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- EMDB-25982: Cryo-EM structure of hMPV preF bound by Fabs MPE8 and SAN32-2 -

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Basic information

Entry
Database: EMDB / ID: EMD-25982
TitleCryo-EM structure of hMPV preF bound by Fabs MPE8 and SAN32-2
Map data
Sample
  • Complex: Trimeric prefusion hMPV F glycoprotein bound by three molecules of Fabs SAN32-2 and MPE8
    • Protein or peptide: Fusion glycoprotein F0
    • Protein or peptide: SAN32-2 Fab heavy chain
    • Protein or peptide: SAN32-2 Fab light chain
    • Protein or peptide: MPE8 Fab heavy chain
    • Protein or peptide: MPE8 Fab light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homologyPrecursor fusion glycoprotein F0, Paramyxoviridae / Fusion glycoprotein F0 / 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 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsRush SA / Hsieh C-L / McLellan JS
Funding support United States, 1 items
OrganizationGrant numberCountry
Welch FoundationF-0003-19620604 United States
CitationJournal: Cell Rep / Year: 2022
Title: Characterization of prefusion-F-specific antibodies elicited by natural infection with human metapneumovirus.
Authors: Scott A Rush / Gurpreet Brar / Ching-Lin Hsieh / Emilie Chautard / Jennifer N Rainho-Tomko / Chris D Slade / Christine A Bricault / Ana Kume / James Kearns / Rachel Groppo / Sophia T Mundle ...Authors: Scott A Rush / Gurpreet Brar / Ching-Lin Hsieh / Emilie Chautard / Jennifer N Rainho-Tomko / Chris D Slade / Christine A Bricault / Ana Kume / James Kearns / Rachel Groppo / Sophia T Mundle / Linong Zhang / Danilo Casimiro / Tong-Ming Fu / Joshua M DiNapoli / Jason S McLellan /
Abstract: Human metapneumovirus (hMPV) is a major cause of acute respiratory infections in infants and older adults, for which no vaccines or therapeutics are available. The viral fusion (F) glycoprotein is ...Human metapneumovirus (hMPV) is a major cause of acute respiratory infections in infants and older adults, for which no vaccines or therapeutics are available. The viral fusion (F) glycoprotein is required for entry and is the primary target of neutralizing antibodies; however, little is known about the humoral immune response generated from natural infection. Here, using prefusion-stabilized F proteins to interrogate memory B cells from two older adults, we obtain over 700 paired non-IgM antibody sequences representing 563 clonotypes, indicative of a highly polyclonal response. Characterization of 136 monoclonal antibodies reveals broad recognition of the protein surface, with potently neutralizing antibodies targeting each antigenic site. Cryo-EM studies further reveal two non-canonical sites and the molecular basis for recognition of the apex of hMPV F by two prefusion-specific neutralizing antibodies. Collectively, these results provide insight into the humoral response to hMPV infection in older adults and will help guide vaccine development.
History
DepositionJan 17, 2022-
Header (metadata) releaseSep 14, 2022-
Map releaseSep 14, 2022-
UpdateNov 23, 2022-
Current statusNov 23, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25982.png

Downloads & links

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Map

FileDownload / File: emd_25982.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.37 Å/pix.
x 256 pix.
= 349.92 Å		1.37 Å/pix.
x 256 pix.
= 349.92 Å		1.37 Å/pix.
x 256 pix.
= 349.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.36688 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.0
Minimum - Maximum-10.731491 - 44.180817
Average (Standard dev.)3.674986e-06 (±1.015123)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 349.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Trimeric prefusion hMPV F glycoprotein bound by three molecules o...

EntireName: Trimeric prefusion hMPV F glycoprotein bound by three molecules of Fabs SAN32-2 and MPE8
Components
  • Complex: Trimeric prefusion hMPV F glycoprotein bound by three molecules of Fabs SAN32-2 and MPE8
    • Protein or peptide: Fusion glycoprotein F0
    • Protein or peptide: SAN32-2 Fab heavy chain
    • Protein or peptide: SAN32-2 Fab light chain
    • Protein or peptide: MPE8 Fab heavy chain
    • Protein or peptide: MPE8 Fab light chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Trimeric prefusion hMPV F glycoprotein bound by three molecules o...

