Research Article

Cryo-EM Structure of a Fully Glycosylated Soluble Cleaved HIV-1 Envelope Trimer

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Science  20 Dec 2013:
Vol. 342, Issue 6165, pp. 1484-1490
DOI: 10.1126/science.1245627

Knowing the Enemy

Infection of host cells by HIV-1 is mediated by an envelope glycoprotein (Env) trimeric spike on the surface of the virus. Proteins comprising the Env trimer must be cleaved for infectivity, and thus viral fusion involves three Env conformations. The flexibility of the Env trimer has made it a challenge to determine a high-resolution structure, although such a structure is key both for understanding trimer function and for guiding vaccine design. Lyumkis et al. (p. 1484) and Julien et al. (p. 1477) studied soluble cleaved trimers stabilized by specific mutations but that have kept a near-native antigenicity profile. Lyumkis et al. present a high-resolution structure of the trimer in complex with a broadly neutralizing antibody, and Julien et al. present a crystal structure of the trimer in complex with another broadly neutralizing antibody.


The HIV-1 envelope glycoprotein (Env) trimer contains the receptor binding sites and membrane fusion machinery that introduce the viral genome into the host cell. As the only target for broadly neutralizing antibodies (bnAbs), Env is a focus for rational vaccine design. We present a cryo–electron microscopy reconstruction and structural model of a cleaved, soluble Env trimer (termed BG505 SOSIP.664 gp140) in complex with a CD4 binding site (CD4bs) bnAb, PGV04, at 5.8 angstrom resolution. The structure reveals the spatial arrangement of Env components, including the V1/V2, V3, HR1, and HR2 domains, as well as shielding glycans. The structure also provides insights into trimer assembly, gp120-gp41 interactions, and the CD4bs epitope cluster for bnAbs, which covers a more extensive area and defines a more complex site of vulnerability than previously described.

HIV-1 currently infects more than 34 million people worldwide and causes AIDS. The availability of antiviral therapies has greatly reduced the death toll, particularly in the Western world, but has not yet reduced the global spread of this deadly pathogen. A successful preventive vaccine would be a large step toward this critical goal. The trimeric viral envelope glycoprotein (Env) spike, a major vaccine development target (1), consists of three gp120 subunits that contain the CD4 receptor and co-receptor binding sites and three gp41 subunits that drive membrane fusion. Immune selection pressure creates extensive Env sequence variation that complicates vaccine development, but trimer-targeting broadly neutralizing antibodies (bnAbs) provide important clues about vulnerable Env sites (1). Critical features of bnAb epitopes have been revealed by x-ray structures of fragment antigen binding (Fab) complexes with the gp120 core, gp120 outer domain, gp41 peptides, and scaffolded epitopes, or from glycan arrays (29). These structures are based on only a subcomponent of the Env spike and do not reveal the full complement of intersubunit contacts and constraints. Low-resolution electron microscopy (EM) structures of the trimer provide an overall architecture (1016) but do not define the molecular details of bnAb epitopes.

Here, we used cryo-EM to study soluble (truncated at residue 664), cleaved recombinant trimers from the BG505 genotype, stabilized by specific substitutions, including a disulfide bond (termed SOS) between residue 501 (HXB2 numbering) and 605, and an Ile-to-Pro mutation at position 559 (termed IP) (17, 18). These BG505 SOSIP.664 gp140 trimers are highly stable and homogeneous, have a near-native antigenicity profile (19), and display a well-defined shape when viewed by negative-stain EM at intermediate resolution (11, 12, 14, 20). We present here the cryo-EM structure at 5.8 Å resolution of this Env trimer in complex with bnAb PGV04 against a CD4 binding site (CD4bs) epitope. The structure reveals the overall organization of Env, the interaction between gp120 and gp41 subunits, and how trimer formation affects the CD4bs and its associated bnAb epitopes.

