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Basic information

Entry
Database: PDB / ID: 5foj
TitleCryo electron microscopy structure of Grapevine Fanleaf Virus complex with Nanobody
Components
  • Nanobody
  • RNA2 polyprotein
KeywordsVIRUS / NANOBODY / COMPLEX
Function / homology
Function and homology information


transport of virus in host, cell to cell / host cell plasmodesma / viral capsid / host cell cytoplasm / host cell nucleus / structural molecule activity
Similarity search - Function
Nepovirus subgroup A, RNA2 polyprotein, Protein 2A / Nepovirus subgroup A polyprotein / Nepovirus coat protein / Nepovirus coat protein, C-terminal / Nepovirus coat protein, N-terminal / Nepovirus coat protein, central domain / Nepovirus coat protein, C-terminal domain / Nepovirus coat protein, N-terminal domain / Jelly Rolls - #20 / Viral coat protein subunit ...Nepovirus subgroup A, RNA2 polyprotein, Protein 2A / Nepovirus subgroup A polyprotein / Nepovirus coat protein / Nepovirus coat protein, C-terminal / Nepovirus coat protein, N-terminal / Nepovirus coat protein, central domain / Nepovirus coat protein, C-terminal domain / Nepovirus coat protein, N-terminal domain / Jelly Rolls - #20 / Viral coat protein subunit / Jelly Rolls / Sandwich / Mainly Beta
Similarity search - Domain/homology
Biological speciesCamelus dromedarius (Arabian camel)
Grapevine fanleaf virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsOrlov, I. / Hemmer, C. / Ackerer, L. / Lorber, B. / Ghannam, A. / Poignavent, V. / Hleibieh, K. / Sauter, C. / Schmitt-Keichinger, C. / Belval, L. ...Orlov, I. / Hemmer, C. / Ackerer, L. / Lorber, B. / Ghannam, A. / Poignavent, V. / Hleibieh, K. / Sauter, C. / Schmitt-Keichinger, C. / Belval, L. / Hily, J.M. / Marmonier, A. / Komar, V. / Gersch, S. / Schellenberger, P. / Bron, P. / Vigne, E. / Muyldermans, S. / Lemaire, O. / Demangeat, G. / Ritzenthaler, C. / Klaholz, B.P.
Funding support1items
OrganizationGrant numberCountry
French National Research Agency
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Structural basis of nanobody recognition of grapevine fanleaf virus and of virus resistance loss.
Authors: Igor Orlov / Caroline Hemmer / Léa Ackerer / Bernard Lorber / Ahmed Ghannam / Vianney Poignavent / Kamal Hleibieh / Claude Sauter / Corinne Schmitt-Keichinger / Lorène Belval / Jean-Michel ...Authors: Igor Orlov / Caroline Hemmer / Léa Ackerer / Bernard Lorber / Ahmed Ghannam / Vianney Poignavent / Kamal Hleibieh / Claude Sauter / Corinne Schmitt-Keichinger / Lorène Belval / Jean-Michel Hily / Aurélie Marmonier / Véronique Komar / Sophie Gersch / Pascale Schellenberger / Patrick Bron / Emmanuelle Vigne / Serge Muyldermans / Olivier Lemaire / Gérard Demangeat / Christophe Ritzenthaler / Bruno P Klaholz /
Abstract: Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently, and ...Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently, and shown to be highly effective in plants, including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo electron microscopy structure of the GFLV-Nb23 complex, which provides the basis for molecular recognition by the Nb. The structure reveals a composite binding site bridging over three domains of one capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced-fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.
History
DepositionNov 22, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 20, 2016Provider: repository / Type: Initial release
Revision 1.1Apr 13, 2016Group: Source and taxonomy
Revision 1.2Aug 30, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id
Revision 2.0May 13, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Data processing / Database references / Derived calculations / Experimental preparation / Other / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / audit_author ...atom_site / audit_author / cell / citation / citation_author / em_3d_fitting / em_3d_reconstruction / em_buffer / em_crystal_formation / em_ctf_correction / em_entity_assembly / em_entity_assembly_naturalsource / em_experiment / em_image_recording / em_image_scans / em_imaging / em_imaging_optics / em_software / em_virus_entity / em_virus_shell / em_vitrification / entity / entity_name_com / entity_src_gen / entity_src_nat / pdbx_audit_support / pdbx_database_status / pdbx_poly_seq_scheme / pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop / pdbx_struct_oper_list / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_rmsd_angle / pdbx_validate_torsion / pdbx_version / refine_ls_restr / software / struct / struct_conf / struct_conn / struct_mon_prot_cis / struct_ref / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _cell.Z_PDB / _citation.journal_abbrev ..._cell.Z_PDB / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.title / _citation.year / _em_3d_fitting.details / _em_3d_reconstruction.actual_pixel_size / _em_3d_reconstruction.algorithm / _em_3d_reconstruction.magnification_calibration / _em_3d_reconstruction.method / _em_3d_reconstruction.nominal_pixel_size / _em_3d_reconstruction.num_particles / _em_3d_reconstruction.resolution_method / _em_3d_reconstruction.software / _em_3d_reconstruction.symmetry_type / _em_buffer.details / _em_buffer.name / _em_ctf_correction.details / _em_ctf_correction.em_image_processing_id / _em_ctf_correction.type / _em_entity_assembly.entity_id_list / _em_entity_assembly.name / _em_entity_assembly.source / _em_experiment.specimen_type / _em_image_recording.average_exposure_time / _em_image_recording.detector_mode / _em_image_scans.dimension_height / _em_image_scans.dimension_width / _em_image_scans.frames_per_image / _em_image_scans.number_digital_images / _em_imaging.alignment_procedure / _em_imaging.calibrated_magnification / _em_imaging.cryogen / _em_imaging.date / _em_imaging.details / _em_imaging.electron_dose / _em_imaging.specimen_holder_model / _em_vitrification.details / _entity.pdbx_description / _entity.src_method / _entity_name_com.name / _pdbx_database_status.SG_entry / _pdbx_poly_seq_scheme.auth_mon_id / _pdbx_poly_seq_scheme.auth_seq_num / _pdbx_poly_seq_scheme.pdb_mon_id / _struct.pdbx_CASP_flag / _struct.title / _struct_conn.pdbx_dist_value / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_seq_one_letter_code
Description: Polymer geometry
Details: Further atomic model refinement including temperature factors
Provider: author / Type: Coordinate replacement
Revision 2.1May 20, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 3.0Aug 11, 2021Group: Atomic model / Data collection ...Atomic model / Data collection / Database references / Derived calculations / Refinement description
Category: atom_site / database_2 ...atom_site / database_2 / em_software / pdbx_struct_sheet_hbond / pdbx_validate_torsion / refine / struct_conf / struct_mon_prot_cis / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_software.category / _em_software.image_processing_id / _em_software.imaging_id / _em_software.name / _pdbx_validate_torsion.phi / _pdbx_validate_torsion.psi / _refine.pdbx_diffrn_id / _struct_mon_prot_cis.pdbx_omega_angle / _struct_sheet_range.beg_auth_comp_id / _struct_sheet_range.beg_auth_seq_id / _struct_sheet_range.beg_label_comp_id / _struct_sheet_range.beg_label_seq_id / _struct_sheet_range.end_auth_comp_id / _struct_sheet_range.end_auth_seq_id / _struct_sheet_range.end_label_comp_id / _struct_sheet_range.end_label_seq_id / _struct_sheet_range.id / _struct_sheet_range.sheet_id
Description: Model orientation/position / Provider: author / Type: Coordinate replacement
Revision 3.1Nov 20, 2024Group: Data collection / Derived calculations ...Data collection / Derived calculations / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature / pdbx_struct_oper_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation

