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- EMDB-42517: HIV-1 JR-FL NFL.664 soluble trimer in complex with polyclonal Fab... -

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Basic information

Entry
Database: EMDB / ID: EMD-42517
TitleHIV-1 JR-FL NFL.664 soluble trimer in complex with polyclonal Fab from rabbit U5756
Map datamain map
Sample
  • Complex: HIV-1 JR-FL NFL.664 soluble trimer in complex with polyclonal Fab from rabbit U5756
    • Complex: HIV-1 JR-FL NFL.664 soluble trimer in complex with polyclonal Fab from rabbit U5756
KeywordsEMPEM / polyclonal antibodies / rabbit / HIV vaccine candidate / HIV-1 / Env / VIRAL PROTEIN
Biological speciesHuman immunodeficiency virus 1 / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / negative staining / Resolution: 32.0 Å
AuthorsLee W-H / Ozorowski G / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01 AI157299 United States
CitationJournal: Front Immunol / Year: 2024
Title: mRNA lipid nanoparticles expressing cell-surface cleavage independent HIV Env trimers elicit autologous tier-2 neutralizing antibodies.
Authors: Javier Guenaga / Mehrdad Alirezaei / Yu Feng / Mohamad-Gabriel Alameh / Wen-Hsin Lee / Sabyasachi Baboo / Jocelyn Cluff / Richard Wilson / Shridhar Bale / Gabriel Ozorowski / Paulo Lin / ...Authors: Javier Guenaga / Mehrdad Alirezaei / Yu Feng / Mohamad-Gabriel Alameh / Wen-Hsin Lee / Sabyasachi Baboo / Jocelyn Cluff / Richard Wilson / Shridhar Bale / Gabriel Ozorowski / Paulo Lin / Ying Tam / Jolene K Diedrich / John R Yates / James C Paulson / Andrew B Ward / Drew Weissman / Richard T Wyatt /
Abstract: The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the surface of the virus. The Env gp120 and gp41 subunits mediate receptor binding and membrane fusion and are generated ...The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the surface of the virus. The Env gp120 and gp41 subunits mediate receptor binding and membrane fusion and are generated from the gp160 precursor by cellular furins. This cleavage event is required for viral entry. One approach to generate HIV-1 neutralizing antibodies following immunization is to express membrane-bound Env anchored on the cell-surface by genetic means using the natural HIV gp41 transmembrane (TM) spanning domain. To simplify the process of Env trimer membrane expression we sought to remove the need for Env precursor cleavage while maintaining native-like conformation following genetic expression. To accomplish these objectives, we selected our previously developed 'native flexibly linked' (NFL) stabilized soluble trimers that are both near-native in conformation and cleavage-independent. We genetically fused the NFL construct to the HIV TM domain by using a short linker or by restoring the native membrane external proximal region, absent in soluble trimers, to express the full HIV Env ectodomain on the plasma membrane. Both forms of cell-surface NFL trimers, without and with the MPER, displayed favorable antigenic profiles by flow cytometry when expressed from plasmid DNA or mRNA. These results were consistent with the presence of well-ordered cell surface native-like trimeric Env, a necessary requirement to generate neutralizing antibodies by vaccination. Inoculation of rabbits with mRNA lipid nanoparticles (LNP) expressing membrane-bound stabilized HIV Env NFL trimers generated tier 2 neutralizing antibody serum titers in immunized animals. Multiple inoculations of mRNA LNPs generated similar neutralizing antibody titers compared to immunizations of matched NFL soluble proteins in adjuvant. Given the recent success of mRNA vaccines to prevent severe COVID, these are important developments for genetic expression of native-like HIV Env trimers in animals and potentially in humans.
History
DepositionOct 30, 2023-
Header (metadata) releaseJul 3, 2024-
Map releaseJul 3, 2024-
UpdateAug 21, 2024-
Current statusAug 21, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42517.png

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Map

FileDownload / File: emd_42517.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.68 Å/pix.
x 192 pix.
= 322.56 Å		1.68 Å/pix.
x 192 pix.
= 322.56 Å		1.68 Å/pix.
x 192 pix.
= 322.56 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.68 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01
Minimum - Maximum-0.028665893 - 0.058035005
Average (Standard dev.)-0.000049449984 (±0.004535354)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 322.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map B

Fileemd_42517_half_map_1.map
Annotationhalf map B
Projections & Slices
AxesZYX

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Half map: half map A

Fileemd_42517_half_map_2.map
Annotationhalf map A
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Sample components

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Entire : HIV-1 JR-FL NFL.664 soluble trimer in complex with polyclonal Fab...

EntireName: HIV-1 JR-FL NFL.664 soluble trimer in complex with polyclonal Fab from rabbit U5756
Components
  • Complex: HIV-1 JR-FL NFL.664 soluble trimer in complex with polyclonal Fab from rabbit U5756
    • Complex: HIV-1 JR-FL NFL.664 soluble trimer in complex with polyclonal Fab from rabbit U5756

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Supramolecule #1: HIV-1 JR-FL NFL.664 soluble trimer in complex with polyclonal Fab...

SupramoleculeName: HIV-1 JR-FL NFL.664 soluble trimer in complex with polyclonal Fab from rabbit U5756
type: complex / ID: 1 / Parent: 0 / Details: HIV-1 JR-FL NFL.664 soluble trimer
Source (natural)Organism: Human immunodeficiency virus 1

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Supramolecule #2: HIV-1 JR-FL NFL.664 soluble trimer in complex with polyclonal Fab...

SupramoleculeName: HIV-1 JR-FL NFL.664 soluble trimer in complex with polyclonal Fab from rabbit U5756
type: complex / ID: 2 / Parent: 1 / Details: polyclonal Fab from rabbit U5756
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 7.4
StainingType: NEGATIVE / Material: uranyl formate / Details: 2% w/v

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6v0r


Details: EM map simulated from coordinates using molmap in UCSF Chimera and low pass filtered
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 3282
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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