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- EMDB-40045: Cryo-EM structure of hSlo1 in digitonin, Ca2+-free and EDTA-free -

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Basic information

Entry
Database: EMDB / ID: EMD-40045
TitleCryo-EM structure of hSlo1 in digitonin, Ca2+-free and EDTA-free
Map dataunsharpened cryo-EM map of hSlo1 in digitonin, Ca2 -free and EDTA-free
Sample
  • Complex: ALFA-hSlo1 tetrameric channel
    • Protein or peptide: Calcium-activated potassium channel subunit alpha-1
KeywordsSlo1 / BK channel / Ca2+- and voltage-activated K+ channel / ion channel / TRANSPORT PROTEIN
Function / homology
Function and homology information


Acetylcholine inhibits contraction of outer hair cells / micturition / Ca2+ activated K+ channels / large conductance calcium-activated potassium channel activity / response to carbon monoxide / calcium-activated potassium channel activity / negative regulation of cell volume / smooth muscle contraction involved in micturition / intracellular potassium ion homeostasis / Sensory processing of sound by inner hair cells of the cochlea ...Acetylcholine inhibits contraction of outer hair cells / micturition / Ca2+ activated K+ channels / large conductance calcium-activated potassium channel activity / response to carbon monoxide / calcium-activated potassium channel activity / negative regulation of cell volume / smooth muscle contraction involved in micturition / intracellular potassium ion homeostasis / Sensory processing of sound by inner hair cells of the cochlea / response to osmotic stress / cGMP effects / voltage-gated potassium channel activity / voltage-gated potassium channel complex / potassium ion transmembrane transport / regulation of membrane potential / potassium ion transport / caveola / response to calcium ion / vasodilation / actin binding / postsynaptic membrane / response to hypoxia / positive regulation of apoptotic process / apical plasma membrane / identical protein binding / membrane / metal ion binding / plasma membrane
Similarity search - Function
: / Ca2+-activated K+ channel Slowpoke, TrkA_C like domain / 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. / Ion transport domain / Ion transport protein / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Calcium-activated potassium channel subunit alpha-1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsTao X / Zhao C / MacKinnon R
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
Jane Coffin Childs (JCC) Fund United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Membrane protein isolation and structure determination in cell-derived membrane vesicles.
Authors: Xiao Tao / Chen Zhao / Roderick MacKinnon /
Abstract: Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of ...Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of proteins in membrane vesicles derived directly from cells. Structures of the ion channel Slo1 from total cell membranes and from cell plasma membranes were determined at 3.8 Å and 2.7 Å resolution, respectively. The plasma membrane environment stabilizes Slo1, revealing an alteration of global helical packing, polar lipid, and cholesterol interactions that stabilize previously unresolved regions of the channel and an additional ion binding site in the Ca regulatory domain. The two methods presented enable structural analysis of both internal and plasma membrane proteins without disrupting weakly interacting proteins, lipids, and cofactors that are essential to biological function.
History
DepositionMar 10, 2023-
Header (metadata) releaseMay 10, 2023-
Map releaseMay 10, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40045.png

Downloads & links

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Map

FileDownload / File: emd_40045.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened cryo-EM map of hSlo1 in digitonin, Ca2 -free and EDTA-free
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.28
Minimum - Maximum-0.31359535 - 0.96702486
Average (Standard dev.)0.015829392 (±0.060817406)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: cryo-EM map of hSlo1 in digitonin, Ca2 -free...

