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- EMDB-0342: CryoEM structure of Nav1.7 VSD2 (deactived state) in complex with... -

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Entry
Database: EMDB / ID: 0342
TitleCryoEM structure of Nav1.7 VSD2 (deactived state) in complex with the gating modifier toxin ProTx2
Map dataMap generated with a mask including Nav (with truncated C-terminal coiled-coil), ProTx2 and Fv fragment.
SampleNav1.7 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 / homologyCytoplasmic 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
SourceHomo sapiens (human) / Arcobacter butzleri (strain RM4018) (bacteria) / Thrixopelma pruriens (green velvet) / mouse (mice)
Methodsingle particle reconstruction / cryo EM / 4.2 Å resolution
AuthorsXu H / Rohou A / Arthur CP / Estevez A / Ciferri C / Payandeh J / Koth CM
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 / 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 ReportPDB-ID: 6n4r

SummaryFull reportAbout validation report
DateDeposition: Nov 20, 2018 / Header (metadata) release: Dec 26, 2018 / Map release: Jan 23, 2019 / Last update: Feb 6, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6n4r
  • Surface level: 3.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0342.map.gz (map file in CCP4 format, 186625 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
360 pix
1.2 Å/pix.
= 432. Å
360 pix
1.2 Å/pix.
= 432. Å
360 pix
1.2 Å/pix.
= 432. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2 Å
Density
Contour Level:3.5 (by author), 3.5 (movie #1):
Minimum - Maximum-25.596209000000002 - 39.945180000000001
Average (Standard dev.)0.0033436231 (0.4687166)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions360360360
Origin0.00.00.0
Limit359.0359.0359.0
Spacing360360360
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.21.21.2
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z432.000432.000432.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-25.59639.9450.003

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Supplemental data

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Sample components

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Entire Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab

EntireName: Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab
Number of components: 5

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Component #1: protein, Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab

ProteinName: Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab
Recombinant expression: No
MassTheoretical: 245 kDa
SourceSpecies: Homo sapiens (human)

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Component #2: protein, Nav1.7 VSD2-NavAb chimera

ProteinName: Nav1.7 VSD2-NavAb chimera / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 33.453512 kDa
SourceSpecies: Arcobacter butzleri (strain RM4018) (bacteria) / Strain: RM4018
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #3: protein, Beta/omega-theraphotoxin-Tp2a

ProteinName: Beta/omega-theraphotoxin-Tp2a / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 3.839687 kDa
SourceSpecies: Thrixopelma pruriens (green velvet)

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Component #4: protein, Fab light chain

ProteinName: Fab light chainFragment antigen-binding / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 23.48391 kDa
SourceSpecies: mouse (mice)

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Component #5: protein, Fab heavy chain

ProteinName: Fab heavy chainFragment antigen-binding / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 24.523518 kDa
SourceSpecies: mouse (mice)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen 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
pH: 8
Support filmGrid was coated with a thin layer of gold
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %
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
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 41 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 165000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 2500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 25084

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 53206
3D reconstructionSoftware: cisTEM / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF
Details: Spatial frequencies higher than 8 Angstroms were not used during refinement.

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