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- EMDB-44474: HIV-1 Env 16055 dGly4 NFL -

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Basic information

Entry
Database: EMDB / ID: EMD-44474
TitleHIV-1 Env 16055 dGly4 NFL
Map datasharpened map
Sample
  • Complex: HIV-1 Env 16055 dGly4 NFL
    • Protein or peptide: Envelope glycoprotein gp140
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsHIV-1 / NFL / Env / vaccine design / VIRAL PROTEIN
Function / homology
Function and homology information


positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane / structural molecule activity / membrane
Similarity search - Function
Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120
Similarity search - Domain/homology
Envelope glycoprotein gp160
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsOzorowski G / Lee W-H / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)UM1 AI144462 United States
CitationJournal: NPJ Vaccines / Year: 2025
Title: Accelerated cGMP production of near-native HIV-1 Env trimers following electroporation transfection and immunogenicity analysis.
Authors: Shridhar Bale / Elena Gustchina / Javier Guenaga / Victor Ayala / Wen-Hsin Lee / Gabriel Ozorowski / Stephen Whitney / Richard Wilson / Sabyasachi Baboo / Jolene K Diedrich / Esmeralda D ...Authors: Shridhar Bale / Elena Gustchina / Javier Guenaga / Victor Ayala / Wen-Hsin Lee / Gabriel Ozorowski / Stephen Whitney / Richard Wilson / Sabyasachi Baboo / Jolene K Diedrich / Esmeralda D Doyle / Lauren Hudacik / Elana Ben-Akiva / Kristen A Rodrigues / Darrell J Irvine / John R Yates / James C Paulson / Andrew B Ward / Timothy Fouts / Richard T Wyatt /
Abstract: Evaluation of recombinant HIV-1 surface glycoproteins (Env) as vaccine candidates for Phase I human experimental trials often requires production of cGMP-grade well-ordered Env trimers. Here, we ...Evaluation of recombinant HIV-1 surface glycoproteins (Env) as vaccine candidates for Phase I human experimental trials often requires production of cGMP-grade well-ordered Env trimers. Here, we report an accelerated cGMP compatible approach for expression and purification of a stabilized HIV clade C-derived trimer '16055 DG4 NFL' (for native flexibly linked). This recombinant trimer was expressed from CHO-S™ cells using a MaxCyte® VLX™ electroporation-based transient transfection process. The 16055 DG4 NFL was designed with multiple internal stabilizing mutations and, as well, deletion of four N-linked glycans (DG4) proximal to the CD4 binding site (CD4bs) engineered to improve B cell recognition of this conserved neutralizing determinant. The transient process circumvents the need to develop stable cell lines expressing the Env trimers that is often the most time-consuming step impacting vaccine development timelines. The 16055 DG4 NFL trimer was purified by immunoaffinity chromatography using the broadly neutralizing antibody (bNAb), PGT145. Following additional downstream processing steps, purified trimer was vialed, frozen and stored at -80 °C. Upon thaw and analysis, the trimer displayed homogeneity and a near-native conformation as determined by size-exclusion chromatography (SEC), negative stain and cryo-electron microscopy (EM), differential scanning calorimetry (DSC) and biolayer interferometry (BLI). The immunogenicity of the trimer was tested in rabbits with bolus, escalating dose and divided dose immunization regimens. Rabbits from all three regimens elicited tier 2 autologous neutralizing antibodies that targeted the exposed protein region at the CD4bs. The trimer is currently under investigation in a human clinical trial (NCT06332339) for safety, tolerability and as a priming candidate followed by heterologous boosting to potentially elicit cross-neutralizing antibodies.
History
DepositionApr 15, 2024-
Header (metadata) releaseSep 3, 2025-
Map releaseSep 3, 2025-
UpdateSep 3, 2025-
Current statusSep 3, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44474.png

Downloads & links

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Map

FileDownload / File: emd_44474.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 384 pix.
= 401.28 Å		1.05 Å/pix.
x 384 pix.
= 401.28 Å		1.05 Å/pix.
x 384 pix.
= 401.28 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.045 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.27
Minimum - Maximum-0.91769266 - 1.5054265
Average (Standard dev.)0.0006714102 (±0.026263418)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 401.27997 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44474_msk_1.map
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Half map: half map A

Fileemd_44474_half_map_1.map
Annotationhalf map A
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Half map: half map B

Fileemd_44474_half_map_2.map
Annotationhalf map B
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Sample components

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Entire : HIV-1 Env 16055 dGly4 NFL

EntireName: HIV-1 Env 16055 dGly4 NFL
Components
  • Complex: HIV-1 Env 16055 dGly4 NFL
    • Protein or peptide: Envelope glycoprotein gp140
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: HIV-1 Env 16055 dGly4 NFL

SupramoleculeName: HIV-1 Env 16055 dGly4 NFL / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Human immunodeficiency virus 1

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Macromolecule #1: Envelope glycoprotein gp140

MacromoleculeName: Envelope glycoprotein gp140 / type: protein_or_peptide / ID: 1 / Details: HIV-1 Env 16055 dGly4 NFL gp140 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 71.212586 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: NGNLWVTVYY GVPVWKDAET TLFCASDAKA YEKEKHNVWA THACVPTDPN PQEMVLENVT ENFNMWKNDM VEQMHTDVIS LWDQSLKPC VKLTPLCVTL ECRQVNTTNA TSSVNVTNGE EIKNCSFNAT TELRDKKQKV YALFYRLDIV PLEEERKGNS S KYRLINCN ...String:
NGNLWVTVYY GVPVWKDAET TLFCASDAKA YEKEKHNVWA THACVPTDPN PQEMVLENVT ENFNMWKNDM VEQMHTDVIS LWDQSLKPC VKLTPLCVTL ECRQVNTTNA TSSVNVTNGE EIKNCSFNAT TELRDKKQKV YALFYRLDIV PLEEERKGNS S KYRLINCN TSACTQACPK VTFDPIPIHY CAPAGYAILK CNNKTFNGTG PCNNVSTVQC THGIKPVVST QLLLNGSLAE GE IIIRSEQ LTNNVKTIIV HLNESVEIVC TRPNQYTRKS IRIGPGQTFY AMGDIIGNIR QAYCNISKDD WIRTLQRVGK KLA EHFPRR IIQFTSPAGG DLEITTHSFN CRGEFFYCNT SSLFNSTYNP NDTNSNSSSS NSSLDITIPC RIKQIINMWQ RVGQ CMYAP PIEGNITCKS NITGLLLVRD GGVESQETEI FRPGGGDMRN NWRSELYKYK VVEIKPLGIA PTRAKRRVVE GGGGS GGGG SAVGLGAVRR GFLGAAGSTM GAASITLTVQ ARQLLSGIVQ QQSNLLKAPE AQQHLLQLGV WGIKQLQTRV LAIERY LKD QQLLGIWGCS GKLICTTAVP WNSSWSNKSH DEIWGNMTWM QWDREIGNYT NTIYRLLEDS QNQQEQNEKD LLALD

UniProtKB: Envelope glycoprotein gp160

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 30 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.4 mg/mL
BufferpH: 7.4 / Details: Added LMNG to 0.005 mM shortly before freezing
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: ab initio model
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 91115
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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