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- EMDB-21173: De novo designed octahedral nanoparticle O43_dn18 -

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Basic information

Entry
Database: EMDB / ID: EMD-21173
TitleDe novo designed octahedral nanoparticle O43_dn18
Map dataDe novo designed octahedral nanoparticle O43_dn18, Cryo EM map
Sample
  • Complex: De novo designed nanoparticle of octahedral symmetry O43_dn18_NP
    • Protein or peptide: O43_dn18B
    • Protein or peptide: O43_dn18A
KeywordsDe novo / Nanoparticle / DE NOVO PROTEIN
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.54 Å
AuthorsAntanasijevic A / Ward AB
Funding support United States, 1 items
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, 2020-
Header (metadata) releaseJan 29, 2020-
Map releaseAug 12, 2020-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6vfi
  • Surface level: 0.02
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6vfi
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21173.png

Downloads & links

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Map

FileDownload / File: emd_21173.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDe novo designed octahedral nanoparticle O43_dn18, Cryo EM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 300 pix.
= 309. Å		1.03 Å/pix.
x 300 pix.
= 309. Å		1.03 Å/pix.
x 300 pix.
= 309. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.027051566 - 0.057740707
Average (Standard dev.)0.00050589954 (±0.004457786)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 309.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z309.000309.000309.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-205-205-205
NX/NY/NZ411411411
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0270.0580.001

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Supplemental data

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Mask #1

Fileemd_21173_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: De novo designed octahedral nanoparticle O43 dn18, Cryo EM Half-map 1

Fileemd_21173_half_map_1.map
AnnotationDe novo designed octahedral nanoparticle O43_dn18, Cryo EM Half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: De novo designed octahedral nanoparticle O43 dn18, Cryo EM Half-map 2

Fileemd_21173_half_map_2.map
AnnotationDe novo designed octahedral nanoparticle O43_dn18, Cryo EM Half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : De novo designed nanoparticle of octahedral symmetry O43_dn18_NP

EntireName: De novo designed nanoparticle of octahedral symmetry O43_dn18_NP
Components
  • Complex: De novo designed nanoparticle of octahedral symmetry O43_dn18_NP
    • Protein or peptide: O43_dn18B
    • Protein or peptide: O43_dn18A

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Supramolecule #1: De novo designed nanoparticle of octahedral symmetry O43_dn18_NP

SupramoleculeName: De novo designed nanoparticle of octahedral symmetry O43_dn18_NP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Self-assembling nanoparticle of octahedral symmetry
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 810 KDa

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Macromolecule #1: O43_dn18B

MacromoleculeName: O43_dn18B / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.828147 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MGEEAELAYL LGELAYKLGE YRIAIRAYRI ALKRDPNNAE AWYNLGNAYY KQGDYDEAIE YYQKALELDP NNAEAWYNLG NAYYKQGDY DEAIEYYQKA LELDPSNLDA AVNLGAATML TS

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Macromolecule #2: O43_dn18A

MacromoleculeName: O43_dn18A / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 22.722627 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MDRCEELARR IAEVVERAKR AGTSEDEIAE SVARVISLVI RALKLSGSSY EVICECVARI VAEIVEALKR SGTSAVEIAK IVARVISEV IRTLKESGSS YEVICECVAR IVAEIVEALK RSGTSAAIIA LIVALVISEV IRTLKESGSS FEVILECVIR I VLEIIEAL ...String:
MDRCEELARR IAEVVERAKR AGTSEDEIAE SVARVISLVI RALKLSGSSY EVICECVARI VAEIVEALKR SGTSAVEIAK IVARVISEV IRTLKESGSS YEVICECVAR IVAEIVEALK RSGTSAAIIA LIVALVISEV IRTLKESGSS FEVILECVIR I VLEIIEAL KRSGTSEQDV MLIVMAVLLV VLATLQLSGS GGWLEHHHHH H

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.0 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
25.0 mMTris-HCl
150.0 mMSodium ChlorideNaCl

Details: TBS buffer, 0.2um filtered, 0.06mM DDM detergent added immediately before freezing
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 11 / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Details: 0.06mM DDM detergent (from an 8X stock) added immediately before freezing.
DetailsNanoparticles were generated by co-expression of the two components (A and B) in E coli. Assembled particles are purified by a combination of Ni-affinity and gel filtration chromatography.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 70.0 K / Max: 90.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1336 / Average electron dose: 50.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 330095
Startup modelType of model: OTHER / Details: Ab Initio generated model from cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: O (octahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.54 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 5050
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsOctahedral nanoparticle O43_dn18 model was fit into the map using UCSF Chimera. A combination of Rosetta relaxed refinement and manual refinement in Coot was used to relax the model into the map.
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6vfi:
De novo designed octahedral nanoparticle O43_dn18

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