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- PDB-6nt3: Cryo-EM structure of a human-cockroach hybrid Nav channel. -

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Basic information

Entry
Database: PDB / ID: 6nt3
TitleCryo-EM structure of a human-cockroach hybrid Nav channel.
ComponentsSodium channel protein type 9 subunit alpha, Sodium channel protein PaFPC1, chimeric construct
KeywordsMEMBRANE PROTEIN / Sodium channel / scorpion toxin / electrical signaling / fast inactivation
Function / homology
Function and homology information


detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / high voltage-gated calcium channel activity / voltage-gated sodium channel activity / Interaction between L1 and Ankyrins / sodium ion transport / detection of temperature stimulus involved in sensory perception of pain / voltage-gated calcium channel complex / behavioral response to pain / Phase 0 - rapid depolarisation ...detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / high voltage-gated calcium channel activity / voltage-gated sodium channel activity / Interaction between L1 and Ankyrins / sodium ion transport / detection of temperature stimulus involved in sensory perception of pain / voltage-gated calcium channel complex / behavioral response to pain / Phase 0 - rapid depolarisation / calcium ion import across plasma membrane / sodium ion transmembrane transport / sensory perception of pain / post-embryonic development / response to toxic substance / Sensory perception of sweet, bitter, and umami (glutamate) taste / circadian rhythm / inflammatory response / axon / plasma membrane
Similarity search - Function
Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Voltage-gated sodium channel alpha subunit, inactivation gate / Voltage-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-associated / Sodium ion transport-associated / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit / Voltage-gated cation channel calcium and sodium / Short calmodulin-binding motif containing conserved Ile and Gln residues. ...Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Voltage-gated sodium channel alpha subunit, inactivation gate / Voltage-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-associated / Sodium ion transport-associated / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit / Voltage-gated cation channel calcium and sodium / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif, EF-hand binding site / Voltage-dependent channel domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Chem-76F / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / CHOLESTEROL HEMISUCCINATE / Sodium channel protein PaFPC1 / Sodium channel protein type 9 subunit alpha
Similarity search - Component
Biological speciesPeriplaneta americana (American cockroach)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsClairfeuille, T. / Rohou, A. / Payandeh, J.
CitationJournal: Science / Year: 2019
Title: Structural basis of α-scorpion toxin action on Na channels.
Authors: Thomas Clairfeuille / Alexander Cloake / Daniel T Infield / José P Llongueras / Christopher P Arthur / Zhong Rong Li / Yuwen Jian / Marie-France Martin-Eauclaire / Pierre E Bougis / Claudio ...Authors: Thomas Clairfeuille / Alexander Cloake / Daniel T Infield / José P Llongueras / Christopher P Arthur / Zhong Rong Li / Yuwen Jian / Marie-France Martin-Eauclaire / Pierre E Bougis / Claudio Ciferri / Christopher A Ahern / Frank Bosmans / David H Hackos / Alexis Rohou / Jian Payandeh /
Abstract: Fast inactivation of voltage-gated sodium (Na) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Na ...Fast inactivation of voltage-gated sodium (Na) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Na channel alone and in complex with a lethal α-scorpion toxin, AaH2, by electron microscopy, both at 3.5-angstrom resolution. AaH2 wedges into voltage-sensing domain IV (VSD4) to impede fast activation by trapping a deactivated state in which gating charge interactions bridge to the acidic intracellular carboxyl-terminal domain. In the absence of AaH2, the S4 helix of VSD4 undergoes a ~13-angstrom translation to unlatch the intracellular fast-inactivation gating machinery. Highlighting the polypharmacology of α-scorpion toxins, AaH2 also targets an unanticipated receptor site on VSD1 and a pore glycan adjacent to VSD4. Overall, this work provides key insights into fast inactivation, electromechanical coupling, and pathogenic mutations in Na channels.
History
DepositionJan 28, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 20, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
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 / 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 / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _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 / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_unobs_or_zero_occ_atoms.label_asym_id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 3.0Aug 18, 2021Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / database_2 / entity / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_unobs_or_zero_occ_atoms / struct_conn
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.pdbx_formal_charge / _atom_site.type_symbol / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.pdbx_synonyms / _chem_comp.type / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _entity.formula_weight / _entity.pdbx_description / _pdbx_entity_nonpoly.comp_id / _pdbx_entity_nonpoly.name / _pdbx_nonpoly_scheme.mon_id / _pdbx_nonpoly_scheme.pdb_mon_id / _pdbx_unobs_or_zero_occ_atoms.auth_atom_id / _pdbx_unobs_or_zero_occ_atoms.auth_comp_id / _pdbx_unobs_or_zero_occ_atoms.label_atom_id / _pdbx_unobs_or_zero_occ_atoms.label_comp_id / _struct_conn.pdbx_leaving_atom_flag
Revision 4.0Aug 25, 2021Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Non-polymer description / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / database_PDB_caveat / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_validate_chiral
Item: _atom_site.auth_comp_id / _atom_site.label_comp_id ..._atom_site.auth_comp_id / _atom_site.label_comp_id / _chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.pdbx_synonyms / _chem_comp.type / _pdbx_entity_nonpoly.comp_id / _pdbx_nonpoly_scheme.mon_id / _pdbx_nonpoly_scheme.pdb_mon_id

