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- EMDB-40044: cryo-EM structure of hSlo1 in plasma membrane vesicles -

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Basic information

Entry
Database: EMDB / ID: EMD-40044
Titlecryo-EM structure of hSlo1 in plasma membrane vesicles
Map datacryo-EM map of hSlo1 in plasma membrane vesicles, sharpened with a B-factor of 59.1
Sample
  • Complex: ALFA-hSlo1-eGFP ion channel
    • Protein or peptide: Calcium-activated potassium channel subunit alpha-1
  • Ligand: MAGNESIUM ION
  • Ligand: CALCIUM IONCalcium
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: CHOLESTEROL
  • Ligand: SODIUM IONSodium
Function / homology
Function and homology information


Acetylcholine inhibits contraction of outer hair cells / large conductance calcium-activated potassium channel activity / micturition / Ca2+ activated K+ channels / 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 / large conductance calcium-activated potassium channel activity / micturition / Ca2+ activated K+ channels / 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 / caveola / potassium ion transport / response to calcium ion / vasodilation / actin binding / postsynaptic membrane / response to hypoxia / positive regulation of apoptotic process / apical plasma membrane / membrane / identical protein binding / 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 / 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: 2.7 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-
UpdateMay 10, 2023-
Current statusMay 10, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40044.png

Downloads & links

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Map

FileDownload / File: emd_40044.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM map of hSlo1 in plasma membrane vesicles, sharpened with a B-factor of 59.1
Voxel sizeX=Y=Z: 0.743 Å
Density
Contour LevelBy AUTHOR: 0.11
Minimum - Maximum-0.508687 - 0.9203312
Average (Standard dev.)0.0013251947 (±0.030779773)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 309.08798 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: unsharpened cryo-EM map of hSlo1 in plasma membrane vesicles

Fileemd_40044_additional_1.map
Annotationunsharpened cryo-EM map of hSlo1 in plasma membrane vesicles
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 plasma membrane vesicles

Fileemd_40044_half_map_1.map
Annotationhalf map 1 of hSlo1 in plasma membrane vesicles
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 plasma membrane vesicles

Fileemd_40044_half_map_2.map
Annotationhalf map 2 of hSlo1 in plasma membrane vesicles
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-eGFP ion channel

EntireName: ALFA-hSlo1-eGFP ion channel
Components
  • Complex: ALFA-hSlo1-eGFP ion channel
    • Protein or peptide: Calcium-activated potassium channel subunit alpha-1
  • Ligand: MAGNESIUM ION
  • Ligand: CALCIUM IONCalcium
  • Ligand: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
  • Ligand: CHOLESTEROL
  • Ligand: SODIUM IONSodium

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Supramolecule #1: ALFA-hSlo1-eGFP ion channel

SupramoleculeName: ALFA-hSlo1-eGFP ion channel / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
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: 122.440102 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(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 8 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(tri...

MacromoleculeName: (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate
type: ligand / ID: 4 / Number of copies: 88 / Formula: POV
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-POV:
(2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / phospholipid*YM / POPC

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Macromolecule #5: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 5 / Number of copies: 24 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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Macromolecule #6: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 6 / Number of copies: 4
Molecular weightTheoretical: 22.99 Da

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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
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 22 sec.
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Specialist opticsEnergy filter - Slit width: 6 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: ab-initio model by cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 32063
FSC plot (resolution estimation)

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