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- PDB-9cdd: Kalium channelrhodopsin 1 C110A mutant from Hyphochytrium catenoi... -

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Basic information

Entry
Database: PDB / ID: 9cdd
TitleKalium channelrhodopsin 1 C110A mutant from Hyphochytrium catenoides, Laser-Flash-Illuminated
ComponentsKalium Channelrhodopsin 1
KeywordsMEMBRANE PROTEIN / Retinal protein / Channelrhodopsin / Cation channel / Peptidisc / Optogenetics / Transport Protein
Function / homologyCHOLESTEROL / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / RETINAL
Function and homology information
Biological speciesHyphochytrium catenoides (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsMorizumi, T. / Kim, K. / Ernst, O.P.
Funding support United States, Canada, 4items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Kalium Channelrhodopsin 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,9049
Polymers30,4841
Non-polymers4,4218
Water48627
1
A: Kalium Channelrhodopsin 1
hetero molecules

A: Kalium Channelrhodopsin 1
hetero molecules

A: Kalium Channelrhodopsin 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,71327
Polymers91,4513
Non-polymers13,26224
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation2

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Components

#1: Protein Kalium Channelrhodopsin 1


Mass: 30483.678 Da / Num. of mol.: 1 / Mutation: C110A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hyphochytrium catenoides (eukaryote) / Production host: Komagataella pastoris (fungus)
#2: Chemical ChemComp-RET / RETINAL


Mass: 284.436 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H28O / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C27H46O
#4: Chemical
ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE


Mass: 744.034 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 27 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Kalium Channelrhodopin 1 C110A Reconstituted in Peptidisc, Laser-Flash-Illuminated
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 91.385 MDa / Experimental value: NO
Source (natural)Organism: Hyphochytrium catenoides (eukaryote)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2150 mMSodium ChlorideNaCl1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was monodisperse
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 400 divisions/in. / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 7.5 sec. / Electron dose: 40 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7683

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4particle selection
2EPUimage acquisition
4cryoSPARC4.4CTF correction
7PHENIX1.2model fitting
9cryoSPARC4.4initial Euler assignment
10cryoSPARC4.4final Euler assignment
11cryoSPARC4.4classification
12cryoSPARC4.43D reconstruction
13Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1589866
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 230267 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 109.7 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Cross-correlation
Atomic model buildingPDB-ID: 8GI8

8gi8


Accession code: 8GI8 / Source name: PDB / Type: experimental model

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