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- PDB-6n4r: CryoEM structure of Nav1.7 VSD2 (deactived state) in complex with... -

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Basic information

Entry
Database: PDB / ID: 6n4r
TitleCryoEM structure of Nav1.7 VSD2 (deactived state) in complex with the gating modifier toxin ProTx2
Components
  • Beta/omega-theraphotoxin-Tp2a
  • Fab heavy chain
  • Fab light chain
  • Nav1.7 VSD2-NavAb chimera
KeywordsMEMBRANE PROTEIN / voltage-gated sodium channel / gating modifier toxin
Function / homology
Function and homology information


detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / cardiac muscle cell action potential involved in contraction / Interaction between L1 and Ankyrins / voltage-gated sodium channel activity / sodium ion transport / Phase 0 - rapid depolarisation / detection of temperature stimulus involved in sensory perception of pain / behavioral response to pain / calcium channel regulator activity ...detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / cardiac muscle cell action potential involved in contraction / Interaction between L1 and Ankyrins / voltage-gated sodium channel activity / sodium ion transport / Phase 0 - rapid depolarisation / detection of temperature stimulus involved in sensory perception of pain / behavioral response to pain / calcium channel regulator activity / sodium channel regulator activity / sodium ion transmembrane transport / sensory perception of pain / post-embryonic development / Sensory perception of sweet, bitter, and umami (glutamate) taste / response to toxic substance / circadian rhythm / toxin activity / inflammatory response / axon / lipid binding / extracellular region / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Sodium ion transport-associated / Voltage-gated sodium channel alpha subunit, inactivation gate / 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 ...Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Sodium ion transport-associated / Voltage-gated sodium channel alpha subunit, inactivation gate / 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
Ion transport protein / Beta/omega-theraphotoxin-Tp2a / Sodium channel protein type 9 subunit alpha
Similarity search - Component
Biological speciesArcobacter butzleri (bacteria)
Homo sapiens (human)
Mus musculus (house mouse)
Thrixopelma pruriens (green velvet)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsXu, H. / Rohou, A. / Arthur, C.P. / Estevez, A. / Ciferri, C. / Payandeh, J. / Koth, C.M.
CitationJournal: Cell / Year: 2019
Title: Structural Basis of Nav1.7 Inhibition by a Gating-Modifier Spider Toxin.
Authors: Hui Xu / Tianbo Li / Alexis Rohou / Christopher P Arthur / Foteini Tzakoniati / Evera Wong / Alberto Estevez / Christine Kugel / Yvonne Franke / Jun Chen / Claudio Ciferri / David H Hackos / ...Authors: Hui Xu / Tianbo Li / Alexis Rohou / Christopher P Arthur / Foteini Tzakoniati / Evera Wong / Alberto Estevez / Christine Kugel / Yvonne Franke / Jun Chen / Claudio Ciferri / David H Hackos / Christopher M Koth / Jian Payandeh /
Abstract: Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and ...Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable ∼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.
History
DepositionNov 20, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 23, 2019Provider: repository / Type: Initial release
Revision 1.1Feb 6, 2019Group: Data collection / 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 1.2Feb 20, 2019Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Dec 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 1.4Oct 23, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
B: Nav1.7 VSD2-NavAb chimera
C: Nav1.7 VSD2-NavAb chimera
D: Nav1.7 VSD2-NavAb chimera
E: Beta/omega-theraphotoxin-Tp2a
F: Beta/omega-theraphotoxin-Tp2a
G: Beta/omega-theraphotoxin-Tp2a
H: Beta/omega-theraphotoxin-Tp2a
I: Fab light chain
J: Fab heavy chain
K: Fab light chain
L: Fab heavy chain
A: Nav1.7 VSD2-NavAb chimera


Theoretical massNumber of molelcules
Total (without water)245,18812
Polymers245,18812
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Nav1.7 VSD2-NavAb chimera / Neuroendocrine sodium channel / hNE-Na / Peripheral sodium channel 1 / PN1 / Sodium channel protein ...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


Mass: 33453.512 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arcobacter butzleri (strain RM4018) (bacteria), (gene. exp.) Homo sapiens (human)
Strain: RM4018 / Gene: Abu_1752, SCN9A, NENA / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A8EVM5, UniProt: Q15858
#2: Protein/peptide
Beta/omega-theraphotoxin-Tp2a / Beta/omega-TRTX-Tp2a / ProTx-II / PT-II / Protoxin-2 / ProTx2


Mass: 3839.687 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) Thrixopelma pruriens (green velvet) / References: UniProt: P83476
#3: Antibody Fab light chain


Mass: 23483.910 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)
#4: Antibody Fab heavy chain


Mass: 24523.518 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse)
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: Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weightValue: 0.245 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
Details: 10 mM Tris pH 8.0, 100 mM NaCl, 0.06% FA3, 0.1 mg/ml POPC:POPE:POPG mixed at molar ratio 3:1:1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Grid was coated with a thin layer of gold / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Apply 3 uL, blot 2.5s. Ted Pella 595 filter paper.

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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: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 41 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 25084
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1cisTEM1.0.0particle selection
2SerialEMimage acquisition
4cisTEM1.0.0CTF correction
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: C2 (2 fold cyclic)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53206
Details: Spatial frequencies higher than 8 Angstroms were not used during refinement.
Symmetry type: POINT

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