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

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Entry
Database: PDB / ID: 2xky
TitleSingle particle analysis of Kir2.1NC_4 in negative stain
ComponentsINWARD RECTIFIER POTASSIUM CHANNEL 2Inward-rectifier potassium channel
KeywordsMETAL TRANSPORT / ION CHANNEL / MEMBRANE PROTEIN
Function / homology
Function and homology information


Classical Kir channels / regulation of skeletal muscle contraction via regulation of action potential / cardiac muscle cell action potential / relaxation of skeletal muscle / Phase 4 - resting membrane potential / magnesium ion transport / voltage-gated potassium channel activity involved in cardiac muscle cell action potential repolarization / membrane repolarization during action potential / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits ...Classical Kir channels / regulation of skeletal muscle contraction via regulation of action potential / cardiac muscle cell action potential / relaxation of skeletal muscle / Phase 4 - resting membrane potential / magnesium ion transport / voltage-gated potassium channel activity involved in cardiac muscle cell action potential repolarization / membrane repolarization during action potential / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / membrane repolarization during cardiac muscle cell action potential / regulation of membrane repolarization / positive regulation of potassium ion transmembrane transport / inward rectifier potassium channel activity / cardiac muscle cell action potential involved in contraction / regulation of cardiac muscle cell contraction / regulation of monoatomic ion transmembrane transport / relaxation of cardiac muscle / potassium ion import across plasma membrane / regulation of heart rate by cardiac conduction / intercalated disc / voltage-gated potassium channel complex / potassium ion transmembrane transport / phosphatidylinositol-4,5-bisphosphate binding / T-tubule / potassium ion transport / cellular response to mechanical stimulus / postsynaptic membrane / protein homotetramerization / dendritic spine / dendrite / neuronal cell body / glutamatergic synapse / membrane / identical protein binding / plasma membrane
Similarity search - Function
Potassium channel, inwardly rectifying, Kir2.1 / Potassium channel, inwardly rectifying, Kir, N-terminal / Inward rectifier potassium channel N-terminal / Potassium channel, inwardly rectifying, transmembrane domain / Inward rectifier potassium channel transmembrane domain / Potassium channel, inwardly rectifying, Kir, cytoplasmic / Potassium channel, inwardly rectifying, Kir / Inward rectifier potassium channel, C-terminal / Inward rectifier potassium channel C-terminal domain / Immunoglobulin E-set
Similarity search - Domain/homology
Inward rectifier potassium channel 2
Similarity search - Component
Biological speciesMUS MUSCULUS (house mouse)
MethodELECTRON MICROSCOPY / SOLUTION SCATTERING / single particle reconstruction / negative staining / Resolution: 17.2 Å
Model type detailsCA ATOMS ONLY, CHAIN I, J, K, L
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.
Citation
Journal: Biochim Biophys Acta / Year: 2011
Title: Self-directed assembly and clustering of the cytoplasmic domains of inwardly rectifying Kir2.1 potassium channels on association with PSD-95.
Authors: 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 /
Abstract: The interaction of the extra-membranous domain of tetrameric inwardly rectifying Kir2.1 ion channels (Kir2.1NC(4)) with the membrane associated guanylate kinase protein PSD-95 has been studied using ...The interaction of the extra-membranous domain of tetrameric inwardly rectifying Kir2.1 ion channels (Kir2.1NC(4)) with the membrane associated guanylate kinase protein PSD-95 has been studied using Transmission Electron Microscopy in negative stain. Three types of complexes were observed in electron micrographs corresponding to a 1:1 complex, a large self-enclosed tetrad complex and extended chains of linked channel domains. Using models derived from small angle X-ray scattering experiments in which high resolution structures from X-ray crystallographic and Nuclear Magnetic Resonance studies are positioned, the envelopes from single particle analysis can be resolved as a Kir2.1NC(4):PSD-95 complex and a tetrad of this unit (Kir2.1NC(4):PSD-95)(4). The tetrad complex shows the close association of the Kir2.1 cytoplasmic domains and the influence of PSD-95 mediated self-assembly on the clustering of these channels.
#1: Journal: Nat Neurosci / Year: 2005
Title: Cytoplasmic domain structures of Kir2.1 and Kir3.1 show sites for modulating gating and rectification.
Authors: Scott Pegan / Christine Arrabit / Wei Zhou / Witek Kwiatkowski / Anthony Collins / Paul A Slesinger / Senyon Choe /
Abstract: N- and C-terminal cytoplasmic domains of inwardly rectifying K (Kir) channels control the ion-permeation pathway through diverse interactions with small molecules and protein ligands in the cytoplasm. ...N- and C-terminal cytoplasmic domains of inwardly rectifying K (Kir) channels control the ion-permeation pathway through diverse interactions with small molecules and protein ligands in the cytoplasm. Two new crystal structures of the cytoplasmic domains of Kir2.1 (Kir2.1(L)) and the G protein-sensitive Kir3.1 (Kir3.1(S)) channels in the absence of PIP(2) show the cytoplasmic ion-permeation pathways occluded by four cytoplasmic loops that form a girdle around the central pore (G-loop). Significant flexibility of the pore-facing G-loop of Kir2.1(L) and Kir3.1(S) suggests a possible role as a diffusion barrier between cytoplasmic and transmembrane pores. Consistent with this, mutations of the G-loop disrupted gating or inward rectification. Structural comparison shows a di-aspartate cluster on the distal end of the cytoplasmic pore of Kir2.1(L) that is important for modulating inward rectification. Taken together, these results suggest the cytoplasmic domains of Kir channels undergo structural changes to modulate gating and inward rectification.
History
DepositionJul 15, 2010Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 20, 2011Provider: repository / Type: Initial release
Revision 1.1Aug 10, 2011Group: Database references
Revision 1.2Apr 19, 2017Group: Other
Revision 1.3Aug 30, 2017Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id
Revision 1.4Nov 13, 2019Group: Data collection / Other / Category: cell / Item: _cell.Z_PDB

