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- EMDB-21185: De novo designed tetrahedral nanoparticle T33_dn10 displaying 4 c... -

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Basic information

Entry
Database: EMDB / ID: EMD-21185
TitleDe novo designed tetrahedral nanoparticle T33_dn10 displaying 4 copies of BG505-SOSIP trimer on the surface
Map dataDe novo designed tetrahedral nanoparticle T33_dn10 presenting BG505-SOSIP, CryoEM Map
Sample
  • Complex: 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)
    • Protein or peptide: T33_dn10B
    • Protein or peptide: BG505-SOSIP-T33_dn10A
KeywordsNanoparticle / Rosetta / De novo protein design / HIV Env / DE NOVO PROTEIN
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.25 Å
AuthorsAntanasijevic A / Ward AB
Funding support United States, 1 items
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, 2020-
Header (metadata) releaseJan 29, 2020-
Map releaseJul 22, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vfk
  • Surface level: 0.022
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6vfk
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21185.png

Downloads & links

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Map

FileDownload / File: emd_21185.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDe novo designed tetrahedral nanoparticle T33_dn10 presenting BG505-SOSIP, CryoEM Map
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.044927433 - 0.08914533
Average (Standard dev.)0.000027195401 (±0.0026569706)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 506.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z440440440
origin x/y/z0.0000.0000.000
length x/y/z506.000506.000506.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS440440440
D min/max/mean-0.0450.0890.000

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Supplemental data

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Mask #1

Fileemd_21185_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 tetrahedral nanoparticle T33 dn10 presenting BG505-SOSIP,...

Fileemd_21185_half_map_1.map
AnnotationDe novo designed tetrahedral nanoparticle T33_dn10 presenting BG505-SOSIP, CryoEM Half-Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: De novo designed tetrahedral nanoparticle T33 dn10 presenting BG505-SOSIP,...

Fileemd_21185_half_map_2.map
AnnotationDe novo designed tetrahedral nanoparticle T33_dn10 presenting BG505-SOSIP, CryoEM Half-Map 1
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 tetrahedral nanoparticle T33_dn10 displaying 4 c...

EntireName: 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)
Components
  • Complex: 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)
    • Protein or peptide: T33_dn10B
    • Protein or peptide: BG505-SOSIP-T33_dn10A

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Supramolecule #1: De novo designed tetrahedral nanoparticle T33_dn10 displaying 4 c...

SupramoleculeName: 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 / ID: 1 / Parent: 0 / Macromolecule list: all
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
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: T33_dn10B

MacromoleculeName: T33_dn10B / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 31.46683 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MIEEVVAEMI DILAESSKKS IEELARAADN KTTEKAVAEA IEEIARLATA AIQLIEALAK NLASEEFMAR AISAIAELAK KAIEAIYRL ADNHTTDTFM ARAIAAIANL AVTAILAIAA LASNHTTEEF MARAISAIAE LAKKAIEAIY RLADNHTTDK F MAAAIEAI ...String:
MIEEVVAEMI DILAESSKKS IEELARAADN KTTEKAVAEA IEEIARLATA AIQLIEALAK NLASEEFMAR AISAIAELAK KAIEAIYRL ADNHTTDTFM ARAIAAIANL AVTAILAIAA LASNHTTEEF MARAISAIAE LAKKAIEAIY RLADNHTTDK F MAAAIEAI ALLATLAILA IALLASNHTT EKFMARAIMA IAILAAKAIE AIYRLADNHT SPTYIEKAIE AIEKIARKAI KA IEMLAKN ITTEEYKEKA KKIIDIIRKL AKMAIKKLED NRTLEHHHHH H

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Macromolecule #2: BG505-SOSIP-T33_dn10A

MacromoleculeName: BG505-SOSIP-T33_dn10A / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 88.855289 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKRGLCCVLL LCGAVFVSPS QEIHARFRRG ARAENLWVTV YYGVPVWKDA ETTLFCASDA KAYETKKHNV WATHCCVPTD PNPQEIHLE NVTEEFNMWK NNMVEQMHTD IISLWDQSLK PCVKLTPLCV TLQCTNVTNN ITDDMRGELK NCSFNMTTEL R DKKQKVYS ...String:
MKRGLCCVLL LCGAVFVSPS QEIHARFRRG ARAENLWVTV YYGVPVWKDA ETTLFCASDA KAYETKKHNV WATHCCVPTD PNPQEIHLE NVTEEFNMWK NNMVEQMHTD IISLWDQSLK PCVKLTPLCV TLQCTNVTNN ITDDMRGELK NCSFNMTTEL R DKKQKVYS LFYRLDVVQI NENQGNRSNN SNKEYRLINC NTSAITQACP KVSFEPIPIH YCAPAGFAIL KCKDKKFNGT GP CTNVSTV QCTHGIKPVV STQLLLNGSL AEEEVIIRSE NITNNAKNIL VQLNESVQIN CTRPNNNTVK SIRIGPGQWF YYT GDIIGD IRQAHCNVSK ATWNETLGKV VKQLRKHFGN NTIIRFANSS GGDLEVTTHS FNCGGEFFYC NTSGLFNSTW ISNT SVQGS NSTGSNDSIT LPCRIKQIIN MWQRIGQAMY APPIQGVIRC VSNITGLILT RDGGSTNSTT ETFRPGGGDM RDNWR SELY KYKVVKIEPL GVAPTRCKRR VVGRRRRRRA VGIGAVSLGF LGAAGSTMGA ASMTLTVQAR NLLSGIVQQQ SNLLRA PEC QQHLLKDTHW GIKQLQARVL AVEHYLRDQQ LLGIWGCSGK LICCTNVPWN SSWSNRNLSE IWDNMTWLQW DKEISNY TQ IIYGLLEESQ NQQEKNEQGS GSGSGSGGEE AELAYLLGEL AYKLGEYRIA IRAYRIALKR DPNNAEAWYN LGNAYYKQ G DYDEAIEYYQ KALELDPNNA EAWYNLGNAY YKQGDYDEAI EYYEKALELD PENLEALQNL LNAMDKQG

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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

Concentration1.6 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
25.0 mMTris-HClTris
150.0 mMsodium chlorideNaClSodium chloride

Details: TBS buffer, pH 7.4
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 11 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 10 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
Details: Blotting time varied in the 3-7 s range, Blotting force set to 0, Wait time of 10s..
DetailsNanoparticle 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.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 798 / Average exposure time: 11.25 sec. / Average electron dose: 49.39 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 25791 / Details: Template picking in cryoSPARC
Startup modelType of model: OTHER / Details: Ab Initio map from cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: T (tetrahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 81105
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel refinement was performed by iterative rounds of Rosetta relaxed refinement and manual refinement in Coot.
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6vfk:
De novo designed tetrahedral nanoparticle T33_dn10 displaying 4 copies of BG505-SOSIP trimer on the surface

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