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- PDB-8vt2: cryo-EM structure of HMPV (MPV-2c) -

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Basic information

Entry
Database: PDB / ID: 8vt2
Titlecryo-EM structure of HMPV (MPV-2c)
ComponentsPrefusion of HMPV (MPV-2c)
KeywordsVIRAL PROTEIN / Prefusion / HMPV / Cryo-EM / VIRUS
Biological speciesHuman metapneumovirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsYu, X. / Langedijk, J.P.M.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2024
Title: Efficacious human metapneumovirus vaccine based on AI-guided engineering of a closed prefusion trimer.
Authors: Mark J G Bakkers / Tina Ritschel / Machteld Tiemessen / Jacobus Dijkman / Angelo A Zuffianò / Xiaodi Yu / Daan van Overveld / Lam Le / Richard Voorzaat / Marlies M van Haaren / Martijn de ...Authors: Mark J G Bakkers / Tina Ritschel / Machteld Tiemessen / Jacobus Dijkman / Angelo A Zuffianò / Xiaodi Yu / Daan van Overveld / Lam Le / Richard Voorzaat / Marlies M van Haaren / Martijn de Man / Sem Tamara / Leslie van der Fits / Roland Zahn / Jarek Juraszek / Johannes P M Langedijk /
Abstract: The prefusion conformation of human metapneumovirus fusion protein (hMPV Pre-F) is critical for eliciting the most potent neutralizing antibodies and is the preferred immunogen for an efficacious ...The prefusion conformation of human metapneumovirus fusion protein (hMPV Pre-F) is critical for eliciting the most potent neutralizing antibodies and is the preferred immunogen for an efficacious vaccine against hMPV respiratory infections. Here we show that an additional cleavage event in the F protein allows closure and correct folding of the trimer. We therefore engineered the F protein to undergo double cleavage, which enabled screening for Pre-F stabilizing substitutions at the natively folded protomer interfaces. To identify these substitutions, we developed an AI convolutional classifier that successfully predicts complex polar interactions often overlooked by physics-based methods and visual inspection. The combination of additional processing, stabilization of interface regions and stabilization of the membrane-proximal stem, resulted in a Pre-F protein vaccine candidate without the need for a heterologous trimerization domain that exhibited high expression yields and thermostability. Cryo-EM analysis shows the complete ectodomain structure, including the stem, and a specific interaction of the newly identified cleaved C-terminus with the adjacent protomer. Importantly, the protein induces high and cross-neutralizing antibody responses resulting in near complete protection against hMPV challenge in cotton rats, making the highly stable, double-cleaved hMPV Pre-F trimer an attractive vaccine candidate.
History
DepositionJan 25, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 31, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 7, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Prefusion of HMPV (MPV-2c)
B: Prefusion of HMPV (MPV-2c)
C: Prefusion of HMPV (MPV-2c)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)173,35712
Polymers169,6613
Non-polymers3,6969
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Prefusion of HMPV (MPV-2c)


Mass: 56553.586 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human metapneumovirus / Production host: Homo sapiens (human)
#2: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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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: Prefusion of HMPV (MPV-2cREKR) / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Human metapneumovirus
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97386 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01310824
ELECTRON MICROSCOPYf_angle_d0.89214661
ELECTRON MICROSCOPYf_dihedral_angle_d10.2161584
ELECTRON MICROSCOPYf_chiral_restr0.0621784
ELECTRON MICROSCOPYf_plane_restr0.0061857

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