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-1764
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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)139,9214
Polymers139,9214
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrix
1given(-1), (-1), (1)
2given(-1), (1), (1)
3given(1), (-1), (1)

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Components

#1: Protein
INWARD RECTIFIER POTASSIUM CHANNEL 2 / Inward-rectifier potassium channel / POTASSIUM CHANNEL / INWARDLY RECTIFYING SUBFAMILY J MEMBER 2 / INWARD RECTIFIER K(+) CHANNEL KIR2.1 / IRK-1 / Coordinate model: Cα atoms only


Mass: 34980.273 Da / Num. of mol.: 4 / Fragment: KIR2.1 CYTOPLASMIC DOMAIN, RESIDUES 1-67,189-428
Source method: isolated from a genetically manipulated source
Details: HOMOTETRAMER OF FUSED N, C TERMINI / Source: (gene. exp.) MUS MUSCULUS (house mouse) / Plasmid: PET15B / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21 / References: UniProt: P35561

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Experimental details

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Experiment

Experiment
Method
ELECTRON MICROSCOPY
SOLUTION SCATTERING
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: MOUSE KIR2.1, CYTOPLASMIC DOMAIN / Type: COMPLEX
Buffer solutionName: 20MM TRIS/HCL, 150MM NACL, 1MM REDUCED GSH, 1MM EDTA, 50MM L-GLUTAMIC ACID, 50MM L-ARGININE
pH: 7.5
Details: 20MM TRIS/HCL, 150MM NACL, 1MM REDUCED GSH, 1MM EDTA, 50MM L-GLUTAMIC ACID, 50MM L-ARGININE
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE / Material: Uranyl Acetate
Specimen supportDetails: CARBON

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Data collection

MicroscopyModel: FEI TECNAI 10 / Details: LOW DOSE
Electron gunElectron source: TUNGSTEN HAIRPIN / Accelerating voltage: 100 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1250 nm / Nominal defocus min: 600 nm / Cs: 2 mm
Specimen holderTilt angle max: 0.1 ° / Tilt angle min: 0 °
Image recordingFilm or detector model: GENERIC GATAN
Image scansNum. digital images: 22
Radiation wavelengthRelative weight: 1
Soln scatter
TypeIDBuffer nameConc. range (mg/ml)Data reduction software listDetector typeMean guiner radius (nm)Num. of time framesProtein lengthSource beamlineSource classSource typeTemperature (K)
x-ray120MM TRIS/HCL, 150MM NACL, 1MM REDUCED GSH, 1MM EDTA, 50MM L-GLUTAMIC ACID, 50MM L-ARGININE0.5-4.8OTOKO/GNOMMULTIWIRE 2-D4.536016STATION 2.1YSRS277
modelling2

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2EMAN3D reconstruction
CTF correctionDetails: PARAMETERS DETERMINED USING SCATTERING CURVE
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 17.2 Å / Num. of particles: 49012 / Nominal pixel size: 2.93 Å / Actual pixel size: 2.93 Å
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, ...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).
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: METHOD--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- ...Details: METHOD--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. REFINEMENT PROTOCOL--DOCKED USING CHIMERA
Atomic model buildingPDB-ID: 1U4F

1u4f

RefinementHighest resolution: 17.2 Å
Refinement stepCycle: LAST / Highest resolution: 17.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1236 0 0 0 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
Num. of conformers calculated: 20 / Num. of conformers submitted: 1 / Software author list: PETOUKHOV, M. V. & SVERGUN, D. I. / Software list: SASREF7/BUNCH8

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