|Entry||Database: EMDB / ID: 0341|
|Title||CryoEM structure of Nav1.7 VSD2 (actived state) in complex with the gating modifier toxin ProTx2|
|Sample||Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab:|
Nav1.7 VSD2-NavAb chimera / Beta/omega-theraphotoxin-Tp2a / Fab light chainFragment antigen-binding / Fab heavy chainFragment antigen-binding
|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
|Source||Homo sapiens (human) / Arcobacter butzleri (strain RM4018) (bacteria) / Thrixopelma pruriens (green velvet) / mouse (mice) / Mus musculus (house mouse)|
|Method||single particle reconstruction / cryo EM / 3.6 Å resolution|
|Authors||Xu H / Rohou A / Arthur CP / Estevez A / Ciferri C / Payandeh J / Koth CM|
|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.
|Validation Report||PDB-ID: 6n4q|
SummaryFull reportAbout validation report
|Date||Deposition: Nov 20, 2018 / Header (metadata) release: Dec 26, 2018 / Map release: Jan 23, 2019 / Last update: Feb 6, 2019|
|Structure viewer||EM map: |
Downloads & links
|File||emd_0341.map.gz (map file in CCP4 format, 186625 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.2 Å|
CCP4 map header:
+Entire Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab
|Entire||Name: Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab|
Number of components: 5
+Component #1: protein, Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab
|Protein||Name: Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab|
Recombinant expression: No
|Mass||Theoretical: 245 kDa|
|Source||Species: Homo sapiens (human)|
+Component #2: protein, Nav1.7 VSD2-NavAb chimera
|Protein||Name: Nav1.7 VSD2-NavAb chimera / Number of Copies: 4 / Recombinant expression: No|
|Mass||Theoretical: 33.453512 kDa|
|Source||Species: Arcobacter butzleri (strain RM4018) (bacteria) / Strain: RM4018|
|Source (engineered)||Expression System: Trichoplusia ni (cabbage looper)|
+Component #3: protein, Beta/omega-theraphotoxin-Tp2a
|Protein||Name: Beta/omega-theraphotoxin-Tp2a / Number of Copies: 4 / Recombinant expression: No|
|Mass||Theoretical: 3.839687 kDa|
|Source||Species: Thrixopelma pruriens (green velvet)|
+Component #4: protein, Fab light chain
|Protein||Name: Fab light chainFragment antigen-binding / Number of Copies: 2 / Recombinant expression: No|
|Mass||Theoretical: 23.48391 kDa|
|Source||Species: mouse (mice)|
+Component #5: protein, Fab heavy chain
|Protein||Name: Fab heavy chainFragment antigen-binding / Number of Copies: 2 / Recombinant expression: No|
|Mass||Theoretical: 24.523518 kDa|
|Source||Species: Mus musculus (house mouse)|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 2 mg/ml|
Buffer solution: 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
|Support film||Grid was coated with a thin layer of gold|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %|
Details: Apply 3 uL, blot 2.5s. Ted Pella 595 filter paper..
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 41 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 165000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 2500.0 nm / Energy filter: GIF Bioquantum|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Image acquisition||Number of digital images: 25084|
|Processing||Method: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 187192|
|3D reconstruction||Software: cisTEM / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF|
Details: Spatial frequencies higher than 8 Angstroms were not used during refinement.
-Atomic model buiding
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