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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 dataCryo-EM map of disulfide crosslinked MBP-NavAb N49K/L109A/M116V/G9C/Q150C in the resting state
Sample
  • Complex: Fusion of maltose-binding protein and voltage-gated sodium channel NavAb in the resting state
    • Protein or peptide: Fusion of Maltose-binding protein and voltage-gated sodium channel NavAb
Function / homology
Function and homology information


detection of maltose stimulus / maltose binding / maltose transport complex / maltose transport / maltodextrin transmembrane transport / carbohydrate transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / carbohydrate transport / monoatomic cation channel activity / ATP-binding cassette (ABC) transporter complex ...detection of maltose stimulus / maltose binding / maltose transport complex / maltose transport / maltodextrin transmembrane transport / carbohydrate transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / carbohydrate transport / monoatomic cation channel activity / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / outer membrane-bounded periplasmic space / periplasmic space / DNA damage response / membrane / identical protein binding / plasma membrane
Similarity search - Function
Voltage-gated cation channel calcium and sodium / 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
Maltodextrin-binding protein / Ion transport protein / Maltose/maltodextrin-binding periplasmic protein
Similarity search - Component
Biological speciesArcobacter butzleri RM4018 (bacteria) / Arcobacter butzleri (strain RM4018) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 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 Stroke (NIH/NINDS)R01 NS015751 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM007750 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01 HL112808 United States
Howard Hughes Medical Institute (HHMI) 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.
History
DepositionJun 4, 2019-
Header (metadata) releaseJun 26, 2019-
Map releaseAug 14, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 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
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  • 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

emd_20265.png

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)
AnnotationCryo-EM map of disulfide crosslinked MBP-NavAb N49K/L109A/M116V/G9C/Q150C in the resting state
Voxel sizeX=Y=Z: 1.056 Å
Density
Contour LevelBy AUTHOR: 3 / Movie #1: 3
Minimum - Maximum-12.836589 - 15.613651
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
Components
  • Complex: Fusion of maltose-binding protein and voltage-gated sodium channel NavAb in the resting state
    • Protein or peptide: Fusion of Maltose-binding protein and voltage-gated sodium channel NavAb

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Supramolecule #1: Fusion of maltose-binding protein and voltage-gated sodium channe...

SupramoleculeName: Fusion of maltose-binding protein and voltage-gated sodium channel NavAb in the resting state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Arcobacter butzleri RM4018 (bacteria)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
Molecular weightExperimental: 300 KDa

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Macromolecule #1: Fusion of Maltose-binding protein and voltage-gated sodium channe...

MacromoleculeName: Fusion of Maltose-binding protein and voltage-gated sodium channel NavAb
type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Arcobacter butzleri (strain RM4018) (bacteria) / Strain: RM4018
Molecular weightTheoretical: 74.072797 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MKIKTGARIL ALSALTTMMF SASALAKIEE GKLVIWINGD KGYNGLAEVG KKFEKDTGIK VTVEHPDKLE EKFPQVAATG DGPDIIFWA HDRFGGYAQS GLLAEITPDK AFQDKLYPFT WDAVRYNGKL IAYPIAVEAL SLIYNKDLLP NPPKTWEEIP A LDKELKAK ...String:
MKIKTGARIL ALSALTTMMF SASALAKIEE GKLVIWINGD KGYNGLAEVG KKFEKDTGIK VTVEHPDKLE EKFPQVAATG DGPDIIFWA HDRFGGYAQS GLLAEITPDK AFQDKLYPFT WDAVRYNGKL IAYPIAVEAL SLIYNKDLLP NPPKTWEEIP A LDKELKAK GKSALMFNLQ EPYFTWPLIA ADGGYAFKYE NGKYDIKDVG VDNAGAKAGL TFLVDLIKNK HMNADTDYSI AE AAFNKGE TAMTINGPWA WSNIDTSKVN YGVTVLPTFK GQPSKPFVGV LSAGINAASP NKELAKEFLE NYLLTDEGLE AVN KDKPLG AVALKSYEEE LAKDPRIAAT MENAQKGEIM PNIPQMSAFW YAVRTAVINA ASGRQTVDEA LKDAQTNAMY LAIT NIVES SFFTKFIIYL IVLNGITMGL ETSKTFMQSF GVYTTLFKQI VITIFTIEII LRIYVHRISF FKDPWSLFDF FVVAI SLVP TSSCFEILRV LRVLRLFRAV TAVPQVRKIV SALISVIPGM LSVIALMTLF FYIFAIMATC LFGERFPEWF GTLGES FYT LFQVMTLESW SMGIVRPLME VYPYAWVFFI PFIFVVTFVM INLVVAIIVD AMAILNQKEE QHIIDEVQSH EDNINNE II KLREEIVELK ELIKTSLKN

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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

Concentration8 mg/mL
BufferpH: 8
Component:
ConcentrationName
100.0 mMSodium chloride
10.0 mMTris HCl
0.12 %Digitonin
10.0 mMMaltose
GridModel: C-flat-1.2/1.3 4C / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.04 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 40 % / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 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 opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 90.8 K / Max: 103.5 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-43 / Number grids imaged: 9 / Number real images: 5000 / 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: 476000
Details: Auto-particle picking using RELION 3.0 beta and manual particle removals of bad particles.
CTF correctionSoftware - Name: Gctf
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3rvy


Details: Pore module only
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.0 beta)
Final 3D classificationNumber classes: 100 / Avg.num./class: 4760 / Software - Name: RELION (ver. 3.0 beta)
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) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0-beta) / Number images used: 333899
DetailsMovie frames were aligned and 2x binned to a pixel size off 1.056 A using MotionCor2.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3rvy


Chain - Chain ID: A / Chain - Residue range: 1001-1221
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6p6w:
Cryo-EM structure of voltage-gated sodium channel NavAb N49K/L109A/M116V/G94C/Q150C disulfide crosslinked mutant in the resting state

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