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- PDB-8yej: Cryo-EM structure of the channelrhodopsin GtCCR2 focused on the m... -

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Basic information

Entry
Database: PDB / ID: 8yej
TitleCryo-EM structure of the channelrhodopsin GtCCR2 focused on the monomer
ComponentsGtCCR2
KeywordsMEMBRANE PROTEIN / Microbial rhodopsin
Function / homologyBacteriorhodopsin-like protein / Archaeal/bacterial/fungal rhodopsins / Bacteriorhodopsin-like protein / photoreceptor activity / phototransduction / membrane / RETINAL / Uncharacterized protein
Function and homology information
Biological speciesGuillardia theta CCMP2712 (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 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 20, 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: GtCCR2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)53,1102
Polymers52,8251
Non-polymers2841
Water1086
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein GtCCR2


Mass: 52825.227 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Guillardia theta CCMP2712 (eukaryote) / Strain: CCMP2712 / Gene: GUITHDRAFT_99928 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: L1K1K8
#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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / 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: GtCCR2 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Guillardia theta (eukaryote)
Source (recombinant)Organism: Homo sapiens (human)
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
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: DARK 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: 9775

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
4cryoSPARC3.3.2CTF correction
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5791254
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 808581 / Algorithm: FOURIER SPACE / Symmetry type: POINT
RefinementResolution: 2.86→2.86 Å / Cor.coef. Fo:Fc: 0.832 / SU B: 3.779 / SU ML: 0.119 / ESU R: 0.156
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.4692 --
obs0.4692 78016 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 74.938 Å2
Refinement stepCycle: 1 / Total: 1946
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.0131998
ELECTRON MICROSCOPYr_bond_other_d0.0310.0171926
ELECTRON MICROSCOPYr_angle_refined_deg1.0841.622725
ELECTRON MICROSCOPYr_angle_other_deg1.5761.5614401
ELECTRON MICROSCOPYr_dihedral_angle_1_deg3.2885239
ELECTRON MICROSCOPYr_dihedral_angle_2_deg31.09420.79588
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.0315313
ELECTRON MICROSCOPYr_dihedral_angle_4_deg11.761155
ELECTRON MICROSCOPYr_chiral_restr0.0620.2264
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.022203
ELECTRON MICROSCOPYr_gen_planes_other0.0030.02502
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_it2.8227.881959
ELECTRON MICROSCOPYr_mcbond_other2.827.866958
ELECTRON MICROSCOPYr_mcangle_it4.86911.8111197
ELECTRON MICROSCOPYr_mcangle_other4.86711.8281198
ELECTRON MICROSCOPYr_scbond_it2.7238.1941039
ELECTRON MICROSCOPYr_scbond_other2.7228.1951040
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other4.69312.1421529
ELECTRON MICROSCOPYr_long_range_B_refined12.1728337
ELECTRON MICROSCOPYr_long_range_B_other12.1738336
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 -
Rwork0.59 5805 -
obs--100 %

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