title: Exploiting radiation damage to map proteins in nucleoprotein complexes: the internal structure of bacteriophage T7.
abstract: In the final stage of radiation damage in cryo-electron microscopy of proteins, bubbles of hydrogen gas are generated. Proteins embedded in DNA bubble sooner than free-standing proteins and DNA does not bubble under the same conditions. These properties make it possible to distinguish protein from DNA. Here we explored the scope of this technique ("bubblegram imaging") by applying it to bacteriophage T7, viewed as a partially defined model system. T7 has a thin-walled icosahedral capsid, 60 nm in diameter, with a barrel-shaped protein core under one of its twelve vertices (the portal vertex). The core is densely wrapped with DNA but details of their interaction and how their injection into a host bacterium is coordinated are lacking. With short (10 s) intervals between exposures of 17 electrons/Å(2) each, bubbling starts in the third exposure, with 1-4 bubbles nucleating in the core: in subsequent exposures, these bubbles grow and merge. A 3D reconstruction from fifth-exposure images depicts a bipartite cylindrical gas cloud in the core. In its portal-proximal half, the axial region is gaseous whereas in the portal-distal half, it is occupied by a 3 nm-wide dense rod. We propose that they respectively represent core protein and an end of the packaged genome, poised for injection into a host cell. Single bubbles at other sites may represent residual scaffolding protein. Thus, bubbling depends on dose rate, protein amount, and tightness of the DNA seal.
authors.affiliation: Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Naiqian
authors.initials: N
authors.lastname: Cheng
authors.affiliation: Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Weimin
authors.initials: W
authors.lastname: Wu
authors.affiliation: Protein Expression Laboratory, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Norman R
authors.initials: NR
authors.lastname: Watts
authors.affiliation: Laboratory of Structural Biology Research, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: stevena@mail.nih.gov.
authors.forename: Alasdair C
authors.initials: AC
authors.lastname: Steven
mesh_terms: D017123:Bacteriophage T7 / Q000648:ultrastructure*; D036022:Capsid Proteins / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D009698:Nucleoproteins / Q000648:ultrastructure*
chemical_list: D036022:Capsid Proteins; D009698:Nucleoproteins
keywords: 3-dimensional image reconstruction; Cryo-electron microscopy; DNA ejection; Differential mass mapping; Nucleocapsid structure; Radiation biology