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- EMDB-21163: De novo designed tetrahedral nanoparticle T33_dn5 -

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Basic information

Entry
Database: EMDB / ID: EMD-21163
TitleDe novo designed tetrahedral nanoparticle T33_dn5
Map dataDe novo designed tetrahedral nanoparticle T33_dn5, Negative stain EM map
Sample
  • Complex: De novo designed tetrahedral nanoparticle T33_dn5, Negative Stain EM Map
    • Protein or peptide: T33_dn5A
    • Protein or peptide: T33_dn5B
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / negative staining / Resolution: 19.68 Å
AuthorsWard AB / Antanasijevic A
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-
UpdateAug 19, 2020-
Current statusAug 19, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21163.png

Downloads & links

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Map

FileDownload / File: emd_21163.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDe novo designed tetrahedral nanoparticle T33_dn5, Negative stain EM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.1 Å/pix.
x 120 pix.
= 492. Å		4.1 Å/pix.
x 120 pix.
= 492. Å		4.1 Å/pix.
x 120 pix.
= 492. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.03945905 - 0.17908229
Average (Standard dev.)-0.00005523943 (±0.007416304)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 492.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z492.000492.000492.000
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/NS120120120
D min/max/mean-0.0390.179-0.000

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Supplemental data

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Sample components

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Entire : De novo designed tetrahedral nanoparticle T33_dn5, Negative Stain...

EntireName: De novo designed tetrahedral nanoparticle T33_dn5, Negative Stain EM Map
Components
  • Complex: De novo designed tetrahedral nanoparticle T33_dn5, Negative Stain EM Map
    • Protein or peptide: T33_dn5A
    • Protein or peptide: T33_dn5B

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Supramolecule #1: De novo designed tetrahedral nanoparticle T33_dn5, Negative Stain...

SupramoleculeName: De novo designed tetrahedral nanoparticle T33_dn5, Negative Stain EM Map
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Nanoparticles were generated by co-expression of the two components (A and B) in E coli. Assembled particles were purified using a combination of Ni-affinity chromatography and gel filtration chromatography.
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET28b

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Macromolecule #1: T33_dn5A

MacromoleculeName: T33_dn5A / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MGNSAEAMYK MGNAAYKQGD YILAIIAYLL ALEKDPNNAE AWYNLGNAAY KQGDYDEAIE YYQKALELDP NNAEAWYNLG NAYYKQGDYD EAIEYYEKAL ELDPNNAEAL KNLLEAIAEQ D

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Macromolecule #2: T33_dn5B

MacromoleculeName: T33_dn5B / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MHHHHTDPLA VILYIAILKA EKSIARAKAA EALGKIGDER AVEPLIKALK DEDALVRAAA ADALGQIGDE RAVEPLIKAL KDEEGLVRAS AAIALGQIGD ERAVQPLIKA LTDERDLVRV AAAVALGRIG DEKAVRPLII VLKDEEGEVR EAAAIALGSI GGERVRAAME ...String:
MHHHHTDPLA VILYIAILKA EKSIARAKAA EALGKIGDER AVEPLIKALK DEDALVRAAA ADALGQIGDE RAVEPLIKAL KDEEGLVRAS AAIALGQIGD ERAVQPLIKA LTDERDLVRV AAAVALGRIG DEKAVRPLII VLKDEEGEVR EAAAIALGSI GGERVRAAME KLAERGTGFA RKVAVNYLET HKLEHHHHHH

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
25.0 mMTris-HCl
150.0 mMsodium chlorideNaCl

Details: TBS buffer, pH 7.4
StainingType: NEGATIVE / Material: Uranyl Formate
Details: Sample diluted to 0.05 mg/mL. 3 uL was applied onto the grid, blotted off, and then stained with 2% uranyl formate for 60 seconds.
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
DetailsT33_dn5 nanoparticle was purified by SEC, diluted to 0.05mg/ml and loaded onto a grid.

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Particle selectionDetails: Appion manual picker
Startup modelType of model: OTHER
Details: Negative stain EM map of the non-liganded nanoparticle
Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 19.68 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 5868
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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