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- EMDB-23520: Cryo-EM structure of RSV preF bound by Fabs 32.4K and 01.4B -

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Basic information

Entry
Database: EMDB / ID: EMD-23520
TitleCryo-EM structure of RSV preF bound by Fabs 32.4K and 01.4B
Map dataFull map, sharpened with DeepEMhancer
Sample
  • Complex: Prefusion RSV F bound by Fabs 01.4B and 32.4K
    • Protein or peptide: Fusion glycoprotein F0
    • Protein or peptide: 01.4B Fab Heavy chain
    • Protein or peptide: 01.4B Fab Light chain
    • Protein or peptide: 32.4K Fab Heavy chain
    • Protein or peptide: 32.4K Fab Light chain
Function / homologyPrecursor fusion glycoprotein F0, Paramyxoviridae / Fusion glycoprotein F0 / 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 / Fusion glycoprotein F0
Function and homology information
Biological speciesRespiratory syncytial virus / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.21 Å
AuthorsWrapp D / 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) releaseApr 21, 2021-
Map releaseApr 21, 2021-
UpdateApr 28, 2021-
Current statusApr 28, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.127
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.127
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7luc
  • Surface level: 0.127
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7luc
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23520.png

Downloads & links

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Map

FileDownload / File: emd_23520.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map, sharpened with DeepEMhancer
Voxel sizeX=Y=Z: 1.073 Å
Density
Contour LevelBy AUTHOR: 0.127 / Movie #1: 0.127
Minimum - Maximum-0.029057795 - 2.3664036
Average (Standard dev.)0.0012512992 (±0.023640186)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 360.52798 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0731.0731.073
M x/y/z336336336
origin x/y/z0.0000.0000.000
length x/y/z360.528360.528360.528
α/β/γ90.00090.00090.000
start NX/NY/NZ1219875
NX/NY/NZ141223232
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS336336336
D min/max/mean-0.0292.3660.001

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Supplemental data

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Additional map: Focused map (C1)

Fileemd_23520_additional_1.map
AnnotationFocused map (C1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_23520_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_23520_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Prefusion RSV F bound by Fabs 01.4B and 32.4K

EntireName: Prefusion RSV F bound by Fabs 01.4B and 32.4K
Components
  • Complex: Prefusion RSV F bound by Fabs 01.4B and 32.4K
    • Protein or peptide: Fusion glycoprotein F0
    • Protein or peptide: 01.4B Fab Heavy chain
    • Protein or peptide: 01.4B Fab Light chain
    • Protein or peptide: 32.4K Fab Heavy chain
    • Protein or peptide: 32.4K Fab Light chain

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Supramolecule #1: Prefusion RSV F bound by Fabs 01.4B and 32.4K

SupramoleculeName: Prefusion RSV F bound by Fabs 01.4B and 32.4K / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Respiratory syncytial virus
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightExperimental: 440 KDa

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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
Molecular weightTheoretical: 58.615941 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 LFQ

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Macromolecule #2: 01.4B Fab Heavy chain

MacromoleculeName: 01.4B Fab Heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.154789 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
QVQLVQSGAE VKKPGASVKL SCQASGYTFN NYGVSWLRQA PGQGLEWMGW ISAYNGNKKY APKFQGRLTL TTVTSTGTAY MELRSLKSD DTALYFCARD PPAVAAAMFD FWGQGTQVTV SS

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Macromolecule #3: 01.4B Fab Light chain

MacromoleculeName: 01.4B Fab Light chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.499002 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
DVVLTQSPLS LPVTLGQPAS ISCRSSQSLV LSDGNTYLSW FHQRPGHSPR RLIYRISHRD SGVPDRFSGS ESGTDFTLKI SRVEAEDVG IYYCMQGTHW PRTFGQGTKV EIK

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Macromolecule #4: 32.4K Fab Heavy chain

MacromoleculeName: 32.4K Fab Heavy chain / type: protein_or_peptide / ID: 4 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.140781 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
EVQLVQSGAE VKKPGESLKI SCKGSADSFT NHWIGWVRQT PGKGLEWMGM IYPGDSDTRY SPSFQGQVTL SVDKSVTTVY LQWNSLKAS DTAIYYCARQ VGGVVVAEPP PYYYYGMDAW GQGTTVTVSS

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Macromolecule #5: 32.4K Fab Light chain

MacromoleculeName: 32.4K Fab Light chain / type: protein_or_peptide / ID: 5 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.664103 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
DIQLTQSPSS LSASVGDRVT LTCRASQSIA TFLNWFQQRP GKAPKLLMFD ASKLQTGVPS RFSGSGSGTH FTLTISTLQP EDFATYYCQ QSYDLPLTFG PGTKVEIK

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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

Concentration0.7 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
2.0 mMC4H11NO3Tris
200.0 mMNaClSodium chloridesodium chloride
0.2 %NaN3sodium azide
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 37.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: OTHER
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.21 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 526804
FSC plot (resolution estimation)

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