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- EMDB-0500: Cryo-EM structure of a human-cockroach hybrid Nav channel. -

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Basic information

Entry
Database: EMDB / ID: EMD-0500
TitleCryo-EM structure of a human-cockroach hybrid Nav channel.
Map dataSharpened and masked map used for model building.
Sample
  • Complex: NavPas-VSD4
    • Protein or peptide: Sodium channel protein type 9 subunit alpha, Sodium channel protein PaFPC1, chimeric construct
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: (7E,21R,24S)-27-amino-24-hydroxy-18,24-dioxo-19,23,25-trioxa-24lambda~5~-phosphaheptacos-7-en-21-yl (9Z,12E)-octadeca-9,12-dienoate
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE
  • Ligand: Digitonin
Function / homology
Function and homology information


detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / high voltage-gated calcium channel activity / voltage-gated sodium channel activity / Interaction between L1 and Ankyrins / sodium ion transport / voltage-gated calcium channel complex / behavioral response to pain / Phase 0 - rapid depolarisation / calcium ion import across plasma membrane ...detection of mechanical stimulus involved in sensory perception / voltage-gated sodium channel complex / high voltage-gated calcium channel activity / voltage-gated sodium channel activity / Interaction between L1 and Ankyrins / sodium ion transport / voltage-gated calcium channel complex / behavioral response to pain / Phase 0 - rapid depolarisation / calcium ion import across plasma membrane / detection of temperature stimulus involved in sensory perception of pain / 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 / inflammatory response / axon / plasma membrane
Similarity search - Function
Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Voltage-gated sodium channel alpha subunit, inactivation gate / Voltage-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-associated / 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. ...Voltage-gated Na+ ion channel, cytoplasmic domain / Cytoplasmic domain of voltage-gated Na+ ion channel / Voltage-gated sodium channel alpha subunit, inactivation gate / Voltage-dependent L-type calcium channel, IQ-associated domain / Voltage-dependent L-type calcium channel, IQ-associated / 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 / Voltage-dependent channel domain superfamily / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Sodium channel protein PaFPC1 / Sodium channel protein type 9 subunit alpha
Similarity search - Component
Biological speciesPeriplaneta americana (American cockroach)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsClairfeuille T / Rohou A / Payandeh J
CitationJournal: Science / Year: 2019
Title: Structural basis of α-scorpion toxin action on Na channels.
Authors: Thomas Clairfeuille / Alexander Cloake / Daniel T Infield / José P Llongueras / Christopher P Arthur / Zhong Rong Li / Yuwen Jian / Marie-France Martin-Eauclaire / Pierre E Bougis / Claudio ...Authors: Thomas Clairfeuille / Alexander Cloake / Daniel T Infield / José P Llongueras / Christopher P Arthur / Zhong Rong Li / Yuwen Jian / Marie-France Martin-Eauclaire / Pierre E Bougis / Claudio Ciferri / Christopher A Ahern / Frank Bosmans / David H Hackos / Alexis Rohou / Jian Payandeh /
Abstract: Fast inactivation of voltage-gated sodium (Na) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Na ...Fast inactivation of voltage-gated sodium (Na) channels is essential for electrical signaling, but its mechanism remains poorly understood. Here we determined the structures of a eukaryotic Na channel alone and in complex with a lethal α-scorpion toxin, AaH2, by electron microscopy, both at 3.5-angstrom resolution. AaH2 wedges into voltage-sensing domain IV (VSD4) to impede fast activation by trapping a deactivated state in which gating charge interactions bridge to the acidic intracellular carboxyl-terminal domain. In the absence of AaH2, the S4 helix of VSD4 undergoes a ~13-angstrom translation to unlatch the intracellular fast-inactivation gating machinery. Highlighting the polypharmacology of α-scorpion toxins, AaH2 also targets an unanticipated receptor site on VSD1 and a pore glycan adjacent to VSD4. Overall, this work provides key insights into fast inactivation, electromechanical coupling, and pathogenic mutations in Na channels.
History
DepositionJan 28, 2019-
Header (metadata) releaseFeb 6, 2019-
Map releaseFeb 20, 2019-
UpdateAug 25, 2021-
Current statusAug 25, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 3.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nt3
  • Surface level: 3.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0500.png

