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- PDB-6orq: Modified BG505 SOSIP-based immunogen RC1 in complex with the elic... -

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Entry
Database: PDB / ID: 6orq
TitleModified BG505 SOSIP-based immunogen RC1 in complex with the elicited V3-glycan patch antibody Ab275MUR
Components
  • (Ab275MUR antibody Fab ...) x 2
  • (RC1 variant of HIV-1 Env glycoprotein ...) x 2
KeywordsVIRAL PROTEIN/Immune System / HIV-1 broadly-neutralizing antibody / Env trimer structure / V3-glycan patch / cryo-EM / RC1 / immunogen design / VIRAL PROTEIN-IMMUNE SYSTEM complex / ANTIVIRAL PROTEIN
Biological speciesHuman immunodeficiency virus 1
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsAbernathy, M.E. / Gristick, H.B. / Bjorkman, P.J.
Funding supportUnited States , 2件
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious DiseasesP01 AI100148United States
National Institutes of Health/National Institute of General Medical SciencesP50 GM082545-06United States
CitationJournal: Nature / Year: 2019
Title: Immunization expands B cells specific to HIV-1 V3 glycan in mice and macaques.
Authors: Amelia Escolano / Harry B Gristick / Morgan E Abernathy / Julia Merkenschlager / Rajeev Gautam / Thiago Y Oliveira / Joy Pai / Anthony P West / Christopher O Barnes / Alexander A Cohen / Haoqing Wang / Jovana Golijanin / Daniel Yost / Jennifer R Keeffe / Zijun Wang / Peng Zhao / Kai-Hui Yao / Jens Bauer / Lilian Nogueira / Han Gao / Alisa V Voll / David C Montefiori / Michael S Seaman / Anna Gazumyan / Murillo Silva / Andrew T McGuire / Leonidas Stamatatos / Darrell J Irvine / Lance Wells / Malcolm A Martin / Pamela J Bjorkman / Michel C Nussenzweig /
Abstract: Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it ...Broadly neutralizing monoclonal antibodies protect against infection with HIV-1 in animal models, suggesting that a vaccine that elicits these antibodies would be protective in humans. However, it has not yet been possible to induce adequate serological responses by vaccination. Here, to activate B cells that express precursors of broadly neutralizing antibodies within polyclonal repertoires, we developed an immunogen, RC1, that facilitates the recognition of the variable loop 3 (V3)-glycan patch on the envelope protein of HIV-1. RC1 conceals non-conserved immunodominant regions by the addition of glycans and/or multimerization on virus-like particles. Immunization of mice, rabbits and rhesus macaques with RC1 elicited serological responses that targeted the V3-glycan patch. Antibody cloning and cryo-electron microscopy structures of antibody-envelope complexes confirmed that immunization with RC1 expands clones of B cells that carry the anti-V3-glycan patch antibodies, which resemble precursors of human broadly neutralizing antibodies. Thus, RC1 may be a suitable priming immunogen for sequential vaccination strategies in the context of polyclonal repertoires.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 30, 2019 / Release: Jun 12, 2019
RevisionDateData content typeProviderType
1.0Jun 12, 2019Structure modelrepositoryInitial release

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Assembly

Deposited unit
A: RC1 variant of HIV-1 Env glycoprotein gp41
B: RC1 variant of HIV-1 Env glycoprotein gp120
C: RC1 variant of HIV-1 Env glycoprotein gp41
D: Ab275MUR antibody Fab heavy chain
E: Ab275MUR antibody Fab light chain
F: RC1 variant of HIV-1 Env glycoprotein gp41
G: RC1 variant of HIV-1 Env glycoprotein gp120
H: Ab275MUR antibody Fab heavy chain
I: RC1 variant of HIV-1 Env glycoprotein gp120
J: Ab275MUR antibody Fab heavy chain
K: Ab275MUR antibody Fab light chain
L: Ab275MUR antibody Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)355,36395
Polymers337,41412
Non-polymers17,95083
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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RC1 variant of HIV-1 Env glycoprotein ... , 2 types, 6 molecules ACFBGI

#1: Protein/peptide RC1 variant of HIV-1 Env glycoprotein gp41


Mass: 17346.428 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Cell (production host): HEK 293 Expi / Production host: Homo sapiens (human)
#2: Protein/peptide RC1 variant of HIV-1 Env glycoprotein gp120


Mass: 57762.633 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus 1 / Cell (production host): 293 6E / Production host: Homo sapiens (human)

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Ab275MUR antibody Fab ... , 2 types, 6 molecules DHJEKL

#3: Protein/peptide Ab275MUR antibody Fab heavy chain


Mass: 13418.949 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell (production host): Expi293 / Production host: Homo sapiens (human)
#4: Protein/peptide Ab275MUR antibody Fab light chain


Mass: 23943.238 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell (production host): HEK 293 Expi / Production host: Homo sapiens (human)

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Non-polymers , 3 types, 83 molecules

#5: Chemical...
ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


Mass: 221.208 Da / Num. of mol.: 73
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6 / N-Acetylglucosamine
#6: Chemical
ChemComp-BMA / BETA-D-MANNOSE


Mass: 180.156 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Formula: C6H12O6 / Mannose
#7: Chemical
ChemComp-MAN / ALPHA-D-MANNOSE


Mass: 180.156 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Formula: C6H12O6 / Mannose

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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

Type: COMPLEX

IDNameDetailsEntity IDParent-IDSource
1Complex of RC1 variant of BG505 SOSIP.664 trimer with three Ab275MUR Fabs and three 8ANC195 FabsFab fragments generated by recombinant expression and complexed with the RC1 variant of BG505 SOSIP.664 trimer1, 2, 3, 40MULTIPLE SOURCES
2RC1 variant of BG505 SOSIP.664 trimer1,21RECOMBINANT
3Ab275MUR FabFab fragment generated by proteolytic cleavage of IgG antibody using Ficin3,41RECOMBINANT
Molecular weightValue: 0.6 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Human immunodeficiency virus 111676
21Mus musculoides (Temminck's mouse)60742
32Human immunodeficiency virus 111676
43Mus musculoides (Temminck's mouse)60742
Source (recombinant)

Ncbi tax-ID: 9606 / Organism: Homo sapiens (human)

IDEntity assembly-IDCell
11293 6E
22Expi293
33Expi293
Buffer solutionpH: 8
Buffer component

Buffer-ID: 1

IDConc.NameFormula
120 mMTris-HClTris
2120 mMsodium chlorideNaClSodium chloride
SpecimenConc.: 1.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K
Details: 0 blot force, 3 second blot time, 3 uL sample added to freshly glow-discharged grids

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 73000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 328
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.14_3219: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.06CTF correction
7UCSF Chimeramodel fitting
9cryoSPARC2.1initial Euler assignment
10cryoSPARC2.1final Euler assignment
11cryoSPARC2.1classification
12cryoSPARC2.13D reconstruction
13PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 49308 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-ID
15T3ZG
25T3ZB
34FQQB
44FQQA
RefinementHighest resolution: 4.4 Å
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.006920442
f_angle_d1.136427855
f_chiral_restr0.06273252
f_plane_restr0.00813474
f_dihedral_angle_d10.379912042

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