Fig. 5: The N-whisker is essential for capsid assembly.

a, b Functional single and three amino acid substitution mutants in N-whisker from CRISPR combinatorial libraries. c Frequencies of recombination that resulted in infectious phage using various deletion mutants in N-whisker. d, e SDS-polyacrylamide gel electrophoresis of lysates of infected E. coli cells (panel d) and purified proheads (panel e). Most of the major capsid protein gp23 was uncleaved in the 4-del portal mutant (lane 2) and remained (unassembled) in the supernatant upon high speed centrifugation (lane 4) whereas it was cleaved to gp23* which occurs only following capsid assembly (lane 1) and very little of it remained in the supernatant (lane 3). e Expanded, partially expanded, and unexpanded proheads purified by CsCl gradient centrifugation and DEAE ion-exchange chromatography. The samples were concentrated and approximately 2 × 10¹⁰ prohead particles were loaded in each lane. “+” and “−” represent with and without boiling, respectively, of the purified prohead samples isolated from the ion-exchange column. The expanded heads are stable and remain intact in SDS without boiling (lane 1; no gp23* enters the gel), whereas the unexpanded capsids dissociate (lane 3). The 4-del mutant showed a partially expanded peak (lanes 5–6) but no fully expanded prohead peak as in the WT (lanes 1–2) (Supplementary Fig. 5). Note the presence of small amounts of uncleaved gp23 present in the 4-del mutant proheads. f, g Cryo-EM images of the expanded heads purified from WT and 4-del mutant infections as shown in panel e. Yellow and red arrows identify prolate, icosahedral capsids in side and top views, respectively. Blue arrows identify round capsids of different sizes. See Methods for more details.