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Structure visualization

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Assembly

Deposited unit
A: Sodium channel protein type 9 subunit alpha, Sodium channel protein PaFPC1, chimeric construct
hetero molecules


Theoretical massNumber of molelcules
Total (without water)186,81515
Polymers178,7641
Non-polymers8,05214
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Sodium channel protein type 9 subunit alpha, Sodium channel protein PaFPC1, chimeric construct / / PaFPC1 / NavPaS / Neuroendocrine sodium channel / hNE-Na / Peripheral sodium channel 1 / PN1 / ...PaFPC1 / NavPaS / Neuroendocrine sodium channel / hNE-Na / Peripheral sodium channel 1 / PN1 / Sodium channel protein type IX subunit alpha / Voltage-gated sodium channel subunit alpha Nav1.7 / PaFPC1 / NavPaS


Mass: 178763.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Periplaneta americana (American cockroach), (gene. exp.) Homo sapiens (human)
Gene: SCN9A, NENA / Cell line (production host): Expi293 / Production host: Homo sapiens (human) / References: UniProt: D0E0C2, UniProt: Q15858

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Sugars , 3 types, 7 molecules

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


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-3[DManpb1-6]DManpb1-3DGlcpNAcb1-3DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2-2-2/a3-b1_b3-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(3+1)][b-D-GlcpNAc]{[(3+1)][b-D-Manp]{[(3+1)][b-D-Manp]{}[(6+1)][b-D-Manp]{}}}}}LINUCSPDB-CARE
#3: 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.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#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 / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 4
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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Non-polymers , 4 types, 7 molecules

#5: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#6: Chemical
ChemComp-76F / (7E,21R,24S)-27-amino-24-hydroxy-18,24-dioxo-19,23,25-trioxa-24lambda~5~-phosphaheptacos-7-en-21-yl (9Z,12E)-octadeca-9,12-dienoate / DOPE / Discrete optimized protein energy


Mass: 742.018 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C41H76NO8P
#7: Chemical ChemComp-LHG / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / Phosphatidylglycerol


Mass: 722.970 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C38H75O10P / Comment: phospholipid*YM
#8: Chemical ChemComp-AJP / Digitonin / Digitonin


Mass: 1229.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C56H92O29 / Comment: detergent*YM

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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: NavPas-VSD4 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.155 MDa / Experimental value: NO
Source (natural)Organism: Periplaneta americana (American cockroach)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293
Buffer solutionpH: 7.5
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grids were plasma etched using the Solarus plasma cleaner (Gatan) in the hydrogen-oxygen setting. Grids were etched for 4 minutes on each side to remove burrs from hole edges. The grids were ...Details: Grids were plasma etched using the Solarus plasma cleaner (Gatan) in the hydrogen-oxygen setting. Grids were etched for 4 minutes on each side to remove burrs from hole edges. The grids were then coated on both sides with 5nm of Au/Pd which was plasma deposited using the Leica ACE600 (Leica).
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / 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 FIELDBright-field microscopy / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10421
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameCategory
1cisTEMparticle selection
2SerialEMimage acquisition
4cisTEMCTF correction
10cisTEMinitial Euler assignment
11cisTEMfinal Euler assignment
12cisTEMclassification
13cisTEM3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: Automated picking using a soft-edged disc as a template yielded 591739 candidate coordinates. 2D classification led to the selection of 334055 particles for further analysis.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 150583 / Algorithm: FOURIER SPACE
Details: No information from spatial frequencies higher than 1/5A were ever used during refinement.
Num. of class averages: 1 / Symmetry type: POINT

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