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- EMDB-8517: Chicken Slo2.2 in a closed conformation vitrified in the presence... -

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Basic information

Entry
Database: EMDB / ID: EMD-8517
TitleChicken Slo2.2 in a closed conformation vitrified in the presence of 300 mM NaCl
Map dataClosed Slo2.2 density map
Sample
  • Organelle or cellular component: Chicken Slo2.2 in closed conformation
    • Protein or peptide: Potassium channel subfamily T member 1
KeywordsIon channel / potassium channel / TRANSPORT PROTEIN
Function / homology
Function and homology information


intracellular sodium-activated potassium channel activity / outward rectifier potassium channel activity / potassium ion transmembrane transport / plasma membrane
Similarity search - Function
Calcium-activated potassium channel BK, alpha subunit / : / Calcium-activated BK potassium channel alpha subunit / Calcium-activated potassium channel slowpoke-like RCK domain / RCK N-terminal domain profile. / Potassium channel domain / Ion channel
Similarity search - Domain/homology
Potassium channel subfamily T member 1
Similarity search - Component
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.76 Å
AuthorsHite RK / MacKinnon R
CitationJournal: Cell / Year: 2017
Title: Structural Titration of Slo2.2, a Na-Dependent K Channel.
Authors: Richard K Hite / Roderick MacKinnon /
Abstract: The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of ...The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of structures of Slo2.2, a neuronal Na-activated K channel, as a function of the Na concentration. We find that Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Na concentration. An open conformation emerges from an ensemble of closed conformations in a highly Na-dependent manner, without evidence of Na-dependent intermediates. In other words, channel opening is a highly concerted, switch-like process. The midpoint of the structural titration matches that of the functional titration. A maximum open conformation probability approaching 1.0 and maximum functional open probability approaching 0.7 imply that, within the class of open channels, there is a subclass that is not permeable to ions.
History
DepositionDec 11, 2016-
Header (metadata) releaseFeb 8, 2017-
Map releaseFeb 8, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5u76
  • Surface level: 4
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8517.png

Downloads & links

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Map

FileDownload / File: emd_8517.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClosed Slo2.2 density map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 256 pix.
= 332.8 Å		1.3 Å/pix.
x 256 pix.
= 332.8 Å		1.3 Å/pix.
x 256 pix.
= 332.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0 / Movie #1: 4
Minimum - Maximum-6.7269893 - 14.454624000000001
Average (Standard dev.)-0.1761945 (±0.6271196)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 332.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z332.800332.800332.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-6.72714.455-0.176

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Supplemental data

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Sample components

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Entire : Chicken Slo2.2 in closed conformation

EntireName: Chicken Slo2.2 in closed conformation
Components
  • Organelle or cellular component: Chicken Slo2.2 in closed conformation
    • Protein or peptide: Potassium channel subfamily T member 1

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Supramolecule #1: Chicken Slo2.2 in closed conformation

SupramoleculeName: Chicken Slo2.2 in closed conformation / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Gallus gallus (chicken)

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Macromolecule #1: Potassium channel subfamily T member 1

MacromoleculeName: Potassium channel subfamily T member 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 137.409812 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MARAKLKNSP SESNSHVKTV PPATTEDVRG VSPLLPARRM GSLGSDVGQR PHAEDFSMDS SFSQVQVEFY VNENTFKERL KLFFIKNQR SSLRIRLFNF SLKLLTCLLY IVRVLLDNPE EGIGCWECEK QNYTLFNQST KINWSHIFWV DRKLPLWAVQ V SIALISFL ...String:
MARAKLKNSP SESNSHVKTV PPATTEDVRG VSPLLPARRM GSLGSDVGQR PHAEDFSMDS SFSQVQVEFY VNENTFKERL KLFFIKNQR SSLRIRLFNF SLKLLTCLLY IVRVLLDNPE EGIGCWECEK QNYTLFNQST KINWSHIFWV DRKLPLWAVQ V SIALISFL ETMLLIYLSY KGNIWEQIFR ISFILEMINT VPFIITIFWP PLRNLFIPVF LNCWLAKYAL ENMINDLHRA IQ RTQSAMF NQVLILICTL LCLVFTGTCG IQHLERAGEK LSLFKSFYFC IVTFSTVGYG DVTPKIWPSQ LLVVIMICVA LVV LPLQFE ELVYLWMERQ KSGGNYSRHR AQTEKHVVLC VSSLKIDLLM DFLNEFYAHP RLQDYYVVIL CPTEMDIQVR RVLQ IPLWS QRVIYLQGSA LKDQDLMRAK MDNGEACFIL SSRNEVDRTA ADHQTILRAW AVKDFAPNCP LYVQILKPEN KFHVK FADH VVCEEECKYA MLALNCVCPA TSTLITLLVH TSRGQEGQES PEQWQRMYGR CSGNEVYHIR MGDSKFFMEY EGKSFT YAA FHAHKKYGVC LIGIRREENK SILLNPGPRH IMAASDTCFY INITKEENSA FIFKQAEKQK KKGFAGRGTY DGPSRLP VH SIIASMGTVA MDLQNTECRP TNSSKLALPA ENGSGNRRPS IAPVLELADT SSLLPCDLLS DQSEDEMTQS DEEGSAVV E YVKGYPPNSP YIGSSPTLCH LLPEKAPFCC LRLDKGCKHN SFEDAKAYGF KNKLIIVSAE TAGNGLYNFI VPLRAYYRS RKELNPIVLL LDNKPEHHFL EAICCFPMVY YMEGTIDNLD SLLQCGIIYA DNLVVVDKES TMSAEEDYMA DAKTIVNVQT MFRLFPSLS IITELTHPSN MRFMQFRAKD SYSLALSKLE KKERENGSNL AFMFRLPFAA GRVFSISMLD TLLYQSFVKD Y MITITRLL LGLDTTPGSG YLCAMKITED DLWIRTYGRL FQKLCSSSAE IPIGIYRTES HMFATSEPHD IRAQSQISIN VE DCEDTKD VKEHWGIKTG HHRNSCSSDQ SEHPLLRRKS MQWARRLSRK GNKHSGKTAE WISQQRLSLY RRSERQELSE LVK NRMKHL GLPTTGYDEM NDHQNTLSYV LINPPPDTRL ELNDIVYLIR SDPLAHVAND GHSRKSSCSN KLGPCNPETR DETQ L

UniProtKB: Potassium channel subfamily T member 1

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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

Concentration6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
300.0 mMKClpotassium chloride
300.0 mMNaClsodium chloride
1.5 mMdodecyl maltoside
0.075 mg/ml1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
0.025 mg/ml1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 88 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2000 / Average exposure time: 1.2 sec. / Average electron dose: 59.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 160000 / Details: Manual particle selection in RELION
Startup modelType of model: OTHER / Details: Eman2 used for initial model generation
Final reconstructionNumber classes used: 8 / Applied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN / Number images used: 130000
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN
Final 3D classificationNumber classes: 10 / Avg.num./class: 16000 / Software - Name: RELION (ver. 2.0)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-5u76:
Chicken Slo2.2 in a closed conformation vitrified in the presence of 300 mM NaCl

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