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- PDB-5va1: Cryo-EM structure of the human ether-a-go-go related K+ channel -

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Basic information

Entry
Database: PDB / ID: 5va1
TitleCryo-EM structure of the human ether-a-go-go related K+ channel
DescriptorPotassium voltage-gated channel
subfamily H (Eag-related)
member 2
KeywordsTRANSPORT PROTEIN / K+ channel / PAS / CNBHD / voltage sensor / selectivity filter
Specimen sourceHomo sapiens / human
MethodElectron microscopy (3.7 Å resolution / Particle / Single particle)
AuthorsWang, W.W. / MacKinnon, R.
CitationCell, 2017, 169, 422-430.e10

Cell, 2017, 169, 422-430.e10 StrPapers
Cryo-EM Structure of the Open Human Ether-à-go-go-Related K(+) Channel hERG.
Weiwei Wang / Roderick MacKinnon

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Mar 24, 2017 / Release: May 3, 2017

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Assembly

Deposited unit
A: Potassium voltage-gated channel subfamily H member 2


Theoretical massNumber of molelcules
Total (without water)88,9021
Polyers88,9021
Non-polymers00
Water0
#1
A: Potassium voltage-gated channel subfamily H member 2

A: Potassium voltage-gated channel subfamily H member 2

A: Potassium voltage-gated channel subfamily H member 2

A: Potassium voltage-gated channel subfamily H member 2


Theoretical massNumber of molelcules
Total (without water)355,6074
Polyers355,6074
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation4
#2


  • idetical with deposited unit in distinct coordinate
  • point asymmetric unit
TypeNameSymmetry operationNumber
point symmetry operation1
#3


  • idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1

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Components

#1: Polypeptide(L)Potassium voltage-gated channel subfamily H member 2 / Eag homolog / Ether-a-go-go-related gene potassium channel 1 / hERG1 / Voltage-gated potassium channel subunit Kv11.1


Mass: 88901.664 Da / Num. of mol.: 1 / Fragment: UNP residues 1-140,381-870,1006-1159 / Source: (gene. exp.) Homo sapiens / human / References: UniProt: Q12809

Cellular component

Molecular function

  • C3HC4-type RING finger domain binding (GO: 0055131)
  • delayed rectifier potassium channel activity (GO: 0005251)
  • identical protein binding (GO: 0042802)
  • inward rectifier potassium channel activity (GO: 0005242)
  • phosphorelay sensor kinase activity (GO: 0000155)
  • protein homodimerization activity (GO: 0042803)
  • scaffold protein binding (GO: 0097110)
  • ubiquitin protein ligase binding (GO: 0031625)
  • voltage-gated potassium channel activity (GO: 0005249)
  • voltage-gated potassium channel activity involved in cardiac muscle cell action potential repolarization (GO: 0086008)
  • voltage-gated potassium channel activity involved in ventricular cardiac muscle cell action potential repolarization (GO: 1902282)

Biological process

  • cardiac conduction (GO: 0061337)
  • cardiac muscle contraction (GO: 0060048)
  • cellular response to drug (GO: 0035690)
  • membrane depolarization during action potential (GO: 0086010)
  • membrane repolarization during action potential (GO: 0086011)
  • membrane repolarization during cardiac muscle cell action potential (GO: 0086013)
  • membrane repolarization during ventricular cardiac muscle cell action potential (GO: 0098915)
  • negative regulation of potassium ion export (GO: 1902303)
  • negative regulation of potassium ion transmembrane transport (GO: 1901380)
  • positive regulation of potassium ion transmembrane transport (GO: 1901381)
  • potassium ion export (GO: 0071435)
  • potassium ion export across plasma membrane (GO: 0097623)
  • potassium ion homeostasis (GO: 0055075)
  • potassium ion transmembrane transport (GO: 0071805)
  • regulation of heart rate by cardiac conduction (GO: 0086091)
  • regulation of heart rate by hormone (GO: 0003064)
  • regulation of membrane potential (GO: 0042391)
  • regulation of membrane repolarization (GO: 0060306)
  • regulation of potassium ion transmembrane transport (GO: 1901379)
  • regulation of ventricular cardiac muscle cell membrane repolarization (GO: 0060307)
  • ventricular cardiac muscle cell action potential (GO: 0086005)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: human ether-a-go-go related K+ channel hERG
Details: Truncated hERG construct hERGTs (amino acid residues 141-380 and 871-1005 deleted)
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: 1 / Source: RECOMBINANT
Molecular weightExperimental flag: NO
Source (natural)Organism: Homo sapiens
Source (recombinant)Cell: HEK293S GnTI- / Organism: Homo sapiens / Plasmid: BacMam
Buffer solutionDetails: pH 7.4, adjusted with NaOH / pH: 7.4
Buffer component
IDConc.Conc. unitsNameFormulaBuffer ID
120mMHEPESC8H18N2O4S1
2300mMpotassium chlorideKCl1
310mMDithiothreitolC4H10O2S21
40.025%n-Dodecyl beta-D-maltosideC24H46O111
50.005%Cholesteryl hemisuccinateC31H50O41
60.025mg/mLPOPE:POPC:POPA 5:5:11
70.5mMtris(2-carboxyethyl)phosphineC9H15O6P1
SpecimenConc.: 6 mg/ml
Details: A 1 mL peak fraction was collected and concentrated ~3x to obtain the final ~6 mg/mL sample.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 98 % / Details: one blot: 3 second blot time, 0 blot force / Chamber temperature: 298 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 38461 / Nominal defocus max: 3500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 85 e/Å2
Details: 50 0.3-second frames were collected for each movie at a dose rate of ~1.8 e-/A2/frame
Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 3 / Number of real images: 4100
Image scansDimension width: 7420 / Dimension height: 7676 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategoryImage processing IDImaging IDFitting ID
1RELION AUTOPICK1.4PARTICLE SELECTION1
2SerialEM3.5IMAGE ACQUISITION1
3Digital Micrograph3IMAGE ACQUISITION1
5RELION1.4CTF CORRECTION1
6CTFFIND4CTF CORRECTION1
9UCSF Chimera1.10.2MODEL FITTING1
10Coot0.8.2MODEL FITTING1
12REFMAC5.8MODEL REFINEMENT1
13RELION1.4INITIAL EULER ASSIGNMENT1
14RELION1.4FINAL EULER ASSIGNMENT1
15RELION1.4CLASSIFICATION1
16RELION1.4RECONSTRUCTION1
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: ~830k particles from Autopick / Number of particles selected: 830000
SymmetryPoint symmetry: C4
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 213255 / Algorithm: FOURIER SPACE / Number of class averages: 3 / Symmetry type: POINT
Atomic model buildingOverall b value: 140 / Ref protocol: AB INITIO MODEL / Ref space: RECIPROCAL / Target criteria: Fourier Shell Correlation

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