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- EMDB-21183: De novo designed icosahedral nanoparticle I53_dn5 presenting BG50... -

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Basic information

Entry
Database: EMDB / ID: EMD-21183
TitleDe novo designed icosahedral nanoparticle I53_dn5 presenting BG505-SOSIP, Cryo-EM Map
Map dataBG505-SOSIP-I53_dn5 Icosahedral Nanoparticle, Cryo EM map
Sample
  • Complex: De novo designed icosahedral nanoparticle I53_dn5 displaying 20 copies of BG505-SOSIP trimer on the surface. Nanoparticle consists of 60 copies of both subunits (BG505-SOSIP-I53_dn5B and I53_dn5A)
    • Protein or peptide: BG505-SOSIP-I53_dn5B
    • Protein or peptide: I53_dn5A
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.46 Å
AuthorsWard AB / Antanasijevic A
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-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.003
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.003
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21183.png

Downloads & links

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Map

FileDownload / File: emd_21183.map.gz / Format: CCP4 / Size: 536.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBG505-SOSIP-I53_dn5 Icosahedral Nanoparticle, Cryo EM map
Voxel sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.003 / Movie #1: 0.003
Minimum - Maximum-0.0026518067 - 0.012264347
Average (Standard dev.)0.00053777447 (±0.0014047732)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions520520520
Spacing520520520
CellA=B=C: 598.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z520520520
origin x/y/z0.0000.0000.000
length x/y/z598.000598.000598.000
α/β/γ90.00090.00090.000
start NX/NY/NZ79740
NX/NY/NZ93103213
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS520520520
D min/max/mean-0.0030.0120.001

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Supplemental data

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Half map: BG505-SOSIP-I53 dn5 Icosahedral Nanoparticle, Cryo EM Half-map 1

Fileemd_21183_half_map_1.map
AnnotationBG505-SOSIP-I53_dn5 Icosahedral Nanoparticle, Cryo EM Half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: BG505-SOSIP-I53 dn5 Icosahedral Nanoparticle, Cryo EM Half-map 2

Fileemd_21183_half_map_2.map
AnnotationBG505-SOSIP-I53_dn5 Icosahedral Nanoparticle, 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 icosahedral nanoparticle I53_dn5 displaying 20 c...

EntireName: De novo designed icosahedral nanoparticle I53_dn5 displaying 20 copies of BG505-SOSIP trimer on the surface. Nanoparticle consists of 60 copies of both subunits (BG505-SOSIP-I53_dn5B and I53_dn5A)
Components
  • Complex: De novo designed icosahedral nanoparticle I53_dn5 displaying 20 copies of BG505-SOSIP trimer on the surface. Nanoparticle consists of 60 copies of both subunits (BG505-SOSIP-I53_dn5B and I53_dn5A)
    • Protein or peptide: BG505-SOSIP-I53_dn5B
    • Protein or peptide: I53_dn5A

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Supramolecule #1: De novo designed icosahedral nanoparticle I53_dn5 displaying 20 c...

SupramoleculeName: De novo designed icosahedral nanoparticle I53_dn5 displaying 20 copies of BG505-SOSIP trimer on the surface. Nanoparticle consists of 60 copies of both subunits (BG505-SOSIP-I53_dn5B and I53_dn5A)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Nanoparticle is assembled by combining equimolar amounts of BG505-SOSIP-I53_dn5B and I53_dn5A and subsequent incubation. Nanoparticle is purified from unassembled components by SEC
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293F / Recombinant plasmid: pPPI4

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Macromolecule #1: BG505-SOSIP-I53_dn5B

MacromoleculeName: BG505-SOSIP-I53_dn5B / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKRGLCCVLL LCGAVFVSPS QEIHARFRRG ARAENLWVTV YYGVPVWKDA ETTLFCASDA KAYETKKHNV WATHCCVPTD PNPQEIHLEN VTEEFNMWKN NMVEQMHTDI ISLWDQSLKP CVKLTPLCVT LQCTNVTNNI TDDMRGELKN CSFNMTTELR DKKQKVYSLF ...String:
MKRGLCCVLL LCGAVFVSPS QEIHARFRRG ARAENLWVTV YYGVPVWKDA ETTLFCASDA KAYETKKHNV WATHCCVPTD PNPQEIHLEN VTEEFNMWKN NMVEQMHTDI ISLWDQSLKP CVKLTPLCVT LQCTNVTNNI TDDMRGELKN CSFNMTTELR DKKQKVYSLF YRLDVVQINE NQGNRSNNSN KEYRLINCNT SAITQACPKV SFEPIPIHYC APAGFAILKC KDKKFNGTGP CTNVSTVQCT HGIKPVVSTQ LLLNGSLAEE EVIIRSENIT NNAKNILVQL NESVQINCTR PNNNTVKSIR IGPGQWFYYT GDIIGDIRQA HCNVSKATWN ETLGKVVKQL RKHFGNNTII RFANSSGGDL EVTTHSFNCG GEFFYCNTSG LFNSTWISNT SVQGSNSTGS NDSITLPCRI KQIINMWQRI GQAMYAPPIQ GVIRCVSNIT GLILTRDGGS TNSTTETFRP GGGDMRDNWR SELYKYKVVK IEPLGVAPTR CKRRVVGRRR RRRAVGIGAV SLGFLGAAGS TMGAASMTLT VQARNLLSGI VQQQSNLLRA PECQQHLLKD THWGIKQLQA RVLAVEHYLR DQQLLGIWGC SGKLICCTNV PWNSSWSNRN LSEIWDNMTW LQWDKEISNY TQIIYGLLEE SQNQQEKNEQ GSGSGSGSGG EEAELAYLLG ELAYKLGEYR IAIRAYRIAL KRDPNNAEAW YNLGNAYYKQ GRYREAIEYY QKALELDPNN AEAWYNLGNA YYERGEYEEA IEYYRKALRL DPNNADAMQN LLNAKMREEL EAS

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Macromolecule #2: I53_dn5A

MacromoleculeName: I53_dn5A / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MGKYDGSKLR IGILHARWNA EIILALVLGA LKRLQEFGVK RENIIIETVP GSFELPYGSK LFVEKQKRLG KPLDAIIPIG VLIKGSTMHF EYICDSTTHQ LMKLNFELGI PVIFGVLTCL TDEQAEARAG LIEGKMHNHG EDWGAAAVEM ATKFNLEHHH HHH

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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

Concentration1.7 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-I53_dn5B and I53_dn5A and subsequent incubation. Nanoparticle is purified from unassembled components by SEC. Sample is then concentrated to 1.7mg/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: 0.8 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: 2 / Number real images: 2999 / 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: Manual picking in Relion
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)
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: I (icosahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 12.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 9525

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Atomic model buiding 1

DetailsModel refinement was not performed due to low resolution.

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