National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
UM1AI100663
United States
Bill & Melinda Gates Foundation
OPP1115782
United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
UM1AI144462
United States
Citation
Journal: Cell Rep / Year: 2020 Title: HIV-1 Envelope and MPER Antibody Structures in Lipid Assemblies. Authors: Kimmo Rantalainen / Zachary T Berndsen / Aleksandar Antanasijevic / Torben Schiffner / Xi Zhang / Wen-Hsin Lee / Jonathan L Torres / Lei Zhang / Adriana Irimia / Jeffrey Copps / Kenneth H ...Authors: Kimmo Rantalainen / Zachary T Berndsen / Aleksandar Antanasijevic / Torben Schiffner / Xi Zhang / Wen-Hsin Lee / Jonathan L Torres / Lei Zhang / Adriana Irimia / Jeffrey Copps / Kenneth H Zhou / Young D Kwon / William H Law / Chaim A Schramm / Raffaello Verardi / Shelly J Krebs / Peter D Kwong / Nicole A Doria-Rose / Ian A Wilson / Michael B Zwick / John R Yates / William R Schief / Andrew B Ward / Abstract: Structural and functional studies of HIV envelope glycoprotein (Env) as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the ...Structural and functional studies of HIV envelope glycoprotein (Env) as a transmembrane protein have long been complicated by challenges associated with inherent flexibility of the molecule and the membrane-embedded hydrophobic regions. Here, we present approaches for incorporating full-length, wild-type HIV-1 Env, as well as C-terminally truncated and stabilized versions, into lipid assemblies, providing a modular platform for Env structural studies by single particle electron microscopy. We reconstitute a full-length Env clone into a nanodisc, complex it with a membrane-proximal external region (MPER) targeting antibody 10E8, and structurally define the full quaternary epitope of 10E8 consisting of lipid, MPER, and ectodomain contacts. By aligning this and other Env-MPER antibody complex reconstructions with the lipid bilayer, we observe evidence of Env tilting as part of the neutralization mechanism for MPER-targeting antibodies. We also adapt the platform toward vaccine design purposes by introducing stabilizing mutations that allow purification of unliganded Env with a peptidisc scaffold.
History
Deposition
Feb 4, 2020
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Header (metadata) release
Apr 22, 2020
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Map release
Apr 22, 2020
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Update
May 13, 2020
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Current status
May 13, 2020
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_21330.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation
Ectodomain from nanodisc of C-terminally truncated HIV-1 Envelope glycoprotein clone BG505 in complex with PGT151 Fab
Voxel size
X=Y=Z: 1.15 Å
Density
Contour Level
By AUTHOR: 0.623 / Movie #1: 0.623
Minimum - Maximum
-1.3360683 - 3.9776995
Average (Standard dev.)
0.003749174 (±0.045142528)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
420
420
420
Spacing
420
420
420
Cell
A=B=C: 483.0 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.15
1.15
1.15
M x/y/z
420
420
420
origin x/y/z
0.000
0.000
0.000
length x/y/z
483.000
483.000
483.000
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
420
420
420
D min/max/mean
-1.336
3.978
0.004
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Supplemental data
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Sample components
-
Entire : Ectodomain from nanodisc of C-terminally truncated HIV-1 Envelope...
Entire
Name: Ectodomain from nanodisc of C-terminally truncated HIV-1 Envelope glycoprotein clone BG505 in complex with PGT151 Fab
Components
Complex: Ectodomain from nanodisc of C-terminally truncated HIV-1 Envelope glycoprotein clone BG505 in complex with PGT151 Fab
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Supramolecule #1: Ectodomain from nanodisc of C-terminally truncated HIV-1 Envelope...
Supramolecule
Name: Ectodomain from nanodisc of C-terminally truncated HIV-1 Envelope glycoprotein clone BG505 in complex with PGT151 Fab type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#11
Source (natural)
Organism: Homo sapiens (human)
Recombinant expression
Organism: Homo sapiens (human)
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
-
Sample preparation
Concentration
0.18 mg/mL
Buffer
pH: 7.4
Grid
Support film - Material: CARBON / Support film - topology: CONTINUOUS / Details: unspecified
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
Details
C-terminally truncated Env purified with stabilizing PGT151 Fab. Assembled to DOPC+CHS nanodisc with MSP1D1 scaffold by detergent removal with biobeads.
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Electron microscopy
Microscope
FEI TALOS ARCTICA
Image recording
#0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #0 - Detector mode: COUNTING / #0 - Number real images: 2343 / #0 - Average electron dose: 50.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #1 - Average electron dose: 50.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stage
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
+
Image processing
Image recording ID
1
Particle selection
Number selected: 117554
CTF correction
Software - Name: Gctf (ver. 1.06)
Startup model
Type of model: OTHER / Details: Unliganded Env ectodomain low-pass filtered to 15A
Final reconstruction
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 27027
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