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- EMDB-21168: BG505-SOSIP-T33_dn2A nanoparticle fusion component in complex wit... -

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Basic information

Entry
Database: EMDB / ID: EMD-21168
TitleBG505-SOSIP-T33_dn2A nanoparticle fusion component in complex with VRC01-Fab
Map dataBG505-SOSIP-T33_dn2A fusion component in complex with VRC01-Fab - Negative Stain Map
Sample
  • Complex: BG505-SOSIP-T33_dn2A Nanoparticle Fusion Construct in complex with VRC01-Fab
    • Protein or peptide: BG505-SOSIP-T33_dn2A nanoparticle fusion component
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / negative staining / Resolution: 15.62 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: 0.015
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21168.png

Downloads & links

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Map

FileDownload / File: emd_21168.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBG505-SOSIP-T33_dn2A fusion component in complex with VRC01-Fab - Negative Stain Map
Voxel sizeX=Y=Z: 2.05 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.046146616 - 0.08171026
Average (Standard dev.)-0.0002522363 (±0.0057265414)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 328.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.052.052.05
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z328.000328.000328.000
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/NS160160160
D min/max/mean-0.0460.082-0.000

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Supplemental data

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Sample components

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Entire : BG505-SOSIP-T33_dn2A Nanoparticle Fusion Construct in complex wit...

EntireName: BG505-SOSIP-T33_dn2A Nanoparticle Fusion Construct in complex with VRC01-Fab
Components
  • Complex: BG505-SOSIP-T33_dn2A Nanoparticle Fusion Construct in complex with VRC01-Fab
    • Protein or peptide: BG505-SOSIP-T33_dn2A nanoparticle fusion component

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Supramolecule #1: BG505-SOSIP-T33_dn2A Nanoparticle Fusion Construct in complex wit...

SupramoleculeName: BG505-SOSIP-T33_dn2A Nanoparticle Fusion Construct in complex with VRC01-Fab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Nanoparticle Fusion Construct in complex with 3 copies of VRC01 Fab Fragment
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-T33_dn2A nanoparticle fusion component

MacromoleculeName: BG505-SOSIP-T33_dn2A nanoparticle fusion component / 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 DLLELDKWAS LWGSMGNLAE KMYKAGNAMY RKGQYTIAII AYTLALLKDP NNAEAWYNLG NAAYKKGEYD EAIEAYQKAL ELDPNNAEAW YNLGNAYYKQ GDYDEAIEYY KKALRLDPRN VDAIENLIEA EEKQGAS

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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

Concentration0.02 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
25.0 mMTris-HClTris
150.0 mMsodium chlorideNaClSodium chloride

Details: TBS buffer, pH 7.4
StainingType: NEGATIVE / Material: Uranyl Formate
Details: Sample diluted to 0.02 mg/mL. 3 uL was applied onto the grid, blotted off, and then stained with 2% uranyl formate for 60 seconds.
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
DetailsBG505-SOSIP-T33_dn2A and VRC01 Fab samples purified by SEC. BG505-SOSIP-T33_dn2A was incubated with 6-fold molar excess of VRC01-Fab for 1 hour, diluted to 0.02mg/ml and imaged.

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Electron microscopy

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

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Image processing

Startup modelType of model: OTHER / Details: Negative Stain EM map of BG505-SOSIP trimer
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: C3 (3 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 15.62 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 15252

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