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- EMDB-21176: EM-Based Polyclonal Epitope Mapping. ConM-SOSIP Complexed with Pu... -

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Basic information

Entry
Database: EMDB / ID: EMD-21176
TitleEM-Based Polyclonal Epitope Mapping. ConM-SOSIP Complexed with Purified Polyclonal Fab (Grp 1, ID r2382, Wk4)
Map dataEM-Based Polyclonal Epitope Mapping. ConM-SOSIP Complexed with Purified Polyclonal Fab (Grp 1, ID r2382 Wk4), Negative Stain EM map
Sample
  • Complex: ConM-SOSIP.v9 complexed with purified polyclonal Fab from an immunized rabbit (Grp 1, ID r2382, Wk4)
    • Protein or peptide: ConM-SOSIPv9
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / negative staining / Resolution: 16.52 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-
UpdateAug 26, 2020-
Current statusAug 26, 2020Processing 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
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21176.png

Downloads & links

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Map

FileDownload / File: emd_21176.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM-Based Polyclonal Epitope Mapping. ConM-SOSIP Complexed with Purified Polyclonal Fab (Grp 1, ID r2382 Wk4), Negative Stain EM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.77 Å/pix.
x 224 pix.
= 396.48 Å		1.77 Å/pix.
x 224 pix.
= 396.48 Å		1.77 Å/pix.
x 224 pix.
= 396.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.77 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.027319307 - 0.06622066
Average (Standard dev.)0.00001359268 (±0.002764895)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 396.47998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.771.771.77
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z396.480396.480396.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.0270.0660.000

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Supplemental data

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Sample components

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Entire : ConM-SOSIP.v9 complexed with purified polyclonal Fab from an immu...

EntireName: ConM-SOSIP.v9 complexed with purified polyclonal Fab from an immunized rabbit (Grp 1, ID r2382, Wk4)
Components
  • Complex: ConM-SOSIP.v9 complexed with purified polyclonal Fab from an immunized rabbit (Grp 1, ID r2382, Wk4)
    • Protein or peptide: ConM-SOSIPv9

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Supramolecule #1: ConM-SOSIP.v9 complexed with purified polyclonal Fab from an immu...

SupramoleculeName: ConM-SOSIP.v9 complexed with purified polyclonal Fab from an immunized rabbit (Grp 1, ID r2382, Wk4)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293F / Recombinant plasmid: pPPI4

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Macromolecule #1: ConM-SOSIPv9

MacromoleculeName: ConM-SOSIPv9 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDAMKRGLCC VLLLCGAVFV SPSQEIHARF RRGARAENLW VTVYYGVPVW KDACTTLFCA SDAKAYDTEK RNVWATHCCV PTDPNPQEIV LENVTENFNM WKNNMVEQMH EDIISLWDQS LKPCVKLTPL CVTLNCTDVN ATNNTTNNEE IKNCSFNITT ELRDKKKKVY ...String:
MDAMKRGLCC VLLLCGAVFV SPSQEIHARF RRGARAENLW VTVYYGVPVW KDACTTLFCA SDAKAYDTEK RNVWATHCCV PTDPNPQEIV LENVTENFNM WKNNMVEQMH EDIISLWDQS LKPCVKLTPL CVTLNCTDVN ATNNTTNNEE IKNCSFNITT ELRDKKKKVY ALFYKLDVVP IDDNNSYRLI NCNTSAITQA CPKVSFEPIP IHYCAPAGFA ILKCNDKKFN GTGPCKNVST VQCTHGIKPV VSTQLLLNGS LAEEEIIIRS ENITNNAKTI IVQLNESVEI NCTRPNNNTV KSIRIGPGQW FYYTGDIIGD IRQAHCNISR TKWNKTLQQV AKKLREHFNK TIIFNQSSGG DLEITTHSFN CGGEFFYCNT SELFNSTWNG TNNTITLPCR IKQIINMWQR VGQAMYAPPI EGKIRCTSNI TGLLLTRDGG NNNTETFRPG GGDMRDNWRS ELYKYKVVKI EPLGVAPTRC KRRVVERRRR RRAVGIGAVS LGFLGAAGST MGAASMTLTV QARNLLSGIV QQQSNLLCAP ECQQHLLQDT HWGIKQLQAR VLAVEHYLKD QQLLGIWGCS GKLICCTNVP WNSSWSNKSQ DEIWDNMTWM EWDKEINNYT DIIYSLIEES QNQQEKNEQE LLALD

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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 / Pretreatment - Type: GLOW DISCHARGE
DetailsConM-SOSIP.v9 was complexed with the purified polyclonal Fab overnight. The complex was purified using SEC and concentrated. The complex was then diluted in TBS to 0.05mg/ml to be loaded onto an imaging grid.

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number grids imaged: 1 / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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 / Nominal magnification: 62000
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Negative Stain EM map of SOSIP trimer
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 16.52 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 86565
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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