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- PDB-7sge: I53-50 nanoparticle core reconstructed from GPC-I53-50NP by focus... -

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Basic information

Entry
Database: PDB / ID: 7sge
TitleI53-50 nanoparticle core reconstructed from GPC-I53-50NP by focused refinement
Components
  • I53-50B component
  • Josiah GPCysR4-I53-50A - nanoparticle component
KeywordsVIRAL PROTEIN / glycoprotein / Lassa / Josiah / vaccine design / nanoparticle / protein design
Biological speciessynthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.67 Å
AuthorsAntanasijevic, A. / Brouwer, P.J.M. / Ward, A.B.
Funding support United States, 1items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 12, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 30, 2022Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Dec 28, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Josiah GPCysR4-I53-50A - nanoparticle component
B: I53-50B component


Theoretical massNumber of molelcules
Total (without water)92,0822
Polymers92,0822
Non-polymers00
Water0
1
A: Josiah GPCysR4-I53-50A - nanoparticle component
B: I53-50B component
x 60


Theoretical massNumber of molelcules
Total (without water)5,524,947120
Polymers5,524,947120
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: Josiah GPCysR4-I53-50A - nanoparticle component
B: I53-50B component
x 5


  • icosahedral pentamer
  • 460 kDa, 10 polymers
Theoretical massNumber of molelcules
Total (without water)460,41210
Polymers460,41210
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Josiah GPCysR4-I53-50A - nanoparticle component
B: I53-50B component
x 6


  • icosahedral 23 hexamer
  • 552 kDa, 12 polymers
Theoretical massNumber of molelcules
Total (without water)552,49512
Polymers552,49512
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

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


Mass: 73845.570 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: De novo designed / Source: (gene. exp.) synthetic construct (others) / Plasmid: pPPI4 / Cell line (production host): HEK293F / Production host: Homo sapiens (human)
#2: Protein I53-50B component


Mass: 18236.877 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Plasmid: pET28b / Production host: Escherichia coli BL21 (bacteria)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: I53-50 nanoparticle core recovered from GPC-I53-50 nanoparticle
Type: COMPLEX
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.
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: synthetic construct (others)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F
Buffer solutionpH: 7 / Details: TBS
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTrisTris-HClTris1
2150 mMSodium chlorideNaClSodium chloride1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: I53-50 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.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K / Details: Blot time 4s, Wait time 10s, Blot force 0

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 29000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10.5 sec. / Electron dose: 50.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 2337
Image scansMovie frames/image: 42

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2particle selection
2Leginonimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13RosettaEMmodel refinement
14Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 145508 / Details: Template picker
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 3.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 86411 / Algorithm: BACK PROJECTION
Details: Icosahedral symmetry imposed for refinement. Solvent mask around the nanoparticle core used for refinement.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6P6F

6p6f

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