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- PDB-7ek2: Cryo-EM structure of VCCN1 in lipid nanodisc -

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Basic information

Entry
Database: PDB / ID: 7ek2
TitleCryo-EM structure of VCCN1 in lipid nanodisc
ComponentsBestrophin-like protein
KeywordsMEMBRANE PROTEIN / Ion channel / Thylakoid / Photosynthesis / Bestrophin family
Function / homologyregulation of photosynthesis, light reaction / UPF0187 protein At3g61320-like / UPF0187 family / Bestrophin/UPF0187 / Bestrophin, RFP-TM, chloride channel / voltage-gated chloride channel activity / membrane / Uncharacterized protein
Function and homology information
Biological speciesMalus domestica (apple)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsHagino, T. / Kato, T. / Kasuya, G. / Kobayashi, K. / Kusakizako, T. / Yamashita, K. / Nishizawa, T. / Nureki, O.
CitationJournal: Nat Commun / Year: 2022
Title: Cryo-EM structures of thylakoid-located voltage-dependent chloride channel VCCN1.
Authors: Tatsuya Hagino / Takafumi Kato / Go Kasuya / Kan Kobayashi / Tsukasa Kusakizako / Shin Hamamoto / Tomoaki Sobajima / Yuichiro Fujiwara / Keitaro Yamashita / Hisashi Kawasaki / Andrés D ...Authors: Tatsuya Hagino / Takafumi Kato / Go Kasuya / Kan Kobayashi / Tsukasa Kusakizako / Shin Hamamoto / Tomoaki Sobajima / Yuichiro Fujiwara / Keitaro Yamashita / Hisashi Kawasaki / Andrés D Maturana / Tomohiro Nishizawa / Osamu Nureki /
Abstract: In the light reaction of plant photosynthesis, modulation of electron transport chain reactions is important to maintain the efficiency of photosynthesis under a broad range of light intensities. ...In the light reaction of plant photosynthesis, modulation of electron transport chain reactions is important to maintain the efficiency of photosynthesis under a broad range of light intensities. VCCN1 was recently identified as a voltage-gated chloride channel residing in the thylakoid membrane, where it plays a key role in photoreaction tuning to avoid the generation of reactive oxygen species (ROS). Here, we present the cryo-EM structures of Malus domestica VCCN1 (MdVCCN1) in nanodiscs and detergent at 2.7 Å and 3.0 Å resolutions, respectively, and the structure-based electrophysiological analyses. VCCN1 structurally resembles its animal homolog, bestrophin, a Ca-gated anion channel. However, unlike bestrophin channels, VCCN1 lacks the Ca-binding motif but instead contains an N-terminal charged helix that is anchored to the lipid membrane through an additional amphipathic helix. Electrophysiological experiments demonstrate that these structural elements are essential for the channel activity, thus revealing the distinct activation mechanism of VCCN1.
History
DepositionApr 3, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 6, 2022Provider: repository / Type: Initial release
Revision 1.1May 18, 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
Revision 1.2Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Bestrophin-like protein


Theoretical massNumber of molelcules
Total (without water)42,0581
Polymers42,0581
Non-polymers00
Water00
1
A: Bestrophin-like protein
x 5


Theoretical massNumber of molelcules
Total (without water)210,2895
Polymers210,2895
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
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: C5 (5 fold cyclic))
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(0.309016994, -0.951056516), (0.951056516, 0.309016994), (1)130.837709, -20.7226573
3generate(-0.809016994, -0.587785252), (0.587785252, -0.809016994), (1)190.977203, 97.3077452
4generate(-0.809016994, 0.587785252), (-0.587785252, -0.809016994), (1)97.3077452, 190.977203
5generate(0.309016994, 0.951056516), (-0.951056516, 0.309016994), (1)-20.7226573, 130.837709

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Components

#1: Protein Bestrophin-like protein


Mass: 42057.738 Da / Num. of mol.: 1 / Mutation: A207S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Malus domestica (apple) / Gene: DVH24_014265 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: A0A498JCY7
Sequence detailsSER 207 is the natural mutation, which appeared in the sequence amplified from the cDNA library.

