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- PDB-2xky: Single particle analysis of Kir2.1NC_4 in negative stain -

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Basic information

Entry
Database: PDB / ID: 2xky
TitleSingle particle analysis of Kir2.1NC_4 in negative stain
DescriptorINWARD RECTIFIER POTASSIUM CHANNEL 2
KeywordsMETAL TRANSPORT / ION CHANNEL / MEMBRANE PROTEIN
Specimen sourceMus musculus / mammal / MOUSE / ハツカネズミ, はつかねずみ /
MethodElectron microscopy + Solution scattering (17.2 A resolution / Single particle / Negative stain)
AuthorsFomina, S. / Howard, T.D. / Sleator, O.K. / Golovanova, M. / O'Ryan, L. / Leyland, M.L. / Grossmann, J.G. / Collins, R.F. / Prince, S.M.
CitationBiochim. Biophys. Acta, 2011, 1808, 2374-2389

primary. Biochim. Biophys. Acta, 2011, 1808, 2374-2389 StrPapers
Self-directed assembly and clustering of the cytoplasmic domains of inwardly rectifying Kir2.1 potassium channels on association with PSD-95.
Svetlana Fomina / Tina D Howard / Olivia K Sleator / Marina Golovanova / Liam O'Ryan / Mark L Leyland / J Günter Grossmann / Richard F Collins / Stephen M Prince

#1. Nat.Neurosci., 2005, 8, 279- StrPapers
Cytoplasmic Domain Structures of Kir2.1 And Kir3.1 Show Sites for Modulating Gating and Rectification.
Pegan, S. / Arrabit, C. / Zhou, W. / Kwiatkowski, W. / Collins, A. / Slesinger, P.A. / Choe, S.

DateDeposition: Jul 15, 2010 / Release: Jul 20, 2011 / Last modification: Aug 10, 2011

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Assembly

Deposited unit
I: INWARD RECTIFIER POTASSIUM CHANNEL 2
J: INWARD RECTIFIER POTASSIUM CHANNEL 2
K: INWARD RECTIFIER POTASSIUM CHANNEL 2
L: INWARD RECTIFIER POTASSIUM CHANNEL 2


Theoretical massNumber of molelcules
Total (without water)139,9224
Polyers139,9224
Non-polymers00
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Polypeptide(L)
INWARD RECTIFIER POTASSIUM CHANNEL 2 / POTASSIUM CHANNEL / INWARDLY RECTIFYING SUBFAMILY J MEMBER 2 / INWARD RECTIFIER K(+) CHANNEL KIR2.1 / IRK-1


Mass: 34980.535 Da / Num. of mol.: 4 / Details: HOMOTETRAMER OF FUSED N, C TERMINI / Fragment: KIR2.1 CYTOPLASMIC DOMAIN, RESIDUES 1-67,189-428
Source: (gene. exp.) Mus musculus / mammal / ハツカネズミ, はつかねずみ /
References: UniProt: P35561

Cellular component

Molecular function

  • identical protein binding (GO: 0042802)
  • inward rectifier potassium channel activity (GO: 0005242)
  • phosphatidylinositol-4,5-bisphosphate binding (GO: 0005546)
  • voltage-gated potassium channel activity involved in cardiac muscle cell action potential repolarization (GO: 0086008)

Biological process

  • cardiac muscle cell action potential (GO: 0086001)
  • cardiac muscle cell action potential involved in contraction (GO: 0086002)
  • cellular response to mechanical stimulus (GO: 0071260)
  • magnesium ion transport (GO: 0015693)
  • membrane repolarization during action potential (GO: 0086011)
  • membrane repolarization during cardiac muscle cell action potential (GO: 0086013)
  • positive regulation of potassium ion transmembrane transport (GO: 1901381)
  • potassium ion import (GO: 0010107)
  • potassium ion transmembrane transport (GO: 0071805)
  • potassium ion transport (GO: 0006813)
  • protein homotetramerization (GO: 0051289)
  • regulation of cardiac muscle cell contraction (GO: 0086004)
  • regulation of heart rate by cardiac conduction (GO: 0086091)
  • regulation of membrane repolarization (GO: 0060306)
  • regulation of skeletal muscle contraction via regulation of action potential (GO: 0014861)
  • relaxation of cardiac muscle (GO: 0055119)
  • relaxation of skeletal muscle (GO: 0090076)

