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- PDB-7mpg: Cryo-EM structure of Prefusion-stabilized RSV F (DS-Cav1) in comp... -

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Basic information

Entry
Database: PDB / ID: 7mpg
TitleCryo-EM structure of Prefusion-stabilized RSV F (DS-Cav1) in complex with Fab AM14
Components
  • AM14 Fab Heavy Chain
  • AM14 Fab Light Chain
  • Fusion glycoprotein F0,Envelope glycoprotein
KeywordsIMMUNE SYSTEM / antigen / antibody / epitope / vaccine / structural vaccinology
Function / homology
Function and homology information


host cell Golgi membrane / entry receptor-mediated virion attachment to host cell / membrane => GO:0016020 / 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 / Fibritin C-terminal / Fibritin C-terminal region
Similarity search - Domain/homology
Fusion glycoprotein F0 / Envelope glycoprotein
Similarity search - Component
Biological speciesHuman respiratory syncytial virus
Human immunodeficiency virus 1
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsAbeyrathne, P.D. / Malito, E. / Harshbarger, W.D.
CitationJournal: MAbs / Year: 2021
Title: Improved epitope resolution of the prefusion trimer-specific antibody AM14 bound to the RSV F glycoprotein.
Authors: Wayne Harshbarger / Priyanka D Abeyrathne / Sai Tian / Ying Huang / Sumana Chandramouli / Matthew James Bottomley / Enrico Malito /
Abstract: Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and ...Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and vaccination against RSV remains a public health priority. The RSV F glycoprotein is a major target of neutralizing antibodies, and the prefusion stabilized form of F (DS-Cav1) is under investigation as a vaccine antigen. AM14 is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F (PreF), which spans two F protomers. The quality of recognizing a trimer-specific epitope makes AM14 valuable for probing PreF-based immunogen conformation and functionality during vaccine production. Currently, only a low-resolution (5.5 Å) X-ray structure is available of the PreF-AM14 complex, revealing few reliable details of the interface. Here, we perform complementary structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) to provide improved resolution structures at 3.6 Å and 3.4 Å resolutions, respectively. Both X-ray and cryo-EM structures provide clear side-chain densities, which allow for accurate mapping of the AM14 epitope on DS-Cav1. The structures help rationalize the molecular basis for AM14 loss of binding to RSV F monoclonal antibody-resistant mutants and reveal flexibility for the side chain of a key antigenic residue on PreF. This work provides the basis for a comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens.
History
DepositionMay 4, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 8, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Fusion glycoprotein F0,Envelope glycoprotein
B: Fusion glycoprotein F0,Envelope glycoprotein
C: Fusion glycoprotein F0,Envelope glycoprotein
F: AM14 Fab Heavy Chain
G: AM14 Fab Light Chain
H: AM14 Fab Heavy Chain
I: AM14 Fab Light Chain
D: AM14 Fab Heavy Chain
E: AM14 Fab Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)321,83712
Polymers321,1739
Non-polymers6643
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_1ens_2chain "E"
d_2ens_2chain "G"
d_3ens_2chain "I"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1GLNLEUA1 - 451
d_12ens_1NAGNAGB
d_21ens_1GLNLEUC1 - 451
d_22ens_1NAGNAGD
d_31ens_1GLNLEUE1 - 451
d_32ens_1NAGNAGF
d_11ens_2ASPGLYL1 - 213
d_21ens_2ASPGLYH1 - 213
d_31ens_2ASPGLYJ1 - 213

NCS ensembles :
ID
ens_1
ens_2

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Components

#1: Protein Fusion glycoprotein F0,Envelope glycoprotein / Fusion glycoprotein F1 / Fusion glycoprotein F2


Mass: 54806.496 Da / Num. of mol.: 3 / Mutation: S155C,S190F,V207L,S290C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human respiratory syncytial virus, (gene. exp.) Human immunodeficiency virus 1
Gene: RSV F / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: A0A0X8XQD7, UniProt: M1E1E4
#2: Antibody AM14 Fab Heavy Chain


Mass: 26380.352 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#3: Antibody AM14 Fab Light Chain


Mass: 25870.906 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Homo sapiens respiratory syncytial virus ACOMPLEX#1-#30MULTIPLE SOURCES
2Fusion glycoprotein F1 and F2 fused with Fibritin trimerization domain.COMPLEX#11RECOMBINANT
3Fab AM14 heavy and light chainCOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.285 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Respiratory syncytial virus A21972429
23Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Cricetulus griseus (Chinese hamster)10029
23Homo sapiens (human)9606
Details of virusEmpty: NO / Enveloped: YES / Isolate: OTHER / Type: VIRION
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
110 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES1
2150 mMSodium chlorideNaCl1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: DS-Cav1-FabAM14.
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 26.7 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1078

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4cisTEM1.0.0CTF correctionCTFFIND4
10cisTEM1.0.0initial Euler assignment
11cisTEM1.0.0final Euler assignment
12cisTEM1.0.0classification
13cisTEM1.0.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23737 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 87.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00320744
ELECTRON MICROSCOPYf_angle_d0.63928157
ELECTRON MICROSCOPYf_dihedral_angle_d8.5362829
ELECTRON MICROSCOPYf_chiral_restr0.0583286
ELECTRON MICROSCOPYf_plane_restr0.0043579
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.000712167852585
ens_1d_3AELECTRON MICROSCOPYNCS constraints0.000706849514556
ens_2d_2EELECTRON MICROSCOPYNCS constraints0.000709555071327
ens_2d_3EELECTRON MICROSCOPYNCS constraints0.068224507246

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