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- PDB-8t4d: MD65 N332-GT5 SOSIP in complex with RM_N332_08 Fab and RM20A3 Fab -

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Basic information

Entry
Database: PDB / ID: 8t4d
TitleMD65 N332-GT5 SOSIP in complex with RM_N332_08 Fab and RM20A3 Fab
Components
  • (MD65 N332-GT5 SOSIP ...) x 2
  • RM20A3 heavy chain Fv
  • RM20A3 light chain Fv
  • RM_N332_08 heavy chain Fv
  • RM_N332_08 light chain Fv
KeywordsVIRAL PROTEIN/Immune System / germline targeting / NHP / SOSIP / Env / HIV / BG18 / immunogen design / antibody / VIRAL PROTEIN / VIRAL PROTEIN-Immune System complex
Biological speciesHuman immunodeficiency virus 1
Macaca mulatta (Rhesus monkey)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsOzorowski, G. / Torres, J.L. / Ward, A.B.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)UM1 Al100663 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)UM1 AI144462 United States
CitationJournal: Science / Year: 2024
Title: Vaccine priming of rare HIV broadly neutralizing antibody precursors in nonhuman primates.
Authors: Jon M Steichen / Ivy Phung / Eugenia Salcedo / Gabriel Ozorowski / Jordan R Willis / Sabyasachi Baboo / Alessia Liguori / Christopher A Cottrell / Jonathan L Torres / Patrick J Madden / ...Authors: Jon M Steichen / Ivy Phung / Eugenia Salcedo / Gabriel Ozorowski / Jordan R Willis / Sabyasachi Baboo / Alessia Liguori / Christopher A Cottrell / Jonathan L Torres / Patrick J Madden / Krystal M Ma / Henry J Sutton / Jeong Hyun Lee / Oleksandr Kalyuzhniy / Joel D Allen / Oscar L Rodriguez / Yumiko Adachi / Tina-Marie Mullen / Erik Georgeson / Michael Kubitz / Alison Burns / Shawn Barman / Rohini Mopuri / Amanda Metz / Tasha K Altheide / Jolene K Diedrich / Swati Saha / Kaitlyn Shields / Steven E Schultze / Melissa L Smith / Torben Schiffner / Dennis R Burton / Corey T Watson / Steven E Bosinger / Max Crispin / John R Yates / James C Paulson / Andrew B Ward / Devin Sok / Shane Crotty / William R Schief /
Abstract: Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically ...Germline-targeting immunogens hold promise for initiating the induction of broadly neutralizing antibodies (bnAbs) to HIV and other pathogens. However, antibody-antigen recognition is typically dominated by heavy chain complementarity determining region 3 (HCDR3) interactions, and vaccine priming of HCDR3-dominant bnAbs by germline-targeting immunogens has not been demonstrated in humans or outbred animals. In this work, immunization with N332-GT5, an HIV envelope trimer designed to target precursors of the HCDR3-dominant bnAb BG18, primed bnAb-precursor B cells in eight of eight rhesus macaques to substantial frequencies and with diverse lineages in germinal center and memory B cells. We confirmed bnAb-mimicking, HCDR3-dominant, trimer-binding interactions with cryo-electron microscopy. Our results demonstrate proof of principle for HCDR3-dominant bnAb-precursor priming in outbred animals and suggest that N332-GT5 holds promise for the induction of similar responses in humans.
History
DepositionJun 9, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 8, 2024Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.0May 8, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.0May 8, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: Mask / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: Mask / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 8, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1May 29, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification
Revision 1.3Jun 4, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jun 4, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: MD65 N332-GT5 SOSIP gp120
C: RM20A3 heavy chain Fv
D: RM20A3 light chain Fv
B: MD65 N332-GT5 SOSIP gp41
H: RM_N332_08 heavy chain Fv
L: RM_N332_08 light chain Fv
E: MD65 N332-GT5 SOSIP gp120
J: RM20A3 heavy chain Fv
M: RM20A3 light chain Fv
G: MD65 N332-GT5 SOSIP gp41
O: RM_N332_08 heavy chain Fv
Q: RM_N332_08 light chain Fv
F: MD65 N332-GT5 SOSIP gp120
K: RM20A3 heavy chain Fv
N: RM20A3 light chain Fv
I: MD65 N332-GT5 SOSIP gp41
P: RM_N332_08 heavy chain Fv
R: RM_N332_08 light chain Fv
hetero molecules


Theoretical massNumber of molelcules
Total (without water)389,38869
Polymers375,79218
Non-polymers13,59751
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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MD65 N332-GT5 SOSIP ... , 2 types, 6 molecules AEFBGI

#1: Protein MD65 N332-GT5 SOSIP gp120


Mass: 54286.773 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Production host: Homo sapiens (human)
#4: Protein MD65 N332-GT5 SOSIP gp41


Mass: 17096.148 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Production host: Homo sapiens (human)

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Antibody , 4 types, 12 molecules CJKDMNHOPLQR

#2: Antibody RM20A3 heavy chain Fv


Mass: 13511.111 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macaca mulatta (Rhesus monkey) / Production host: Homo sapiens (human)
#3: Antibody RM20A3 light chain Fv


Mass: 13508.800 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macaca mulatta (Rhesus monkey) / Production host: Homo sapiens (human)
#5: Antibody RM_N332_08 heavy chain Fv


Mass: 14394.084 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Macaca mulatta (Rhesus monkey) / Production host: Homo sapiens (human)
#6: Antibody RM_N332_08 light chain Fv


Mass: 12466.945 Da / Num. of mol.: 3
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, 51 molecules

#7: 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.: 6
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
#8: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#9: 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.: 42 / 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

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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

ComponentName: MD65 N332-GT5 SOSIP in complex with RM_N332_08 Fab and RM20A3 Fab
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Macaca mulatta (Rhesus monkey)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 190000 X / Nominal defocus max: 1400 nm / Nominal defocus min: 500 nm / Alignment procedure: COMA FREE
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameCategory
2EPUimage acquisition
8PHENIXmodel refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 99087 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00525284
ELECTRON MICROSCOPYf_angle_d0.97534293
ELECTRON MICROSCOPYf_dihedral_angle_d10.0379207
ELECTRON MICROSCOPYf_chiral_restr0.0563963
ELECTRON MICROSCOPYf_plane_restr0.0054302

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