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- EMDB-26562: Cryo-EM structure of Human respiratory syncytial virus F variant ... -

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Basic information

Entry
Database: EMDB / ID: EMD-26562
TitleCryo-EM structure of Human respiratory syncytial virus F variant (construct pXCS847A)
Map data
Sample
  • Complex: Human respiratory syncytial virus F variant (construct pXCS847A) with Fabs AM14 and AM22
    • Protein or peptide: RSV variant (construct pXCS847A) F1
    • Protein or peptide: AM14 Fab heavy chain
    • Protein or peptide: AM14 Fab light chain
    • Protein or peptide: AM22 Fab heavy chain
    • Protein or peptide: AM22 Fab light chain
    • Protein or peptide: RSV variant (construct pXCS847A) F2
  • 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 speciesRespiratory syncytial virus / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsLees JA / Ammirati M / Han S
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Sci Transl Med / Year: 2023
Title: Rational design of a highly immunogenic prefusion-stabilized F glycoprotein antigen for a respiratory syncytial virus vaccine.
Authors: Ye Che / Alexey V Gribenko / Xi Song / Luke D Handke / Kari S Efferen / Kristin Tompkins / Srinivas Kodali / Lorna Nunez / A Krishna Prasad / Lynn M Phelan / Mark Ammirati / Xiaodi Yu / ...Authors: Ye Che / Alexey V Gribenko / Xi Song / Luke D Handke / Kari S Efferen / Kristin Tompkins / Srinivas Kodali / Lorna Nunez / A Krishna Prasad / Lynn M Phelan / Mark Ammirati / Xiaodi Yu / Joshua A Lees / Wei Chen / Lyndsey Martinez / Vidia Roopchand / Seungil Han / Xiayang Qiu / John P DeVincenzo / Kathrin U Jansen / Philip R Dormitzer / Kena A Swanson /
Abstract: Respiratory syncytial virus (RSV) is the leading, global cause of serious respiratory disease in infants and is an important cause of respiratory illness in older adults. No RSV vaccine is currently ...Respiratory syncytial virus (RSV) is the leading, global cause of serious respiratory disease in infants and is an important cause of respiratory illness in older adults. No RSV vaccine is currently available. The RSV fusion (F) glycoprotein is a key antigen for vaccine development, and its prefusion conformation is the target of the most potent neutralizing antibodies. Here, we describe a computational and experimental strategy for designing immunogens that enhance the conformational stability and immunogenicity of RSV prefusion F. We obtained an optimized vaccine antigen after screening nearly 400 engineered F constructs. Through in vitro and in vivo characterization studies, we identified F constructs that are more stable in the prefusion conformation and elicit ~10-fold higher serum-neutralizing titers in cotton rats than DS-Cav1. The stabilizing mutations of the lead construct (847) were introduced onto F glycoprotein backbones of strains representing the dominant circulating genotypes of the two major RSV subgroups, A and B. Immunization of cotton rats with a bivalent vaccine formulation of these antigens conferred complete protection against RSV challenge, with no evidence of disease enhancement. The resulting bivalent RSV prefusion F investigational vaccine has recently been shown to be efficacious against RSV disease in two pivotal phase 3 efficacy trials, one for passive protection of infants by immunization of pregnant women and the second for active protection of older adults by direct immunization.
History
DepositionMar 30, 2022-
Header (metadata) releaseApr 19, 2023-
Map releaseApr 19, 2023-
UpdateMay 10, 2023-
Current statusMay 10, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26562.png

Downloads & links

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Map

FileDownload / File: emd_26562.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.37 Å
Density
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.17849936 - 0.29863864
Average (Standard dev.)6.7627094e-05 (±0.00811417)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 383.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_26562_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_26562_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Human respiratory syncytial virus F variant (construct pXCS847A) ...

EntireName: Human respiratory syncytial virus F variant (construct pXCS847A) with Fabs AM14 and AM22
Components
  • Complex: Human respiratory syncytial virus F variant (construct pXCS847A) with Fabs AM14 and AM22
    • Protein or peptide: RSV variant (construct pXCS847A) F1
    • Protein or peptide: AM14 Fab heavy chain
    • Protein or peptide: AM14 Fab light chain
    • Protein or peptide: AM22 Fab heavy chain
    • Protein or peptide: AM22 Fab light chain
    • Protein or peptide: RSV variant (construct pXCS847A) F2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Human respiratory syncytial virus F variant (construct pXCS847A) ...

SupramoleculeName: Human respiratory syncytial virus F variant (construct pXCS847A) with Fabs AM14 and AM22
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Respiratory syncytial virus

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Macromolecule #1: RSV variant (construct pXCS847A) F1

