States of phage T3/T7 capsids: buoyant density centrifugation and cryo-EM
Mature double-stranded DNA bacteriophages have capsids with symmetrical shells that typically resist disruption, as they must to survive in the wild. However, flexibility and associated dynamism assist function. We describe biochemistry-oriented procedures used to find previously obscure flexibility for capsids of the related phages, T3 and T7. The primary procedures are hydration-based buoyant density ultracentrifugation and purified particle-based cryo-electron microscopy (cryo-EM). We review the buoyant density centrifugation in detail. The mature, stable T3/T7 capsid is a shell flexibility-derived conversion product of an initially assembled procapsid (capsid I). During DNA packaging, capsid I expands and loses a scaffolding protein to form capsid II. The following are observations made with capsid II. (1) The in vivo DNA packaging of wild type T3 generates capsid II that has a slight (1.4%), cryo-EM-detected hyper-expansion relative to the mature phage capsid. (2) DNA packaging in some altered conditions generates more extensive hyper-expansion of capsid II, initially detected by hydration-based preparative buoyant density centrifugation in Nycodenz density gradients. (3) Capsid contraction sometimes occurs, e.g., during quantized leakage of DNA from mature T3 capsids without a tail.
KeywordsBacteriophage assembly Capsid flexibility DNA packaging DNA injection Nycodenz
Compliance with ethical standards
This study was funded by NIH (R01-GM120600 to BD and R01-AI072035 to WJ) and the Welch Foundation (AQ-764 to PS).
Conflict of interest
Philip Serwer declares that he has no conflict of interest. Elena T. Wright declares that she has no conflict of interest. Borries Demeler declares that he has no conflict of interest. Wen Jiang declares that he has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
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