(A) The CAP-1 binding region in the CA hexamer. Sidechains involved in critical interactions are shown explicitly and labeled. Water molecules that form bridging hydrogen bonds are shown as magenta spheres. Putative hydrogen bonds are shown as yellow lines.
(B) Superposition of a representative NTD within the hexamer and the NTD/CAP-1 complex (NTD is pale yellow, CAP-1 is represented by green translucent sphere) (Kelly et al., 2007). The black arrows indicate how the inhibitor displaces key residues from their positions in hexameric CA.
(C) The CA-I binding region. Shown is a representative NTD-CTD contact site (NTD is colored orange, CTD is blue) superimposed with the CTD/CA-I co-crystal structure (CTD is cyan, CA-I is magenta) (Ternois et al., 2005).

(A-C) Cylinders assembled in vitro from pure wildtype HIV-1 CA (A), A14C/E45C (B), and A14C/E45C/W184A/M185A (C), stained with uranyl acetate and visualized via transmission electron microscopy. The black bars represent 100 nm.
(D) Non-reducing SDS-PAGE analysis of in vitro assembly reactions, with wildtype CA (lanes 1 and 4), A14C/E45C (lanes 2 and 5), and A14C/E45C/W184A/M185A (lanes 3 and 6) after assembly under reducing conditions (lanes 1-3), and after crosslinking (lanes 4-5). Molecular weight markers are labeled on the left, and the positions of crosslinked CA oligomers (n = 1-6) are indicated on the right. Note that n > 6 oligomers were not observed, supporting the conclusion that disulfide bond formation is not driven by random diffusional encounters, but by specific interactions within the assembled cylinders.
(E) Size exclusion chromatographic profile of discrete hexamers of crosslinked A14C/E45C/W184A/M185A. Elution volumes of protein standards are indicated.

(A) Side view of a crosslinked hexamer. Each protomer is in a different color, with the NTDs in muted shades and the CTDs in brighter shades. The NTD and CTD layers are indicated.
(B) Top view of a crosslinked hexamer, colored as in panel A. The positions of the first three helices of each protomer are indicated by numbered circles. These form a helical barrel at the core of the hexamer.
(C) Top view of one sheet in the CcmK4-templated CA crystals, which recapitulates the hexameric lattice of authentic capsids at its planar limit. The NTDs are colored orange, and the CTDs blue. This view emphasizes that interactions between neighboring hexamers are mediated only by the CTD.
(D) Top view of the CTD-CTD interface that connects neighboring hexamers, as seen in the CcmK4-templated (cyan) and crosslinked hexagonal crystals (blue), and superimposed with the isolated full-affinity CTD dimer (pink) (Worthylake et al., 1999). The black oval represents the two-fold symmetry axis. We speculate that the slight differences in domain orientations across the dyad arise from the W184A and M185A mutations in the crystallized constructs. The average deviations for all Cα positions are: cyan/pink = 2.4 Å, blue/pink = 2.8 Å, cyan/blue = 0.9 Å.

(A) Side view of one representative protomer (colored blue) and its interaction with the adjacent NTD subunit (orange), as seen in the crosslinked hexamers. Secondary structural elements are labeled. The engineered disulfide is marked by the black asterisk.
(B) Hydrophobic contacts. The sidechain atoms of hydrophobic interfacial residues are represented in stick and translucent space-filling representations and labeled.
(C) Polar and water-mediated contacts. Selected sidechains are shown explicitly and labeled. Green mesh shows unbiased Fo-Fc density contoured at +3σ. These were modeled as water molecules (magenta spheres) in the structure derived from hexagonal crystals. Putative hydrogen bonds are represented by yellow lines. Note that the region around the salt-bridge between P1 and D51 (red asterisk), which forms only upon maturation of CA, is particularly water-rich. These two residues coordinate water-mediated hydrogen bonds with H12, T48, and Q50. We speculate that the missing E45 sidechain (mutated to cysteine in this construct) would participate in this network.
(D) Helix-capping hydrogen bonds at the NTD-CTD interface. Relevant sidechains are shown explicitly and labeled. Hydrogen bonds are shown as yellow lines. An intermolecular C-cap for helix 7 in the blue protomer is not shown. The most critical of these caps appear to be R173 (to helix 3), since it is located in the middle of the hexamerization interface, and is conserved in 1,668/1,670 sequences in the Los Alamos HIV database, with the remaining two conservatively substituted with lysine.

(A) Conformational variation in the tertiary structure of the NTD. The thirteen crystallographically independent high-resolution structures were superimposed on the NTD and shown in Cα trace (orange). Cα deviations were then calculated for each residue, and mapped onto a translucent sausage representation (NTD only). The width and coloring of the sausage are directly proportional to the deviation, with the minimum in white and maximum in red. The superposition also reveals substantial rigid-body motions between the two domains. The intramolecular pivot point is at the flexible linker (black arrowhead).
(B) Conformational variation in the tertiary structure of the CTD (blue), analyzed and displayed as in panel A. The locations of the W814A and M185A mutations are indicated by spheres.
(C) Conformational variation in the quaternary structure of the CA hexamer. The four crystallographically independent models were superimposed on NTD helices 1, 2, 3, 4, and 7 at each of the rotationally equivalent positions and represented as in panels A and B (viewed from the bottom). Only the globular regions that are invariant in tertiary structure are shown in the sausage representation. The hexamers closely obey six-fold rotational symmetry, and there is little variation in the NTD core across all four hexamers. The restricted mobility of the NTD is not simply a consequence of the engineered disulfides, because the templated hexamer was not crosslinked and the NTD rings in all four X-ray models are very similar to the uncrosslinked cryoEM structure (Ganser-Pornillos et al., 2007) (see also Fig. S4).
(D) Stereoview of the thirteen independent high-resolution structures of the NTD-CTD interface, superimposed on the NTD. Lever-like motions of the CTD maintain helix-capping hydrogen bonds (yellow lines) at energetically favorable distances, while producing substantial linear displacements at distal regions of the domain. Hydrogen bonds are shown as yellow lines. Capping residues are shown in stick representation, as are sidechains for Y145 and R162, which form a pi-cation stack that may be energetically significant (black asterisk).