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- EMDB-21186: BG505-SOSIP model reconstructed by subparticle extraction and ref... -

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Basic information

Entry
Database: EMDB / ID: EMD-21186
TitleBG505-SOSIP model reconstructed by subparticle extraction and refinement from a tetrahedral nanoparticle T33_dn10
Map dataBG505-SOSIP subparticle reconstruction following extraction from BG505-SOSIP-T33_dn10 nanoparticle, CryoEM Map
Sample
  • Complex: BG505-SOSIP reconstructed from the tetrahedral nanoparticle T33_dn10 by subparticle extraction and refinement
    • Protein or peptide: BG505-SOSIPv5.2(7S) - gp120
    • Protein or peptide: BG505-SOSIPv5.2(7S) - gp41
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.14 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-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vfl
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21186.png

Downloads & links

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Map

FileDownload / File: emd_21186.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBG505-SOSIP subparticle reconstruction following extraction from BG505-SOSIP-T33_dn10 nanoparticle, CryoEM Map
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.09490436 - 0.16213804
Average (Standard dev.)0.0016676999 (±0.007868744)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 207.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z207.000207.000207.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.0950.1620.002

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Supplemental data

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Mask #1

Fileemd_21186_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: BG505-SOSIP subparticle reconstruction following extraction from BG505-SOSIP-T33 dn10 nanoparticle,...

Fileemd_21186_half_map_1.map
AnnotationBG505-SOSIP subparticle reconstruction following extraction from BG505-SOSIP-T33_dn10 nanoparticle, CryoEM Halfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: BG505-SOSIP subparticle reconstruction following extraction from BG505-SOSIP-T33 dn10 nanoparticle,...

Fileemd_21186_half_map_2.map
AnnotationBG505-SOSIP subparticle reconstruction following extraction from BG505-SOSIP-T33_dn10 nanoparticle, CryoEM Halfmap 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 : BG505-SOSIP reconstructed from the tetrahedral nanoparticle T33_d...

EntireName: BG505-SOSIP reconstructed from the tetrahedral nanoparticle T33_dn10 by subparticle extraction and refinement
Components
  • Complex: BG505-SOSIP reconstructed from the tetrahedral nanoparticle T33_dn10 by subparticle extraction and refinement
    • Protein or peptide: BG505-SOSIPv5.2(7S) - gp120
    • Protein or peptide: BG505-SOSIPv5.2(7S) - gp41
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: BG505-SOSIP reconstructed from the tetrahedral nanoparticle T33_d...

SupramoleculeName: BG505-SOSIP reconstructed from the tetrahedral nanoparticle T33_dn10 by subparticle extraction and refinement
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Nanoparticle is assembled by combining equimolar amounts of BG505-SOSIP-T33_dn10A and T33_dn10B and subsequent incubation. EM map of the full nanoparticle can be found elsewhere (D_ ...Details: Nanoparticle is assembled by combining equimolar amounts of BG505-SOSIP-T33_dn10A and T33_dn10B and subsequent incubation. EM map of the full nanoparticle can be found elsewhere (D_1000246183). Signal originating from the nanoparticle was subtracted and BG505-SOSIP antigens are extracted as independent subparticles
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: BG505-SOSIPv5.2(7S) - gp120

MacromoleculeName: BG505-SOSIPv5.2(7S) - gp120 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 56.863668 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 VVG

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Macromolecule #2: BG505-SOSIPv5.2(7S) - gp41

MacromoleculeName: BG505-SOSIPv5.2(7S) - gp41 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 17.198387 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
AVGIGAVSLG FLGAAGSTMG AASMTLTVQA RNLLSGIVQQ QSNLLRAPEC QQHLLKDTHW GIKQLQARVL AVEHYLRDQQ LLGIWGCSG KLICCTNVPW NSSWSNRNLS EIWDNMTWLQ WDKEISNYTQ IIYGLLEESQ NQQEKNEQDL LELD

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 33 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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: 11.0 nm / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
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: 324420
Details: 81105 T33_dn10 tetrahedral nanoparticles (each carrying 4 copies of BG505-SOSIP antigen) have been 3D aligned in Relion. Particles have been aligned and refined using tetrahedral symmetry. ...Details: 81105 T33_dn10 tetrahedral nanoparticles (each carrying 4 copies of BG505-SOSIP antigen) have been 3D aligned in Relion. Particles have been aligned and refined using tetrahedral symmetry. Localized reconstruction v1.2 was then applied to extract the BG505-SOSIP trimeric subparticles, resulting in a total of 324420 starting particles. The full nanoparticle reconstruction can be found elsewhere (EMD-21185).
CTF correctionSoftware - Name: RELION (ver. 3.0)
Startup modelType of model: OTHER / Details: Ab Initio map from cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Details: Particle 2D aligned in cryoSPARC and then in Relion
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: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.14 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 84435
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5cez

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

PDB-6vfl:
BG505-SOSIP model reconstructed by subparticle extraction and refinement from a tetrahedral nanoparticle T33_dn10

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