Specimen Preparation, EM Data Acquisition, and Image Processing of SOSIP Trimers

We produced BG505 SOSIP.664 gp140 trimers in human embryonic kidney (HEK) 293T cells, and therefore they have a typical human cell glycosylation profile. The Env trimer is relatively small by EM standards (~425 kD, of which almost half is glycan) and lacks features that facilitate high-resolution image processing (21). We therefore adopted a cryo-EM feature enhancement strategy, as recently described (22), by adding PGV04 Fabs as fiducial markers for computational alignment of the trimer. We recorded the EM data on a direct electron detector, which improves the signal relative to conventional methods and enables correction for beam-induced motion and specimen drift (23). Image-processing algorithms similar to those that have recently provided near-atomic resolution characterization of select macromolecular complexes (24, 25) were used in the analysis. Together, these cryo-EM technical advances, combined with design and production of a stable soluble Env trimer, enabled reconstruction of the SOSIP.664-PGV04 complex to 5.8 Å resolution (Fig. 1 and fig. S1). The reconstructed electron potential map provided sufficient detail for modeling most of gp120, including the variable loops and the heptad repeat 1 (HR1) and HR2 components of gp41 (movie S1). The EM reconstruction was validated by the Fab and gp120 densities that were in excellent agreement with the previously determined structures, by several recently described quantitative metrics for EM (21, 26, 27), and also by an independently obtained x-ray structure of the same trimer (from HEK 293S GnTI−/− cells and with a simpler glycan profile) in complex with the PGT122 bnAb at a similar resolution (fig. S2H) (28). The EM map presented here is substantially improved in resolution and in features relative to previous trimer reconstructions; it also revealed partially discontinuous density surrounding the periphery of the trimer that is consistent with N-linked glycans on both gp120 and gp41 (fig. S4) (29).

Fig. 1 5.8 Å EM reconstruction and model of Env trimer in complex with PGV04.

(A and B) Side (A) and top (B) views of BG505 SOSIP trimer EM reconstruction (left) and corresponding model (right). Segmentation and color coding: gray, PGV04; blue, gp120; orange, gp41; purple, V1/V2; green, V3. (C) The center panel shows a side view of the EM map alone with the Fab density removed. The outer panels show the modeled portion corresponding to the boxed region in the middle panel. The viral membrane would be at the bottom of the figure.

Structural Arrangement of gp120 and Variable Loops V1, V2, and V3

The gp120 core crystal structure in complex with PGV04 [PDB ID: 3SE9 (30)] was docked into the EM map and further refined (31). The crystal structure of a scaffolded V1/V2 protein [PDB ID: 3U4E (9)] could also be fitted into density at the trimer apex (Fig. 2A and fig. S5A). Densities corresponding to N-acetylglucosamine moieties of the glycans at the Asn156 and Asn160 sites (numbering relative to the reference strain HXB2) within V1/V2 are apparent at the trimer apex (Fig. 2B and figs. S4 and S5A) and are consistent with the arrangement of the V1/V2 Asn156 and Asn160 glycans predicted by a low-resolution model of the same trimer in complex with bnAb PG9 (14). The V1 loop is in a slightly different conformation from that seen in scaffolded CAP45 V1/V2 (9). The V2 loop could not be built in its entirety but was localized above the CD4bs (fig. S5, A and B) in a position that restricts access to the epitope. The V3 loop is situated directly beneath the V1/V2 hairpin with the tip pointing toward the trimer axis and interacting with the V2 base from the adjacent protomer, in a different configuration than previously observed (Fig. 2C and fig. S5, A and C) (8). The Asn197 glycan at the V2 base is at the interprotomer interface (Fig. 2B and fig. S5A) and may shield V3 epitopes on the trimer; various V3 antibodies have higher affinities for monomeric than for trimeric Env, consistent with such steric constraints (32, 33). Overall, the gp120 subunits have a compact globular configuration and are assembled into a trimer through intra- and interprotomer interactions at the apex that involve the V1/V2 and V3 loop structures (Fig. 1 and Fig. 2B), consistent with previous observations (34, 35). This arrangement exposes several basic residues at the trimer apex that are important targets for bnAbs such as PG9, PG16, PGT145, and CH01, which have acquired negatively charged sulfated tyrosine moieties in their paratopes (9) (Fig. 2D).

Fig. 2 Structures of gp120 V1/V2/V3 variable loops.

