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- PDB-7ek1: Cryo-EM structure of VCCN1 in detergent -

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Basic information

Entry
Database: PDB / ID: 7ek1
TitleCryo-EM structure of VCCN1 in detergent
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: 3 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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Bestrophin-like protein


Theoretical massNumber of molelcules
Total (without water)42,0581
Polymers42,0581
Non-polymers00
Water0
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
2generate(0.309016994, -0.951056516), (0.951056516, 0.309016994), (1)139.17927, -22.0438308
3generate(-0.809016994, -0.587785252), (0.587785252, -0.809016994), (1)203.152958, 103.511602
4generate(-0.809016994, 0.587785252), (-0.587785252, -0.809016994), (1)103.511602, 203.152958
5generate(0.309016994, 0.951056516), (-0.951056516, 0.309016994), (1)-22.0438308, 139.17927

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Components

#1: Protein Bestrophin-like protein


Mass: 42057.738 Da / Num. of mol.: 1
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 FIELDBright-field microscopy
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1161

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Processing

SoftwareName: REFMAC / Version: 5.8.0274 / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4.1.13CTF correction
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 322637
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 117986 / Symmetry type: POINT
RefinementResolution: 3→119.974 Å / Cor.coef. Fo:Fc: 0.785 / WRfactor Rwork: 0.3593 / SU B: 8.766 / SU ML: 0.163 / Average fsc overall: 0.9037 / Average fsc work: 0.9037 / ESU R: 0.19
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3593 96335 -
all0.3593 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: BABINET MODEL
Displacement parametersBiso mean: 95.418 Å2
Baniso -1Baniso -2Baniso -3
1-1.787 Å20.002 Å20.002 Å2
2--1.723 Å2-0.016 Å2
3----3.51 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0150.0132732
ELECTRON MICROSCOPYr_bond_other_d0.050.0172700
ELECTRON MICROSCOPYr_ext_dist_refined_d0.3180.1225
ELECTRON MICROSCOPYr_angle_refined_deg1.831.6243713
ELECTRON MICROSCOPYr_angle_other_deg1.6791.5676190
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.3615335
ELECTRON MICROSCOPYr_dihedral_angle_2_deg31.07120.072138
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.1315483
ELECTRON MICROSCOPYr_dihedral_angle_4_deg20.481525
ELECTRON MICROSCOPYr_chiral_restr0.1180.2368
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.023003
ELECTRON MICROSCOPYr_gen_planes_other0.0020.02635
ELECTRON MICROSCOPYr_nbd_refined0.1960.21078
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1620.24680
ELECTRON MICROSCOPYr_nbtor_refined0.1620.22810
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0740.23104
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.2290.248
ELECTRON MICROSCOPYr_symmetry_nbd_refined0.1480.234
ELECTRON MICROSCOPYr_nbd_other0.1930.2168
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_refined0.3470.26
ELECTRON MICROSCOPYr_mcbond_it12.9149.1171343
ELECTRON MICROSCOPYr_mcbond_other12.919.1061342
ELECTRON MICROSCOPYr_mcangle_it16.80713.7471677
ELECTRON MICROSCOPYr_mcangle_other16.80513.7621678
ELECTRON MICROSCOPYr_scbond_it16.10310.7441389
ELECTRON MICROSCOPYr_scbond_other16.09710.7571390
ELECTRON MICROSCOPYr_scangle_it22.72115.4992036
ELECTRON MICROSCOPYr_scangle_other22.71515.5122037
ELECTRON MICROSCOPYr_lrange_it27.851179.36910653
ELECTRON MICROSCOPYr_lrange_other27.85179.37110654
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: 0 / Total num. of bins used: 20 / % reflection obs: 100 %

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
3-3.0781.55571611.55571610.731.555
3.078-3.1620.82569410.82569410.8290.825
3.162-3.2540.63967190.63967190.8890.639
3.254-3.3540.50965770.50965770.920.509
3.354-3.4640.44363410.44363410.9310.443
3.464-3.5850.3461430.3461430.9350.34
3.585-3.720.21359490.21359490.9610.213
3.72-3.8720.18456780.18456780.9630.184
3.872-4.0440.18155050.18155050.9680.181
4.044-4.2410.22452830.22452830.970.224
4.241-4.470.27849640.27849640.9690.278
4.47-4.7410.27446410.27446410.9680.274
4.741-5.0680.31945020.31945020.9560.319
5.068-5.4730.28840920.28840920.9350.288
5.473-5.9940.28438090.28438090.8970.284
5.994-6.70.33833990.33833990.840.338
6.7-7.7320.3629720.3629720.8310.36
7.732-9.460.3325970.3325970.8760.33
9.46-13.3380.33119740.33119740.9270.331
13.338-119.9741.17810871.17810870.4441.178

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