|Entry||Database: PDB / ID: 6n4q|
|Title||CryoEM structure of Nav1.7 VSD2 (actived state) in complex with the gating modifier toxin ProTx2|
|Keywords||MEMBRANE PROTEIN / voltage-gated sodium channel / gating modifier toxin|
|Function / homology||Cytoplasmic domain of voltage-gated Na+ ion channel / Interaction between L1 and Ankyrins / Sodium ion transport-associated / Ion transport protein / Voltage gated sodium channel, alpha-9 subunit / Phase 0 - rapid depolarisation / Voltage-dependent channel domain superfamily / Voltage-gated Na+ ion channel, cytoplasmic domain / Sodium ion transport-associated / Ion transport domain ...Cytoplasmic domain of voltage-gated Na+ ion channel / Interaction between L1 and Ankyrins / Sodium ion transport-associated / Ion transport protein / Voltage gated sodium channel, alpha-9 subunit / Phase 0 - rapid depolarisation / Voltage-dependent channel domain superfamily / Voltage-gated Na+ ion channel, cytoplasmic domain / Sodium ion transport-associated / Ion transport domain / Voltage gated sodium channel, alpha subunit / IQ motif, EF-hand binding site / voltage-gated sodium channel complex / membrane depolarization during action potential / behavioral response to pain / voltage-gated sodium channel activity / sodium ion binding / neuronal action potential / sodium ion transmembrane transport / voltage-gated ion channel activity / sodium ion transport / regulation of ion transmembrane transport / sensory perception of pain / post-embryonic development / response to toxic substance / ion channel activity / toxin activity / lipid binding / inflammatory response / axon / integral component of plasma membrane / integral component of membrane / extracellular region / identical protein binding / plasma membrane / Ion transport protein / Beta/omega-theraphotoxin-Tp2a / Sodium channel protein type 9 subunit alpha|
Function and homology information
|Specimen source||Arcobacter butzleri (bacteria)|
Homo sapiens (human)
Mus musculus (house mouse)
Thrixopelma pruriens (green velvet)
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.6 Å resolution|
|Authors||Xu, H. / Rohou, A. / Arthur, C.P. / Estevez, A. / Ciferri, C. / Payandeh, J. / Koth, C.M.|
|Citation||Journal: 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 / 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.
SummaryFull reportAbout validation report
|Date||Deposition: Nov 20, 2018 / Release: Jan 23, 2019|
|Structure viewer||Molecule: |
Downloads & links
A: Nav1.7 VSD2-NavAb chimera
B: Nav1.7 VSD2-NavAb chimera
C: Nav1.7 VSD2-NavAb chimera
D: Nav1.7 VSD2-NavAb chimera
I: Fab light chain
J: Fab heavy chain
K: Fab light chain
L: Fab heavy chain
Mass: 33453.512 Da / Num. of mol.: 4
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
Mass: 3839.687 Da / Num. of mol.: 4 / Source: (synth.) Thrixopelma pruriens (green velvet) / References: UniProt: P83476
Mass: 23483.910 Da / Num. of mol.: 2 / Source: (natural) Mus musculus (house mouse)
Mass: 24523.518 Da / Num. of mol.: 2 / Source: (natural) Mus musculus (house mouse)
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: single particle reconstruction|
|Component||Name: Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab|
Type: COMPLEX / Entity ID: 1,
|Molecular weight||Value: 0.245 MDa / Experimental value: NO||Source (natural)||Organism: Homo sapiens (human)||Source (recombinant)||Organism: Trichoplusia ni (cabbage looper)||Buffer solution||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|
|Specimen||Conc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES||Specimen support||Details: Grid was coated with a thin layer of gold / Grid material: COPPER / Grid mesh size: 300 / Grid type: C-flat-1.2/1.3||Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins / Details: Apply 3 uL, blot 2.5s. Ted Pella 595 filter paper.|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 microns / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Image recording||Average exposure time: 10 sec. / Electron dose: 41 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1 / Number of real images: 25084|
|EM imaging optics||Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV|
|Image scans||Movie frames/image: 40|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Symmetry||Point symmetry: C2|
|3D reconstruction||Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 187192|
Details: Spatial frequencies higher than 8 Angstroms were not used during refinement.
Symmetry type: POINT
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