(A) Comparison of the V1/V2 domain from the EM model (purple) and the previously published x-ray structure (3U4E, white) with key residues labeled (N, Asn). The structures are nearly identical except for the base of V1/V2 (strands A and D) and the V1 loop. (B) Quaternary arrangement of V1, V2, and V3 regions of gp120 at the top of the trimer. The positions of glycans at Asn156, Asn160, and Asn197 have been highlighted as red spheres. Lines denote approximate protomer boundaries. (C) Superposition of gp120 and the V3 loop from the EM model (blue/green) onto a previous x-ray structure containing a V3 loop (2B4C, gray/white). Whereas gp120 is structurally conserved, the V3 β-hairpin loop exhibits a different configuration. Specifically, V3 bends around a hinge formed near residues 302 and 326. The N-terminal residues of V3 up to residue 301 and the C-terminal residues after residue 327 are structurally similar. Residues 1 to 90 and 126 to 196 are omitted for clarity. (D) Surface view of the modeled portion in (B), with the basic residues (Arg and Lys) colored blue. A large number of basic amino acids are localized to the trimer apex.

Structural Arrangement of gp41

gp41 forms a pedestal at the base of the trimer (Fig. 3, A and B). Each protomer is characterized by two long, prominent helices. The first (HR1) forms a three-helix bundle with the neighboring protomers in the trimer core; the second (HR2) wraps around the outer periphery of the trimer base, angling downward and with its C terminus proximal to the viral membrane (Fig. 3, A and B). The three-helix bundle is formed by the C-terminal half of HR1 and resembles the arrangement in the crystal structure of postfusion gp41 (36), and also in a proposed open, intermediate state as observed by single-particle EM (37) (Fig. 3, C and D). To assess whether the three-helix bundle is a feature induced by PGV04 binding, we calculated an independent 12.7 Å reconstruction of the unliganded trimer (fig. S6). The three-helix bundle was present in this unliganded structure; hence, it is likely that the gp41 conformation that we describe here represents the closed, prefusion state, and not an activated intermediate as previously suggested (37). Just above the three-helix bundle and below the trimer apex, we observe a small opening that is continuous with the exterior (fig. S7). The large hole that is a reported feature of the trimer core in some previous models is likely a result of lower-resolution reconstructions (>20 Å) (38), as illustrated in fig. S3.

Fig. 3 Structure of gp41.

(A) Segmented EM density map of the gp41 trimer with gp120 removed. The C-terminal half of HR1 (rust) forms a three-helix bundle at the center of the trimer; the C-terminal half of HR2 (yellow) forms a helical structure that wraps around the trimer base. Additional density that is not assigned in the model (beige) likely corresponds to the intervening region between HR1 and HR2, including the disulfide loop, as well as C1 and C5 from gp120. Density parallel to HR1 (brown) likely corresponds to the N-terminal half of HR1, the fusion peptide proximal region (FPPR), and the fusion peptide (FP). (B) Modeled portion corresponding to the same views of the EM density maps in (A). (C) EM density of the three-helix bundle formed by HR1 in the PGV04-bound trimer structure. (D) Overlay of the EM density of the three-helix bundle formed by HR1 in the PGV04-bound structure that is filtered to 9.5 Å (orange) with the 9 Å reconstruction of a 17b-bound SOSIP gp140 trimer (gray, EMDB-5462) (37). (E) An 8.2 Å reconstruction of a SOSIP trimer from which the last 14 amino acids were deleted (SOSIP.650-PGV04). The difference between the SOSIP.650 and SOSIP.664 maps corresponds to a short helical segment (red) at the end of HR2 that projects toward the adjacent protomer (see also fig. S8).

To identify the gp41 C terminus, we calculated an ~8 Å reconstruction of a similar trimer but with the last 14 amino acids deleted (designated SOSIP.650) (Fig. 3E). Difference maps showed that a segment corresponding to ~3.5 helical turns, or ~14 amino acids, was absent from the SOSIP.650 reconstruction (Fig. 3E and fig. S8). Additionally, our data indicate that residues around position 650 in HR2 interact strongly with the bottom of HR1 (fig. S9).

