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- EMDB-23605: Cryo-EM Structure of disulfide stabilized HMPV F v4-B -

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Basic information

Entry
Database: EMDB / ID: EMD-23605
TitleCryo-EM Structure of disulfide stabilized HMPV F v4-B
Map dataSharpened map
Sample
  • Complex: HMPV F v4-B
    • Protein or peptide: Fusion glycoprotein F0
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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
Methodsingle particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsGorman J / Kwong PD
Funding support United States, 2 items
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, 2021-
Header (metadata) releaseAug 25, 2021-
Map releaseAug 25, 2021-
UpdateOct 6, 2021-
Current statusOct 6, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lze
  • Surface level: 0.6
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23605.png

Downloads & links

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Map

FileDownload / File: emd_23605.map.gz / Format: CCP4 / Size: 70.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 1.0961 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.6
Minimum - Maximum-1.867353 - 3.992982
Average (Standard dev.)0.0056837033 (±0.09117918)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions264264264
Spacing264264264
CellA=B=C: 289.3704 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.09609848484851.09609848484851.0960984848485
M x/y/z264264264
origin x/y/z0.0000.0000.000
length x/y/z289.370289.370289.370
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS264264264
D min/max/mean-1.8673.9930.006

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Supplemental data

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Mask #1

Fileemd_23605_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map

Fileemd_23605_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_23605_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_23605_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

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Sample components

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Entire : HMPV F v4-B

EntireName: HMPV F v4-B
Components
  • Complex: HMPV F v4-B
    • Protein or peptide: Fusion glycoprotein F0
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: HMPV F v4-B

SupramoleculeName: HMPV F v4-B / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Human metapneumovirus
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Fusion glycoprotein F0

MacromoleculeName: Fusion glycoprotein F0 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Human metapneumovirus
Molecular weightTheoretical: 54.473844 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: LKESYLEESC STITEGYLSV LRTGWYTNVF TLEVGDVENL TCTDCPSLIK TELDLTKSAL RELKTVSADQ LAREEQIEGG GGGGFVLGA IALGVATAAA VTAGIAIAKT IRLESEVNAI KGCLKTTNEC VSTLGNGVRV LATAVRELKE FVSKNLTSAI N KNKCDIAD ...String:
LKESYLEESC STITEGYLSV LRTGWYTNVF TLEVGDVENL TCTDCPSLIK TELDLTKSAL RELKTVSADQ LAREEQIEGG GGGGFVLGA IALGVATAAA VTAGIAIAKT IRLESEVNAI KGCLKTTNEC VSTLGNGVRV LATAVRELKE FVSKNLTSAI N KNKCDIAD LCMAVSFSQF NRRFLNVVRQ FSDNAGITPA ISLDLMTDAE LARAVSYMPT SAGQIKLMLE NRAMVRRKGF GI LIGVYGS SVIYMVQLPI FGVIDTPCWI IKAAPSCSEK DGNYACLLRE DQGWYCKNAG STVYYPNDKD CETRGDHVFC DTA AGINVA EQSRECNINI STTNYPCKVS TGRHPISMVA LSPLGALVAC YKGVSCSIGS NRVGIIKQLP KGCSYITNQD ADTV TIDNT VYQLSKVEGE QHVIKGRPVS SSFDPICFPE DQFNVALDQV FESIENCQAL VDQSNKILNS AESAIGGYIP EAPRD GQAY VRKDGEWVLL STFLGGLVPR

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration1.0 mg/mL
BufferpH: 7.4 / Component - Formula: PBS
GridModel: C-flat-1.2/1.3 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average exposure time: 10.0 sec. / Average electron dose: 64.05 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationSoftware - Name: cryoSPARC (ver. 2.15)
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 59432

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Atomic model buiding 1

Initial modelPDB ID:

5l1x

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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