Cryoelectron tomography of enveloped human cytomegalovirus particles

The compartmentalization of eukaryotic cells presents considerable challenges to the herpesvirus life cycle. The herpesvirus tegument, a bulky proteinaceous aggregate sandwiched between herpesviruses’ capsid and envelope, is uniquely evolved to address these challenges, yet tegument structure and organization remain poorly characterized. Here we use deep-learning–enhanced cryoEM to investigate the tegument of human cytomegalovirus virions and noninfectious enveloped particles (NIEPs; a genome packaging-aborted state), revealing a portal-biased tegumentation scheme. We resolve atomic structures of portal vertex-associated tegument (PVAT) and identify multiple configurations of PVAT arising from layered reorganization of pUL77, pUL48 (large tegument protein), and pUL47 (inner tegument protein) assemblies. Analyses show pUL77 seals the last-packaged viral genome end through electrostatic interactions, pUL77 and pUL48 harbor a head–linker–capsid-binding motif conducive to PVAT reconfigurati..., HCMV strain AD169 particles were mixed with 5-nm gold beads and applied onto freshly glow-discharged Quantifoil Holey Carbon grids, then plunge frozen into liquid ethane. Tilt series were acquired using the SerialEM software suite on a ThermoFisher Titan Krios electron microscope operated at 300 kV and equipped with a Gatan imaging filter, Gatan K2 Summit direct electron detector, and Volta phase plate (VPP). Prior to collecting each tilt series, we advanced and pre-conditioned the VPP by electron illumination at a dose of 12 nC for 60s to ensure a phase shift of ~0.3π. Movies were recorded with the K2 Summit camera operated in counting mode at 5 frames/s with a dose rate of ~8-10 e-/pixel/s. Tilt series were collected from -66° to +60° at 2° intervals at 53,000x nominal magnification (for a calibrated pixel size of 2.6 Å) with a cumulative electron dose of approximately 100 e-/Å2. Defocus was targeted to -0.6 μm.
Collected movies of raw tilt series were aligned and averaged using Motio..., , # Cryoelectron tomography of enveloped human cytomegalovirus particles

Human herpesviruses possess a proteinaceous tegument important for survival in the complicated intracellular environment. Despite tegument's many functions, its structure remains poorly characterized. In this study, we use a combination of deep learning-enhanced cryoelectron tomography (cryoET) and single-particle cryoelectron microscopy (cryoEM) to investigate human cytomegalovirus (HCMV; a betaherpesvirus) tegument structure. The dataset here contains raw data images of enveloped HCMV particles from our cryoET experiments, which we reconstructed into tomograms and then processed using *IsoNet*, our deep learning-based missing-wedge correction algorithm. Final *IsoNet*-processed tomograms are included with each image stack.

## Description of the data and file structure

Each unique file string prefix (e.g., HCMV_cryoET_pp###) corresponds to one of eleven tomograms. Each individual tomogram has 66 associated file...