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- EMDB-21019: Cryo-EM Structure of the Hyperpolarization-Activated Potassium Ch... -

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Basic information

Entry
Database: EMDB / ID: EMD-21019
TitleCryo-EM Structure of the Hyperpolarization-Activated Potassium Channel KAT1: Octamer
Map dataStructure of the Hyperpolarization-Activated Potassium Channel KAT1
Sample
  • Complex: Hyperpolarization-Activated Potassium Channel KAT1
    • Protein or peptide: Potassium channel KAT1
  • Ligand: (2S)-1-(nonanoyloxy)-3-(phosphonooxy)propan-2-yl tetradecanoate
  • Ligand: (3beta,5beta,14beta,17alpha)-cholestan-3-ol
Keywordsmembrane protein / voltage-gated ion channel / potassium channel / TRANSPORT PROTEIN
Function / homology
Function and homology information


inward rectifier potassium channel activity / monoatomic ion channel complex / identical protein binding / plasma membrane
Similarity search - Function
Potassium channel KAT/AKT / KHA domain / KHA, dimerisation domain of potassium ion channel / KHA domain profile. / Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily ...Potassium channel KAT/AKT / KHA domain / KHA, dimerisation domain of potassium ion channel / KHA domain profile. / Potassium channel, voltage-dependent, EAG/ELK/ERG / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / RmlC-like jelly roll fold / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
Potassium channel KAT1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsClark MD / Contreras GF
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM088406 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)F30MH116647 United States
CitationJournal: Nature / Year: 2020
Title: Electromechanical coupling in the hyperpolarization-activated K channel KAT1.
Authors: Michael David Clark / Gustavo F Contreras / Rong Shen / Eduardo Perozo /
Abstract: Voltage-gated potassium (K) channels coordinate electrical signalling and control cell volume by gating in response to membrane depolarization or hyperpolarization. However, although voltage-sensing ...Voltage-gated potassium (K) channels coordinate electrical signalling and control cell volume by gating in response to membrane depolarization or hyperpolarization. However, although voltage-sensing domains transduce transmembrane electric field changes by a common mechanism involving the outward or inward translocation of gating charges, the general determinants of channel gating polarity remain poorly understood. Here we suggest a molecular mechanism for electromechanical coupling and gating polarity in non-domain-swapped K channels on the basis of the cryo-electron microscopy structure of KAT1, the hyperpolarization-activated K channel from Arabidopsis thaliana. KAT1 displays a depolarized voltage sensor, which interacts with a closed pore domain directly via two interfaces and indirectly via an intercalated phospholipid. Functional evaluation of KAT1 structure-guided mutants at the sensor-pore interfaces suggests a mechanism in which direct interaction between the sensor and the C-linker hairpin in the adjacent pore subunit is the primary determinant of gating polarity. We suggest that an inward motion of the S4 sensor helix of approximately 5-7 Å can underlie a direct-coupling mechanism, driving a conformational reorientation of the C-linker and ultimately opening the activation gate formed by the S6 intracellular bundle. This direct-coupling mechanism contrasts with allosteric mechanisms proposed for hyperpolarization-activated cyclic nucleotide-gated channels, and may represent an unexpected link between depolarization- and hyperpolarization-activated channels.
History
DepositionNov 21, 2019-
Header (metadata) releaseDec 18, 2019-
Map releaseJun 3, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0366
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0366
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6v1y
  • Surface level: 0.0366
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21019.png

Downloads & links

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Map

FileDownload / File: emd_21019.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the Hyperpolarization-Activated Potassium Channel KAT1
Voxel sizeX=Y=Z: 1.064 Å
Density
Contour LevelBy AUTHOR: 0.0366 / Movie #1: 0.0366
Minimum - Maximum-0.08383638 - 0.17692013
Average (Standard dev.)0.00087307056 (±0.005323131)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 361.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0641.0641.064
M x/y/z340340340
origin x/y/z0.0000.0000.000
length x/y/z361.760361.760361.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS340340340
D min/max/mean-0.0840.1770.001

