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- EMDB-25091: ChRmine in MSP1E3D1 lipid nanodisc -

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Basic information

Entry
Database: EMDB / ID: EMD-25091
TitleChRmine in MSP1E3D1 lipid nanodisc
Map dataChRmine in MSP1E3D1 lipid nanodisc
Sample
  • Complex: ChRmine trimer
    • Protein or peptide: ChRmine
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: RETINAL
  • Ligand: water
Biological speciesTiarina fusa (eukaryote) / Rhodomonas lens (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsTucker K / Brohawn S
Funding support United States, 1 items
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, 2021-
Header (metadata) releaseDec 1, 2021-
Map releaseDec 1, 2021-
UpdateAug 31, 2022-
Current statusAug 31, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7sfk
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25091.png

Downloads & links

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Map

FileDownload / File: emd_25091.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationChRmine in MSP1E3D1 lipid nanodisc
Voxel sizeX=Y=Z: 1.137 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-5.4301043 - 9.7369375
Average (Standard dev.)-9.067203e-05 (±0.20197977)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 250.14 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1371.1371.137
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z250.140250.140250.140
α/β/γ90.00090.00090.000
start NX/NY/NZ-140-140-140
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-5.4309.737-0.000

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Supplemental data

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Sample components

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Entire : ChRmine trimer

EntireName: ChRmine trimer
Components
  • Complex: ChRmine trimer
    • Protein or peptide: ChRmine
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: RETINAL
  • Ligand: water

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Supramolecule #1: ChRmine trimer

SupramoleculeName: ChRmine trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Tiarina fusa (eukaryote)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
Molecular weightTheoretical: 110 KDa

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Macromolecule #1: ChRmine

MacromoleculeName: ChRmine / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Rhodomonas lens (eukaryote)
Molecular weightTheoretical: 36.602957 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: M(AYA)HAPGTDQM FYVGTMDGWY LDTKLNSVAI GAHWSCFIVL TITTFYLGYE SWTSRGPSKR TSFYAGYQEE QNLALF VNF FAMLSYFGKI VADTLGHNFG DVGPFIIGFG NYRYADYMLT CPMLVYDLLY QLRAPYRVSC SAIIFAILMS GVLAEFY AE GDPRLRNGAY ...String:
M(AYA)HAPGTDQM FYVGTMDGWY LDTKLNSVAI GAHWSCFIVL TITTFYLGYE SWTSRGPSKR TSFYAGYQEE QNLALF VNF FAMLSYFGKI VADTLGHNFG DVGPFIIGFG NYRYADYMLT CPMLVYDLLY QLRAPYRVSC SAIIFAILMS GVLAEFY AE GDPRLRNGAY AWYGFGCFWF IFAYSIVMSI VAKQYSRLAQ LAQDTGAEHS LHVLKFAVFT FSMLWILFPL VWAICPRG F GWIDDNWTEV AHCVCDIVAK SCYGFALARF RKTYDEELFR LLEQLGHDED EFQKLELDMR LSSNGERLRR LSLNSLEVL FQ

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Macromolecule #2: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 2 / Number of copies: 21 / Formula: PEE
Molecular weightTheoretical: 744.034 Da
Chemical component information

ChemComp-PEE:
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE, phospholipid*YM / Discrete optimized protein energy

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Macromolecule #3: RETINAL

MacromoleculeName: RETINAL / type: ligand / ID: 3 / Number of copies: 3 / Formula: RET
Molecular weightTheoretical: 284.436 Da
Chemical component information

ChemComp-RET:
RETINAL / Retinal

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 57 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration14.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3TRIS
150.0 mMNaClSodium chlorideSodium Chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS TALOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2

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Image processing

Particle selectionNumber selected: 8444523
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 81839

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