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Experimental details

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Experiment

Experiment
Method
ELECTRON MICROSCOPY
SOLUTION SCATTERING
EM experimentReconstruction method: SINGLE PARTICLE / Specimen type: NEGATIVE STAIN

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Sample preparation

Assembly of specimenName: MOUSE KIR2.1, CYTOPLASMIC DOMAIN / Aggregation state: PARTICLE
Buffer solutionName: 20MM TRIS/HCL, 150MM NACL, 1MM REDUCED GSH, 1MM EDTA, 50MM L-GLUTAMIC ACID, 50MM L-ARGININE
Sample preparationpH: 7.5 / Sample conc.: 1 mg/ml
Specimen supportDetails: CARBON

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Data collection

MicroscopyMicroscope model: OTHER / Details: LOW DOSE
Electron gunElectron source: TUNGSTEN HAIRPIN / Accelerating voltage: 100 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1250 nm / Nominal defocus min: 600 nm / Cs: 2 mm
Specimen holderTilt angle max: 0.1 deg. / Tilt angle min: 0 deg.
CameraType: GATAN ORIUS CCD CAMERA
EM image scansNumber digital images: 22
Radiation wavelengthRelative weight: 1
Soln scatter

Buffer name: 20MM TRIS/HCL, 150MM NACL, 1MM REDUCED GSH, 1MM EDTA, 50MM L-GLUTAMIC ACID, 50MM L-ARGININE / Conc. range: 0.5-4.8 / Data reduction software list: OTOKO/GNOM / Detector type: MULTIWIRE 2-D / Mean guiner radius: 4.53 nm / Number of time frames: 60 / Protein length: 16 / Source beamline: STATION 2.1 / Source class: Y / Source type: SRS / Temperature: 277 K

TypeID
x-ray1
modelling2

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Processing

Image selectionSoftware name: EMAN / Number of particles: 49012
3D reconstructionResolution: 17.2 A / Nominal pixel size: 2.93 A/pix / Actual pixel size: 2.93 A/pix
CTF correction method: PARAMETERS DETERMINED USING SCATTERING CURVE
Details: THE COORDINATES DEPOSITED ARE FROM A COMBINED SAXS/EM STUDY. THE DOMAINS IN THE PROTEINS (HIGH RESOLUTION STRUCTURES FROM THE PDB) ARE POSITIONED RELATIVE TO ONE ANOTHER USING A SAXS CURVE, THIS COMPOSITE STRUCTURE IS THEN FITTED INTO AN EM MAP. MODEL GENERATED FROM SAXS REFINEMENT USING BUNCH. PETOUKHOV, M. V. AND SVERGUN, D. I. (2005). BIOPHYS J 89, 1237-50. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-1764. (DEPOSITION ID: 7401).
Atomic model buildingMethod: A MAP WAS GENERATED FROM THE SAXS MODEL COORDINATES AT A RESOLUTION MATCHING THE EXPERIMENTAL MAP. T HIS CALCULATED MAP WAS FITTED INTO THE EXPERIMENTAL MAP BY MAXIMIZING THE CROSS-CORRELATION WITH THE EXPERIMENTAL MAP. THE COORDINATES WERE THEN REPLACED IN THE CALCULATED MAP TO GENERATE THE FINAL ENTRY.
Ref protocol: DOCKED USING CHIMERA / Ref space: REAL
Atomic model buildingPDB-ID: 1U4F
Least-squares processHighest resolution: 17.2 A
Refine hist #LASTHighest resolution: 17.2 A
Number of atoms included #LASTProtein: 1236 / Nucleic acid: 0 / Ligand: 0 / Solvent: 0 / Total: 1236
Soln scatter modelMethod: RIGID BODY MODELLING
Conformer selection criteria: CONFORMERS WERE CONSISTENT, BEST AGREEMENT WITH EXPERIMENTAL DATA DEPOSITED (CHI=2.6).
Details: NUMBER OF TIME FRAMES USED 25(60S, 4.25M CAMERA), 35(60S, 1M CAMERA). PROTEIN CONCENTRATION 0.5 MG/ML (4.25M CAMERA), 4.8 MG/ML (1M CAMERA)
Entry fitting list: PROGRAM PRE-BUNCH, 1U4F, C_4 SYMMETRY + SEQUENCE DATA
Number of conformers calculated: 20 / Number of conformers submitted: 1 / Software author list: PETOUKHOV, M. V. & SVERGUN, D. I. / Software list: SASREF7/BUNCH8

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