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- EMDB-45467: Kalium channelrhodopsin 1 C110A mutant from Hyphochytrium catenoi... -

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Basic information

Entry
Database: EMDB / ID: EMD-45467
TitleKalium channelrhodopsin 1 C110A mutant from Hyphochytrium catenoides, Dark State
Map dataKalium channelrhodopsin 1 C110A mutant from Hyphochytrium catenoides, Dark State
Sample
  • Complex: Kalium Channelrhodopin 1 C110A Reconstituted in Peptidisc, Dark State
    • Protein or peptide: Kalium channelrhodopsin 1 C110A
  • Ligand: RETINAL
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: water
KeywordsRetinal Protein / Channelrhodopsin / Cation Channel / Peptidisc / Optogenetics / Transport Protein / MEMBRANE PROTEIN
Biological speciesHyphochytrium catenoides (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsMorizumi T / Kim K / Ernst OP
Funding support United States, Canada, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM140838 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)RF1NS133657 United States
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2023-05764 Canada
Canadian Institutes of Health Research (CIHR)PJT-159464 Canada
CitationJournal: Nat Commun / Year: 2025
Title: Structural insights into light-gating of potassium-selective channelrhodopsin.
Authors: Takefumi Morizumi / Kyumhyuk Kim / Hai Li / Probal Nag / Tal Dogon / Oleg A Sineshchekov / Yumei Wang / Leonid S Brown / Songhwan Hwang / Han Sun / Ana-Nicoleta Bondar / Igor Schapiro / ...Authors: Takefumi Morizumi / Kyumhyuk Kim / Hai Li / Probal Nag / Tal Dogon / Oleg A Sineshchekov / Yumei Wang / Leonid S Brown / Songhwan Hwang / Han Sun / Ana-Nicoleta Bondar / Igor Schapiro / Elena G Govorunova / John L Spudich / Oliver P Ernst /
Abstract: Structural information on channelrhodopsins' mechanism of light-gated ion conductance is scarce, limiting its engineering as optogenetic tools. Here, we use single-particle cryo-electron microscopy ...Structural information on channelrhodopsins' mechanism of light-gated ion conductance is scarce, limiting its engineering as optogenetic tools. Here, we use single-particle cryo-electron microscopy of peptidisc-incorporated protein samples to determine the structures of the slow-cycling mutant C110A of kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) in the dark and upon laser flash excitation. Upon photoisomerization of the retinal chromophore, the retinylidene Schiff base NH-bond reorients from the extracellular to the cytoplasmic side. This switch triggers a series of side chain reorientations and merges intramolecular cavities into a transmembrane K conduction pathway. Molecular dynamics simulations confirm K flux through the illuminated state but not through the resting state. The overall displacement between the closed and the open structure is small, involving mainly side chain rearrangements. Asp105 and Asp116 play a key role in K conductance. Structure-guided mutagenesis and patch-clamp analysis reveal the roles of the pathway-forming residues in channel gating and selectivity.
History
DepositionJun 24, 2024-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45467.png

Downloads & links

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Map

FileDownload / File: emd_45467.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationKalium channelrhodopsin 1 C110A mutant from Hyphochytrium catenoides, Dark State
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 256 pix.
= 263.68 Å		1.03 Å/pix.
x 256 pix.
= 263.68 Å		1.03 Å/pix.
x 256 pix.
= 263.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.03 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-0.5207128 - 0.63724154
Average (Standard dev.)-0.000043323293 (±0.012326106)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 263.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map B

Fileemd_45467_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_45467_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Kalium Channelrhodopin 1 C110A Reconstituted in Peptidisc, Dark State

EntireName: Kalium Channelrhodopin 1 C110A Reconstituted in Peptidisc, Dark State
Components
  • Complex: Kalium Channelrhodopin 1 C110A Reconstituted in Peptidisc, Dark State
    • Protein or peptide: Kalium channelrhodopsin 1 C110A
  • Ligand: RETINAL
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: water

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Supramolecule #1: Kalium Channelrhodopin 1 C110A Reconstituted in Peptidisc, Dark State

SupramoleculeName: Kalium Channelrhodopin 1 C110A Reconstituted in Peptidisc, Dark State
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Hyphochytrium catenoides (eukaryote)
Molecular weightTheoretical: 91.385 MDa

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Macromolecule #1: Kalium channelrhodopsin 1 C110A

MacromoleculeName: Kalium channelrhodopsin 1 C110A / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Hyphochytrium catenoides (eukaryote)
Molecular weightTheoretical: 30.483678 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MPFYDSRPPE GWPKGSINDM DYPLLGSICA VCCVFVAGSG IWMLYRLDLG MGYSCKPYKS GRAPEVNSLS GIICLLCGTM YAAKSFDFF DGGGTPFSLN WYWYLDYVFT APLLILDFAF TLDLPHKIRY FFAVFLTLWC GVAAFVTPSA YRFAYYALGC C WFTPFALS ...String:
MPFYDSRPPE GWPKGSINDM DYPLLGSICA VCCVFVAGSG IWMLYRLDLG MGYSCKPYKS GRAPEVNSLS GIICLLCGTM YAAKSFDFF DGGGTPFSLN WYWYLDYVFT APLLILDFAF TLDLPHKIRY FFAVFLTLWC GVAAFVTPSA YRFAYYALGC C WFTPFALS LMRHVKERYL VYPPKCQRWL FWACVIFFGF WPMFPILFIF SWLGTGHISQ QAFYIIHAFL DLTCKSIFGI LM TVFRLEL EEHTEVQGLP LNEPETLS

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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

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Macromolecule #3: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 3 / Number of copies: 3 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #4: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 4 / Number of copies: 4 / Formula: PEE
Molecular weightTheoretical: 744.034 Da
Chemical component information

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

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 20 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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

Concentration0.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMNaClSodium Chloride
GridModel: Homemade / Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 35 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV
DetailsSample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Number grids imaged: 1 / Number real images: 5483 / Average exposure time: 7.5 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 75000
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

Particle selectionNumber selected: 1320264
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 86551
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4)
Final 3D classificationNumber classes: 3 / Avg.num./class: 175695 / Software - Name: cryoSPARC (ver. 4.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8gi8


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 109.6 / Target criteria: Cross-correlation
Output model

PDB-9cdc:
Kalium channelrhodopsin 1 C110A mutant from Hyphochytrium catenoides, Dark State

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