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- EMDB-23521: Prefusion RSV F glycoprotein bound by neutralizing site V-directe... -

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Basic information

Entry
Database: EMDB / ID: EMD-23521
TitlePrefusion RSV F glycoprotein bound by neutralizing site V-directed antibody ADI-14442
Map dataTrimeric prefusion RSV F protein bound by three copies of Fab ADI-14442
Sample
  • Complex: Trimeric prefusion RSV F glycoprotein bound by three molecules of Fab ADI-14442.
    • Complex: Trimeric prefusion RSV F glycoprotein
      • Protein or peptide: Fusion glycoprotein F0
    • Complex: Fab ADI-14442
      • Protein or peptide: Heavy chain of human antibody Fab ADI-14442
      • Protein or peptide: Light chain of human antibody Fab ADI-14442
Function / homology
Function and homology information


positive regulation of syncytium formation by virus / host cell Golgi membrane / entry receptor-mediated virion attachment to host cell / fusion of virus membrane with host plasma membrane / host cell plasma membrane / virion membrane / plasma membrane
Similarity search - Function
Precursor fusion glycoprotein F0, Paramyxoviridae / Fusion glycoprotein F0
Similarity search - Domain/homology
Fusion glycoprotein F0
Similarity search - Component
Biological speciesRespiratory syncytial virus A2 / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsGilman MSA / McLellan JS
CitationJournal: Immunity / Year: 2021
Title: Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein induces genetically and antigenically diverse antibody responses.
Authors: Maryam Mukhamedova / Daniel Wrapp / Chen-Hsiang Shen / Morgan S A Gilman / Tracy J Ruckwardt / Chaim A Schramm / Larissa Ault / Lauren Chang / Alexandrine Derrien-Colemyn / Sarah A M Lucas / ...Authors: Maryam Mukhamedova / Daniel Wrapp / Chen-Hsiang Shen / Morgan S A Gilman / Tracy J Ruckwardt / Chaim A Schramm / Larissa Ault / Lauren Chang / Alexandrine Derrien-Colemyn / Sarah A M Lucas / Amy Ransier / Samuel Darko / Emily Phung / Lingshu Wang / Yi Zhang / Scott A Rush / Bharat Madan / Guillaume B E Stewart-Jones / Pamela J Costner / LaSonji A Holman / Somia P Hickman / Nina M Berkowitz / Nicole A Doria-Rose / Kaitlyn M Morabito / Brandon J DeKosky / Martin R Gaudinski / Grace L Chen / Michelle C Crank / John Misasi / Nancy J Sullivan / Daniel C Douek / Peter D Kwong / Barney S Graham / Jason S McLellan / John R Mascola /
Abstract: An effective vaccine for respiratory syncytial virus (RSV) is an unrealized public health goal. A single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) ...An effective vaccine for respiratory syncytial virus (RSV) is an unrealized public health goal. A single dose of the prefusion-stabilized fusion (F) glycoprotein subunit vaccine (DS-Cav1) substantially increases serum-neutralizing activity in healthy adults. We sought to determine whether DS-Cav1 vaccination induces a repertoire mirroring the pre-existing diversity from natural infection or whether antibody lineages targeting specific epitopes predominate. We evaluated RSV F-specific B cell responses before and after vaccination in six participants using complementary B cell sequencing methodologies and identified 555 clonal lineages. DS-Cav1-induced lineages recognized the prefusion conformation of F (pre-F) and were genetically diverse. Expressed antibodies recognized all six antigenic sites on the pre-F trimer. We identified 34 public clonotypes, and structural analysis of two antibodies from a predominant clonotype revealed a common mode of recognition. Thus, vaccination with DS-Cav1 generates a diverse polyclonal response targeting the antigenic sites on pre-F, supporting the development and advanced testing of pre-F-based vaccines against RSV.
History
DepositionFeb 22, 2021-
Header (metadata) releaseJun 16, 2021-
Map releaseJun 16, 2021-
UpdateJun 16, 2021-
Current statusJun 16, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lue
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7lue
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23521.png

Downloads & links

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Map

FileDownload / File: emd_23521.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTrimeric prefusion RSV F protein bound by three copies of Fab ADI-14442
Voxel sizeX=Y=Z: 1.075 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.16903993 - 0.24221794
Average (Standard dev.)0.00013314525 (±0.004291453)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 322.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0751.0751.075
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z322.500322.500322.500
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1690.2420.000

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Supplemental data

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Sample components

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Entire : Trimeric prefusion RSV F glycoprotein bound by three molecules of...

EntireName: Trimeric prefusion RSV F glycoprotein bound by three molecules of Fab ADI-14442.
Components
  • Complex: Trimeric prefusion RSV F glycoprotein bound by three molecules of Fab ADI-14442.
    • Complex: Trimeric prefusion RSV F glycoprotein
      • Protein or peptide: Fusion glycoprotein F0
    • Complex: Fab ADI-14442
      • Protein or peptide: Heavy chain of human antibody Fab ADI-14442
      • Protein or peptide: Light chain of human antibody Fab ADI-14442

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Supramolecule #1: Trimeric prefusion RSV F glycoprotein bound by three molecules of...

