Journal: bioRxiv

Article Title: HIV-1 Envelope and MPER antibody structures in lipid assemblies

doi: 10.1101/838912

Figure Lengend Snippet: Stabilizing PGT151 Fab highlighted in blue. Different assembly types are presented with cartoons next to negative stain and cryo-EM 3D reconstructions. Env ectodomain is highlighted in gray, PGT151 in blue, micelle and lipid bilayer in yellow. A) The ectodomain can be reconstructed to 3 – 5Å resolution from all lipid-detergent micelle samples. B and C) Cryo-EM reconstructions of nanodiscs with height of the ectodomain from the bilayer surface indicated and density that passes through the disc of PC64FL is highlighted in purple. D) In bicelles, additional compositional heterogeneity limits the analysis to negative-stain EM reconstructions. E) Summary of the rotational, positional and compositional heterogeneity.

Article Snippet: Cryo-EM data were collected on a Titan Krios and Talos Arctica operating at 300 keV or 200 keV, respectively, both equipped with a K2 direct electron detector (Gatan) using Leginon.

Techniques: Staining, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: HIV-1 Envelope and MPER antibody structures in lipid assemblies

doi: 10.1101/838912

Figure Lengend Snippet: Related to , , and . A) negative stain EM, and B) cryo-EM.

Article Snippet: Cryo-EM data were collected on a Titan Krios and Talos Arctica operating at 300 keV or 200 keV, respectively, both equipped with a K2 direct electron detector (Gatan) using Leginon.

Techniques: Staining, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: HIV-1 Envelope and MPER antibody structures in lipid assemblies

doi: 10.1101/838912

Figure Lengend Snippet: A) Representative 2D class averages from negative-stain EM data from lipid assemblies of PC64FL and AMC011FL in complex with MPER-targeting antibodies showing tilting of ectodomain and estimated Fab angles in relation to bilayer. Lipid bilayer is highlighted in yellow and MPER Fab in red. B) Comparison of VRC42.01 Fab binding angle in nanodisc and micelle. PGT151 Fab is highlighted in blue throughout the figure. C) Low-pass filtered cryo-EM reconstructions of AMC011FL nanodisc in complex with 10E8 Fab with different Fab occupancies. Highest resolution and particle count were obtained with complex containing one copy of PGT151 Fab and three copies of 10E8 Fab, which is used for panels D through I. D) Low-pass filtered reconstructions with one, two or three copies of 10E8 Fab showing first tilting and then lifting of Env with addition of third 10E8 Fab. Approximate tilt degree of Env in relation to bilayer surface is indicated as well as change from vertical position. E) Highest resolution reconstruction and epitopes of the three Fabs with 10E8 Fab crystal structure docked in (PDB ID:5T80). Epitope components are highlighted as indicated in the panel below. Distance from bilayer surface is also indicated. F) Local resolution estimation showing up to 5Å resolution in the ectodomain and stabilized 10E8 Fab epitope. G) Horizontal (Fab A) and tilted (Fab C) Fab stabilized TMD orientation are highlighted in purple. H) Two Fab orientations and dependency of Fab C on the PGT151 position is highlighted in red in low-pass filtered maps. I) Comparison of 10E8 Fab docking in AMC011FL nanodisc and JRFL∆CT micelle reconstructions (EMD-3312) showing a ~10 Å change in the distance between the MPER peptides (residue Gln135 in PDB 5T80).

Article Snippet: Cryo-EM data were collected on a Titan Krios and Talos Arctica operating at 300 keV or 200 keV, respectively, both equipped with a K2 direct electron detector (Gatan) using Leginon.

Techniques: Staining, Binding Assay, Cryo-EM Sample Prep