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- EMDB-22661: Structure of NavAb/Nav1.7-VS2A chimera trapped in the resting sta... -

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Basic information

Entry
Database: EMDB / ID: EMD-22661
TitleStructure of NavAb/Nav1.7-VS2A chimera trapped in the resting state by tarantula toxin m3-Huwentoxin-IV
Map dataEM map without post-cut-off B-factor sharpening
Sample
  • Complex: Complex of NavAb/Nav1.7-VS2A chimera and m3-Huwentoxin-IV
    • Complex: NavAb/Nav1.7-VS2A chimera
      • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein,Ion transport protein,Sodium channel protein type 9 subunit alpha chimera
    • Complex: m3-Huwentoxin-IV
      • Protein or peptide: Mu-theraphotoxin-Hs2a
Function / homology
Function and homology information


detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / host cell presynaptic membrane / high voltage-gated calcium channel activity / voltage-gated sodium channel activity / Interaction between L1 and Ankyrins / ion channel inhibitor activity / detection of maltose stimulus / sodium ion transport / maltose binding ...detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / host cell presynaptic membrane / high voltage-gated calcium channel activity / voltage-gated sodium channel activity / Interaction between L1 and Ankyrins / ion channel inhibitor activity / detection of maltose stimulus / sodium ion transport / maltose binding / maltose transport complex / maltose transport / voltage-gated calcium channel complex / maltodextrin transmembrane transport / behavioral response to pain / Phase 0 - rapid depolarisation / detection of temperature stimulus involved in sensory perception of pain / calcium ion import across plasma membrane / carbohydrate transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / sodium channel regulator activity / carbohydrate transport / sodium ion transmembrane transport / monoatomic cation channel activity / sensory perception of pain / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / post-embryonic development / response to toxic substance / Sensory perception of sweet, bitter, and umami (glutamate) taste / circadian rhythm / outer membrane-bounded periplasmic space / toxin activity / periplasmic space / inflammatory response / axon / DNA damage response / extracellular region / membrane / identical protein binding / plasma membrane
Similarity search - Function
Huwentoxin-1 family signature. / Huwentoxin, conserved site-1 / Huwentoxin-1 family / Ion channel inhibitory toxin / Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Voltage-gated sodium channel alpha subunit, inactivation gate / Sodium ion transport-associated / Sodium ion transport-associated / Voltage gated sodium channel, alpha subunit ...Huwentoxin-1 family signature. / Huwentoxin, conserved site-1 / Huwentoxin-1 family / Ion channel inhibitory toxin / Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Voltage-gated sodium channel alpha subunit, inactivation gate / Sodium ion transport-associated / 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 / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Voltage-dependent channel domain superfamily / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Ion transport protein / Maltose/maltodextrin-binding periplasmic protein / Huwentoxin-IV / Sodium channel protein type 9 subunit alpha
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Haplopelma schmidti (Chinese earth tiger) / Arcobacter butzleri (strain RM4018) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsWisedchaisri G / Tonggu L / Gamal El-Din TM / McCord E / Zheng N / Catterall WA
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01 NS015751 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01 HL112808 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R35 NS111573 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Mol Cell / Year: 2021
Title: Structural Basis for High-Affinity Trapping of the Na1.7 Channel in Its Resting State by Tarantula Toxin.
Authors: Goragot Wisedchaisri / Lige Tonggu / Tamer M Gamal El-Din / Eedann McCord / Ning Zheng / William A Catterall /
Abstract: Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting ...Voltage-gated sodium channels initiate electrical signals and are frequently targeted by deadly gating-modifier neurotoxins, including tarantula toxins, which trap the voltage sensor in its resting state. The structural basis for tarantula-toxin action remains elusive because of the difficulty of capturing the functionally relevant form of the toxin-channel complex. Here, we engineered the model sodium channel NaAb with voltage-shifting mutations and the toxin-binding site of human Na1.7, an attractive pain target. This mutant chimera enabled us to determine the cryoelectron microscopy (cryo-EM) structure of the channel functionally arrested by tarantula toxin. Our structure reveals a high-affinity resting-state-specific toxin-channel interaction between a key lysine residue that serves as a "stinger" and penetrates a triad of carboxyl groups in the S3-S4 linker of the voltage sensor. By unveiling this high-affinity binding mode, our studies establish a high-resolution channel-docking and resting-state locking mechanism for huwentoxin-IV and provide guidance for developing future resting-state-targeted analgesic drugs.
History
DepositionSep 15, 2020-
Header (metadata) releaseDec 2, 2020-
Map releaseDec 2, 2020-
UpdateJan 20, 2021-
Current statusJan 20, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 4
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7k48
  • Surface level: 4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22661.png

Downloads & links

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Map

FileDownload / File: emd_22661.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEM map without post-cut-off B-factor sharpening
Voxel sizeX=Y=Z: 1.056 Å
Density
Contour LevelBy AUTHOR: 3.0 / Movie #1: 4
Minimum - Maximum-11.073463 - 18.740183
Average (Standard dev.)0.069499016 (±0.92631894)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 270.336 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0561.0561.056
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z270.336270.336270.336
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-11.07318.7400.069

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Supplemental data

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Mask #1

Fileemd_22661_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: EM map with post-cut-off B-factor sharpening of -50 A2.

