![]() The threefold symmetric (C3) reconstruction of the free spike trimer resulted in a 3.6 Å–resolution map, whereas the asymmetric reconstruction (C1) was refined to 3.8-Å resolution ( Fig. ![]() Initial two-dimensional (2D) classification revealed the presence of two distinct classes: free spike trimers and dimers of trimers ( Fig. We next performed single-particle cryo–electron microscopy (cryo-EM) on the spike formulated in PS 80 detergent ( Fig. Matrix-M does not appear to interact with the spike nanoparticles. 2D classes showing individual spikes, higher-order spike nanoparticles, and Matrix-M cages of different sizes. ![]() In the raw micrograph, spike rosettes are circled in yellow and Matrix-M adjuvant cages are circled in white. ( B) Representative negative-stain EM images and 2D classes of SARS-CoV-2 3Q-2P-FL, formulated in PS 80 detergent in the presence of Matrix-M adjuvant. A, Ala D, Asp E, Glu K, Lys L, Leu N, Asn P, Pro Q, Gln R, Arg S, Ser V, Val. The native furin cleavage site was mutated (RRAR→QQAQ) to be protease resistant and stabilized by introducing two proline (2P) substitutions at positions K986P and V987P to produce SARS-CoV-2 3Q-2P-FL spike. Structural elements include a cleavable signal sequence (SS, white), NTD (blue), RBD (green), SD1 and SD2 (light blue), protease cleavage site 2′ (S2′, arrow), fusion peptide (FP, red), heptad repeat 1 (HR1, yellow), central helix (CH, brown), heptad repeat 2 (HR2, purple), TM domain (black), and CT (white). ( A) Linear diagram of the sequence and structure elements of the FL SARS-CoV-2 spike protein showing the S1 and S2 ectodomain. Tight clustering of the spikes in the NVAX-CoV2373 nanoparticle formulation may lead to stronger immune responses over soluble trimers alone, similar to other viral glycoprotein immunogens (hemagglutinin and respiratory syncytial virus F) ( 12, 13). ![]() Imaging revealed trimeric spike proteins present as free trimers or as multitrimer rosettes, containing as many as 14 trimers with their TM domains enclosed in micellar cores of PS 80 detergent ( Fig. To characterize the structural integrity of the 3Q-2P-FL immunogen, we performed negative-stain electron microscopy of the FL spike constituted in PS 80 in the presence of Matrix-M adjuvant, recapitulating the vaccine formulation being tested in humans. ![]() The FL spikes, expressed and purified from insect cells, were formulated in 0.01% (v/v) polysorbate 80 (PS 80) detergent. The final construct, SARS-CoV-2-3Q-2P, was also modified at the S1/S2 polybasic cleavage site from RRAR to QQAQ to render it protease resistant, along with two proline substitutions at residues K986 and V987 in the S2 fusion machinery core for enhanced stability ( Fig. Here, we describe the structure of a leading SARS-CoV-2 S vaccine candidate (NVAX-CoV2373) based on a full-length (FL) S, residues 1 to 1273, which includes the transmembrane (TM) and the cytoplasmic tail (CT) ( Fig. The structure of the stabilized SARS-CoV-2 spike ectodomain has been solved in its prefusion conformation and resembles the SARS-CoV spike ( 9– 11). Like other type 1 fusion proteins, the SARS-CoV-2 S prefusion trimer is metastable and undergoes structural rearrangement from a prefusion to a postfusion conformation upon S-protein receptor binding and cleavage ( 7, 8). ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |