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- PDB-6p6w: Cryo-EM structure of voltage-gated sodium channel NavAb N49K/L109... -

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Basic information

Entry
Database: PDB / ID: 6p6w
TitleCryo-EM structure of voltage-gated sodium channel NavAb N49K/L109A/M116V/G94C/Q150C disulfide crosslinked mutant in the resting state
ComponentsFusion of Maltose-binding protein and voltage-gated sodium channel NavAb
Keywordsmembrane 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 speciesEscherichia coli K-12 (bacteria)
Arcobacter butzleri (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsWisedchaisri, G. / Tonggu, L. / McCord, E. / Gamal El-Din, T.M. / Wang, L. / Zheng, N. / Catterall, W.A.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and StrokeR01 NS015751 United States
National Institutes of Health/National Heart, Lung, and Blood InstituteR01 HL112808 United States
National Institutes of Health/National Institute of General Medical SciencesT32 GM007750 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 Report
SummaryFull reportAbout validation report
History
DepositionJun 4, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 14, 2019Provider: repository / Type: Initial release
Revision 1.1Aug 21, 2019Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Structure visualization

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Assembly

Deposited unit
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


Theoretical massNumber of molelcules
Total (without water)296,2914
Polymers296,2914
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: cross-linking, gel filtration
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15540 Å2
ΔGint-178 kcal/mol
Surface area49960 Å2
MethodPISA

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Components

#1: Protein/peptide
Fusion of Maltose-binding protein and voltage-gated sodium channel NavAb / dihydrodipicolinate synthase


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

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Fusion of maltose-binding protein and voltage-gated sodium channel NavAb in the resting state
Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: YES
Source (natural)Organism: Arcobacter butzleri RM4018 (bacteria)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
Buffer component

Buffer-ID: 1

IDConc.Name
1100 mMSodium chloride
210 mMTris HCl
30.12 %Digitonin
410 mMMaltose
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 40 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 103.5 K / Temperature (min): 90.8 K
Image recordingAverage 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 opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 43 / Used frames/image: 1-43

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.0 betaparticle selection
2Leginon3.3image acquisition
4GctfCTF correction
7Coot0.8.9.1model fitting
9PHENIX1.14-3260model refinement
10RELION3.0 betainitial Euler assignment
11cisTEM1.0.0-betafinal Euler assignment
12RELION3.0 betaclassification
13cisTEM1.0.0-beta3D reconstruction
Image processingDetails: Movie frames were aligned and 2x binned to a pixel size off 1.056 A using MotionCor2.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 476000
Details: Auto-particle picking using RELION 3.0 beta and manual particle removals of bad particles.
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 333899 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 3RVY
Pdb chain-ID: A / Pdb chain residue range: 1001-1221

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