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- EMDB-20265: Cryo-EM structure of voltage-gated sodium channel NavAb N49K/L109... -

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Basic information

Entry
Database: EMDB / ID: EMD-20265
TitleCryo-EM structure of voltage-gated sodium channel NavAb N49K/L109A/M116V/G94C/Q150C disulfide crosslinked mutant in the resting state
Map data
SampleFusion of maltose-binding protein and voltage-gated sodium channel NavAb in the resting state
  • Fusion of Maltose-binding protein and voltage-gated sodium channel NavAb
Function / homology
Function and homology information


carbohydrate transmembrane transporter activity / ion channel activity / periplasmic space / integral component of membrane / identical protein binding
Bacterial extracellular solute-binding protein / Ion transport domain / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Voltage-dependent channel domain superfamily / Ion transport protein / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding proteins, family 1 signature.
Maltodextrin-binding protein / Ion transport protein
Biological speciesArcobacter butzleri RM4018 (bacteria) / Arcobacter butzleri (strain RM4018) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsWisedchaisri G / Tonggu L / McCord E / Gamal El-Din TM / Wang L / Zheng N / Catterall WA
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and StrokeR01 NS015751 United States
National Institutes of Health/National Institute of General Medical SciencesT32 GM007750 United States
National Institutes of Health/National Heart, Lung, and Blood InstituteR01 HL112808 United States
Howard Hughes Medical Institute United States
CitationJournal: Cell / Year: 2019
Title: Resting-State Structure and Gating Mechanism of a Voltage-Gated Sodium Channel.
Authors: Goragot Wisedchaisri / Lige Tonggu / Eedann McCord / Tamer M Gamal El-Din / Liguo Wang / Ning Zheng / William A Catterall /
Abstract: Voltage-gated sodium (Na) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state ...Voltage-gated sodium (Na) channels initiate action potentials in nerve, muscle, and other electrically excitable cells. The structural basis of voltage gating is uncertain because the resting state exists only at deeply negative membrane potentials. To stabilize the resting conformation, we inserted voltage-shifting mutations and introduced a disulfide crosslink in the VS of the ancestral bacterial sodium channel NaAb. Here, we present a cryo-EM structure of the resting state and a complete voltage-dependent gating mechanism. The S4 segment of the VS is drawn intracellularly, with three gating charges passing through the transmembrane electric field. This movement forms an elbow connecting S4 to the S4-S5 linker, tightens the collar around the S6 activation gate, and prevents its opening. Our structure supports the classical "sliding helix" mechanism of voltage sensing and provides a complete gating mechanism for voltage sensor function, pore opening, and activation-gate closure based on high-resolution structures of a single sodium channel protein.
Validation ReportPDB-ID: 6p6w

SummaryFull reportAbout validation report
History
DepositionJun 4, 2019-
Header (metadata) releaseJun 26, 2019-
Map releaseAug 14, 2019-
UpdateAug 21, 2019-
Current statusAug 21, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6p6w
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20265.map.gz / Format: CCP4 / Size: 107.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 304 pix.
= 321.024 Å
1.06 Å/pix.
x 304 pix.
= 321.024 Å
1.06 Å/pix.
x 304 pix.
= 321.024 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.056 Å
Density
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-12.836589 - 15.613651000000001
Average (Standard dev.)0.010954351 (±0.2788101)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-152-152-152
Dimensions304304304
Spacing304304304
CellA=B=C: 321.024 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0561.0561.056
M x/y/z304304304
origin x/y/z0.0000.0000.000
length x/y/z321.024321.024321.024
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-152-152-152
NC/NR/NS304304304
D min/max/mean-12.83715.6140.011

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Supplemental data

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Sample components

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Entire Fusion of maltose-binding protein and voltage-gated sodium channe...

EntireName: Fusion of maltose-binding protein and voltage-gated sodium channel NavAb in the resting state
Number of components: 2

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Component #1: protein, Fusion of maltose-binding protein and voltage-gated sodi...

ProteinName: Fusion of maltose-binding protein and voltage-gated sodium channel NavAb in the resting state
Recombinant expression: No
MassExperimental: 300 kDa
SourceSpecies: Arcobacter butzleri RM4018 (bacteria)
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Component #2: protein, Fusion of Maltose-binding protein and voltage-gated sodi...

ProteinName: Fusion of Maltose-binding protein and voltage-gated sodium channel NavAb
Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 74.072797 kDa
SourceSpecies: Arcobacter butzleri (strain RM4018) (bacteria) / Strain: RM4018
Source (engineered)Expression System: Trichoplusia ni (cabbage looper)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 8 mg/mL / pH: 8
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 40 %

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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: 60 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 500.0 - 2500.0 nm / Energy filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: (90.8 - 103.5 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 5000

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C4 (4 fold cyclic) / Number of projections: 333899
Details: Movie frames were aligned and 2x binned to a pixel size off 1.056 A using MotionCor2.
3D reconstructionSoftware: cisTEM / Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Input PDB model: 3RVY
Chain ID: A
Output model

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