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Supplemental data

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Sample components

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Entire : Hyperpolarization-Activated Potassium Channel KAT1

EntireName: Hyperpolarization-Activated Potassium Channel KAT1
Components
  • Complex: Hyperpolarization-Activated Potassium Channel KAT1
    • Protein or peptide: Potassium channel KAT1
  • Ligand: (2S)-1-(nonanoyloxy)-3-(phosphonooxy)propan-2-yl tetradecanoate
  • Ligand: (3beta,5beta,14beta,17alpha)-cholestan-3-ol

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Supramolecule #1: Hyperpolarization-Activated Potassium Channel KAT1

SupramoleculeName: Hyperpolarization-Activated Potassium Channel KAT1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: Potassium channel KAT1

MacromoleculeName: Potassium channel KAT1 / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 59.639594 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSISWTRNFF ERFCVEEYNI DTIKQSSFLS ADLLPSLGAR INQSTKLRKH IISPFNPRYR AWEMWLVLLV IYSAWICPFQ FAFITYKKD AIFIIDNIVN GFFAIDIILT FFVAYLDSHS YLLVDSPKKI AIRYLSTWFA FDVCSTAPFQ PLSLLFNYNG S ELGFRILS ...String:
MSISWTRNFF ERFCVEEYNI DTIKQSSFLS ADLLPSLGAR INQSTKLRKH IISPFNPRYR AWEMWLVLLV IYSAWICPFQ FAFITYKKD AIFIIDNIVN GFFAIDIILT FFVAYLDSHS YLLVDSPKKI AIRYLSTWFA FDVCSTAPFQ PLSLLFNYNG S ELGFRILS MLRLWRLRRV SSLFARLEKD IRFNYFWIRC TKLISVTLFA IHCAGCFNYL IADRYPNPRK TWIGAVYPNF KE ASLWNRY VTALYWSITT LTTTGYGDFH AENPREMLFD IFFMMFNLGL TAYLIGNMTN LVVHWTSRTR TFRDSVRAAS EFA SRNQLP HDIQDQMLSH ICLKFKTEGL KQQETLNNLP KAIRSSIANY LFFPIVHNIY LFQGVSRNFL FQLVSDIDAE YFPP KEDII LQNEAPTDLY ILVSGAVDFT VYVDGHDQFQ GKAVIGETFG EVGVLYYRPQ PFTVRTTELS QILRISRTSL MSAMH AHAD DGRVIMNNLF MKLRGQQSSR GSLEVLFQ

UniProtKB: Potassium channel KAT1

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Macromolecule #2: (2S)-1-(nonanoyloxy)-3-(phosphonooxy)propan-2-yl tetradecanoate

MacromoleculeName: (2S)-1-(nonanoyloxy)-3-(phosphonooxy)propan-2-yl tetradecanoate
type: ligand / ID: 2 / Number of copies: 8 / Formula: QNP
Molecular weightTheoretical: 522.652 Da
Chemical component information

ChemComp-QNP:
(2S)-1-(nonanoyloxy)-3-(phosphonooxy)propan-2-yl tetradecanoate

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Macromolecule #3: (3beta,5beta,14beta,17alpha)-cholestan-3-ol

MacromoleculeName: (3beta,5beta,14beta,17alpha)-cholestan-3-ol / type: ligand / ID: 3 / Number of copies: 8 / Formula: QNJ
Molecular weightTheoretical: 388.669 Da
Chemical component information

ChemComp-QNJ:
(3beta,5beta,14beta,17alpha)-cholestan-3-ol / Coprostanol

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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

Concentration5 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
50.0 mMHEPES
200.0 mMpotassium chlorideKCl
2.0 mMcalcium chlorideCaCl2
0.05 %(w/v)digitonin
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 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 / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy 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: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 1502 / Average exposure time: 12.0 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Initial model generated within Relion
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2)
Final 3D classificationSoftware - Name: RELION (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2)
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2) / Number images used: 91689
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-6v1y:
Cryo-EM Structure of the Hyperpolarization-Activated Potassium Channel KAT1: Octamer

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