|Entry||Database: PDB / ID: 6p6w|
|Title||Cryo-EM structure of voltage-gated sodium channel NavAb N49K/L109A/M116V/G94C/Q150C disulfide crosslinked mutant in the resting state|
|Components||Fusion of Maltose-binding protein and voltage-gated sodium channel NavAb|
|Keywords||membrane protein / metal transport / Ion channel / ion transport protein|
|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 species||Escherichia coli K-12 (bacteria)|
Arcobacter butzleri (bacteria)
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å|
|Authors||Wisedchaisri, G. / Tonggu, L. / McCord, E. / Gamal El-Din, T.M. / Wang, L. / Zheng, N. / Catterall, W.A.|
|Funding support|| United States, 4items |
|Citation||Journal: 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.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
Downloads & links
A: Fusion of Maltose-binding protein and voltage-gated sodium channel NavAb
B: Fusion of Maltose-binding protein and voltage-gated sodium channel NavAb
C: Fusion of Maltose-binding protein and voltage-gated sodium channel NavAb
D: Fusion of Maltose-binding protein and voltage-gated sodium channel NavAb
Mass: 74072.797 Da / Num. of mol.: 4
Mutation: R4A, N49K, L109A, M116V, G94C, Q150C,R4A, N49K, L109A, M116V, G94C, Q150C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria), (gene. exp.) Arcobacter butzleri (strain RM4018) (bacteria)
Gene: malE, BX06_05300, Abu_1752 / Strain: RM4018 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A028ANC5, UniProt: A8EVM5
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: Fusion of maltose-binding protein and voltage-gated sodium channel NavAb in the resting state|
Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
|Molecular weight||Value: 0.3 MDa / Experimental value: YES|
|Source (natural)||Organism: Arcobacter butzleri RM4018 (bacteria)|
|Source (recombinant)||Organism: Trichoplusia ni (cabbage looper)|
|Buffer solution||pH: 8|
|Specimen||Conc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 40 % / Chamber temperature: 298 K|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µns / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 103.5 K / Temperature (min): 90.8 K|
|Image recording||Average exposure time: 8.6 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 9 / Num. of real images: 5000|
|EM imaging optics||Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV|
|Image scans||Width: 3838 / Height: 3710 / Movie frames/image: 43 / Used frames/image: 1-43|
|Software||Name: PHENIX / Version: 1.14_3260: / Classification: refinement|
|Image processing||Details: Movie frames were aligned and 2x binned to a pixel size off 1.056 A using MotionCor2.|
|CTF correction||Type: PHASE FLIPPING AND AMPLITUDE CORRECTION|
|Particle selection||Num. of particles selected: 476000 |
Details: Auto-particle picking using RELION 3.0 beta and manual particle removals of bad particles.
|Symmetry||Point symmetry: C4 (4 fold cyclic)|
|3D reconstruction||Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 333899 / Num. of class averages: 1 / Symmetry type: POINT|
|Atomic model building||Protocol: FLEXIBLE FIT / Space: REAL|
|Atomic model building||PDB-ID: 3RVY|
Pdb chain-ID: A / Pdb chain residue range: 1001-1221
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