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- PDB-7w9w: 2.02 angstrom cryo-EM structure of the pump-like channelrhodopsin... -

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Entry
Database: PDB / ID: 7w9w
Title2.02 angstrom cryo-EM structure of the pump-like channelrhodopsin ChRmine
ComponentsChRmine
KeywordsMEMBRANE PROTEIN / channelrhodopsin / ion channel / photoreceptor / cryo-EM
Function / homologyRhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins / Up-down Bundle / Mainly Alpha / CHOLESTEROL / PALMITIC ACID / RETINAL
Function and homology information
Biological speciesRhodomonas lens (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.02 Å
AuthorsKishi, K.E. / Kim, Y. / Fukuda, M. / Yamashita, K. / Deisseroth, K. / Kato, H.E.
Funding support Japan, 8items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP21wm0525018 Japan
Japan Society for the Promotion of Science (JSPS)21H05142 Japan
Japan Science and TechnologyJPMJPR1782 Japan
Japan Science and TechnologyJPMJFR204S Japan
Japan Science and TechnologyJPMJCR21P3 Japan
Other private Japan
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)R01MH075957 Japan
Other private Japan
CitationJournal: Cell / Year: 2022
Title: Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine.
Authors: Koichiro E Kishi / Yoon Seok Kim / Masahiro Fukuda / Masatoshi Inoue / Tsukasa Kusakizako / Peter Y Wang / Charu Ramakrishnan / Eamon F X Byrne / Elina Thadhani / Joseph M Paggi / Toshiki E ...Authors: Koichiro E Kishi / Yoon Seok Kim / Masahiro Fukuda / Masatoshi Inoue / Tsukasa Kusakizako / Peter Y Wang / Charu Ramakrishnan / Eamon F X Byrne / Elina Thadhani / Joseph M Paggi / Toshiki E Matsui / Keitaro Yamashita / Takashi Nagata / Masae Konno / Sean Quirin / Maisie Lo / Tyler Benster / Tomoko Uemura / Kehong Liu / Mikihiro Shibata / Norimichi Nomura / So Iwata / Osamu Nureki / Ron O Dror / Keiichi Inoue / Karl Deisseroth / Hideaki E Kato /
Abstract: ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created ...ChRmine, a recently discovered pump-like cation-conducting channelrhodopsin, exhibits puzzling properties (large photocurrents, red-shifted spectrum, and extreme light sensitivity) that have created new opportunities in optogenetics. ChRmine and its homologs function as ion channels but, by primary sequence, more closely resemble ion pump rhodopsins; mechanisms for passive channel conduction in this family have remained mysterious. Here, we present the 2.0 Å resolution cryo-EM structure of ChRmine, revealing architectural features atypical for channelrhodopsins: trimeric assembly, a short transmembrane-helix 3, a twisting extracellular-loop 1, large vestibules within the monomer, and an opening at the trimer interface. We applied this structure to design three proteins (rsChRmine and hsChRmine, conferring further red-shifted and high-speed properties, respectively, and frChRmine, combining faster and more red-shifted performance) suitable for fundamental neuroscience opportunities. These results illuminate the conduction and gating of pump-like channelrhodopsins and point the way toward further structure-guided creation of channelrhodopsins for applications across biology.
History
DepositionDec 11, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 2, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: 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.pdbx_database_id_PubMed
Revision 1.2Mar 9, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Nov 13, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature / refine
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification ..._em_admin.last_update / _pdbx_entry_details.has_protein_modification / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

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Assembly

Deposited unit
A: ChRmine
hetero molecules


Theoretical massNumber of molelcules
Total (without water)38,1139
Polymers35,7731
Non-polymers2,3408
Water86548
1
A: ChRmine
hetero molecules

A: ChRmine
hetero molecules

A: ChRmine
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,33827
Polymers107,3193
Non-polymers7,02024
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, -0.866025404), (0.866025404, -0.5), (1)180.758982, 48.4342232
3generate(-0.5, 0.866025404), (-0.866025404, -0.5), (1)48.4342232, 180.758982