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Structure visualization

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  • Biological unit as author_defined_assembly
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-3246
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  • Superimposition on EM map
  • EMDB-3246
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Nanobody
B: RNA2 polyprotein


Theoretical massNumber of molelcules
Total (without water)70,8892
Polymers70,8892
Non-polymers00
Water00
1
A: Nanobody
B: RNA2 polyprotein
x 60


Theoretical massNumber of molelcules
Total (without water)4,253,369120
Polymers4,253,369120
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
Buried area1620 Å2
ΔGint-3 kcal/mol
Surface area29990 Å2
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Antibody Nanobody


Mass: 14816.341 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelus dromedarius (Arabian camel) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): WK6
#2: Protein RNA2 polyprotein / P2


Mass: 56073.141 Da / Num. of mol.: 1 / Fragment: UNP RESIDUES 606-1109 / Source method: isolated from a natural source / Source: (natural) Grapevine fanleaf virus / References: UniProt: P18474
Has protein modificationY

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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

ComponentName: Grapevine fanleaf virus / Type: VIRUS / Entity ID: all / Source: MULTIPLE SOURCES
Source (natural)Organism: Grapevine fanleaf virus
Details of virusEmpty: NO / Enveloped: NO / Isolate: OTHER / Type: VIRION
Virus shellName: pseudo-T = 3 / Triangulation number (T number): 3
Buffer solutionpH: 8.3 / Details: 100 mM TRIS pH 8.3, 50 MM NaCl.
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 95, INSTRUMENT- FEI VITROBOT MARK III, METHOD- BLOT FOR 2 SECONDS BEFORE PLUNGING

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Cs-corrected microscope
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 120000 X / Calibrated magnification: 127272 X / Nominal defocus max: 6000 nm / Nominal defocus min: 600 nm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON II (4k x 4k)
EM imaging opticsSpherical aberration corrector: Cs-corrected microscope
Image scansWidth: 4096 / Height: 4096 / Movie frames/image: 7

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameCategory
2UCSF Chimeramodel fitting
5IMAGICCTF correction
9IMAGIC3D reconstruction
10PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Algorithm: BACK PROJECTION
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3246. (DEPOSITION ID: 14064).
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Initial atomic model building was done by a rigid body fitting, followed by extensive manual model building in Coot and real space refinement of the atomic model against the experimental map ...Details: Initial atomic model building was done by a rigid body fitting, followed by extensive manual model building in Coot and real space refinement of the atomic model against the experimental map using Phenix including restrained temperature factor refinement.
Atomic model buildingPDB-ID: 4V5T

4v5t


Accession code: 4V5T / Source name: PDB / Type: experimental model
RefinementHighest resolution: 2.8 Å
Refinement stepCycle: LAST / Highest resolution: 2.8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4929 0 0 0 4929
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0095056
ELECTRON MICROSCOPYf_angle_d0.8256872
ELECTRON MICROSCOPYf_dihedral_angle_d13.3311791
ELECTRON MICROSCOPYf_chiral_restr0.052760
ELECTRON MICROSCOPYf_plane_restr0.006877

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