Sulfolobus turreted icosahedral virus (STIV) is a model archaeal virus with a pseudo‐T=31 icosahedral virion with a mass of ~64 mega‐Daltons. Although STIV employs pyramidal lysis structures to exit the host, knowledge of the viral entry process is lacking. We therefore initiated crystallographic and cryo‐electron tomographic (CET) studies on STIV attachment and entry. Cryoelectron micrographs showed virion attachment to pili‐like structures emanating from the Sulfolobus host. Tomographic reconstruction and sub‐tomogram averaging revealed pili recognition by the STIV C381 turret protein. Specifically, the triple jelly roll structure of C381 determined by X‐ray crystallography shows that pilus recognition is mediated by conserved surface residues in the second and third domains. In addition, the STIV petal protein (C557), when present, occludes the pili binding site, suggesting that it functions as a maturation protein. Combined, these results demonstrate a role for the namesake STIV turrets in initial cellular attachment and provide the first molecular model for viral attachment in the archaeal domain of life. The work also nicely illustrates the synergistic power of hybrid structural studies utilizing cryo‐electron tomography, single particle analysis and crystallography to model cellular structures at the molecular level.Support or Funding InformationThis work was supported by the National Science Foundation, grants DEB‐1342876 to M.J.Y. and MCB‐1413534 to C.M.L.Tomographic structure of the STIV pilus interaction. (A–J)2.25 nm thick sections at 10 nm spacings through the tomogram. (K) Subvolume averages are shown. The virus/pilus interaction is at the viral turret.(L) The turret highlighted in K rotated 90°, looking into a pilus cross section. C381 is light, medium and dark purple showing the 1st, 2nd and 3rd domains. M)Pilus recognition is mediated by the 2nd and 3rd domains. N)Surface exposed, strictly conserved residues mediate C381/pili recognition.Figure 1