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- PDB-7lze: Cryo-EM Structure of disulfide stabilized HMPV F v4-B -

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Basic information

Entry
Database: PDB / ID: 7lze
TitleCryo-EM Structure of disulfide stabilized HMPV F v4-B
ComponentsFusion glycoprotein F0
KeywordsVIRAL PROTEIN / HMPV F / postfusion / prefusion / immunization
Function / homologyPrecursor fusion glycoprotein F0, Paramyxoviridae / Fusion glycoprotein F0 / membrane => GO:0016020 / fusion of virus membrane with host plasma membrane / host cell plasma membrane / virion membrane / plasma membrane / Fusion glycoprotein F0
Function and homology information
Biological speciesHuman metapneumovirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsGorman, J. / Kwong, P.D.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
Other privateSimons Foundation (SF349247) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Interprotomer disulfide-stabilized variants of the human metapneumovirus fusion glycoprotein induce high titer-neutralizing responses.
Authors: Guillaume B E Stewart-Jones / Jason Gorman / Li Ou / Baoshan Zhang / M Gordon Joyce / Lijuan Yang / Cheng Cheng / Gwo-Yu Chuang / Kathryn E Foulds / Wing-Pui Kong / Adam S Olia / Mallika ...Authors: Guillaume B E Stewart-Jones / Jason Gorman / Li Ou / Baoshan Zhang / M Gordon Joyce / Lijuan Yang / Cheng Cheng / Gwo-Yu Chuang / Kathryn E Foulds / Wing-Pui Kong / Adam S Olia / Mallika Sastry / Chen-Hsiang Shen / John-Paul Todd / Yaroslav Tsybovsky / Raffaello Verardi / Yongping Yang / Peter L Collins / Davide Corti / Antonio Lanzavecchia / Diana G Scorpio / John R Mascola / Ursula J Buchholz / Peter D Kwong /
Abstract: Human metapneumovirus (HMPV) is a major cause of respiratory disease worldwide, particularly among children and the elderly. Although there is no licensed HMPV vaccine, promising candidates have been ...Human metapneumovirus (HMPV) is a major cause of respiratory disease worldwide, particularly among children and the elderly. Although there is no licensed HMPV vaccine, promising candidates have been identified for related pneumoviruses based on the structure-based stabilization of the fusion (F) glycoprotein trimer, with prefusion-stabilized F glycoprotein trimers eliciting significantly higher neutralizing responses than their postfusion F counterparts. However, immunization with HMPV F trimers in either prefusion or postfusion conformations has been reported to elicit equivalent neutralization responses. Here we investigate the impact of stabilizing disulfides, especially interprotomer disulfides (IP-DSs) linking protomers of the F trimer, on the elicitation of HMPV-neutralizing responses. We designed F trimer disulfides, screened for their expression, and used electron microscopy (EM) to confirm their formation, including that of an unexpected postfusion variant. In mice, IP-DS-stabilized prefusion and postfusion HMPV F elicited significantly higher neutralizing responses than non-IP-DS-stabilized HMPV Fs. In macaques, the impact of IP-DS stabilization was more measured, although IP-DS-stabilized variants of either prefusion or postfusion HMPV F induced neutralizing responses many times the average titers observed in a healthy human cohort. Serological and absorption-based analyses of macaque responses revealed elicited HMPV-neutralizing responses to be absorbed differently by IP-DS-containing and by non-IP-DS-containing postfusion Fs, suggesting IP-DS stabilization to alter not only the immunogenicity of select epitopes but their antigenicity as well. We speculate the observed increase in immunogenicity by IP-DS trimers to be related to reduced interprotomer flexibility within the HMPV F trimer.
History
DepositionMar 9, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 25, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 6, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: Fusion glycoprotein F0
B: Fusion glycoprotein F0
C: Fusion glycoprotein F0
hetero molecules


Theoretical massNumber of molelcules
Total (without water)165,3589
Polymers163,4223
Non-polymers1,9376
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17600 Å2
ΔGint-55 kcal/mol
Surface area44210 Å2

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Components

#1: Protein Fusion glycoprotein F0 / Fusion glycoprotein F1 / Fusion glycoprotein F2


Mass: 54473.844 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human metapneumovirus / Production host: Homo sapiens (human) / References: UniProt: Q6WBA7
#2: 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
#3: 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: HMPV F v4-B / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Human metapneumovirus
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Buffer componentFormula: PBS
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 293 K

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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 FIELD / C2 aperture diameter: 70 µm
Image recordingAverage exposure time: 10 sec. / Electron dose: 64.05 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2Leginonimage acquisition
4CTFFIND4CTF correction
11cryoSPARC2.15classification
12cryoSPARC2.153D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59432 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5L1X

5l1x

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027965
ELECTRON MICROSCOPYf_angle_d0.40610800
ELECTRON MICROSCOPYf_dihedral_angle_d3.8931146
ELECTRON MICROSCOPYf_chiral_restr0.0431293
ELECTRON MICROSCOPYf_plane_restr0.0021368

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