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- EMDB-25108: I53-50 nanoparticle core reconstructed from GPC-I53-50NP by focus... -

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Basic information

Entry
Database: EMDB / ID: EMD-25108
TitleI53-50 nanoparticle core reconstructed from GPC-I53-50NP by focused refinement
Map dataI53-50 nanoparticle map reconstructed from GPC-I53-50 nanoparticle by focused refinement - Main map
Sample
  • Complex: I53-50 nanoparticle core recovered from GPC-I53-50 nanoparticle
    • Protein or peptide: Josiah GPCysR4-I53-50A - nanoparticle component
    • Protein or peptide: I53-50B component
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.67 Å
AuthorsAntanasijevic A / Brouwer PJM / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
Bill & Melinda Gates FoundationOPP1170236 United States
CitationJournal: Cell Host Microbe / Year: 2022
Title: Lassa virus glycoprotein nanoparticles elicit neutralizing antibody responses and protection.
Authors: Philip J M Brouwer / Aleksandar Antanasijevic / Adam J Ronk / Helena Müller-Kräuter / Yasunori Watanabe / Mathieu Claireaux / Hailee R Perrett / Tom P L Bijl / Marloes Grobben / Jeffrey C ...Authors: Philip J M Brouwer / Aleksandar Antanasijevic / Adam J Ronk / Helena Müller-Kräuter / Yasunori Watanabe / Mathieu Claireaux / Hailee R Perrett / Tom P L Bijl / Marloes Grobben / Jeffrey C Umotoy / Angela I Schriek / Judith A Burger / Khadija Tejjani / Nicole M Lloyd / Thijs H Steijaert / Marlies M van Haaren / Kwinten Sliepen / Steven W de Taeye / Marit J van Gils / Max Crispin / Thomas Strecker / Alexander Bukreyev / Andrew B Ward / Rogier W Sanders /
Abstract: The Lassa virus is endemic in parts of West Africa, and it causes hemorrhagic fever with high mortality. The development of a recombinant protein vaccine has been hampered by the instability of ...The Lassa virus is endemic in parts of West Africa, and it causes hemorrhagic fever with high mortality. The development of a recombinant protein vaccine has been hampered by the instability of soluble Lassa virus glycoprotein complex (GPC) trimers, which disassemble into monomeric subunits after expression. Here, we use two-component protein nanoparticles consisting of trimeric and pentameric subunits to stabilize GPC in a trimeric conformation. These GPC nanoparticles present twenty prefusion GPC trimers on the surface of an icosahedral particle. Cryo-EM studies of GPC nanoparticles demonstrated a well-ordered structure and yielded a high-resolution structure of an unliganded GPC. These nanoparticles induced potent humoral immune responses in rabbits and protective immunity against the lethal Lassa virus challenge in guinea pigs. Additionally, we isolated a neutralizing antibody that mapped to the putative receptor-binding site, revealing a previously undefined site of vulnerability. Collectively, these findings offer potential approaches to vaccine and therapeutic design for the Lassa virus.
History
DepositionOct 5, 2021-
Header (metadata) releaseOct 12, 2022-
Map releaseOct 12, 2022-
UpdateDec 28, 2022-
Current statusDec 28, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_25108.map.gz / Format: CCP4 / Size: 536.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationI53-50 nanoparticle map reconstructed from GPC-I53-50 nanoparticle by focused refinement - Main map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 520 pix.
= 535.6 Å
1.03 Å/pix.
x 520 pix.
= 535.6 Å
1.03 Å/pix.
x 520 pix.
= 535.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.06968644 - 0.12030742
Average (Standard dev.)3.624253e-05 (±0.0040571783)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions520520520
Spacing520520520
CellA=B=C: 535.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_25108_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: I53-50 nanoparticle map reconstructed from GPC-I53-50 nanoparticle by...

Fileemd_25108_half_map_1.map
AnnotationI53-50 nanoparticle map reconstructed from GPC-I53-50 nanoparticle by focused refinement - Half Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: I53-50 nanoparticle map reconstructed from GPC-I53-50 nanoparticle by...

