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

Abstract

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 SOSIP gp140 trimer in complex with a CD4 binding site (CD4bs) bnAb, PGV04, at 5.8 Å resolution. The structure reveals the spatial arrangement of Env components, including the V1/V2, V3, HR1 and HR2 domains, and 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.

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

“…A total of 3,037 images were collected for RQC ΔR and 2,048 images were collected for RQC ΔR/tae2Δ . For the RQC ΔR/rqc1Δ sample, a Gatan K2 Summit direct detection device was used to record 20 raw frames collected over a 5-s exposure time (250 ms per frame), with each frame receiving a dose of ∼9.4 e − /pixel/s, as previously described (26). A total of 839 such "movies" were automatically collected and recorded at a nominal magnification of 29,000×, corresponding to a pixel size of 1.21 Å at the specimen level.…”

“…A total of 839 such "movies" were automatically collected and recorded at a nominal magnification of 29,000×, corresponding to a pixel size of 1.21 Å at the specimen level. The individual frames were aligned as previously described (26). For all datasets, the estimated underfocus ranged from 0.5 to 4.0 μm and was distributed in an approximately Gaussian manner while centered at 2.0-2.25 μm.…”

“…We performed 2D alignment and clustering using CL2D (30) to eliminate any particles that did not display identifiable features. We then performed another round of 2D alignment and clustering using ISAC (32), outside of Appion to eliminate particles that did not consistently produce stable class averages, as previously described (26). After manual micrograph masking, CL2D, and ISAC, 77,962 particles were retained for all subsequent analyses.…”

“…To obtain a better understanding and quantification of particle occupancies corresponding to heterogeneous regions identified using Frealign9, we performed a 3D sorting strategy, similar to that which has been previously described (26), and is further explained in SI Materials and Methods. The described sorting procedure enabled us to calculate and quantify coassociations between the different components, a procedure that is describe in detail in SI Materials and Methods.…”

Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

“…As a consequence, most of the available high-definition structures of gp120 have been obtained with deglycosylated, variable looptruncated core monomers in complex with stabilizing ligands such as soluble CD4 (sCD4) (6)(7)(8)(9)(10). Important information regarding the overall conformation and ligand interactions of the trimeric spike at intermediate resolution has emerged from the use of increasingly refined cryo-electron microscopy (cryo-EM) technologies (11)(12)(13)(14)(15)(16)(17). Moreover, the crystal structure of a stabilized, soluble, cleaved gp140 trimer (BG505 SOSIP.664) at 4.7-Å resolution was reported recently (18).…”