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- PDB-6vfk: De novo designed tetrahedral nanoparticle T33_dn10 displaying 4 c... -

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Basic information

Entry
Database: PDB / ID: 6vfk
TitleDe novo designed tetrahedral nanoparticle T33_dn10 displaying 4 copies of BG505-SOSIP trimer on the surface
Components
  • BG505-SOSIP-T33_dn10A
  • T33_dn10B
KeywordsDE NOVO PROTEIN / Nanoparticle / Rosetta / De novo protein design / HIV Env
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.25 Å
AuthorsAntanasijevic, A. / Ward, A.B.
Funding support United States, 1items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1115782 United States
CitationJournal: PLoS Pathog / Year: 2020
Title: Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens.
Authors: Aleksandar Antanasijevic / George Ueda / Philip J M Brouwer / Jeffrey Copps / Deli Huang / Joel D Allen / Christopher A Cottrell / Anila Yasmeen / Leigh M Sewall / Ilja Bontjer / Thomas J ...Authors: Aleksandar Antanasijevic / George Ueda / Philip J M Brouwer / Jeffrey Copps / Deli Huang / Joel D Allen / Christopher A Cottrell / Anila Yasmeen / Leigh M Sewall / Ilja Bontjer / Thomas J Ketas / Hannah L Turner / Zachary T Berndsen / David C Montefiori / Per Johan Klasse / Max Crispin / David Nemazee / John P Moore / Rogier W Sanders / Neil P King / David Baker / Andrew B Ward /
Abstract: Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and ...Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and geometries enables combination with antigens of choice to test novel multimerization concepts in immunization strategies where the goal is to improve the induction and maturation of neutralizing antibody lineages. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to an underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. When the immunogenicity of ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle or as soluble proteins were compared in rabbits, the two immunogens elicited similar serum antibody binding titers against the trimer component. Neutralizing antibody titers were slightly elevated in the animals given the nanoparticle immunogen and were initially more focused to the trimer apex. Altogether, our findings indicate that tetrahedral nanoparticles can be successfully applied for presentation of HIV Env trimer immunogens; however, the optimal implementation to different immunization strategies remains to be determined.
History
DepositionJan 5, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 22, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 26, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _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 / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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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  • Biological unit as complete point assembly
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Assembly

Deposited unit
B: T33_dn10B
A: BG505-SOSIP-T33_dn10A


Theoretical massNumber of molelcules
Total (without water)120,3222
Polymers120,3222
Non-polymers00
Water0
1
B: T33_dn10B
A: BG505-SOSIP-T33_dn10A
x 12


Theoretical massNumber of molelcules
Total (without water)1,443,86524
Polymers1,443,86524
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation11
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: T (tetrahedral))

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Components

#1: Protein T33_dn10B


Mass: 31466.830 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pET28b / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein BG505-SOSIP-T33_dn10A


Mass: 88855.289 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pPPI4 / Cell line (production host): HEK293F / Production host: Homo sapiens (human)

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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 T33_dn10 displaying 4 copies of BG505-SOSIP trimer on the surface. Nanoparticle consists of 12 copies of both subunits (BG505-SOSIP-T33_dn10A and T33_dn10B)
Type: COMPLEX
Details: Nanoparticle is assembled by combining equimolar amounts of BG505-SOSIP-T33_dn10A and T33_dn10B and subsequent incubation. Nanoparticle is purified from unassembled components by SEC
Entity ID: all / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293F / Plasmid: pPPI4
Buffer solutionpH: 7.4 / Details: TBS buffer, pH 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HClTris1
2150 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 1.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Nanoparticle is assembled by combining equimolar amounts of BG505-SOSIP-T33_dn10A and T33_dn10B and subsequent incubation. Nanoparticle is purified from unassembled components by SEC. Sample ...Details: Nanoparticle is assembled by combining equimolar amounts of BG505-SOSIP-T33_dn10A and T33_dn10B and subsequent incubation. Nanoparticle is purified from unassembled components by SEC. Sample is then concentrated to 1.6mg/ml in TBS buffer.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K
Details: Blotting time varied in the 3-7 s range, Blotting force set to 0, Wait time of 10s.

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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: 36000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 11.25 sec. / Electron dose: 49.39 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 798
Image scansMovie frames/image: 45

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2Leginonimage acquisition
4RELION3CTF correction
7UCSF Chimeramodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13Rosettamodel refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 25791 / Details: Template picking in cryoSPARC
SymmetryPoint symmetry: T (tetrahedral)
3D reconstructionResolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81105 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Model refinement was performed by iterative rounds of Rosetta relaxed refinement and manual refinement in Coot.

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