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F plasmids circulate widely among the Enterobacteriaceae through encoded type IV secretion systems (T4SS_Fs). Assembly of T4SS_Fs and associated F pili requires 10 VirB/VirD4‐like Tra subunits and eight or more F‐specific subunits. Recently, we presented evidence using in situ cryoelectron tomography (cryoET) that T4SS_Fs undergo structural transitions when activated for pilus production, and that assembled pili are deposited onto alternative basal platforms at the cell surface. Here, we deleted eight conserved F‐specific genes from the MOBF12C plasmid pED208 and quantitated effects on plasmid transfer, pilus production by fluorescence microscopy, and elaboration of T4SS_F structures by in situ cryoET. Mutant phenotypes supported the assignment of F‐specific subunits into three functional Classes: (i) TraF, TraH, and TraW are required for all T4SS_F‐associated activities, (ii) TraU, TraN, and TrbC are …