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- PDB-6vl6: De novo designed tetrahedral nanoparticle T33_dn2 presenting BG50... -

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Basic information

Entry
Database: PDB / ID: 6vl6
TitleDe novo designed tetrahedral nanoparticle T33_dn2 presenting BG505 SOSIP trimers
Components
  • T33_dn2A
  • T33_dn2B
KeywordsDE NOVO PROTEIN / HIV Env / de novo / nanoparticles / vaccine design
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsWard, A.B. / Antanasijevic, A.
Funding support United States, 1items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1115782 United States
CitationJournal: NPJ Vaccines / Year: 2020
Title: Targeting HIV Env immunogens to B cell follicles in nonhuman primates through immune complex or protein nanoparticle formulations.
Authors: Jacob T Martin / Christopher A Cottrell / Aleksandar Antanasijevic / Diane G Carnathan / Benjamin J Cossette / Chiamaka A Enemuo / Etse H Gebru / Yury Choe / Federico Viviano / Stephanie ...Authors: Jacob T Martin / Christopher A Cottrell / Aleksandar Antanasijevic / Diane G Carnathan / Benjamin J Cossette / Chiamaka A Enemuo / Etse H Gebru / Yury Choe / Federico Viviano / Stephanie Fischinger / Talar Tokatlian / Kimberly M Cirelli / George Ueda / Jeffrey Copps / Torben Schiffner / Sergey Menis / Galit Alter / William R Schief / Shane Crotty / Neil P King / David Baker / Guido Silvestri / Andrew B Ward / Darrell J Irvine /
Abstract: Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic ...Following immunization, high-affinity antibody responses develop within germinal centers (GCs), specialized sites within follicles of the lymph node (LN) where B cells proliferate and undergo somatic hypermutation. Antigen availability within GCs is important, as B cells must acquire and present antigen to follicular helper T cells to drive this process. However, recombinant protein immunogens such as soluble human immunodeficiency virus (HIV) envelope (Env) trimers do not efficiently accumulate in follicles following traditional immunization. Here, we demonstrate two strategies to concentrate HIV Env immunogens in follicles, via the formation of immune complexes (ICs) or by employing self-assembling protein nanoparticles for multivalent display of Env antigens. Using rhesus macaques, we show that within a few days following immunization, free trimers were present in a diffuse pattern in draining LNs, while trimer ICs and Env nanoparticles accumulated in B cell follicles. Whole LN imaging strikingly revealed that ICs and trimer nanoparticles concentrated in as many as 500 follicles in a single LN within two days after immunization. Imaging of LNs collected seven days postimmunization showed that Env nanoparticles persisted on follicular dendritic cells in the light zone of nascent GCs. These findings suggest that the form of antigen administered in vaccination can dramatically impact localization in lymphoid tissues and provides a new rationale for the enhanced immune responses observed following immunization with ICs or nanoparticles.
History
DepositionJan 22, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 12, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 2, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _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 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _citation.country / _database_2.pdbx_DOI ..._citation.country / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-21231
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: T33_dn2A
B: T33_dn2B
C: T33_dn2A
N: T33_dn2B
D: T33_dn2A
O: T33_dn2B
E: T33_dn2A
P: T33_dn2B
F: T33_dn2A
Q: T33_dn2B
G: T33_dn2A
R: T33_dn2B
H: T33_dn2A
S: T33_dn2B
I: T33_dn2A
T: T33_dn2B
J: T33_dn2A
U: T33_dn2B
K: T33_dn2A
V: T33_dn2B
L: T33_dn2A
W: T33_dn2B
M: T33_dn2A
X: T33_dn2B


Theoretical massNumber of molelcules
Total (without water)348,50724
Polymers348,50724
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area42580 Å2
ΔGint-344 kcal/mol
Surface area116070 Å2

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Components

#1: Protein
T33_dn2A


Mass: 14136.632 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pPPI4 / Cell line (production host): 293F / Production host: Homo sapiens (human)
#2: Protein
T33_dn2B


Mass: 14905.603 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pET28b / Production host: Escherichia coli BL21(DE3) (bacteria)

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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: De novo designed tetrahedral nanoparticle BG505 SOSIP-T33_dn2
Type: COMPLEX
Details: De novo designed T33_dn2 nanoparticle was engineered to present BG505 SOSIP trimers. 4 trimers per nanoparticle.
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Homo sapiens (human) / Cell: 293F
Buffer solutionpH: 7.4 / Details: Freshly prepared buffer, 0.2 uM filtered
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HCl1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: BG505 SOSIP-T33_dn2 nanoparticles were prepared by combining equimolar amounts of BG505 SOSIP-T33_dn2A and T33_dn2B components and subsequent incubation.
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 10 K
Details: Lauryl maltose neopentyl glycol (LMNG) at a final concentration of 0.005 mM was added to the nanoparticle sample (4.0 mg/mL) and 3 uL was immediately loaded onto plasma-cleaned Quantifoil R ...Details: Lauryl maltose neopentyl glycol (LMNG) at a final concentration of 0.005 mM was added to the nanoparticle sample (4.0 mg/mL) and 3 uL was immediately loaded onto plasma-cleaned Quantifoil R 2/1 holey carbon copper grid (Cu, 400-mesh, Quantifoil Micro Tools GmbH).

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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 FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 11.25 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 2748
Image scansMovie frames/image: 45

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.9.0particle selection
2Leginonimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9Rosettamodel refinement
10Cootmodel refinement
11RELION3initial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 210597 / Details: Template picker in cryoSPARC
SymmetryPoint symmetry: T (tetrahedral)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35521 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 5CEZ

5cez


Accession code: 5CEZ / Source name: PDB / Type: experimental model

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