Fileemd_25108_half_map_2.map
AnnotationI53-50 nanoparticle map reconstructed from GPC-I53-50 nanoparticle by focused refinement - Half Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : I53-50 nanoparticle core recovered from GPC-I53-50 nanoparticle

EntireName: I53-50 nanoparticle core recovered from GPC-I53-50 nanoparticle
Components
  • Complex: I53-50 nanoparticle core recovered from GPC-I53-50 nanoparticle
    • Protein or peptide: Josiah GPCysR4-I53-50A - nanoparticle component
    • Protein or peptide: I53-50B component

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Supramolecule #1: I53-50 nanoparticle core recovered from GPC-I53-50 nanoparticle

SupramoleculeName: I53-50 nanoparticle core recovered from GPC-I53-50 nanoparticle
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Nanoparticle consists of 60 monomeric I53-50A and 60 monomeric I53-50B building blocks. Nanoparticle was assembled by combining equimolar amounts of GPC-I53-50A and I53-50B.
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Josiah GPCysR4-I53-50A - nanoparticle component

MacromoleculeName: Josiah GPCysR4-I53-50A - nanoparticle component / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 73.84557 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGQIVTFFQE VPHVIEEVMN IVLIALSVLA VLKGLYNFAT CGLVGLVTFL LLCGRSCTTS LYKGVYELQT LELNMETLNM TMPLSCTKN NSHHYIMVGN ETGLELTLTN TSIINHKFCN LSDAHKKNLY DHALMSIIST FHLSIPNFNQ YEAMSCDFNG G KISVQYNL ...String:
MGQIVTFFQE VPHVIEEVMN IVLIALSVLA VLKGLYNFAT CGLVGLVTFL LLCGRSCTTS LYKGVYELQT LELNMETLNM TMPLSCTKN NSHHYIMVGN ETGLELTLTN TSIINHKFCN LSDAHKKNLY DHALMSIIST FHLSIPNFNQ YEAMSCDFNG G KISVQYNL SHSYAGDAAN HCGTVANGVL QTFMRMAWGG SYIALDSGCG NWDCIMTSYQ YLIIQNTTWE DHCQFSRPSP IG YLGLLSQ RTRDIYISRR RRGTFTWTLS DSEGKDTPGG YCLTRWMLIE AELKCFGNTA VAKCNEKHDE EFCDMLRLFD FNK QAIQRL KAPAQMSIQL INKAVNALIN DQLIMKNHLR DIMCIPYCNY SKYWYLNHTT TGRTSLPKCW LVSNGSYLNE THFS DDIEQ QADNMITEML QKEYMERQGG SGGSGGSGGS GGSEKAAKAE EAARKMEELF KKHKIVAVLR ANSVEEAIEK AVAVF AGGV HLIEITFTVP DADTVIKALS VLKEKGAIIG AGTVTSVEQC RKAVESGAEF IVSPHLDEEI SQFCKEKGVF YMPGVM TPT ELVKAMKLGH DILKLFPGEV VGPEFVKAMK GPFPNVKFVP TGGVDLDNVC EWFDAGVLAV GVGDALVEGD PDEVREK AK EFVEKIRGCT EGSLEWSHPQ FEK

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Macromolecule #2: I53-50B component

MacromoleculeName: I53-50B component / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.236877 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MNQHSHKDHE TVRIAVVRAR WHAEIVDACV SAFEAAMRDI GGDRFAVDVF DVPGAYEIPL HARTLAETGR YGAVLGTAFV VNGGIYRHE FVASAVINGM MNVQLNTGVP VLSAVLTPHN YDKSKAHTLL FLALFAVKGM EAARACVEIL AAREKIAAGS L EHHHHHH

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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

Concentration2 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
10.0 mMTris-HClTrisTris
150.0 mMNaClSodium chlorideSodium chloride

Details: TBS
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: Blot time 4s, Wait time 10s, Blot force 0.
DetailsI53-50 nanoparticle core recovered from GPC-I53-50 nanoparticle by focused refinement of the core. Nanoparticle was assembled by combining equimolar amounts of GPC-I53-50A and I53-50B.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / 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: 2337 / Average exposure time: 10.5 sec. / Average electron dose: 50.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 145508 / Details: Template picker
Startup modelType of model: OTHER / Details: Map reconstructed from negative stain EM
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0) / Details: Regularized likelihood
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 3.0) / Details: Regularized likelihood
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0)
Details: Icosahedral symmetry imposed for refinement. Solvent mask around the nanoparticle core used for refinement.
Number images used: 86411
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7sge:
I53-50 nanoparticle core reconstructed from GPC-I53-50NP by focused refinement

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