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- PDB-8iu0: Cryo-EM structure of the potassium-selective channelrhodopsin HcK... -

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Basic information

Entry
Database: PDB / ID: 8iu0
TitleCryo-EM structure of the potassium-selective channelrhodopsin HcKCR1 H225F mutant in lipid nanodisc
ComponentsHcKCR1
KeywordsMEMBRANE PROTEIN / Cryo-EM
Function / homologyPALMITIC ACID / Chem-PSC / RETINAL
Function and homology information
Biological speciesHyphochytrium catenoides (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsTajima, S. / Kim, Y. / Nakamura, S. / Yamashita, K. / Fukuda, M. / Deisseroth, K. / Kato, H.E.
Funding support Japan, 7items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP21wm0525018 Japan
Japan Society for the Promotion of Science (JSPS)22H04742 Japan
Japan Society for the Promotion of Science (JSPS)JP20K21383 Japan
Japan Society for the Promotion of Science (JSPS)JP21H01875 Japan
Japan Society for the Promotion of Science (JSPS)21H05142 Japan
Japan Society for the Promotion of Science (JSPS)22H00400 Japan
Japan Society for the Promotion of Science (JSPS)22K19265 Japan
CitationJournal: Cell / Year: 2023
Title: Structural basis for ion selectivity in potassium-selective channelrhodopsins.
Authors: Seiya Tajima / Yoon Seok Kim / Masahiro Fukuda / YoungJu Jo / Peter Y Wang / Joseph M Paggi / Masatoshi Inoue / Eamon F X Byrne / Koichiro E Kishi / Seiwa Nakamura / Charu Ramakrishnan / ...Authors: Seiya Tajima / Yoon Seok Kim / Masahiro Fukuda / YoungJu Jo / Peter Y Wang / Joseph M Paggi / Masatoshi Inoue / Eamon F X Byrne / Koichiro E Kishi / Seiwa Nakamura / Charu Ramakrishnan / Shunki Takaramoto / Takashi Nagata / Masae Konno / Masahiro Sugiura / Kota Katayama / Toshiki E Matsui / Keitaro Yamashita / Suhyang Kim / Hisako Ikeda / Jaeah Kim / Hideki Kandori / Ron O Dror / Keiichi Inoue / Karl Deisseroth / Hideaki E Kato /
Abstract: KCR channelrhodopsins (K-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K ...KCR channelrhodopsins (K-selective light-gated ion channels) have received attention as potential inhibitory optogenetic tools but more broadly pose a fundamental mystery regarding how their K selectivity is achieved. Here, we present 2.5-2.7 Å cryo-electron microscopy structures of HcKCR1 and HcKCR2 and of a structure-guided mutant with enhanced K selectivity. Structural, electrophysiological, computational, spectroscopic, and biochemical analyses reveal a distinctive mechanism for K selectivity; rather than forming the symmetrical filter of canonical K channels achieving both selectivity and dehydration, instead, three extracellular-vestibule residues within each monomer form a flexible asymmetric selectivity gate, while a distinct dehydration pathway extends intracellularly. Structural comparisons reveal a retinal-binding pocket that induces retinal rotation (accounting for HcKCR1/HcKCR2 spectral differences), and design of corresponding KCR variants with increased K selectivity (KALI-1/KALI-2) provides key advantages for optogenetic inhibition in vitro and in vivo. Thus, discovery of a mechanism for ion-channel K selectivity also provides a framework for next-generation optogenetics.
History
DepositionMar 23, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 6, 2023Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: HcKCR1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,31016
Polymers31,4091
Non-polymers8,90115
Water46826
1
A: HcKCR1
hetero molecules

A: HcKCR1
hetero molecules

A: HcKCR1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)120,92948
Polymers94,2263
Non-polymers26,70245
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation2
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C3 (3 fold cyclic))
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(-0.5, -0.866025), (0.866025, -0.5), (1)166.92308, 44.72691
3generate(-0.5, 0.866025), (-0.866025, -0.5), (1)44.72691, 166.92308

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Components

#1: Protein HcKCR1


Mass: 31408.795 Da / Num. of mol.: 1 / Mutation: H225F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hyphochytrium catenoides (eukaryote) / Production host: Spodoptera frugiperda (fall armyworm)
#2: Chemical ChemComp-RET / RETINAL / Retinal


Mass: 284.436 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H28O / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-PSC / (7R,17E,20E)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSA-17,20-DIEN-1-AMINIUM 4-OXIDE / PHOSPHATIDYLCHOLINE / 2-LINOLEOYL-1-PALMITOYL-SN-GYCEROL-3-PHOSPHOCHOLINE / Phosphatidylcholine


Mass: 759.068 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C42H81NO8P / Comment: phospholipid*YM
#4: Chemical
ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C16H32O2
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 26 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: HcKCR1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Hyphochytrium catenoides (eukaryote)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
EM embeddingDetails: nanodisc composed of MSP1D1E3 and soybean lipids / Material: Lipid
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0403 / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 180294 / Symmetry type: POINT
RefinementResolution: 2.66→2.66 Å / Cor.coef. Fo:Fc: 0.806 / SU B: 5.969 / SU ML: 0.115 / ESU R: 0.154
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.35627 --
obs0.35627 87708 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 60.218 Å2
Refinement stepCycle: 1 / Total: 2354
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.0122402
ELECTRON MICROSCOPYr_bond_other_d00.0162317
ELECTRON MICROSCOPYr_angle_refined_deg1.441.6553212
ELECTRON MICROSCOPYr_angle_other_deg0.4661.5475336
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.4135260
ELECTRON MICROSCOPYr_dihedral_angle_2_deg6.531518
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.99210320
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0740.2315
ELECTRON MICROSCOPYr_gen_planes_refined0.0070.022609
ELECTRON MICROSCOPYr_gen_planes_other0.0010.02603
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it6.8666.3771031
ELECTRON MICROSCOPYr_mcbond_other6.8526.3761031
ELECTRON MICROSCOPYr_mcangle_it9.83711.4791291
ELECTRON MICROSCOPYr_mcangle_other9.83911.4921292
ELECTRON MICROSCOPYr_scbond_it8.8047.8871371
ELECTRON MICROSCOPYr_scbond_other8.8017.8911372
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other13.55813.9221921
ELECTRON MICROSCOPYr_long_range_B_refined20.53382.2510219
ELECTRON MICROSCOPYr_long_range_B_other20.54182.2610207
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.5→2.565 Å / Total num. of bins used: 20 /
Num. reflection% reflection
Rfree0 -
Rwork6521 -
obs-100 %

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