The opportunistic pathogenPseudomonas aeruginosais a major cause of antibiotic-tolerant infections in humans.P. aeruginosaevades antibiotics in bacterial biofilms by up-regulating expression of a symbiotic filamentous inoviral prophage, Pf4. We investigated the mechanism of phage-mediated antibiotic tolerance using biochemical reconstitution combined with structural biology and high-resolution cellular imaging. We resolved electron cryomicroscopy atomic structures of Pf4 with and without its linear single-stranded DNA genome, and studied Pf4 assembly into liquid crystalline droplets using optical microscopy and electron cryotomography. By biochemically replicating conditions necessary for antibiotic protection, we found that phage liquid crystalline droplets form phase-separated occlusive compartments around rod-shaped bacteria leading to increased bacterial survival. Encapsulation by these compartments was observed even when inanimate colloidal rods were used to mimic rod-shaped bacteria, suggesting that shape and size complementarity profoundly influences the process. Filamentous inoviruses are pervasive across prokaryotes, and in particular, several Gram-negative bacterial pathogens includingNeisseria meningitidis,Vibrio cholerae,andSalmonella entericaharbor these prophages. We propose that biophysical occlusion mediated by secreted filamentous molecules such as Pf4 may be a general strategy of bacterial survival in harsh environments.

We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are based on 3D data models, we propose to optimise a regularised likelihood target that approximates a function of the 2D experimental images. In addition, analogous to Bayesian polishing and contrast transfer function (CTF) refinement in single-particle analysis, we describe approaches that exploit the increased signal-to-noise ratio in the averaged structure to optimise tilt series alignments, beam-induced motions of the particles throughout the tilt series acquisition, defoci of the individual particles, as well as higher-order optical aberrations of the microscope. Implementation of our approaches in the open-source software package RELION aims to facilitate their general use, in particular for those researchers who are already familiar with its single-particle analysis tools. We illustrate for three applications that our approaches allow structure determination from cryo-ET data to resolutions sufficient for de novo atomic modelling.

Highlights d 3.7 Å cryo-EM structure of S-layer bound to O-antigen of lipopolysaccharide (LPS) d MD simulations and native MS show Ca 2+-dependent S-layer binding to LPS d 4.8 Å in situ cryo-ET structure of the S-layer on Caulobacter crescentus cells d Integrated structural biology reveals principles of LPSmediated S-layer assembly Authors Andriko von Kü gelgen, Haiping Tang,

Despite enormous sequence diversity in surface (S)-layer proteins, structural diversity is much lower than previously thought.S-layer proteins have a bipartite arrangement with a lattice-forming and an anchoring segment.Novel structural biology methods are revealing the architectures of S-layers in situ.S-layer assembly across prokaryotes is tightly coupled to the cell cycle, including the cell division machinery.