The N-terminal half of HR1, including a short helix, and the fusion peptide proximal region (FPPR) and fusion peptide (FP) extend from the top of the internal coiled coil around the three-fold axis and turn down toward the trimer base (Fig. 3A). The internal region at the base of the trimer contains additional density that could not be assigned unambiguously but likely corresponds to the intervening residues between HR1 and HR2, as well as the gp120 C1 and C5 regions (Fig. 3, A and B). This interpretation is consistent with the known intersubunit interactions between gp120 C1/C5 and gp41 residues ~589 to 610 (18). This element of gp41 contains the internal disulfide-bonded loop and also the engineered Cys substitutions that covalently link gp120 residue 501 to gp41 residue 605 via a disulfide bond in SOSIP gp140 (18). The relatively hydrophobic gp120 N and C termini and nearby gp41 residues are sequestered toward the middle of the trimer. We have tentatively assigned the hydrophobic FP to this solvent-inaccessible region (Fig. 3B); there are similarities here to other viral fusion glycoproteins from influenza, respiratory syncytial virus, and Ebola virus (28, 3941). Localization at the gp120-gp41 interface would allow the FP to be released in response to CD4 and co-receptor–induced conformational changes within gp120 and gp41 that drive membrane fusion.

PGV04 Binding Site on the Env Trimer

Subtle differences in the Fab PGV04 interaction with gp120 are apparent when the monomeric and trimeric forms of core gp120 are aligned, which may be explained by trimer-specific contacts (Fig. 4A). On the glycosylated trimer, the Fab light chain interacts extensively with glycan Asn276 (Fig. 4B and fig. S17). The same glycan prevents the VRC01 germline antibody from binding to gp120 monomers (42), so it may be a general impediment to this bNAb class. The glycosylated trimer EM structure also contains additional density for a region corresponding to the V2 loop and/or glycans (Fig. 4B) that likely play a role in recognition of the CD4bs.

Fig. 4 PGV04 interactions with Env trimer.

(A) Superposition of the gp120 portion of the x-ray structure of the gp120:PGV04 complex (3SE9, white/gray) and the EM structure of the SOSIP.664 trimer PGV04 complex (blue/green) illustrates subtle differences in the PGV04:gp120 interaction. Residues 121 to 202 and 395 to 410 are omitted for clarity. (B) Elaborated nature of the CD4 binding site with densities of interest highlighted (red, Asn197; light blue, Asn276; blue, Asn363 and Asn386; purple, V2 and/or portions of neighboring protruding glycans). Densities corresponding to glycans and V2 are positioned to influence how the CD4bs is recognized. A glycan at Asn276 makes extensive interactions with the light chain of PGV04. The full PGV04 epitope on the trimer within 5 Å is denoted by green cross-hatching. (C) Top and side views of EM reconstruction of one BG505 SOSIP.664 trimer (white) bound to two PGV04 Fabs (green) at 7.9 Å resolution. (D) Density representing statistically significant differences between a PGV04-liganded and an unliganded protomer [“t-map” contoured at P < 0.001 (31)]. This view is the same as (B). Raw data are shown in fig. S16. Difference density (blue) in the vicinity of Asn276, Asn363/Asn386 , and V2 is apparent when PGV04 is bound at the CD4bs. (E) Effect of glycosylation on PGV04 binding to the SOSIP.664 gp140 trimer, as evaluated by ITC. PGV04 binds a deglycosylated version of the same trimer with slightly higher affinity and stoichiometry, as well as reduced entropy. Bar graphs represent the means (±SD) for the binding affinity, stoichiometry, entropy, and enthalpy derived from at least two independent titrations. Non-reducing SDS–polyacrylamide gel electrophoresis (PAGE) shows the difference in molecular weight when the trimer is produced in 293T cells (WT) or in 293S GnTI−/− cells before and after deglycosylation with EndoH. (F) Raw data (top) and binding isotherms (bottom) for representative ITC binding experiments.

