterocolitica T3SS. https://doi.org/10.64898/2026.10.28.762456

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Single Molecule Diffusion Studies Reveal Rapid Translations Across Target Membrane Surfaces for both p40Phox PX domain and Class III PI3K VPS34 Complex 1, the Autophagy Lipid Kinase

Single molecule analyses are providing new insights into the molecular mechanisms by which peripheral membrane proteins approach and translate across a target bilayer surface before stable docking. Here we compare and contrast the translational movements of four diffusers on a representative supported lipid bilayer: (i) the fluorescent lipid LRB-(DO)PE, (ii) the G-protein Rab1A anchored to (DO)PE; (iii) the PI(3)P sensor p40Phox PX domain, and (iv) the autophagy lipid kinase VPS34 Complex 1. The latter enzyme is a Class III PI-3-Kinase and its unusual architecture exhibits two distinct membrane-docking "feet" unique to Class III PI3Ks. Notably, the membrane contact surfaces of the PX domain and autophagy lipid kinase each possess multiple positive charges suitable for electrostatic interactions with their anionic target membrane. The present single molecule tracking studies reveal that both the fluorescent lipid and the lipid-anchored G protein Rab1A exhibit 2-dimensional translation typical of simple lipid diffusion in the bilayer. Single molecule density measurements indicate both PX domain and autophagy lipid kinase exhibit significantly reduced membrane docking when the bilayer negative charge is decreased, confirming the importance of their protein-bilayer electrostatic interactions. Single molecule tracking studies reveal that both PX domain and autophagy lipid kinase exhibit hopping movements across the bilayer surface during their initial bilayer contacts, yielding a significantly higher frequency of long range displacements than allowed by lipid 2-D diffusion. After transitioning from hopping to a stable bilayer-docked state, both PX domain and autophagy lipid kinase exhibit 2-D diffusion slower than lipid diffusion indicating the appearance of increased frictional drag against the bilayer due to new protein-bilayer contacts. PX domain requires its target lipid for the transition from electrostatic hopping to stable bilayer docking, while autophagy lipid kinase can dock spontaneously to anionic lipid bilayers even in the absence of its substrate lipid. In principle, the slowly diffusing, bilayer-docked states of PX domain and autophagy lipid kinase could be interrupted by unbinding-hopping-rebinding events. However, such events are not detected for PX domain in the presence of target lipid (DO)PI(3)P nor for autophagy lipid kinase in the presence of substrate lipid (DO)PI. Instead, unbinding-hopping-rebinding events are only observed for autophagy lipid kinase in the absence of substrate lipid, indicating that the presence of a specific binding lipid reduces the frequency or spatial amplitude of such events. Overall, the findings provide evidence that p40Phox PX domain possesses a search mechanism involving electrostatic hopping that speeds its acquisition of target lipid, as previously observed for PH domains. The evidence indicates that the autophagy lipid kinase also exhibits electrostatic hopping/skating behavior that is proposed to play a functional role by speeding regulatory encounters with autophagy lipid kinase activators and effectors. Moreover, the ability of the lipid kinase to spontaneously dock to anionic membranes without a target lipid could facilitate its distribution across broad regions of the growing autophagosome. Finally, the autophagy lipid kinase exhibits multiple translational modes that we hypothesize are enabled by its two membrane docking feet.

biophysics↗
bioRxiv2026

Inferring scaling laws of branching networks from their geometry by global optimization

Optimality principles are widely used to understand branching networks, yet they are tested on individual branchings that cannot reliably distinguish competing scaling laws. We formulate the missing link between the laws and measurements by mapping each law to its predicted branch points through global optimization. Vascular branch points closely match weighted-length optima when each vessel is allowed its own fitted weight. Inferred scaling laws reveal type-specific geometric signatures hidden from local analysis. They beat all tested laws, including Murray's law in 1,679 of 1,744 held-out hepatic and pulmonary trees. For coronary arteries on the heart surface, an exact discrete optimization model extends the framework beyond convex geometry and supports area-preserving scaling. The framework also identifies distinct laws for airways and plant shoots while rejecting weighted-length optimality for neuronal axons. Global optimization turns network geometry into a quantitative test of the principles that govern branching networks.

biophysics↗
bioRxiv2026

Ultra-precision 3D Printed Sample Tubes of Complex Cross Section for Enhanced Electron Paramagnetic Resonance Sensitivity of AqueousSamples

This paper discusses the applications of ultra-precision 3D printing in developing EPR sample tubes for X-band measurements of aqueous samples. The seed idea of this work started from the flat cell idea discussed in Mett and Hyde et al.(2003), Sidabras et al. (2005), and Sidabras et al. (2017), where placing multiple aqueous flat cells perpendicular to the electric field in the microwave cavity reduce RF losses and improve EPR signal sensitivity. In this work, we present the 3D printed aquastar sample holders for dielectric resonators and the aquasun sample holders for cavity resonators. Even though the ultra-precision 3D printing enables the fabrication of complex cross-sections at 25um resolution, the photopolymer material has comparatively higher dielectric losses compared to the conventional materials like glass and PTFE. To reduce the RF losses due to the inherent dielectric properties, we introduced the latticing approach in reducing the amount of plastic without compromising the structural stability. In addition to this, we present the slicing and dielectric approximation approach in reducing the computation cost for these complex cross-sections. The novel sample tubes discussed here present signal improvements through both simulation and experiments, which were validated through power saturation experiments. The sample tube development methodology presented here provide a general framework for designing and implementing 3D printed EPR sample holders, based on resonator electromagnetic field distribution and dielectric properties of the photopolymer material.

biophysics↗