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- PDB-8yel: Cryo-EM structure of the channelrhodopsin GtCCR4 -

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Basic information

Entry
Database: PDB / ID: 8yel
TitleCryo-EM structure of the channelrhodopsin GtCCR4
ComponentsCation channel rhodopsin 4
KeywordsMEMBRANE PROTEIN / Microbial rhodopsin
Function / homologyBacteriorhodopsin-like protein / Archaeal/bacterial/fungal rhodopsins / Bacteriorhodopsin-like protein / membrane / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / RETINAL / Cation channel rhodopsin 4
Function and homology information
Biological speciesGuillardia theta (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.71 Å
AuthorsTanaka, T. / Iida, W. / Sano, F.K. / Oda, K. / Shihoya, W. / Nureki, O.
Funding support Japan, 8items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)22KJ0577 Japan
Japan Society for the Promotion of Science (JSPS)19H05777 Japan
Japan Society for the Promotion of Science (JSPS)22K19371 Japan
Japan Society for the Promotion of Science (JSPS)22H02751 Japan
Japan Society for the Promotion of Science (JSPS)21H05037 Japan
Japan Agency for Medical Research and Development (AMED)JP233fa627001 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121002 Japan
Japan Agency for Medical Research and Development (AMED)JP23ama121012 Japan
CitationJournal: Mol Cell / Year: 2024
Title: The high-light-sensitivity mechanism and optogenetic properties of the bacteriorhodopsin-like channelrhodopsin GtCCR4.
Authors: Tatsuki Tanaka / Shoko Hososhima / Yo Yamashita / Teppei Sugimoto / Toshiki Nakamura / Shunta Shigemura / Wataru Iida / Fumiya K Sano / Kazumasa Oda / Takayuki Uchihashi / Kota Katayama / ...Authors: Tatsuki Tanaka / Shoko Hososhima / Yo Yamashita / Teppei Sugimoto / Toshiki Nakamura / Shunta Shigemura / Wataru Iida / Fumiya K Sano / Kazumasa Oda / Takayuki Uchihashi / Kota Katayama / Yuji Furutani / Satoshi P Tsunoda / Wataru Shihoya / Hideki Kandori / Osamu Nureki /
Abstract: Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), ...Channelrhodopsins are microbial light-gated ion channels that can control the firing of neurons in response to light. Among several cation channelrhodopsins identified in Guillardia theta (GtCCRs), GtCCR4 has higher light sensitivity than typical channelrhodopsins. Furthermore, GtCCR4 shows superior properties as an optogenetic tool, such as minimal desensitization. Our structural analyses of GtCCR2 and GtCCR4 revealed that GtCCR4 has an outwardly bent transmembrane helix, resembling the conformation of activated G-protein-coupled receptors. Spectroscopic and electrophysiological comparisons suggested that this helix bend in GtCCR4 omits channel recovery time and contributes to high light sensitivity. An electrophysiological comparison of GtCCR4 and the well-characterized optogenetic tool ChRmine demonstrated that GtCCR4 has superior current continuity and action-potential spike generation with less invasiveness in neurons. We also identified highly active mutants of GtCCR4. These results shed light on the diverse structures and dynamics of microbial rhodopsins and demonstrate the strong optogenetic potential of GtCCR4.
History
DepositionFeb 22, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Oct 2, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.3Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cation channel rhodopsin 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,5895
Polymers45,9341
Non-polymers2,6554
Water1629
1
A: Cation channel rhodopsin 4
hetero molecules

A: Cation channel rhodopsin 4
hetero molecules

A: Cation channel rhodopsin 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,76615
Polymers137,8013
Non-polymers7,96512
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation2
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(-0.5, -0.866025), (0.866025, -0.5), (1)314.20816, 84.19182
3generate(-0.5, 0.866025), (-0.866025, -0.5), (1)84.19182, 314.20816

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Components

#1: Protein Cation channel rhodopsin 4


Mass: 45933.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Guillardia theta (eukaryote) / Gene: CCR4 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A3G1I4H9
#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-PC1 / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / 3-SN-PHOSPHATIDYLCHOLINE


Mass: 790.145 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C44H88NO8P / Comment: phospholipid*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 9 / 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: GtCCR4 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Guillardia theta (eukaryote)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris(hydroxymethyl)aminomethaneC4H11NO31
2150 mMSodium chlorideNaCl1
32 mMDithiothreitolC4H10O2S21
40.01 %DigitoninC56H92O291
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The protein was reconstituted in lipid nanodiscs.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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 FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 600 nm
Image recordingAverage exposure time: 2 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 16366

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
4cryoSPARC3.3.2CTF correction
7MOLREPmodel fitting
9PHENIXmodel refinement
10Servalcatmodel refinement
11cryoSPARC3.3.2initial Euler assignment
12cryoSPARC3.3.2final Euler assignment
13cryoSPARC3.3.2classification
14cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5403048
3D reconstructionResolution: 2.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 581115 / Algorithm: FOURIER SPACE / Details: With C3 symmetry / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementResolution: 2.71→2.71 Å / Cor.coef. Fo:Fc: 0.83 / SU B: 2.676 / SU ML: 0.085 / ESU R: 0.103
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.45849 --
obs0.45849 189241 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 76.494 Å2
Refinement stepCycle: 1 / Total: 2277
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.0132336
ELECTRON MICROSCOPYr_bond_other_d0.030.0172263
ELECTRON MICROSCOPYr_angle_refined_deg1.1331.6383157
ELECTRON MICROSCOPYr_angle_other_deg1.4961.5625202
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.2875263
ELECTRON MICROSCOPYr_dihedral_angle_2_deg34.08121.091110
ELECTRON MICROSCOPYr_dihedral_angle_3_deg18.90415345
ELECTRON MICROSCOPYr_dihedral_angle_4_deg24.024158
ELECTRON MICROSCOPYr_chiral_restr0.0640.2279
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.022507
ELECTRON MICROSCOPYr_gen_planes_other0.0020.02572
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_it1.3177.8631058
ELECTRON MICROSCOPYr_mcbond_other1.3187.8611057
ELECTRON MICROSCOPYr_mcangle_it2.43711.7791319
ELECTRON MICROSCOPYr_mcangle_other2.43611.7811320
ELECTRON MICROSCOPYr_scbond_it0.8898.4091278
ELECTRON MICROSCOPYr_scbond_other0.8898.4111279
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other1.712.4151839
ELECTRON MICROSCOPYr_long_range_B_refined9.1489356
ELECTRON MICROSCOPYr_long_range_B_other9.1479353
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2→2.052 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.708 14019 -
obs--100 %

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