Extended Data Fig. 2: Purification and oligomeric state of the HCMV gH/UL116/UL141 GATE.

a, Schematic representation of the expression and purification process for the HCMV gH/UL116/UL141 GATE complex. b, Size exclusion chromatography profile of the gH/UL116/UL141 GATE complex. Fractions were analyzed by SDS-PAGE under non-reducing conditions, with the fraction indicated by blue asterisks used for cryo-EM studies. c, Western blot analysis of fraction 8, probed with anti-His, anti-gH, and anti-strep antibodies to detect UL141, gH, and UL116, respectively. d, Fractions from size exclusion chromatography were analyzed to determine the oligomeric distribution of the gH/UL116/UL141 GATE complex. Molecular weight markers (in kDa) are indicated on the left. Two predominant species, ~720 kDa and ~480 kDa, were observed, suggesting different oligomeric states. Fractions containing these species are marked with asterisks. e, Representative 2D class averages from negative stain electron microscopy of the size exclusion chromatography fractions. Particles from the ~720 kDa and ~480 kDa fractions exhibit distinct structural features, resembling either a “plus sign” or an “H” shape, suggesting that the gH/UL116/UL141 GATE complex can exist both as a heterotrimer and as a dimer of heterotrimers (hexamer). These data are representative of five independent experiments.