MacromoleculeName: RSV variant (construct pXCS847A) F1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Respiratory syncytial virus
Molecular weightTheoretical: 45.025391 KDa
Recombinant expressionOrganism: Mammalia (mammals)
SequenceString: FLGFLLGVGS ACASGIAVSK VLHLEGEVNK IKSALLSTNK AVVSLSNGVS VLTIKVLDLK NYIDKQLLPI VNKQSCSISN IETVIEFQQ KNNRLLEITR EFSVNAGVTT PVSTYMLTNS ELLSLINDMP ITNDQKKLMS SNVQIVRQQS YSIMSIIKEE V LAYVVQLP ...String:
FLGFLLGVGS ACASGIAVSK VLHLEGEVNK IKSALLSTNK AVVSLSNGVS VLTIKVLDLK NYIDKQLLPI VNKQSCSISN IETVIEFQQ KNNRLLEITR EFSVNAGVTT PVSTYMLTNS ELLSLINDMP ITNDQKKLMS SNVQIVRQQS YSIMSIIKEE V LAYVVQLP LYGVIDTPCW KLHTSPLCTT NTKEGSNICL TRTDRGWYCD NAGSVSFFPQ AETCKVQSNR VFCDTMNSLT LP SEVNLCN IDIFNPKYDC KIMTSKTDVS SSVITSLGAI VSCYGKTKCT ASNKNRGIIK TFSNGCDYVS NKGVDTVSVG NTL YYVNKQ EGKSLYVKGE PIINFYDPLV FPSSEFDASI SQVNEKINQS LAFIRKSDEL LSAIGGYIPE APRDGQAYVR KDGE WVLLS TFLGG

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Macromolecule #2: AM14 Fab heavy chain

MacromoleculeName: AM14 Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 26.231088 KDa
Recombinant expressionOrganism: Mammalia (mammals)
SequenceString: EVQLVESGGG VVQPGRSLRL SCAASGFSFS HYAMHWVRQA PGKGLEWVAV ISYDGENTYY ADSVKGRFSI SRDNSKNTVS LQMNSLRPE DTALYYCARD RIVDDYYYYG MDVWGQGATV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP V TVSWNSGA ...String:
EVQLVESGGG VVQPGRSLRL SCAASGFSFS HYAMHWVRQA PGKGLEWVAV ISYDGENTYY ADSVKGRFSI SRDNSKNTVS LQMNSLRPE DTALYYCARD RIVDDYYYYG MDVWGQGATV TVSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP V TVSWNSGA LTSGVHTFPA VLQSSGLYSL SSVVTVPSSS LGTQTYICNV NHKPSNTKVD KKVEPKSCDG GGSLVPRGSD YK DDDDK

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Macromolecule #3: AM14 Fab light chain

MacromoleculeName: AM14 Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.972656 KDa
Recombinant expressionOrganism: Mammalia (mammals)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCQASQDIK KYLNWYHQKP GKVPELLMHD ASNLETGVPS RFSGRGSGTD FTLTISSLQP EDIGTYYCQ QYDNLPPLTF GGGTKVEIKR TVRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ S GNSQESVT ...String:
DIQMTQSPSS LSASVGDRVT ITCQASQDIK KYLNWYHQKP GKVPELLMHD ASNLETGVPS RFSGRGSGTD FTLTISSLQP EDIGTYYCQ QYDNLPPLTF GGGTKVEIKR TVRTVAAPSV FIFPPSDEQL KSGTASVVCL LNNFYPREAK VQWKVDNALQ S GNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC

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Macromolecule #4: AM22 Fab heavy chain

MacromoleculeName: AM22 Fab heavy chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.989154 KDa
Recombinant expressionOrganism: Mammalia (mammals)
SequenceString: QVQLVQSGAE VKKPGATVKV SCKISGHTLI KLSIHWVRQA PGKGLEWMGG YEGEVDEIFY AQKFQHRLTV IADTATDTVY MELGRLTSD DTAVYFCGTL GVTVTEAGLG IDDYWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ...String:
QVQLVQSGAE VKKPGATVKV SCKISGHTLI KLSIHWVRQA PGKGLEWMGG YEGEVDEIFY AQKFQHRLTV IADTATDTVY MELGRLTSD DTAVYFCGTL GVTVTEAGLG IDDYWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPKSCD GGGSLVPRGS DY KDDDDK

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Macromolecule #5: AM22 Fab light chain

MacromoleculeName: AM22 Fab light chain / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.273938 KDa
Recombinant expressionOrganism: Mammalia (mammals)
SequenceString: EIVLTQSPGT LSLSPGERAT LSCRASQIVS RNHLAWYQQK PGQAPRLLIF GASSRATGIP VRFSGSGSGT DFTLTINGLA PEDFAVYYC LSSDSSIFTF GPGTKVDFKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
EIVLTQSPGT LSLSPGERAT LSCRASQIVS RNHLAWYQQK PGQAPRLLIF GASSRATGIP VRFSGSGSGT DFTLTINGLA PEDFAVYYC LSSDSSIFTF GPGTKVDFKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #6: RSV variant (construct pXCS847A) F2

MacromoleculeName: RSV variant (construct pXCS847A) F2 / type: protein_or_peptide / ID: 6 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Respiratory syncytial virus
Molecular weightTheoretical: 12.109954 KDa
Recombinant expressionOrganism: Mammalia (mammals)
SequenceString:
MELPILKTNA ITTILAAVTL CFASSQNITE EFYQSTCSAV SKGYLSALRT GWYTSVITIE LSNIKENKCN GTDAKVKLIK QELDKYKNA VTELQLLMQS TPACNSRARR

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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: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration0.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMTris-HClTris Hydrochloride
300.0 mMNaClSodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average exposure time: 20.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 420325
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 183713
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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