Many SOSIP.664 trimer particles had fewer than three PGV04 Fabs bound, even though the Fab was in molar excess of trimer during sample preparation. We used template-based 3D sorting of single-particle images to generate trimer populations with three, two, one, and zero Fabs bound. Each of the subsets was then independently refined to generate maps with resolutions of 5.8 Å, 7.9 Å, 16.9 Å, and 19.1 Å, respectively (figs. S10 to S15). The 7.9 Å, 2-Fab reconstruction (Fig. 4C) sufficed to interpret subtle structural differences created by the presence or absence of a bound Fab. Specifically, PGV04 binding induced positive density corresponding to the putative V2 loop and/or glycans Asn363 and Asn386 (Fig. 4D). Additional density corresponding to glycan Asn301 from the neighboring gp120 protomer and Asn276 from the same gp120 protomer was also seen to interact with PGV04 (fig. S16C) (43). One interpretation is that PGV04 binding stabilizes both the glycans and the flexible loops in the trimer structure.

Solution isothermal titration calorimetry (ITC) measurements also revealed that PGV04 bound BG505 SOSIP.664 trimers in a substoichiometric manner (<2), consistent with the EM results (Fig. 4E). The EM map does not reveal an obvious structural impediment to stoichiometric PGV04 binding (i.e., 3 per trimer). One hypothesis for why some trimers do not bind the bnAb, or do so at a low stoichiometry, is that incomplete or differential glycan processing creates some microheterogeneity. PGV04 binding to 293S cell–produced trimers that bear only high-mannose sugars was again substoichiometric (i.e., <2); however, when trimers were enzymatically deglycosylated, the number of bound Fabs increased from <2 to ~2.5 and the average binding affinity KD improved to 73 nM from 135 nM (Fig. 4F), which suggests that specific glycans of variable composition and size can indeed interfere with antibody binding to the CD4bs. The unfavorable entropic contribution of the binding signal was also lower for the deglycosylated trimers. The entropic cost of locking normally flexible glycans in place may be yet another viral defense against antibodies, potentially one that restricts the stimulation of bnAb germline B cell receptors (42, 44). Eliminating such glycans may facilitate the design of immunogens intended to induce CD4bs antibodies (42, 44).

Quaternary Nature of the CD4 Binding Site

The CD4bs is relatively conserved and immunogenic in the context of HIV-1 infection. Env subunit vaccines, such as monomeric gp120 and uncleaved gp140, have failed to elicit bnAbs against this site while often inducing non-neutralizing or poorly neutralizing Abs that recognize overlapping epitopes (1). This is likely because the CD4bs is presented in a non-native context and thus elicits antibodies that are non-neutralizing because they cannot bind to compact native trimers. The EM structure that we describe delineates the narrow range of approach angles available to CD4bs bnAbs when engaging the trimer, and shows how epitope complexity makes it difficult to target this site effectively via non-native immunogens.

The CD4-induced subdomain known as the bridging sheet is presented on the SOSIP.664 gp140 trimers differently from how it appears on various versions of monomeric gp120 (4). Specifically, the strand arrangement at the base of V1/V2 is reversed on the trimer, such that V2 forms an adjacent/parallel interaction with β20 instead of the antiparallel β-interaction between the V1 base and β20 found in the gp120 core (Fig. 5A and fig. S18). Thus, quaternary interactions mean that the topological relationship between the base of V2 (which includes the Asn197 glycan) and the CD4bs is not the same on trimeric and monomeric gp120.

Fig. 5 Quaternary nature of the CD4 binding site.