Downloads & links

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Map

FileDownload / File: emd_0500.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened and masked map used for model building.
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 3.8 / Movie #1: 3.8
Minimum - Maximum-15.94242 - 23.608826
Average (Standard dev.)0.048209425 (±1.1041741)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 250.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z250250250
origin x/y/z0.0000.0000.000
length x/y/z250.000250.000250.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS250250250
D min/max/mean-15.94223.6090.048

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Supplemental data

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Mask #1

Fileemd_0500_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_0500_additional.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: em-half-volume P1

Fileemd_0500_half_map_1.map
Annotationem-half-volume_P1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: em-half-volume P2

Fileemd_0500_half_map_2.map
Annotationem-half-volume_P2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : NavPas-VSD4

EntireName: NavPas-VSD4
Components
  • Complex: NavPas-VSD4
    • Protein or peptide: Sodium channel protein type 9 subunit alpha, Sodium channel protein PaFPC1, chimeric construct
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: (7E,21R,24S)-27-amino-24-hydroxy-18,24-dioxo-19,23,25-trioxa-24lambda~5~-phosphaheptacos-7-en-21-yl (9Z,12E)-octadeca-9,12-dienoate
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE
  • Ligand: Digitonin

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Supramolecule #1: NavPas-VSD4

SupramoleculeName: NavPas-VSD4 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Periplaneta americana (American cockroach)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: Expi293
Molecular weightTheoretical: 155 KDa

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Macromolecule #1: Sodium channel protein type 9 subunit alpha, Sodium channel prote...

MacromoleculeName: Sodium channel protein type 9 subunit alpha, Sodium channel protein PaFPC1, chimeric construct
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Periplaneta americana (American cockroach)
Molecular weightTheoretical: 178.763656 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: WSHPQFEKGG GSGGGSGGSA WSHPQFEKGG SGGDYKDDDD KGGSGGDYKD DDDKMADNSP LIREERQRLF RPYTRAMLTA PSAQPAKEN GKTEENKDNS RDKGRGANKD RDGSAHPDQA LEQGSRLPAR MRNIFPAELA STPLEDFDPF YKNKKTFVVV T KAGDIFRF ...String:
WSHPQFEKGG GSGGGSGGSA WSHPQFEKGG SGGDYKDDDD KGGSGGDYKD DDDKMADNSP LIREERQRLF RPYTRAMLTA PSAQPAKEN GKTEENKDNS RDKGRGANKD RDGSAHPDQA LEQGSRLPAR MRNIFPAELA STPLEDFDPF YKNKKTFVVV T KAGDIFRF SGEKSLWMLD PFTPIRRVAI STMVQPIFSY FIMITILIHC IFMIMPATQT TYILELVFLS IYTIEVVVKV LA RGFILHP FAYLRDPWNW LDFLVTLIGY ITLVVDLGHL YALRAFRVLR SWRTVTIVPG WRTIVDALSL SITSLKDLVL LLL FSLSVF ALIGLQLFMG NLKHKCVKHF PADGSWGNFT DERWFNYTSN SSHWYIPDDW IEYPLCGNSS GAGMCPPGYT CLQG YGGNP NYGYTSFDTF GWAFLSVFRL VTLDYWEDLY QLALRSAGPW HILFFIIVVF YGTFCFLNFI LAVVVMSYTH MVKRA DEEK AAERELKKEK KAASVANNTA NGQEQTTIEM NGDEAVVIDN NDQAARQQSD PETPAPSVTQ RLTDFLCVWD CCVPWQ KLQ GAIGAVVLSP FFELFIAVII VLNITFMALD HHDMNIEFER ILRTGNYIFT SIYIVEAVLK IIALSPKFYF KDSWNVF DF IIVVFAILEL GLEGVQGLSV FRSFRLLRVF RLAKFWPTLN NFMSVMTKSY GAFVNVMYVM FLLLFIFAII GMQLFGMN Y IDNMERFPDG DLPRWNFTDF LHSFMIVFRA LCGEWIESMW DCMLVGDWSC IPFFVAVFFV GNLVILNLLI ALLLNNYGS FCTSPTSDEE DSKDEDALAQ IVRIFKRFKP NLNAVKLSPM KPDSEDIVES QEIQGNNIAD AEDVLAGEFP PDCCCNAFYK CFPSRPARD SSVQRMWSNI RRVCFLLAKN KYFQKFVTAV LVITSVLLAL EDIYLPQRPV LVNITLYVDY VLTAFFVIEM I IMLFAVGF KKYFTSKWYW LDFIVVVAYL LNFVLMCAGI EALQTLRLLR VFRLFRPLSK VNGMQVVTST LVEAVPHIFN VI LVGIFFW LVFAIMGVQL FAGKFYKCVD ENSTVLSHEI TMDRNDCLHE NYTWENSPMN FDHVGNAYLS LLQVATFKGW LQI MNDAID SREVHKQPIR ETNIYMYLYF IFFIVFGSFF ILKLFVCILI DIFRQQRRKA EGLSATDSRT QLIYRRAVMR TMSA KPVKR IPKPGNKIQG CIFDLVTNQA FDISIMVLIC LNMVTMMVEK EGQSQHMTEV LYWINVVFII LFTGECVLKL ISLRH YYFT VGWNIFDFVV VIISIVGMFL ADLIETYFVS PTLFRVIRLA RIGRILRLVK GAKGIRLLLL ALRKALRTLF NVSFLL FVI MFVYAVFGME FFMHIRDAGA IDDVYNFKTF GQSIILLFQL ATSAGWDGVY FAIANEEDCR APDHELGYPG NCGSRAL GI AYLVSYLIIT CLVVINMYAA VILDYVLEVY EDSKEGLTDD DYDMFFEVWQ QFDPEATQYI RYDQLSELLE ALQPPLQV Q KPNKYKILSM NIPICKDDHI FYKDVLEALV KDVFSRRG