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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: Bestrophin-like protein / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Malus domestica (apple)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 48 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2271

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Processing

SoftwareName: REFMAC / Version: 5.8.0274 / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
9CTFFIND4.1.13CTF correction
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 345938 / Symmetry type: POINT
RefinementResolution: 2.7→119.52 Å / Cor.coef. Fo:Fc: 0.779 / WRfactor Rwork: 0.368 / SU B: 5.162 / SU ML: 0.107 / Average fsc overall: 0.9038 / Average fsc work: 0.9038 / ESU R: 0.128
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3676 173687 -
all0.368 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: BABINET MODEL
Displacement parametersBiso mean: 72.646 Å2
Baniso -1Baniso -2Baniso -3
1-0.428 Å20.009 Å20.004 Å2
2--0.374 Å20.009 Å2
3----0.802 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0130.0132766
ELECTRON MICROSCOPYr_bond_other_d0.050.0172734
ELECTRON MICROSCOPYr_ext_dist_refined_d0.3140.1251
ELECTRON MICROSCOPYr_angle_refined_deg1.7281.6243762
ELECTRON MICROSCOPYr_angle_other_deg1.7041.5666271
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.7345340
ELECTRON MICROSCOPYr_dihedral_angle_2_deg27.39420.292137
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.07315487
ELECTRON MICROSCOPYr_dihedral_angle_4_deg19.2591524
ELECTRON MICROSCOPYr_chiral_restr0.0550.2374
ELECTRON MICROSCOPYr_gen_planes_refined0.0040.023040
ELECTRON MICROSCOPYr_gen_planes_other0.0010.02638
ELECTRON MICROSCOPYr_nbd_refined0.1930.21104
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1530.24880
ELECTRON MICROSCOPYr_nbtor_refined0.1630.22818
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0740.23072
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.2250.254
ELECTRON MICROSCOPYr_symmetry_nbd_refined0.1020.244
ELECTRON MICROSCOPYr_nbd_other0.170.2202
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_refined0.1870.220
ELECTRON MICROSCOPYr_mcbond_it7.4687.0251363
ELECTRON MICROSCOPYr_mcbond_other7.4537.011361
ELECTRON MICROSCOPYr_mcangle_it9.86310.5941702
ELECTRON MICROSCOPYr_mcangle_other9.86710.5931702
ELECTRON MICROSCOPYr_scbond_it9.5878.1071403
ELECTRON MICROSCOPYr_scbond_other9.5848.1131404
ELECTRON MICROSCOPYr_scangle_it14.28711.7492060
ELECTRON MICROSCOPYr_scangle_other14.28711.7562061
ELECTRON MICROSCOPYr_lrange_it18.398138.72411196
ELECTRON MICROSCOPYr_lrange_other18.379138.63111191
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
2.7-2.771.747129111.747129110.7581.747
2.77-2.8461.004124681.004124680.8281.004
2.846-2.9280.869121530.869121530.880.869
2.928-3.0190.69118350.69118350.8980.69
3.019-3.1170.468115540.468115540.9220.468
3.117-3.2270.298110240.298110240.9430.298
3.227-3.3490.228106480.228106480.950.228
3.349-3.4850.205103500.205103500.9520.205
3.485-3.640.21198450.21198450.9640.211
3.64-3.8170.23894680.23894680.9650.238
3.817-4.0240.28889310.28889310.9620.288
4.024-4.2670.30985480.30985480.9570.309
4.267-4.5620.31779280.31779280.9540.317
4.562-4.9270.28774300.28774300.9490.287
4.927-5.3960.27168540.27168540.9320.271
5.396-6.0310.32961590.32961590.880.329
6.031-6.9610.45854500.45854500.8220.458
6.961-8.5190.40346110.40346110.8480.403
8.519-12.0170.27335670.27335670.9290.273
12.017-119.521.05519531.05519530.531.055

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