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- PDB-8h87: Cryo-EM structure of the potassium-selective channelrhodopsin HcK... -

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Basic information

Entry
Database: PDB / ID: 8h87
TitleCryo-EM structure of the potassium-selective channelrhodopsin HcKCR2 in lipid nanodisc
ComponentsHcKCR2
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: 2.53 Å
AuthorsTajima, S. / Kim, Y. / 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
DepositionOct 21, 2022Deposition 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: HcKCR2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)41,17017
Polymers34,0241
Non-polymers7,14716
Water88349
1
A: HcKCR2
hetero molecules

A: HcKCR2
hetero molecules

A: HcKCR2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)123,51151
Polymers102,0713
Non-polymers21,44048
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)166.92311, 44.72691
3generate(-0.5, 0.866025), (-0.866025, -0.5), (1)44.72691, 166.92311

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Components

#1: Protein HcKCR2


Mass: 34023.664 Da / Num. of mol.: 1
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.: 6 / Source method: obtained synthetically / Formula: C42H81NO8P / Comment: phospholipid*YM
#4: Chemical
ChemComp-PLM / PALMITIC ACID / Palmitic acid


Mass: 256.424 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C16H32O2
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 49 / 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: HcKCR2 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
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 composing of MSP1D1E3 and soybean lipid / 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: 51 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0352 / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 682797 / Symmetry type: POINT
RefinementResolution: 2.53→2.53 Å / Cor.coef. Fo:Fc: 0.854 / SU B: 3.999 / SU ML: 0.084 / ESU R: 0.135
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.33604 --
obs0.33604 96784 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 43.708 Å2
Refinement stepCycle: 1 / Total: 2361
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.0122377
ELECTRON MICROSCOPYr_bond_other_d0.0010.0162300
ELECTRON MICROSCOPYr_angle_refined_deg1.5381.6483169
ELECTRON MICROSCOPYr_angle_other_deg0.4781.5435323
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.3515258
ELECTRON MICROSCOPYr_dihedral_angle_2_deg9.431519
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.99410322
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0770.2311
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.022540
ELECTRON MICROSCOPYr_gen_planes_other0.0010.02548
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_it3.9244.3511035
ELECTRON MICROSCOPYr_mcbond_other3.9164.3481035
ELECTRON MICROSCOPYr_mcangle_it5.6756.5451292
ELECTRON MICROSCOPYr_mcangle_other5.7056.5541293
ELECTRON MICROSCOPYr_scbond_it5.4345.5021342
ELECTRON MICROSCOPYr_scbond_other5.4325.5051343
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other8.5987.8021878
ELECTRON MICROSCOPYr_long_range_B_refined14.53789.91910496
ELECTRON MICROSCOPYr_long_range_B_other14.51789.89310469
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.45→2.514 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.336 7217 -
obs--100 %

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