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- EMDB-40063: Cation channelrhodopsin from Hyphochytrium catenoides (HcCCR) emb... -

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Basic information

Entry
Database: EMDB / ID: EMD-40063
TitleCation channelrhodopsin from Hyphochytrium catenoides (HcCCR) embedded in peptidisc
Map data
Sample
  • Complex: Cation channelrhodopsin trimer reconstituted in peptidisc
    • Protein or peptide: Cation Channelrhodopsin
  • Ligand: RETINAL
  • Ligand: SODIUM IONSodium
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: water
KeywordsRetinal Protein / Channelrhodopsin / Cation channel / Peptidisc / Optogenetics / TRANSPORT PROTEIN
Biological speciesHyphochytrium catenoides (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.84 Å
AuthorsMorizumi T / Kim K / Li H / Spudich JL / Ernst OP
Funding support Canada, 3 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2018-04397 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2017-06862 Canada
Canadian Institutes of Health Research (CIHR)PJT-159464 Canada
CitationJournal: Nat Commun / Year: 2023
Title: Structures of channelrhodopsin paralogs in peptidiscs explain their contrasting K and Na selectivities.
Authors: Takefumi Morizumi / Kyumhyuk Kim / Hai Li / Elena G Govorunova / Oleg A Sineshchekov / Yumei Wang / Lei Zheng / Éva Bertalan / Ana-Nicoleta Bondar / Azam Askari / Leonid S Brown / John L ...Authors: Takefumi Morizumi / Kyumhyuk Kim / Hai Li / Elena G Govorunova / Oleg A Sineshchekov / Yumei Wang / Lei Zheng / Éva Bertalan / Ana-Nicoleta Bondar / Azam Askari / Leonid S Brown / John L Spudich / Oliver P Ernst /
Abstract: Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is a light-gated channel used for optogenetic silencing of mammalian neurons. It selects K over Na in the absence of the canonical ...Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is a light-gated channel used for optogenetic silencing of mammalian neurons. It selects K over Na in the absence of the canonical tetrameric K selectivity filter found universally in voltage- and ligand-gated channels. The genome of H. catenoides also encodes a highly homologous cation channelrhodopsin (HcCCR), a Na channel with >100-fold larger Na to K permeability ratio. Here, we use cryo-electron microscopy to determine atomic structures of these two channels embedded in peptidiscs to elucidate structural foundations of their dramatically different cation selectivity. Together with structure-guided mutagenesis, we show that K versus Na selectivity is determined at two distinct sites on the putative ion conduction pathway: in a patch of critical residues in the intracellular segment (Leu69/Phe69, Ile73/Ser73 and Asp116) and within a cluster of aromatic residues in the extracellular segment (primarily, Trp102 and Tyr222). The two filters are on the opposite sides of the photoactive site involved in channel gating.
History
DepositionMar 13, 2023-
Header (metadata) releaseJul 26, 2023-
Map releaseJul 26, 2023-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40063.png

Downloads & links

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Map

FileDownload / File: emd_40063.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-6.9391246 - 9.8733635
Average (Standard dev.)-0.00037979358 (±0.22208038)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 247.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_40063_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_40063_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cation channelrhodopsin trimer reconstituted in peptidisc

EntireName: Cation channelrhodopsin trimer reconstituted in peptidisc
Components
  • Complex: Cation channelrhodopsin trimer reconstituted in peptidisc
    • Protein or peptide: Cation Channelrhodopsin
  • Ligand: RETINAL
  • Ligand: SODIUM IONSodium
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: water

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Supramolecule #1: Cation channelrhodopsin trimer reconstituted in peptidisc

SupramoleculeName: Cation channelrhodopsin trimer reconstituted in peptidisc
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Hyphochytrium catenoides (eukaryote)
Molecular weightTheoretical: 30.197 KDa

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Macromolecule #1: Cation Channelrhodopsin

MacromoleculeName: Cation Channelrhodopsin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Hyphochytrium catenoides (eukaryote)
Molecular weightTheoretical: 30.224213 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MPFCGGRPED GWHHGSIHDM DYPLLGAMAA ICSVFIGGSG AWMLYRLDLG LGYSCKPHHS GYAPEANSFS ALSCLVSGTI YAAKTFDFF DGGGTPFSFN WYWYLDYVFT CPLILLDVLY TLEIPHKLRF VFAVIITLWC GVAAFVTPSA FRFGYYAVGC V WFVPFSFS ...String:
MPFCGGRPED GWHHGSIHDM DYPLLGAMAA ICSVFIGGSG AWMLYRLDLG LGYSCKPHHS GYAPEANSFS ALSCLVSGTI YAAKTFDFF DGGGTPFSFN WYWYLDYVFT CPLILLDVLY TLEIPHKLRF VFAVIITLWC GVAAFVTPSA FRFGYYAVGC V WFVPFSFS LLRHVKQRYQ VYPPKCQKLL FWACTIFFGF WPLFPILFLF SWLGTGHIDQ QAFTIIHAFL DLFCKTVFGL IM TFFRLEL EEHTEVLGLP LNEPKGKH

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Macromolecule #2: RETINAL

MacromoleculeName: RETINAL / type: ligand / ID: 2 / Number of copies: 1 / Formula: RET
Molecular weightTheoretical: 284.436 Da
Chemical component information

ChemComp-RET:
RETINAL / Retinal

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Macromolecule #3: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 3 / Number of copies: 3
Molecular weightTheoretical: 22.99 Da

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Macromolecule #4: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 4 / Number of copies: 7 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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Macromolecule #5: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 5 / Number of copies: 2 / Formula: PEE
Molecular weightTheoretical: 744.034 Da
Chemical component information

ChemComp-PEE:
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE, phospholipid*YM / Discrete optimized protein energy

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 15 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration0.35 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloridesodium chloride
20.0 mMC8H18N2O4SHEPES
GridModel: Homemade / Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 30 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was kept in the dark prior to freezing.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: OTHER / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 5902 / Average electron dose: 40.0 e/Å2 / Details: Falcon 4i detector
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2674318
Details: Blob picking was performed on a subset of micrographs for generating templates for template picking.
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1)
Final 3D classificationNumber classes: 5 / Avg.num./class: 99249 / Software - Name: cryoSPARC (ver. 4.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.84 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1) / Number images used: 298957
DetailsFalcon 4i detector was used for collection. Images were reference corrected.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsInitial fitting done in Phenix
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 151.1 / Target criteria: Cross correlation coefficent
Output model

PDB-8gi9:
Cation channelrhodopsin from Hyphochytrium catenoides (HcCCR) embedded in peptidisc

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