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

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Basic information

Entry
Database: EMDB / ID: EMD-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.
Sample
  • Complex: Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab
    • Protein or peptide: Nav1.7 VSD2-NavAb chimera
    • Protein or peptide: Beta/omega-theraphotoxin-Tp2a
    • Protein or peptide: Fab light chain
    • Protein or peptide: Fab heavy chain
Keywordsvoltage-gated sodium channel / gating modifier toxin / MEMBRANE PROTEIN
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 speciesHomo sapiens (human) / Arcobacter butzleri (strain RM4018) (bacteria) / Thrixopelma pruriens (green velvet) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsXu H / Rohou A
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, 2018-
Header (metadata) releaseDec 26, 2018-
Map releaseJan 23, 2019-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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

emd_0342.png

Downloads & links

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Map

FileDownload / File: emd_0342.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap generated with a mask including Nav (with truncated C-terminal coiled-coil), ProTx2 and Fv fragment.
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.5 / Movie #1: 3.5
Minimum - Maximum-25.596209000000002 - 39.945180000000001
Average (Standard dev.)0.0033436231 (±0.4687166)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 °

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
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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Additional map: Unsharpened map for the main map.

Fileemd_0342_additional.map
AnnotationUnsharpened map for the main map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half map 2

Fileemd_0342_half_map_1.map
AnnotationEM half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half map 1

Fileemd_0342_half_map_2.map
AnnotationEM half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
Components
  • Complex: Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab
    • Protein or peptide: Nav1.7 VSD2-NavAb chimera
    • Protein or peptide: Beta/omega-theraphotoxin-Tp2a
    • Protein or peptide: Fab light chain
    • Protein or peptide: Fab heavy chain

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

SupramoleculeName: Nav1.7 VSD2 in complex with ProTx2 and an anti-Nav Fab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 245 KDa

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Macromolecule #1: Nav1.7 VSD2-NavAb chimera

MacromoleculeName: Nav1.7 VSD2-NavAb chimera / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Arcobacter butzleri (strain RM4018) (bacteria) / Strain: RM4018
Molecular weightTheoretical: 33.453512 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MDYKDDDDKG SLVPRGSHMY LRITNIVESS FFTKFIIYLI VLNTLFMAME HHPMTEEFKN VLAIGNLVFT GIFAIEIILR IYVHRISFF KDPWSLFDSL IVTLSLVELF LADVEGLSVL RSFRLLRVFR LVTAVPQMRK IVSALISVIP GMLSVIALMT L FFYIFAIM ...String:
MDYKDDDDKG SLVPRGSHMY LRITNIVESS FFTKFIIYLI VLNTLFMAME HHPMTEEFKN VLAIGNLVFT GIFAIEIILR IYVHRISFF KDPWSLFDSL IVTLSLVELF LADVEGLSVL RSFRLLRVFR LVTAVPQMRK IVSALISVIP GMLSVIALMT L FFYIFAIM ATQLFGERFP EWFGTLGESF YTLFQVMTLE SWSMGIVRPL MEVYPYAWVF FIPFIFVVTF VMINLVVAIC VD AMAILNQ KEEQHIIDEV QSHEDNINNE IIKLREEIVE LKELIKTSLK N

UniProtKB: Ion transport protein, Sodium channel protein type 9 subunit alpha, Ion transport protein, Sodium channel protein type 9 subunit alpha, Ion transport protein

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Macromolecule #2: Beta/omega-theraphotoxin-Tp2a

MacromoleculeName: Beta/omega-theraphotoxin-Tp2a / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Thrixopelma pruriens (green velvet)
Molecular weightTheoretical: 3.839687 KDa
SequenceString:
YCQKWMWTCD SERKCCEGMV CRLWCKKKLW

UniProtKB: Beta/omega-theraphotoxin-Tp2a

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Macromolecule #3: Fab light chain

MacromoleculeName: Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.48391 KDa
SequenceString: EIVLTQSPAL MAASPGEKVT ITCSVSLSIS SSNLFWYQQK SETSPKPWIY GTSKLASGVP VRFSGSGSGT SYSLTISSME AEDAATYYC QQWSSHSFTF GGGTKLEIKR ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPKDINVKW KIDGSERQNG V LNSWTDQD ...String:
EIVLTQSPAL MAASPGEKVT ITCSVSLSIS SSNLFWYQQK SETSPKPWIY GTSKLASGVP VRFSGSGSGT SYSLTISSME AEDAATYYC QQWSSHSFTF GGGTKLEIKR ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPKDINVKW KIDGSERQNG V LNSWTDQD SKDSTYSMSS TLTLTKDEYE RHNSYTCEAT HKTSTSPIVK SFNRNEC

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Macromolecule #4: Fab heavy chain

MacromoleculeName: Fab heavy chain / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 24.523518 KDa
SequenceString: EVQLVESGGG LVKPGGSLKL SCAASGFTFS NYAMSWVRQT PEKRLEWVAT ISNGGRYTYY PDSVKGRFTI SRDNAKNSLY LQMSSLRSE DTAMYYCARH LYRYDVGGAL DYWGQGTSVT VSSAKTTAPS VYPLAPVCGD TTGSSVTLGC LVKGYFPEPV T LTWNSGSL ...String:
EVQLVESGGG LVKPGGSLKL SCAASGFTFS NYAMSWVRQT PEKRLEWVAT ISNGGRYTYY PDSVKGRFTI SRDNAKNSLY LQMSSLRSE DTAMYYCARH LYRYDVGGAL DYWGQGTSVT VSSAKTTAPS VYPLAPVCGD TTGSSVTLGC LVKGYFPEPV T LTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVTSSTWP SQSITCNVAH PASSTKVDKK IEPRGPTIKP

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 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
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR / Details: Grid was coated with a thin layer of gold
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Apply 3 uL, blot 2.5s. Ted Pella 595 filter paper..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 25084 / Average exposure time: 10.0 sec. / Average electron dose: 41.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0)
Details: Spatial frequencies higher than 8 Angstroms were not used during refinement.
Number images used: 53206
Initial angle assignmentType: OTHER / Software - Name: cisTEM (ver. 1.0.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cisTEM (ver. 1.0.0)
Final 3D classificationSoftware - Name: cisTEM (ver. 1.0.0)

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