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- PDB-5ken: EBOV GP in complex with variable Fab domains of IgGs c4G7 and c13C6 -

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Basic information

Entry
Database: PDB / ID: 5ken
TitleEBOV GP in complex with variable Fab domains of IgGs c4G7 and c13C6
Components
  • (Ebola surface glycoprotein, ...) x 2
  • (c13C6 variable Fab domain ...) x 2
  • (c4G7 variable Fab domain ...) x 2
KeywordsVIRAL PROTEIN/IMMUNE SYSTEM / Ebola virus surface glycoprotein / therapeutic antibody cocktail / ZMapp / VIRAL PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


host cell endoplasmic reticulum / viral budding from plasma membrane / clathrin-dependent endocytosis of virus by host cell / host cell cytoplasm / symbiont-mediated-mediated suppression of host tetherin activity / entry receptor-mediated virion attachment to host cell / symbiont-mediated suppression of host innate immune response / symbiont entry into host cell / membrane raft / fusion of virus membrane with host endosome membrane ...host cell endoplasmic reticulum / viral budding from plasma membrane / clathrin-dependent endocytosis of virus by host cell / host cell cytoplasm / symbiont-mediated-mediated suppression of host tetherin activity / entry receptor-mediated virion attachment to host cell / symbiont-mediated suppression of host innate immune response / symbiont entry into host cell / membrane raft / fusion of virus membrane with host endosome membrane / lipid binding / viral envelope / host cell plasma membrane / virion membrane / extracellular region / identical protein binding
Similarity search - Function
Filoviruses glycoprotein, extracellular domain / Filoviruses glycoprotein / Filovirus glycoprotein / Envelope glycoprotein GP2-like, HR1-HR2
Similarity search - Domain/homology
Envelope glycoprotein
Similarity search - Component
Biological speciesZaire ebolavirus
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsPallesen, J. / Murin, C.D. / de Val, N. / Cottrell, C.A. / Hastie, K.M. / Turner, H.L. / Fusco, M.L. / Flyak, A.I. / Zeitlin, L. / Crowe Jr., J.E. ...Pallesen, J. / Murin, C.D. / de Val, N. / Cottrell, C.A. / Hastie, K.M. / Turner, H.L. / Fusco, M.L. / Flyak, A.I. / Zeitlin, L. / Crowe Jr., J.E. / Andersen, K.G. / Saphire, E.O. / Ward, A.B.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI067927 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19 AI109762 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19 AI109711 United States
CitationJournal: Nat Microbiol / Year: 2016
Title: Structures of Ebola virus GP and sGP in complex with therapeutic antibodies.
Authors: Jesper Pallesen / Charles D Murin / Natalia de Val / Christopher A Cottrell / Kathryn M Hastie / Hannah L Turner / Marnie L Fusco / Andrew I Flyak / Larry Zeitlin / James E Crowe / Kristian ...Authors: Jesper Pallesen / Charles D Murin / Natalia de Val / Christopher A Cottrell / Kathryn M Hastie / Hannah L Turner / Marnie L Fusco / Andrew I Flyak / Larry Zeitlin / James E Crowe / Kristian G Andersen / Erica Ollmann Saphire / Andrew B Ward /
Abstract: The Ebola virus (EBOV) GP gene encodes two glycoproteins. The major product is a soluble, dimeric glycoprotein (sGP) that is secreted abundantly. Despite the abundance of sGP during infection, little ...The Ebola virus (EBOV) GP gene encodes two glycoproteins. The major product is a soluble, dimeric glycoprotein (sGP) that is secreted abundantly. Despite the abundance of sGP during infection, little is known regarding its structure or functional role. A minor product, resulting from transcriptional editing, is the transmembrane-anchored, trimeric viral surface glycoprotein (GP). GP mediates attachment to and entry into host cells, and is the intended target of antibody therapeutics. Because large portions of sequence are shared between GP and sGP, it has been hypothesized that sGP may potentially subvert the immune response or may contribute to pathogenicity. In this study, we present cryo-electron microscopy structures of GP and sGP in complex with GP-specific and GP/sGP cross-reactive antibodies undergoing human clinical trials. The structure of the sGP dimer presented here, in complex with both an sGP-specific antibody and a GP/sGP cross-reactive antibody, permits us to unambiguously assign the oligomeric arrangement of sGP and compare its structure and epitope presentation to those of GP. We also provide biophysical evaluation of naturally occurring GP/sGP mutations that fall within the footprints identified by our high-resolution structures. Taken together, our data provide a detailed and more complete picture of the accessible Ebolavirus glycoprotein landscape and a structural basis to evaluate patient and vaccine antibody responses towards differently structured products of the GP gene.
History
DepositionJun 9, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 7, 2016Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Jul 18, 2018Group: Data collection / Experimental preparation / Category: em_sample_support / em_software / Item: _em_sample_support.grid_type / _em_software.name
Revision 1.3Dec 11, 2019Group: Advisory / Author supporting evidence ...Advisory / Author supporting evidence / Data collection / Derived calculations / Structure summary
Category: em_entity_assembly / pdbx_audit_support ...em_entity_assembly / pdbx_audit_support / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_chiral / pdbx_validate_close_contact / struct_conn / struct_site
Item: _em_entity_assembly.entity_id_list / _pdbx_audit_support.funding_organization / _struct_site.details
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / database_PDB_caveat / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_unobs_or_zero_occ_atoms.auth_asym_id / _pdbx_unobs_or_zero_occ_atoms.auth_seq_id / _pdbx_unobs_or_zero_occ_atoms.label_asym_id / _pdbx_validate_close_contact.auth_asym_id_2 / _pdbx_validate_close_contact.auth_seq_id_2 / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: Ebola surface glycoprotein, GP1
B: Ebola surface glycoprotein, GP2
C: c4G7 variable Fab domain heavy chain
D: c4G7 variable Fab domain light chain
E: Ebola surface glycoprotein, GP1
F: Ebola surface glycoprotein, GP2
G: c4G7 variable Fab domain heavy chain
H: c4G7 variable Fab domain light chain
I: c13C6 variable Fab domain light chain
J: c13C6 variable Fab domain heavy chain
K: Ebola surface glycoprotein, GP1
M: Ebola surface glycoprotein, GP2
N: c4G7 variable Fab domain heavy chain
O: c4G7 variable Fab domain light chain
P: c13C6 variable Fab domain light chain
Q: c13C6 variable Fab domain heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)258,66825
Polymers253,99916
Non-polymers4,6699
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area48700 Å2
ΔGint-156 kcal/mol
Surface area87470 Å2