(A) Relative to gp120 monomer structures (e.g., PDB IDs 1GC1 and 3TGT), the bridging sheet has a different topology in the trimer, as illustrated by the cartoon models. Trimer formation alters how the base of V2 (β2–β3) and β20–β21 are arranged relative to the CD4bs (see also fig. S18). (B) In the EM reconstruction of the trimer, CD4 and CD4bs bnAbs bound to one protomer are in close proximity to the adjacent protomer. The gp120-CD4bs Fab x-ray structures were docked into the EM map via alignment of the gp120 portion of the structures. All regions of the EM map within 5 Å of PGV04, VRC01, and CD4 are colored yellow and are similar to the areas within 5 Å as observed in the corresponding x-ray structures (fig. S20 shows all areas within 2 Å). (C) Major clashes with some CD4bs antibodies differ markedly in the trimeric context. Areas of the EM map within 2 Å of the Fabs are colored red (fig. S20 shows all areas within 5 Å). The minimal clashes with PGV04 serve as a comparator. b12 has only a few clashes with V1/V2, glycans, and V3 from the adjacent protomer, whereas b13 and F105 have extensive clashes. Note that although b12 is typically a bnAb, it does not neutralize the BG505 virus or bind the BG505 SOSIP.664 trimer (19). Thus, the clashes that we visualize here are consistent with each antibody being unable to neutralize the corresponding virus (19). (D) Areas in the EM map within 5 Å of docked Fabs, all of which contain acidic HFR3 insertions, are colored yellow (fig. S20 shows all areas within 2 Å). In addition to their previously known CD4bs epitopes, these bnAbs also interact with the V3 tip and proximal region near the protomer interface (red box). (E) bnAbs from (D) contain acidic HFR3 residues that interact with the basic residues in the highlighted area. Close-up views show the antibody HFR3 interactions with basic residues in the trimer highlighted in the red boxed regions in (D). Acidic residues in the HFR3 are displayed as sticks; regions in the EM map within 3 Å of basic residues are colored blue and labeled accordingly.

The neighboring protomer also plays an important role in restricting access to the CD4bs. When the heavy chain of PGV04 or the related VRC01 bnAb binds the trimer, it is within 5 Å of a loop (residues 61 and 62) that precedes a short α helix (α0) in C1 of a neighboring gp120 protomer (Fig. 5B); likewise, when CD4 (PDB ID: 4JM2) is docked into the CD4bs on the trimer, it also contacts an adjacent protomer (Fig. 5B). The α0 helix is directly downstream from His66, a residue implicated in regulating the sampling of the CD4-bound conformation (45). Thus, the first stage of CD4 binding may require the trimer to flex to an extent, but once initial contact is made, the trimer is further destabilized, and co-receptor binding and fusion can proceed.

Docking CD4bs antibodies with a range of neutralization capabilities into the EM trimer model suggests that the crystal structures of monomeric gp120-Ab complexes do not completely define this epitope cluster. Additional, quaternary structure-dependent contacts are important for CD4bs Fabs to recognize trimeric Env, with the V1/V2 loop structure influencing how they bind and hence neutralize (Fig. 5C). Previous mutagenesis data are now more readily interpretable; for example, the light-chain CDR1 and CDR3 residues are essential for neutralization by the b12 bnAb (46) because they are positioned to interact with V1/V2 (fig. S19). The non-neutralizing Fabs b13 (PDB ID : 3IDX) and F105 (PDB ID: 3HI1) (6) clash with V1/V2 (same protomer) and V3 (adjacent protomer), impeding their ability to bind the trimer (Fig. 5C). These results are consistent with both BG505 virus neutralization and antibody-binding studies using the corresponding SOSIP.664 gp140 trimers (19).

Immunoglobulin framework insertions and mutations are important for broad and potent neutralization (5). Our structure shows that framework region 3 in the heavy chain (HFR3) of CD4bs antibodies 3BNC117 (also 3BNC60), VRC03, and VRC06 interact with basic residues on an adjacent protomer (Fig. 5, D and E). HFR3 insertions contain acidic residues that point toward conserved basic residues near the V3 tip of the adjacent gp120 protomer (Fig. 5E). A properly folded trimer positions these basic residues in an appropriate quaternary orientation for interacting with CD4bs bnAbs. The preference of VRC06 for cleaved over uncleaved trimers (47) is also consistent with previous observations that cleavage is critical for Env to adopt a native-like conformation (20).

Affinity maturation events in the evolution of the CH103 CD4bs bNAb involve the early appearance of acidic residues in the HFR3 loop that then persist (48). However, HFR3 makes no contact with gp120 in the crystal structure of the monomeric gp120-CH103 complex (PDB ID: 4JAN) (48). The trimer structure shows that HFR3 of CH103 is in close proximity to the basic residues on V3 from the adjacent gp120 (Fig. 5E). Thus, the quaternary context of the CH103 epitope helps to explain why somatic hypermutation within HFR3 is critical and why the acidic residues are conserved once they appear.

