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- PDB-6x9u: HIV-1 Envelope Glycoprotein BG505 SOSIP.664, expressed in HEK293S... -

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Basic information

Entry
Database: PDB / ID: 6x9u
TitleHIV-1 Envelope Glycoprotein BG505 SOSIP.664, expressed in HEK293S cells and partially deglycosylated by endoglycosidase H, in complex with RM20A3 Fab
Components
  • (HIV-1 Envelope Glycoprotein BG505 SOSIP.664 ...) x 2
  • RM20A3 Fab Heavy Chain
  • RM20A3 Fab Light Chain
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / HIV-1 / Envelope / glycoprotein / spike / VIRAL PROTEIN-IMMUNE SYSTEM complex
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 / identical protein binding / 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
Macaca mulatta (Rhesus monkey)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBerndsen, Z.T. / Ward, A.B.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)UM1 AI100663 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)UM1 AI144462 United States
Bill & Melinda Gates FoundationOPP1115782 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Visualization of the HIV-1 Env glycan shield across scales.
Authors: Zachary T Berndsen / Srirupa Chakraborty / Xiaoning Wang / Christopher A Cottrell / Jonathan L Torres / Jolene K Diedrich / Cesar A López / John R Yates / Marit J van Gils / James C Paulson ...Authors: Zachary T Berndsen / Srirupa Chakraborty / Xiaoning Wang / Christopher A Cottrell / Jonathan L Torres / Jolene K Diedrich / Cesar A López / John R Yates / Marit J van Gils / James C Paulson / Sandrasegaram Gnanakaran / Andrew B Ward /
Abstract: The dense array of N-linked glycans on the HIV-1 envelope glycoprotein (Env), known as the "glycan shield," is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical ...The dense array of N-linked glycans on the HIV-1 envelope glycoprotein (Env), known as the "glycan shield," is a key determinant of immunogenicity, yet intrinsic heterogeneity confounds typical structure-function analysis. Here, we present an integrated approach of single-particle electron cryomicroscopy (cryo-EM), computational modeling, and site-specific mass spectrometry (MS) to probe glycan shield structure and behavior at multiple levels. We found that dynamics lead to an extensive network of interglycan interactions that drive the formation of higher-order structure within the glycan shield. This structure defines diffuse boundaries between buried and exposed protein surface and creates a mapping of potentially immunogenic sites on Env. Analysis of Env expressed in different cell lines revealed how cryo-EM can detect subtle changes in glycan occupancy, composition, and dynamics that impact glycan shield structure and epitope accessibility. Importantly, this identified unforeseen changes in the glycan shield of Env obtained from expression in the same cell line used for vaccine production. Finally, by capturing the enzymatic deglycosylation of Env in a time-resolved manner, we found that highly connected glycan clusters are resistant to digestion and help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion.
History
DepositionJun 3, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 4, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2Oct 9, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: HIV-1 Envelope Glycoprotein BG505 SOSIP.664 gp120
B: HIV-1 Envelope Glycoprotein BG505 SOSIP.664 gp41
H: RM20A3 Fab Heavy Chain
L: RM20A3 Fab Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)108,31926
Polymers102,1124
Non-polymers6,20722
Water00
1
A: HIV-1 Envelope Glycoprotein BG505 SOSIP.664 gp120
B: HIV-1 Envelope Glycoprotein BG505 SOSIP.664 gp41
H: RM20A3 Fab Heavy Chain
L: RM20A3 Fab Light Chain
hetero molecules

A: HIV-1 Envelope Glycoprotein BG505 SOSIP.664 gp120
B: HIV-1 Envelope Glycoprotein BG505 SOSIP.664 gp41
H: RM20A3 Fab Heavy Chain
L: RM20A3 Fab Light Chain
hetero molecules

A: HIV-1 Envelope Glycoprotein BG505 SOSIP.664 gp120
B: HIV-1 Envelope Glycoprotein BG505 SOSIP.664 gp41
H: RM20A3 Fab Heavy Chain
L: RM20A3 Fab Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)324,95878
Polymers306,33712
Non-polymers18,62066
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation2
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C3 (3 fold cyclic))

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Components

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HIV-1 Envelope Glycoprotein BG505 SOSIP.664 ... , 2 types, 2 molecules AB

#1: Protein HIV-1 Envelope Glycoprotein BG505 SOSIP.664 gp120


Mass: 57945.977 Da / Num. of mol.: 1 / Fragment: UNP residues 30-505 / Mutation: T332N,A501C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: env / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: Q2N0S6
#2: Protein HIV-1 Envelope Glycoprotein BG505 SOSIP.664 gp41


Mass: 17146.482 Da / Num. of mol.: 1 / Mutation: I559P,T605C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: env / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: Q2N0S6

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Antibody , 2 types, 2 molecules HL

#3: Antibody RM20A3 Fab Heavy Chain


Mass: 13511.111 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macaca mulatta (Rhesus monkey) / Production host: Homo sapiens (human)
#4: Antibody RM20A3 Fab Light Chain


Mass: 13508.800 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macaca mulatta (Rhesus monkey) / Production host: Homo sapiens (human)

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Sugars , 3 types, 22 molecules

#5: 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.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#6: Polysaccharide alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#7: 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.: 17
Source method: isolated from a genetically manipulated source
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

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Details

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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1HIV-1 Envelope Glycoprotein BG505 SOSIP.664, expressed in HEK293S cells and partially deglycosylated by endoglycosidase H, in complex with RM20A3 FabCOMPLEX#1-#40MULTIPLE SOURCES
2HIV-1 Envelope Glycoprotein BG505 SOSIP.664COMPLEX#1-#21RECOMBINANTpartially deglycosylated by endoglycosidase H
3RM20A3 FabCOMPLEX#3-#41RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Human immunodeficiency virus 111676
23Macaca mulatta (Rhesus monkey)9544
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
12Homo sapiens (human)9606HEK293S
23Homo sapiens (human)9606
Buffer solutionpH: 7.6
Buffer componentName: TBS
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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: DARK FIELD / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
7UCSF Chimeramodel fitting
9Rosettamodel refinement
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 46104 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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