SupramoleculeName: Trimeric prefusion RSV F glycoprotein bound by three molecules of Fab ADI-14442.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Single molecule of trimeric respiratory syncytial virus fusion protein ectodomain (RSV F) stabilized in the prefusion conformation and fused to the T4 fibritin (foldon) trimerization motif ...Details: Single molecule of trimeric respiratory syncytial virus fusion protein ectodomain (RSV F) stabilized in the prefusion conformation and fused to the T4 fibritin (foldon) trimerization motif is bound by three molecules of Fab ADI-14442.
Source (natural)Organism: Respiratory syncytial virus A2
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: FreeStyle 293-F

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Supramolecule #2: Trimeric prefusion RSV F glycoprotein

SupramoleculeName: Trimeric prefusion RSV F glycoprotein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Details: Single molecule of trimeric respiratory syncytial virus fusion protein ectodomain (RSV F) stabilized in the prefusion conformation and fused to the T4 fibritin (foldon) trimerization motif.
Source (natural)Organism: Respiratory syncytial virus A2
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: FreeStyle 293-F

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Supramolecule #3: Fab ADI-14442

SupramoleculeName: Fab ADI-14442 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Details: Three molecules of Fab ADI-14442, each composed of a heavy and a light chain, are bound to a single molecule of trimeric respiratory syncytial virus fusion protein ectodomain (RSV F) ...Details: Three molecules of Fab ADI-14442, each composed of a heavy and a light chain, are bound to a single molecule of trimeric respiratory syncytial virus fusion protein ectodomain (RSV F) stabilized in the prefusion conformation and fused to the T4 fibritin (foldon) trimerization motif.
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: FreeStyle 293-F

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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: Respiratory syncytial virus A2
Molecular weightTheoretical: 61.132672 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QNITEEFYQS TCSAVSKGYL SALRTGWYTS VITIELSNIK EIKCNGTDAK VKLIKQELDK YKNAVTELQL LMQSTPATNN RARRELPRF MNYTLNNAKK TNVTLSKKRK RRFLGFLLGV GSAIASGVAV SKVLHLEGEV NKIKSALLST NKAVVSLSNG V SVLTSKVL ...String:
QNITEEFYQS TCSAVSKGYL SALRTGWYTS VITIELSNIK EIKCNGTDAK VKLIKQELDK YKNAVTELQL LMQSTPATNN RARRELPRF MNYTLNNAKK TNVTLSKKRK RRFLGFLLGV GSAIASGVAV SKVLHLEGEV NKIKSALLST NKAVVSLSNG V SVLTSKVL DLKNYIDKQL LPIVNKQSCS IPNIETVIEF QQKNNRLLEI TREFSVNAGV TTPVSTYMLT NSELLSLIND MP ITNDQKK LMSNNVQIVR QQSYSIMSII KEEVLAYVVQ LPLYGVIDTP CWKLHTSPLC TTNTKEGSNI CLTRTDRGWY CDN AGSVSF FPQAETCKVQ SNRVFCDTMN SLTLPSEVNL CNVDIFNPKY DCKIMTSKTD VSSSVITSLG AIVSCYGKTK CTAS NKNRG IIKTFSNGCD YVSNKGVDTV SVGNTLYYVN KQEGKSLYVK GEPIINFYDP LVFPSDEFDA SISQVNEKIN QSLAF IRKS DELLSAIGGY IPEAPRDGQA YVRKDGEWVL LSTFLGSLEV LFQGPGHHHH HHHHSAWSHP QFEK

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Macromolecule #2: Heavy chain of human antibody Fab ADI-14442

MacromoleculeName: Heavy chain of human antibody Fab ADI-14442 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.805615 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLVQSGSE VKKPGASVKV SCKASGYRFS NYGISWVRQA PGQGLEWMGW ISAYNGNIKY GNNLQGRVTV TTDTSTATAY MEVRSLTSD DTAVYYCARD VPADGVHFMD VWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT ...String:
QVQLVQSGSE VKKPGASVKV SCKASGYRFS NYGISWVRQA PGQGLEWMGW ISAYNGNIKY GNNLQGRVTV TTDTSTATAY MEVRSLTSD DTAVYYCARD VPADGVHFMD VWGQGTLVTV SSASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCD

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Macromolecule #3: Light chain of human antibody Fab ADI-14442

MacromoleculeName: Light chain of human antibody Fab ADI-14442 / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 24.100822 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DVVMTQSPLS LPVTLGQPAS ISCRSSQSLV HSDTNTYLNW FQQRPGQSPR RLIYKVSNRD SGVPDRFSGS GSGTTFTLKI SRVEAEDVG IYYCMQGSHW APTFGQGTKV EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL Q SGNSQESV ...String:
DVVMTQSPLS LPVTLGQPAS ISCRSSQSLV HSDTNTYLNW FQQRPGQSPR RLIYKVSNRD SGVPDRFSGS GSGTTFTLKI SRVEAEDVG IYYCMQGSHW APTFGQGTKV EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL Q SGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C

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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

Concentration0.8 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chloride
2.0 mMNH2C(CH2OH)3HClTRIS hydrochloride
0.02 %NaN3Sodium azide
GridModel: C-flat-1.2/1.3 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / 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) / Average exposure time: 9.0 sec. / Average electron dose: 48.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 22500
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware:
Namedetails
CTFFIND (ver. 4.1.10)
RELION (ver. 3.0)beta
Startup modelType of model: OTHER
Details: Ab initio model generated using stochastic gradient descent
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Software - details: beta / Number images used: 371323
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3) / Software - details: beta
Details: Ab initio model generated using stochastic gradient descent
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3) / Software - details: beta
FSC plot (resolution estimation)

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