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Components

#1: Protein ChRmine


Mass: 35772.965 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodomonas lens (eukaryote) / Production host: Spodoptera frugiperda (fall armyworm)
#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.: 2 / Source method: obtained synthetically / Formula: C27H46O
#4: Chemical
ChemComp-PLM / PALMITIC ACID


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


Mass: 18.015 Da / Num. of mol.: 48 / 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: ChRmine / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Rhodomonas lens (eukaryote)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid 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: 800 nm
Image recordingElectron dose: 51.533 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0274 / Classification: refinement / Contact author: Garib N. Murshudov / Contact author email: garib[at]mrc-lmb.cam.ac.uk / Date: Dec 3, 2021
Description: (un)restrained refinement or idealisation of macromolecular structures
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.02 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185895 / Symmetry type: POINT
RefinementResolution: 2.02→2.02 Å / Cor.coef. Fo:Fc: 0.853 / WRfactor Rwork: 0.268 / SU B: 2.42 / SU ML: 0.062 / Average fsc overall: 0.8813 / Average fsc work: 0.8813 / ESU R: 0.065
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.2684 174804 -
obs--100 %
Solvent computationSolvent model: BABINET MODEL
Displacement parametersBiso mean: 58.331 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.0132430
ELECTRON MICROSCOPYr_bond_other_d0.0020.0152320
ELECTRON MICROSCOPYr_angle_refined_deg1.6471.6283292
ELECTRON MICROSCOPYr_angle_other_deg1.281.5735321
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.5635273
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.72220.672119
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.57115339
ELECTRON MICROSCOPYr_dihedral_angle_4_deg7.1821512
ELECTRON MICROSCOPYr_chiral_restr0.0840.2297
ELECTRON MICROSCOPYr_gen_planes_refined0.010.022679
ELECTRON MICROSCOPYr_gen_planes_other0.0040.02629
ELECTRON MICROSCOPYr_nbd_refined0.1710.2920
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1160.23798
ELECTRON MICROSCOPYr_nbtor_refined0.1660.22332
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0690.22184
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.1160.2162
ELECTRON MICROSCOPYr_symmetry_nbd_refined0.1940.210
ELECTRON MICROSCOPYr_nbd_other0.1390.2160
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_refined0.1050.28
ELECTRON MICROSCOPYr_mcbond_it4.3145.1941086
ELECTRON MICROSCOPYr_mcbond_other4.2885.1891085
ELECTRON MICROSCOPYr_mcangle_it6.1167.8021358
ELECTRON MICROSCOPYr_mcangle_other6.1147.8071359
ELECTRON MICROSCOPYr_scbond_it5.7566.6361344
ELECTRON MICROSCOPYr_scbond_other5.7546.6381345
ELECTRON MICROSCOPYr_scangle_it9.2679.5471933
ELECTRON MICROSCOPYr_scangle_other9.2659.5491934
ELECTRON MICROSCOPYr_lrange_it15.759104.42810643
ELECTRON MICROSCOPYr_lrange_other15.762104.43910607
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: _ / Total num. of bins used: 20 / % reflection obs: 100 %

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
2-2.0520.805130330.805130330.6010.805
2.052-2.1080.538127290.538127290.7160.538
2.108-2.1690.453121110.453121110.7980.453
2.169-2.2360.39119400.39119400.8510.39
2.236-2.3090.339114890.339114890.8780.339
2.309-2.390.319111290.319111290.8920.319
2.39-2.480.261108350.261108350.9240.261
2.48-2.5820.231102620.231102620.9380.231
2.582-2.6960.20499910.20499910.9510.204
2.696-2.8280.18893970.18893970.960.188
2.828-2.9810.18191120.18191120.9640.181
2.981-3.1610.18485080.18485080.9660.184
3.161-3.3790.18180710.18180710.9680.181
3.379-3.6490.18173840.18173840.970.181
3.649-3.9970.19369260.19369260.9690.193
3.997-4.4680.2162140.2162140.960.21
4.468-5.1570.21754750.21754750.9540.217
5.157-6.310.2946270.2946270.910.29
6.31-8.9010.36135980.36135980.8730.361
8.901-88.6590.63119730.63119730.9560.631

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