Cryo-EM Reconstruction of the Giant Bacteriophage phiK601 Neck

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A group of tailed bacteriophages with large genomes exceeding 200 kbp and huge capsids (~1300 Å), referred to as giant or “jumbo” phages, continues to attract the attention of scientific community worldwide due to their potential applications in various biological-related fields [1]. One of the most important features of this viral group is their potential to be effectively used against multidrug-resistant bacteria, such as Pseudomonas aeruginosa [2]. This approach is known as phage therapy and enables new possibilities for treating severe bacterial infections when existing drugs are ineffective [3]. Jumbo phages possess a unique mechanism for forming a nucleus-like structure after genome injection which helps them to avoid the consequences of various bacterial defense systems, including the CRISPR-Cas system [4]. This feature makes them even more interesting for further research and development of phage therapy. Determining their structure is highly challenging due to the large sizes of virion particles and complex protein composition. Recent advances in cryogenic electron microscopy (cryo-EM) made it possible to achieve high-resolution reconstructions of the capsids of giant phages [5,6]. However, the structure of the entire jumbo virion has not yet been elucidated. In this regard, this study presents a high-resolution investigation of the neck and portal complex of giant phiKZ-like phage phiK601. The phiK601 phage, which infects Pseudomonas aeruginosa, was isolated from a water sample collected in Powai Lake (Mumbai, India). It was purified using CsCl density-gradient ultracentrifugation and collected from the bluish-white layer with a density of approximately 1.4 g/ml. The suspension containing viral particles was dialyzed against SM buffer in order to remove salt particles. The cryo-EM data were collected with a 300 kV TFS Titan Krios G4 microscope and Gatan K3 detector. The parameters were: pixel size 1.05 Å, total exposure dose 45 e/Å2, average defocus 1 µm, and spherical aberration 2.7 mm. The data were processed using CryoSPARC software v4.7.1 [7]. A total of 10,044 raw movie stacks were subjected to motion correction and CTF estimation. Firstly, in order to obtain neck particles, virion heads were automatically picked and used for reconstructing the viral head. Secondly, the volume and the particles were aligned along C5-axis and then centered on a vertex region. Vertex particles were extracted using a box size of 500x500 pixels and subjected to 3D classification with a cylindric mask. A subset of 3,397 particles was selected for 3D reconstruction. The final models with imposed C6 and C12 symmetry were reconstructed at 3.5 Å and 3.4 Å respectively. PhiK601 is a phiKZ-related giant bacteriophage that shares morphological and structural similarity with the other giant phages of Myoviridae family (Fig. 1A). The capsid of ~1300 Å in diameter connects with the tail of ~2000 Å in length. The region connecting these virion components, referred to as the neck, consists of the portal complex and outer decorating proteins. In this study we show two reconstructions of the neck due to the symmetry difference: portal proteins have C12 symmetry, while others, including collar proteins, have C6 symmetry. In order to solve these reconstructions of the neck we applied C6 and C12 symmetry on the same set of particles. The neck structures with 3.5 Å and 3.4 Å resolution according to FSC with 0.143 threshold (Fig. 1B) were obtained as a result of local refinements and local CTF refinements. The reconstruction with C6 applied symmetry presents cryo-EM density for the tail and collar (Fig. 2A), while the reconstruction with C12 applied symmetry shows the density for proteins included in portal complex (Fig. 2B). These three-dimensional reconstructions represent the first high-resolution maps of the neck among phiKZ-like jumbo phages. They provide the new insights into the protein composition and will help to build atomic models of structural proteins [8,9]. (A) CryoEM micrograph displaying the morphology of viral particles. Scale bar = 100 nm; (B) FSC plot indicates the 3.5 Å and 3.6 Å resolution of C6 and C12 reconstructions respectively. Orange – the reconstruction with C12 applied symmetry, blue – the reconstruction with C6 applied symmetry. Neck reconstructions with C6 and C12 applied symmetry with estimated local resolution. (A) The reconstruction with C6 applied symmetry; (B) The reconstruction with C12 applied symmetry. Scale bar = 100 Å.

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