Cryo-EM Reconstruction of Tail and Capsid of Stx Bacteriophage Phi24B

Shiga toxin-encoding bacteriophages (Stx phages) are responsible for converting the pathogenic profiles of their bacterial hosts.Most of Stx phages are lambdoid bacteriophages.Shiga toxin-producing Escherichia coli (STEC), causes severe hemorrhagic colitis disease which is, in some cases, complicated by a life-threatening hemolytic uremic syndrome (HUS) leading to long-term disability and mortality of patients.Although the global incidence of STEC infection is low, severe disease and death occurs in an unacceptably high proportion of infected individuals: 10% and 3-5%, respectively.Bacteriophage vB_EcoP-24 B (hereafter referred to as phi24B) is well-characterized since its initial isolation following induction from a clinical STEC isolate of E. coli O157:H7 [1].The genome and physiology of phi24B-related phages were extensively studied (recently reviewed in [2]), however the structural organization of phi24B virion remains poorly understood, most of the structural proteins encoded by the phage's genome are not identified.Cryo-electron microscopy was performed with Titan Krios 300 kV microscope equipped with a Gatan K3 camera.Samples were applied to the glow discharged Quantifoil 1.2/1.3holey carbon support film and imaged with pixel size 0.83, dose of 53e -/ 2 and -2.5 micrometer defocus.Single particle data processing was carried out with cryoSPARC software.The preprocessing pipeline involved the motion correction, CTF estimation, and template-based particle picking for the capsid particles.The ab-initio reconstruction followed by 3D refinement with icosahedral symmetry yielded the capsid density map with 4.5 overall resolution.The symmetry expansion algorithm followed by masked classification was utilized to resolve the portal and tail located at one of the capsid vertices.The 3D refinement with C6 rotational symmetry resulted in the tail density map with 4 overall resolution.The segmentation of the tail density map allowed us to identify main structural components and assign them to specific bacteriophage genes (Fig. 1).The protein structure models were generated by the Alphafold2 (ColabFold) algorithm [3].Based on the data obtained, atomic models of structural modules of the bacteriophage phi24B virion will be built [4].