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #5: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #6: (7E,21R,24S)-27-amino-24-hydroxy-18,24-dioxo-19,23,25-trioxa-24la...

MacromoleculeName: (7E,21R,24S)-27-amino-24-hydroxy-18,24-dioxo-19,23,25-trioxa-24lambda~5~-phosphaheptacos-7-en-21-yl (9Z,12E)-octadeca-9,12-dienoate
type: ligand / ID: 6 / Number of copies: 4 / Formula: 76F
Molecular weightTheoretical: 742.018 Da
Chemical component information

ChemComp-76F:
(7E,21R,24S)-27-amino-24-hydroxy-18,24-dioxo-19,23,25-trioxa-24lambda~5~-phosphaheptacos-7-en-21-yl (9Z,12E)-octadeca-9,12-dienoate / Discrete optimized protein energy

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Macromolecule #7: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE

MacromoleculeName: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / type: ligand / ID: 7 / Number of copies: 1 / Formula: LHG
Molecular weightTheoretical: 722.97 Da
Chemical component information

ChemComp-LHG:
1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / phospholipid*YM / Phosphatidylglycerol

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Macromolecule #8: Digitonin

MacromoleculeName: Digitonin / type: ligand / ID: 8 / Number of copies: 1 / Formula: AJP
Molecular weightTheoretical: 1.229312 KDa
Chemical component information

ChemComp-AJP:
Digitonin / detergent*YM / Digitonin

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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

Concentration1.5 mg/mL
BufferpH: 7.5
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: PLASMA CLEANING
Details: Grids were plasma etched using the Solarus plasma cleaner (Gatan) in the hydrogen-oxygen setting. Grids were etched for 4 minutes on each side to remove burrs from hole edges. The grids were ...Details: Grids were plasma etched using the Solarus plasma cleaner (Gatan) in the hydrogen-oxygen setting. Grids were etched for 4 minutes on each side to remove burrs from hole edges. The grids were then coated on both sides with 5nm of Au/Pd which was plasma deposited using the Leica ACE600 (Leica).
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 10421 / Average exposure time: 10.0 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionDetails: Automated picking using a soft-edged disc as a template yielded 591739 candidate coordinates. 2D classification led to the selection of 334055 particles for further analysis.
CTF correctionSoftware - Name: cisTEM
Startup modelType of model: EMDB MAP
EMDB ID:

6698


Details: The startup map was low-pass filtered to 40A and resolution-limited automatic refinement in cisTEM was used (beginning at 20A)
Initial angle assignmentType: OTHER / Software - Name: cisTEM
Final 3D classificationSoftware - Name: cisTEM
Final angle assignmentType: OTHER / Software - Name: cisTEM
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM
Details: No information from spatial frequencies higher than 1/5A were ever used during refinement.
Number images used: 150583
FSC plot (resolution estimation)

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