Fileemd_40045_additional_1.map
Annotationcryo-EM map of hSlo1 in digitonin, Ca2 -free and EDTA-free, sharpened with a B-factor of 120.7
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1 of hSlo1 in digitonin, Ca2 -free and EDTA-free

Fileemd_40045_half_map_1.map
Annotationhalf map 1 of hSlo1 in digitonin, Ca2 -free and EDTA-free
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2 of hSlo1 in digitonin, Ca2 -free and EDTA-free

Fileemd_40045_half_map_2.map
Annotationhalf map 2 of hSlo1 in digitonin, Ca2 -free and EDTA-free
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ALFA-hSlo1 tetrameric channel

EntireName: ALFA-hSlo1 tetrameric channel
Components
  • Complex: ALFA-hSlo1 tetrameric channel
    • Protein or peptide: Calcium-activated potassium channel subunit alpha-1

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Supramolecule #1: ALFA-hSlo1 tetrameric channel

SupramoleculeName: ALFA-hSlo1 tetrameric channel / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Calcium-activated potassium channel subunit alpha-1

MacromoleculeName: Calcium-activated potassium channel subunit alpha-1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 120.908125 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAPSRLEEEL RRRLTEPDAL IIPVTMEVPC DSRGQRMWWA FLASSMVTFF GGLFIILLWR TLKYLWTVCC HCGGKTKEAQ KINNGSSQA DGTLKPVDEK EEAVAAEVGW MTSVKDWAGV MISAQTLTGR VLVVLVFALS IGALVIYFID SSNPIESCQN F YKDFTLQI ...String:
MAPSRLEEEL RRRLTEPDAL IIPVTMEVPC DSRGQRMWWA FLASSMVTFF GGLFIILLWR TLKYLWTVCC HCGGKTKEAQ KINNGSSQA DGTLKPVDEK EEAVAAEVGW MTSVKDWAGV MISAQTLTGR VLVVLVFALS IGALVIYFID SSNPIESCQN F YKDFTLQI DMAFNVFFLL YFGLRFIAAN DKLWFWLEVN SVVDFFTVPP VFVSVYLNRS WLGLRFLRAL RLIQFSEILQ FL NILKTSN SIKLVNLLSI FISTWLTAAG FIHLVENSGD PWENFQNNQA LTYWECVYLL MVTMSTVGYG DVYAKTTLGR LFM VFFILG GLAMFASYVP EIIELIGNRK KYGGSYSAVS GRKHIVVCGH ITLESVSNFL KDFLHKDRDD VNVEIVFLHN ISPN LELEA LFKRHFTQVE FYQGSVLNPH DLARVKIESA DACLILANKY CADPDAEDAS NIMRVISIKN YHPKIRIITQ MLQYH NKAH LLNIPSWNWK EGDDAICLAE LKLGFIAQSC LAQGLSTMLA NLFSMRSFIK IEEDTWQKYY LEGVSNEMYT EYLSSA FVG LSFPTVCELC FVKLKLLMIA IEYKSANRES RILINPGNHL KIQEGTLGFF IASDAKEVKR AFFYCKACHD DITDPKR IK KCGCKRLEDE QPSTLSPKKK QRNGGMRNSP NTSPKLMRHD PLLIPGNDQI DNMDSNVKKY DSTGMFHWCA PKEIEKVI L TRSEAAMTVL SGHVVVCIFG DVSSALIGLR NLVMPLRASN FHYHELKHIV FVGSIEYLKR EWETLHNFPK VSILPGTPL SRADLRAVNI NLCDMCVILS ANQNNIDDTS LQDKECILAS LNIKSMQFDD SIGVLQANSQ GFTPPGMDRS SPDNSPVHGM LRQPSITTG VNIPIITELV NDTNVQFLDQ DDDDDPDTEL YLTQPFACGT AFAVSVLDSL MSATYFNDNI LTLIRTLVTG G ATPELEAL IAEENALRGG YSTPQTLANR DRCRVAQLAL LDGPFADLGD GGCYGDLFCK ALKTYNMLCF GIYRLRDAHL ST PSQCTKR YVITNPPYEF ELVPTDLIFC LMQFD

UniProtKB: Calcium-activated potassium channel subunit alpha-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

BufferpH: 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 18495 / Average exposure time: 2.0 sec. / Average electron dose: 51.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.001 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: ab-initio reconstruction by cryoSPARC
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 123286
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: RELION
FSC plot (resolution estimation)

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