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- PDB-8eu8: Cryo-EM structure of CH848 10.17DT DS-SOSIP-2P Env -

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Basic information

Entry
Database: PDB / ID: 8eu8
TitleCryo-EM structure of CH848 10.17DT DS-SOSIP-2P Env
ComponentsCH848 10.17DT SOSIP Envelope glycoprotein gp160
KeywordsVIRAL PROTEIN / HIV-1 / Env / viral fusion protein / glycoprotein
Function / homology
Function and homology information


positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / virus-mediated perturbation of host defense response / fusion of virus membrane with host endosome membrane / viral envelope ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / positive regulation of establishment of T cell polarity / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / virus-mediated perturbation of host defense response / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / apoptotic process / host cell plasma membrane / structural molecule activity / virion membrane / plasma 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
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsWrapp, D. / Acharya, P. / Haynes, B.F.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)5UM1AI144371 United States
CitationJournal: J Virol / Year: 2023
Title: Structure-Based Stabilization of SOSIP Env Enhances Recombinant Ectodomain Durability and Yield.
Authors: Daniel Wrapp / Zekun Mu / Bhishem Thakur / Katarzyna Janowska / Oluwatobi Ajayi / Maggie Barr / Robert Parks / Katayoun Mansouri / Robert J Edwards / Beatrice H Hahn / Priyamvada Acharya / ...Authors: Daniel Wrapp / Zekun Mu / Bhishem Thakur / Katarzyna Janowska / Oluwatobi Ajayi / Maggie Barr / Robert Parks / Katayoun Mansouri / Robert J Edwards / Beatrice H Hahn / Priyamvada Acharya / Kevin O Saunders / Barton F Haynes /
Abstract: The envelope glycoprotein (Env) is the main focus of human immunodeficiency virus type 1 (HIV-1) vaccine development due to its critical role in viral entry. Despite advances in protein engineering, ...The envelope glycoprotein (Env) is the main focus of human immunodeficiency virus type 1 (HIV-1) vaccine development due to its critical role in viral entry. Despite advances in protein engineering, many Env proteins remain recalcitrant to recombinant expression due to their inherent metastability, making biochemical and immunological experiments impractical or impossible. Here, we report a novel proline stabilization strategy to facilitate the production of prefusion Env trimers. This approach, termed "2P," works synergistically with previously described SOSIP mutations and dramatically increases the yield of recombinantly expressed Env ectodomains without altering the antigenic or conformational properties of near-native Env. We determined that the 2P mutations function by enhancing the durability of the prefusion conformation and that this stabilization strategy is broadly applicable to evolutionarily and antigenically diverse Env constructs. These findings provide a new Env stabilization platform to facilitate biochemical research and expand the number of Env variants that can be developed as future HIV-1 vaccine candidates. Recent estimates have placed the number of new human immunodeficiency virus type 1 (HIV-1) infections at approximately 1.5 million per year, emphasizing the ongoing and urgent need for an effective vaccine. The envelope (Env) glycoprotein is the main focus of HIV-1 vaccine development, but, due to its inherent metastability, many Env variants are difficult to recombinantly express in the relatively large quantities that are required for biochemical studies and animal trials. Here, we describe a novel structure-based stabilization strategy that works synergistically with previously described SOSIP mutations to increase the yield of prefusion HIV-1 Env.
History
DepositionOct 18, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 4, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 8, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 9, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CH848 10.17DT SOSIP Envelope glycoprotein gp160
B: CH848 10.17DT SOSIP Envelope glycoprotein gp160
C: CH848 10.17DT SOSIP Envelope glycoprotein gp160
hetero molecules


Theoretical massNumber of molelcules
Total (without water)246,69748
Polymers233,0853
Non-polymers13,61245
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CH848 10.17DT SOSIP Envelope glycoprotein gp160 / Env polyprotein / CH848 10.17DT DS-SOSIP-2P


Mass: 77695.070 Da / Num. of mol.: 3 / Mutation: I543P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: env / Production host: Homo sapiens (human) / References: UniProt: A0A1W6IPB2
#2: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#3: Sugar...
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 27 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CH848 10.17DT DS-SOSIP-2P / Type: COMPLEX
Details: Prefusion conformation of HIV-1 Env (homotrimer of gp120/gp41 heterodimers)
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.233 MDa / Experimental value: YES
Source (natural)Organism: Human immunodeficiency virus 1
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTrisC4H11NO31
2200 mMsodium chlorideNaCl1
30.02 %sodium azideNaN31
40.085 mMDDMC24H46O111
SpecimenConc.: 1.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Glow discharged using an easiGlow Glow Discharge Cleaning System (PELCO).
Grid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 61 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111026 / Symmetry type: POINT

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