Phage liquid crystalline droplets form occlusive sheaths that encapsulate and protect infectious rod-shaped bacteria.

Pf4 Phage Preparation. Native Pf4 phage was initially generated from a static biofilm of a P. aeruginosa PAO1 strain, further amplified by infection of PAO1 on Luria-Bertani plates and isolated by polyethylene glycol precipitation as described previously ( 20 ) and in further detail in SI Appendix , Materials and Methods . Cryo-EM and Cryo-ET Data Collection. Cryo-EM data of the Pf4 phage were collected as movie frame stacks using the EPU software on a Titan Krios microscope (ThermoFisher) operating at 300 kV fitted with a Quantum energy filter and a K2 Summit direct electron detector (Gatan) operating in counting mode. Tilt series data for cryo-ET were collected on the same microscope using the SerialEM software ( 29 ) in two directions starting from 0° between ±60° with 1° tilt increments. Further details on grid preparation and data collection conditions are provided in SI Appendix , Materials and Methods and Table S1 . Cryo-EM Image Processing and Data Analysis. Cryo-EM refinement was performed using the real-space helical reconstruction procedure implemented in Relion 3.0 ( 30 ). Fourier transforms of class averages produced using the Spring software ( 31 ), which showed high-resolution features, were indexed to determine initial helical symmetry parameters used for 3D refinement in Relion. Pf4 compositional variation was accounted for by splitting the dataset into segments from classes showing density at the core of the filament and classes where this density was absent. Model building was performed iteratively using Coot ( 32 ) along with real-space refinement against the density map in PHENIX ( 33 ). Further details are provided in SI Appendix , Materials and Methods . Tomogram Reconstruction from Cryo-ET Data. Tilt series alignment using gold fiducials and tomogram reconstruction were both carried out using a set of image processing, modeling and display programs (IMOD) ( 34 ) as described previously ( 35 ). Visualization of data was performed in IMOD and UCSF Chimera ( 36 ). Fluorescence Microscopy and FRAP. Samples for fluorescence microscopy were either immobilized onto agar pads constructed using Gene Frames (ThermoFisher), or applied directly to glass slides and imaged using a Zeiss AxioImager M2 widefield or Zeiss LSM Exciter confocal microscope, respectively. For FRAP experiments, samples were placed in a capillary on a glass slide and regions of interest were bleached using the Zen software bleaching mode on the Zeiss LSM exciter confocal microscope. Further details on sample preparation, imaging conditions, and image analysis are provided in SI Appendix , Materials and Methods . Materials and Data Availability. Cryo-EM maps reported in this study have been deposited at the Electron Microscopy Data Bank with accession codes EMD-10593 and EMD-10594. Atomic models have been deposited in the Protein Data Bank with PDB ID codes 6TUP and 6TUQ. Reagents generated in this study will be made available on request, but we may require a payment and/or a completed Materials Transfer Agreement if there is potential for commercial application.

Materials and Data Availability. Cryo-EM maps reported in this study have been deposited at the Electron Microscopy Data Bank with accession codes EMD-10593 and EMD-10594. Atomic models have been deposited in the Protein Data Bank with PDB ID codes 6TUP and 6TUQ. Reagents generated in this study will be made available on request, but we may require a payment and/or a completed Materials Transfer Agreement if there is potential for commercial application.

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