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- PDB-7sfj: ChRmine in MSP1E3D1 lipid nanodisc -

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Basic information

Entry
Database: PDB / ID: 7sfj
TitleChRmine in MSP1E3D1 lipid nanodisc
ComponentsChRmine
KeywordsMEMBRANE PROTEIN / Channelrhodopsin / ion channel
Function / homology1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / RETINAL
Function and homology information
Biological speciesRhodomonas lens (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsTucker, K. / Brohawn, S.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Nat Commun / Year: 2022
Title: Cryo-EM structures of the channelrhodopsin ChRmine in lipid nanodiscs.
Authors: Kyle Tucker / Savitha Sridharan / Hillel Adesnik / Stephen G Brohawn /
Abstract: Microbial channelrhodopsins are light-gated ion channels widely used for optogenetic manipulation of neuronal activity. ChRmine is a bacteriorhodopsin-like cation channelrhodopsin (BCCR) more closely ...Microbial channelrhodopsins are light-gated ion channels widely used for optogenetic manipulation of neuronal activity. ChRmine is a bacteriorhodopsin-like cation channelrhodopsin (BCCR) more closely related to ion pump rhodopsins than other channelrhodopsins. ChRmine displays unique properties favorable for optogenetics including high light sensitivity, a broad, red-shifted activation spectrum, cation selectivity, and large photocurrents, while its slow closing kinetics impedes some applications. The structural basis for ChRmine function, or that of any other BCCR, is unknown. Here, we present cryo-EM structures of ChRmine in lipid nanodiscs in apo (opsin) and retinal-bound (rhodopsin) forms. The structures reveal an unprecedented trimeric architecture with a lipid filled central pore. Large electronegative cavities on either side of the membrane facilitate high conductance and selectivity for cations over protons. The retinal binding pocket structure suggests channel properties could be tuned with mutations and we identify ChRmine variants with ten-fold decreased and two-fold increased closing rates. A T119A mutant shows favorable properties relative to wild-type and previously reported ChRmine variants for optogenetics. These results provide insight into structural features that generate an ultra-potent microbial opsin and provide a platform for rational engineering of channelrhodopsins with improved properties that could expand the scale, depth, and precision of optogenetic experiments.
History
DepositionOct 4, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 6, 2022Group: Source and taxonomy / Category: entity_src_gen
Item: _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name
Revision 1.2Aug 31, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: ChRmine
B: ChRmine
C: ChRmine
hetero molecules


Theoretical massNumber of molelcules
Total (without water)126,28727
Polymers109,8093
Non-polymers16,47824
Water543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "C"
d_2ens_1chain "B"
d_3ens_1chain "A"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1AYAASPS1 - 284
d_12ens_1PEEPEET
d_13ens_1PEEPEEU
d_14ens_1PEEPEEV
d_15ens_1PEEPEEW
d_16ens_1PEEPEEX
d_17ens_1PEEPEEY
d_18ens_1PEEPEEZ
d_19ens_1RETRETAA
d_21ens_1AYAASPJ1 - 284
d_22ens_1PEEPEEK
d_23ens_1PEEPEEL
d_24ens_1PEEPEEM
d_25ens_1PEEPEEN
d_26ens_1PEEPEEO
d_27ens_1PEEPEEP
d_28ens_1PEEPEEQ
d_29ens_1RETRETR
d_31ens_1AYAASPA1 - 284
d_32ens_1PEEPEEB
d_33ens_1PEEPEEC
d_34ens_1PEEPEED
d_35ens_1PEEPEEE
d_36ens_1PEEPEEF
d_37ens_1PEEPEEG
d_38ens_1PEEPEEH
d_39ens_1RETRETI

NCS oper:
IDCodeMatrixVector
1given(-0.499079632689, -0.866556015088, -0.000439261248088), (0.866553357404, -0.499079255258, 0.00227502595273), (-0.00219066360037, 0.000754775807537, 0.99999731565)295.908248355, 78.9554778744, 0.217897260328
2given(-0.497924278756, 0.867219945661, -0.000989178128228), (-0.867220472955, -0.497924266205, 0.000276428767672), (-0.000252811252764, 0.000995476118969, 0.999999472557)78.9866805701, 295.716889483, -0.0468995198792

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Components

#1: Protein ChRmine


Mass: 36602.957 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhodomonas lens (eukaryote) / Production host: Spodoptera frugiperda (fall armyworm)
#2: Chemical...
ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE / Discrete optimized protein energy


Mass: 744.034 Da / Num. of mol.: 21 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
#3: Chemical ChemComp-RET / RETINAL / Retinal


Mass: 284.436 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C20H28O
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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 trimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.11 MDa / Experimental value: NO
Source (natural)Organism: Tiarina fusa (eukaryote)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTRISC4H11NO31
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 14.4 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

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
12cryoSPARC3.13D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 8444523
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81839 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 59.74 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00277584
ELECTRON MICROSCOPYf_angle_d0.495310179
ELECTRON MICROSCOPYf_chiral_restr0.03911011
ELECTRON MICROSCOPYf_plane_restr0.00521233
ELECTRON MICROSCOPYf_dihedral_angle_d11.68091281
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2BELECTRON MICROSCOPYNCS constraints0.000700583048083
ens_1d_3AELECTRON MICROSCOPYNCS constraints0.000707096796172

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