Fileemd_22661_additional_1.map
AnnotationEM map with post-cut-off B-factor sharpening of -50 A2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: EM full map

Fileemd_22661_additional_2.map
AnnotationEM full map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half map (odd)

Fileemd_22661_half_map_1.map
AnnotationEM half map (odd)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EM half map (even)

Fileemd_22661_half_map_2.map
AnnotationEM half map (even)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of NavAb/Nav1.7-VS2A chimera and m3-Huwentoxin-IV

EntireName: Complex of NavAb/Nav1.7-VS2A chimera and m3-Huwentoxin-IV
Components
  • Complex: Complex of NavAb/Nav1.7-VS2A chimera and m3-Huwentoxin-IV
    • Complex: NavAb/Nav1.7-VS2A chimera
      • Protein or peptide: Maltose/maltodextrin-binding periplasmic protein,Ion transport protein,Sodium channel protein type 9 subunit alpha chimera
    • Complex: m3-Huwentoxin-IV
      • Protein or peptide: Mu-theraphotoxin-Hs2a

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Supramolecule #1: Complex of NavAb/Nav1.7-VS2A chimera and m3-Huwentoxin-IV

SupramoleculeName: Complex of NavAb/Nav1.7-VS2A chimera and m3-Huwentoxin-IV
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightTheoretical: 300 KDa

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Supramolecule #2: NavAb/Nav1.7-VS2A chimera

SupramoleculeName: NavAb/Nav1.7-VS2A chimera / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12

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Supramolecule #3: m3-Huwentoxin-IV

SupramoleculeName: m3-Huwentoxin-IV / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Haplopelma schmidti (Chinese earth tiger)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Maltose/maltodextrin-binding periplasmic protein,Ion transport pr...

MacromoleculeName: Maltose/maltodextrin-binding periplasmic protein,Ion transport protein,Sodium channel protein type 9 subunit alpha 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: 68.519062 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI IFWAHDRFGG YAQSGLLAEI TPDKAFQDK LYPFTWDAVR YNGKLIAYPI AVEALSLIYN KDLLPNPPKT WEEIPALDKE LKAKGKSALM FNLQEPYFTW P LIAADGGY ...String:
MKIEEGKLVI WINGDKGYNG LAEVGKKFEK DTGIKVTVEH PDKLEEKFPQ VAATGDGPDI IFWAHDRFGG YAQSGLLAEI TPDKAFQDK LYPFTWDAVR YNGKLIAYPI AVEALSLIYN KDLLPNPPKT WEEIPALDKE LKAKGKSALM FNLQEPYFTW P LIAADGGY AFKYENGKYD IKDVGVDNAG AKAGLTFLVD LIKNKHMNAD TDYSIAEAAF NKGETAMTIN GPWAWSNIDT SK VNYGVTV LPTFKGQPSK PFVGVLSAGI NAASPNKELA KEFLENYLLT DEGLEAVNKD KPLGAVALKS YEEELAKDPR IAA TMENAQ KGEIMPNIPQ MSAFWYAVRT AVINAASGRQ TVDEALKDAQ TNAMYLAITN IVESSFFTKF ITICIVLNTL FMAM EHHPM TEEFKNVLAI GNLVFTGIFA AEIILRIYVH RISFFKDPWS LFDFFVVTLS LVELFLADVE GLSVLRSFRL LRLFR AVTA VPQMRKIVSA LISVIPGMLS VIALMTLFFY IFAIMATQLF GERFPEWFGT LGESFYTLFQ VMTLESWSMG IVRPLM EVY PYAWVFFIPF IFVVTFVMIN LVVAIIVDAM AILNQKEEQH IIDEVQSH

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Macromolecule #2: Mu-theraphotoxin-Hs2a

MacromoleculeName: Mu-theraphotoxin-Hs2a / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Haplopelma schmidti (Chinese earth tiger)
Molecular weightTheoretical: 3.999777 KDa
SequenceString:
GCLGIFKACN PSNDQCCKSS KLVCSRKTRW CKWQI

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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

Concentration10 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMNH2C(CH2OH)3HClTris-HClTris
100.0 mMNaClSodium chloridesodium chloride
0.006 %C56H92O25glycol-diosgenin (GDN)
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 2.5-4.0 seconds before plunging.
Detailsm3-HwTx-IV was added in excess to the chimera at 8:1 stoichiometric molar ratio of toxin to channel.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
DetailsPreliminary grid screening was performed manually.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3582 pixel / Digitization - Frames/image: 2-42 / Number real images: 7168 / Average exposure time: 8.6 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1470000
CTF correctionSoftware - Name: Gctf
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6p6w


Details: Low pass filtered to 60 Angstrom
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM (ver. 1.0.0-beta)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0-beta) / Number images used: 501310
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6n4i


Chain - Chain ID: A
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-7k48:
Structure of NavAb/Nav1.7-VS2A chimera trapped in the resting state by tarantula toxin m3-Huwentoxin-IV

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