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Components

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Ebola surface glycoprotein, ... , 2 types, 6 molecules AEKBFM

#1: Protein Ebola surface glycoprotein, GP1


Mass: 30910.719 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Zaire ebolavirus / Strain: Mayinga-76 / Gene: GP / Production host: Drosophila melanogaster (fruit fly) / References: UniProt: Q05320
#2: Protein Ebola surface glycoprotein, GP2


Mass: 12650.368 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Zaire ebolavirus (strain Mayinga-76) / Strain: Mayinga-76 / Gene: GP / Production host: Drosophila melanogaster (fruit fly) / References: UniProt: Q05320

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Antibody , 4 types, 10 molecules CGNDHOIPJQ

#3: Antibody c4G7 variable Fab domain heavy chain


Mass: 12851.230 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Nicotiana benthamiana (plant)
#4: Antibody c4G7 variable Fab domain light chain


Mass: 11733.013 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Nicotiana benthamiana (plant)
#5: Antibody c13C6 variable Fab domain light chain


Mass: 11611.877 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Nicotiana benthamiana (plant)
#6: Antibody c13C6 variable Fab domain heavy chain


Mass: 13169.679 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Nicotiana benthamiana (plant)

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Sugars , 3 types, 9 molecules

#7: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#8: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#9: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
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

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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: Ebola virus trimeric surface glycoprotein in complex with IgG c4G7 and c13C6 Fabs
Type: COMPLEX / Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.54 MDa / Experimental value: YES
Buffer solutionpH: 7.4
Buffer componentName: 1xTBS
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2 4C
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Chamber temperature: 277 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 / Nominal magnification: 22500 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 77 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 57 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2555
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategory
1DoG Pickerparticle selection
2Leginonimage acquisition
4CTFFIND3CTF correction
7UCSF Chimeramodel fitting
8MODELLERmodel fitting
9Rosetta2015.19model fitting
10Coot0.8.2model fitting
12Rosetta2015.19model refinement
13RELION1.4b1initial Euler assignment
14RELION1.4b1final Euler assignment
15RELION1.4b1classification
16RELION1.4b13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73000 / Num. of class averages: 6 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: EMRinger
Details: Model building and refinement were conducted using a combination of software programs. The refinement target was optimizing using the MolProbity score while maintaining a high (good) EMRinger score.

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