SupramoleculeName: Trimeric prefusion hMPV F glycoprotein bound by three molecules of Fabs SAN32-2 and MPE8
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Human metapneumovirus
Molecular weightTheoretical: 457.9 kDa/nm

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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: 60.453852 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSWKVVIIFS LLITPQHGLK ESYLEESCST ITEGYLSVLR TGWYTNVFTL EVGDVENLTC ADGPSLIKTE LDLTKSALRE LRTVSADQL AREEQIENPR RRRFVLGAIA CGVATAAAVT AGVAIAKCIR LESEVTAIKN CLKKTNECVS TLGCGVRVLA T AVRELKDF ...String:
MSWKVVIIFS LLITPQHGLK ESYLEESCST ITEGYLSVLR TGWYTNVFTL EVGDVENLTC ADGPSLIKTE LDLTKSALRE LRTVSADQL AREEQIENPR RRRFVLGAIA CGVATAAAVT AGVAIAKCIR LESEVTAIKN CLKKTNECVS TLGCGVRVLA T AVRELKDF VSKNLTRAIN KNKCDIPDLK MAVSFSQFNR RFLNVVRQFS DNAGITPAIS KDLMTDAELA RAISNMPTSA GQ IKLMLEN RAMVRRKGFG ILIGVYGSSV IYMVQLPIFG VIDTPCWIVK AAPSCSEKKG NYACLLREDQ GWYCQNAGST VYY PCEKDC ETRGDHVFCD TAAGINVAEQ SKECNINIST TNYPCKVSCG RHPISMVALS PLGALVACYK GVSCSIGSNR VGII KQLNK GCSYITNQDA DTVTIDNTVY QLSKVEGEQH VIKGRPVSSS FDPVKFPQDQ FNVALDQCFE SIENSQALVD QSNRI LSSA EKGNTGGGGS GYIPEAPRDG QAYVRKDGEW VLLSTFLGRS LEVLFQGPGH HHHHHHHSAW SHPQFEK

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Macromolecule #2: SAN32-2 Fab heavy chain

MacromoleculeName: SAN32-2 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.099031 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EVQLVQSGAE VKKPGESLRI SCKSSGYTFT TYWITWVRQM PGKGLEWMGR IDPSDSYTDY SPSFKGHVTI SVDKSINTAY LQWGSLKSS DTAIFYCARQ ASSGKIYFDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS ...String:
EVQLVQSGAE VKKPGESLRI SCKSSGYTFT TYWITWVRQM PGKGLEWMGR IDPSDSYTDY SPSFKGHVTI SVDKSINTAY LQWGSLKSS DTAIFYCARQ ASSGKIYFDY WGQGTLVTVS SASTKGPSVF PLAPSSKSTS GGTAALGCLV KDYFPEPVTV S WNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCD

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Macromolecule #3: SAN32-2 Fab light chain

MacromoleculeName: SAN32-2 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.542988 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCQASQGIN YYLNWYQQKP GKAPKVLIYD ASDLETGVPS RFSGGGSGTH FTFTISSLQT EDIGTYYCQ QYDNLPFTFG QGTRLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS ...String:
DIQMTQSPSS LSASVGDRVT ITCQASQGIN YYLNWYQQKP GKAPKVLIYD ASDLETGVPS RFSGGGSGTH FTFTISSLQT EDIGTYYCQ QYDNLPFTFG QGTRLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

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Macromolecule #4: MPE8 Fab heavy chain

MacromoleculeName: MPE8 Fab heavy chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.985793 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISASSSYSDY ADSAKGRFTI SRDNAKTSLF LQMNSLRAE DTAIYFCARA RATGYSSITP YFDIWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ...String:
EVQLVESGGG LVKPGGSLRL SCAASGFTFS SYSMNWVRQA PGKGLEWVSS ISASSSYSDY ADSAKGRFTI SRDNAKTSLF LQMNSLRAE DTAIYFCARA RATGYSSITP YFDIWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD

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Macromolecule #5: MPE8 Fab light chain

MacromoleculeName: MPE8 Fab light chain / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 22.772092 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QSVVTQPPSV SGAPGQRVTI SCTGSSSNIG AGYDVHWYQQ LPGTAPKLLI YDNNNRPSGV PDRFSASKSG TSASLAITGL QAEDEADYY CQSYDRSLSG VFGTGTKVTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT VAWKADSSPV K AGVETTTP ...String:
QSVVTQPPSV SGAPGQRVTI SCTGSSSNIG AGYDVHWYQQ LPGTAPKLLI YDNNNRPSGV PDRFSASKSG TSASLAITGL QAEDEADYY CQSYDRSLSG VFGTGTKVTV LGQPKAAPSV TLFPPSSEEL QANKATLVCL ISDFYPGAVT VAWKADSSPV K AGVETTTP SKQSNNKYAA SSYLSLTPEQ WKSHKSYSCQ VTHEGSTVEK TVAPTECS

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Macromolecule #7: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 7 / Number of copies: 6 / 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

BufferpH: 8
Component:
ConcentrationNameFormula
2.0 mMTris
200.0 mMsodium chlorideNaClSodium chloride
0.02 %sodium azideNaN3
0.025 %amphipolA8-35
GridModel: C-flat-1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 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 opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 29000
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 3636 / Average electron dose: 70.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 772860
CTF correctionSoftware - Name: cryoSPARC (ver. 3.2.0) / Software - details: cryoSPARC Live
Startup modelType of model: NONE / Details: ab-initio
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 134989

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