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- PDB-6vfh: De novo designed tetrahedral nanoparticle T33_dn10 -

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Basic information

Entry
Database: PDB / ID: 6vfh
TitleDe novo designed tetrahedral nanoparticle T33_dn10
Components
  • T33_dn10A
  • T33_dn10B
KeywordsDE NOVO PROTEIN / De novo / Nanoparticle
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.86 Å
AuthorsAntanasijevic, A. / Ward, A.B.
Funding support United States, 1items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1115782 United States
CitationJournal: Elife / Year: 2020
Title: Tailored design of protein nanoparticle scaffolds for multivalent presentation of viral glycoprotein antigens.
Authors: George Ueda / Aleksandar Antanasijevic / Jorge A Fallas / William Sheffler / Jeffrey Copps / Daniel Ellis / Geoffrey B Hutchinson / Adam Moyer / Anila Yasmeen / Yaroslav Tsybovsky / Young- ...Authors: George Ueda / Aleksandar Antanasijevic / Jorge A Fallas / William Sheffler / Jeffrey Copps / Daniel Ellis / Geoffrey B Hutchinson / Adam Moyer / Anila Yasmeen / Yaroslav Tsybovsky / Young-Jun Park / Matthew J Bick / Banumathi Sankaran / Rebecca A Gillespie / Philip Jm Brouwer / Peter H Zwart / David Veesler / Masaru Kanekiyo / Barney S Graham / Rogier W Sanders / John P Moore / Per Johan Klasse / Andrew B Ward / Neil P King / David Baker /
Abstract: Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. To enable a new generation of anti-viral vaccines, we designed self- ...Multivalent presentation of viral glycoproteins can substantially increase the elicitation of antigen-specific antibodies. To enable a new generation of anti-viral vaccines, we designed self-assembling protein nanoparticles with geometries tailored to present the ectodomains of influenza, HIV, and RSV viral glycoprotein trimers. We first designed trimers tailored for antigen fusion, featuring N-terminal helices positioned to match the C termini of the viral glycoproteins. Trimers that experimentally adopted their designed configurations were incorporated as components of tetrahedral, octahedral, and icosahedral nanoparticles, which were characterized by cryo-electron microscopy and assessed for their ability to present viral glycoproteins. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles presented antigenically intact prefusion HIV-1 Env, influenza hemagglutinin, and RSV F trimers in the predicted geometries. This work demonstrates that antigen-displaying protein nanoparticles can be designed from scratch, and provides a systematic way to investigate the influence of antigen presentation geometry on the immune response to vaccination.
History
DepositionJan 5, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 12, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 19, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_DOI ..._citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
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

Movie
  • Biological unit as complete point assembly
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-21172
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  • Superimposition on EM map
  • EMDB-21172
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: T33_dn10A
B: T33_dn10B


Theoretical massNumber of molelcules
Total (without water)45,5492
Polymers45,5492
Non-polymers00
Water00
1
A: T33_dn10A
B: T33_dn10B
x 12


Theoretical massNumber of molelcules
Total (without water)546,59124
Polymers546,59124
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_dn10A


Mass: 14082.408 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: 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) / 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 T33_dn10 / Type: COMPLEX
Details: Self-assembling nanoparticle with tetrahedral symmetry
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.546 MDa / Experimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET28b
Buffer solutionpH: 7.4
Details: TBS buffer, 0.2um filtered, 0.06mM DDM detergent added immediately before freezing
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HCl1
2150 mMSodium ChlorideNaCl1
SpecimenConc.: 4.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Nanoparticles are generated by co-expression of two components (A and B) in E coli. Assembled particles are purified using a combination of Ni-affinity chromatography and gel-filtration chromatography.
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: 0.06mM DDM detergent (from an 8X stock) added immediately before freezing

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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: BRIGHT FIELD / Nominal magnification: 29000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 70 K
Image recordingElectron dose: 50.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 502

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.9.0particle 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: 304308
SymmetryPoint symmetry: T (tetrahedral)
3D reconstructionResolution: 3.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 49961 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Tetrahedral nanoparticle T33_dn10 model was docked into the reconstructed map using UCSF Chimera. The model was then relaxed using a combination of Rosetta relaxed refinement and manual refinement in Coot.

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