The V3 interactions and the evolution of framework mutations essential for neutralization breadth and potency are likely to be relevant to many CD4bs bnAbs. The quaternary constraints on these epitopes will affect how potent CD4bs bnAbs, such as VRC01, are induced and then undergo the affinity maturation process.


The 5.8 Å resolution cryo-EM structure presented here is similar to the x-ray structure of the same SOSIP.664 gp140 trimers in complex with bnAb PGT122 (fig. S2H) (28). Both structures are, however, very different from a 6 Å structure recently reported for a detergent-solubilized, full-length but uncleaved trimer (49). Differences in the design of the Env trimers might contribute to some of these structural inconsistencies. Alternatively, the cryo-EM methodology used to derive the full-length uncleaved trimer structure has been criticized, and the controversy is as yet unresolved (5054). Considered in isolation, our soluble SOSIP.664 gp140 trimer structures are consistent with known Env structure-function relationships and also provide insight into the prefusion form of the trimer. The structures explain how bnAbs recognize their epitopes and why quaternary constraints prevent some non-neutralizing Abs from binding the trimer. Overall, the structures are a step toward understanding trimeric Env at atomic-level resolution and should guide improvements in Env-based vaccines.

Supplementary Materials

Materials and Methods

Figs. S1 to S20

Movie S1

References (5574)

References and Notes

  1. Most of the predicted N-linked glycosylation sites were associated with some additional density in the EM maps (fig. S4). This density is partially discontinuous for the mobile glycan components, providing a spiky appearance surrounding the periphery of the trimer, particularly within gp120 and variable loop regions. Density corresponding to glycans was generally consistent among three independently determined maps, namely BG505 SOSIP.664 bound to three (5.8 Å) or two Fabs (7.9 Å), and BG505 SOSIP.650 bound to three Fabs (8.2 Å). Thus, the spiky appearance of the map is likely due to glycans and not to overrefined or oversharpened data.
  2. See supplementary materials on Science Online.
  3. No such differences were observed in control cases between the Fab-labeled protomers within the 2-Fab reconstruction (fig. S16).
  4. Acknowledgments: We thank Y. Cheng and X. Li for providing raw frame alignment scripts prior to publication, R. Henderson for making the makestack_HRnoise.exe program available for use to assess the overfitting of the EM data, J. Korzun for technical assistance, J.-C. Ducom (TSRI) for support with computational resources, and C. R. King and W. Koff for support and encouragement. Supported by NIH grants HIVRAD P01 AI82362 (J.P.M., I.A.W., and A.B.W.) and R01 AI36082 (I.A.W.); the International AIDS Vaccine Initiative Neutralizing Antibody Consortium (D.R.B., J.P.M., I.A.W., A.B.W.); Scripps CHAVI-ID (UM1 AI100663) (D.R.B., I.A.W., A.B.W.); a Vidi grant from the Netherlands Organization for Scientific Research (R.W.S.); a Starting Investigator Grant from the European Research Council (R.W.S.); and a Canadian Institutes of Health Research fellowship (J.-P.J.). The EM work was conducted at the National Resource for Automated Molecular Microscopy at The Scripps Research Institute, which is supported by the Biomedical Technology Research Center program (GM103310) of the National Institute of General Medical Sciences (B.C., C.S.P.). 3D visualizations were generated using the UCSF Chimera package. The EM reconstructions have been deposited in the Electron Microscopy Data Bank under accession codes EMD-5779, EMD-5780, EMD-5781, and EMD-5782. The structure coordinates have been deposited in the Protein Data Bank under accession code 3J5M. Sharing of other materials will be subject to standard material transfer agreements. Raw EM data will be provided upon request. The content is the responsibility of the authors and does not necessarily reflect the official views of NIGMS or NIH. IAVI has previously filed a patent relating to the BG505 SOSIP.664 trimer: U.S. Prov. Appln. No. 61/772,739, titled “HIV-1 envelope glycoprotein,” with inventors M. Caulfield, A.C., H. Dean, S. Hoffenberg, C. R. King, P.-J.K., A. Marozsan, J.P.M., R.W.S., A.B.W., I.A.W., and J.-P.J. This is manuscript 25060 from The Scripps Research Institute.
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