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- PDB-7ek3: Cryo-EM structure of VCCN1 Y332A mutant in lipid nanodisc -

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Basic information

Entry
Database: PDB / ID: 7ek3
TitleCryo-EM structure of VCCN1 Y332A mutant 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

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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)41,9661
Polymers41,9661
Non-polymers00
Water0
1
A: Bestrophin-like protein
x 5


Theoretical massNumber of molelcules
Total (without water)209,8285
Polymers209,8285
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)133.018346, -21.0680363
3generate(-0.809016994, -0.587785252), (0.587785252, -0.809016994), (1)194.160168, 98.9295471
4generate(-0.809016994, 0.587785252), (-0.587785252, -0.809016994), (1)98.9295471, 194.160168
5generate(0.309016994, 0.951056516), (-0.951056516, 0.309016994), (1)-21.0680363, 133.018346

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Components

#1: Protein Bestrophin-like protein


Mass: 41965.645 Da / Num. of mol.: 1 / Mutation: A207S, Y322A
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: 48 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2835

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Processing

SoftwareName: REFMAC / Version: 5.8.0274 / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
4CTFFIND4.1.13CTF correction
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
13RELION3.13D 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: 107192 / Symmetry type: POINT
RefinementResolution: 2.7→119.52 Å / Cor.coef. Fo:Fc: 0.795 / WRfactor Rwork: 0.323 / SU B: 4.889 / SU ML: 0.102 / Average fsc overall: 0.9184 / Average fsc work: 0.9184 / ESU R: 0.112
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3226 173690 -
all0.323 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: BABINET MODEL
Displacement parametersBiso mean: 61.764 Å2
Baniso -1Baniso -2Baniso -3
1-0.854 Å20 Å2-0 Å2
2--0.83 Å2-0.002 Å2
3----1.684 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0150.0132758
ELECTRON MICROSCOPYr_bond_other_d0.0010.0172729
ELECTRON MICROSCOPYr_ext_dist_refined_d0.3530.1253
ELECTRON MICROSCOPYr_angle_refined_deg1.8711.6243751
ELECTRON MICROSCOPYr_angle_other_deg1.7111.5666260
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.9915340
ELECTRON MICROSCOPYr_dihedral_angle_2_deg29.45820.294136
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.27115486
ELECTRON MICROSCOPYr_dihedral_angle_4_deg20.7721524
ELECTRON MICROSCOPYr_chiral_restr0.0580.2374
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.023032
ELECTRON MICROSCOPYr_gen_planes_other0.0020.02632
ELECTRON MICROSCOPYr_nbd_refined0.1870.21148
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1550.24968
ELECTRON MICROSCOPYr_nbtor_refined0.1690.22774
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0810.23192
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.1990.250
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_other0.0230.22
ELECTRON MICROSCOPYr_symmetry_nbd_refined0.1060.234
ELECTRON MICROSCOPYr_nbd_other0.1570.2222
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_refined0.1540.210
ELECTRON MICROSCOPYr_mcbond_it6.1575.9231363
ELECTRON MICROSCOPYr_mcbond_other6.1485.9121361
ELECTRON MICROSCOPYr_mcangle_it8.5228.9221702
ELECTRON MICROSCOPYr_mcangle_other8.5158.9211702
ELECTRON MICROSCOPYr_scbond_it8.7686.9361395
ELECTRON MICROSCOPYr_scbond_other8.7656.941396
ELECTRON MICROSCOPYr_scangle_it13.10910.0352049
ELECTRON MICROSCOPYr_scangle_other13.10810.0392050
ELECTRON MICROSCOPYr_lrange_it17.524116.36811215
ELECTRON MICROSCOPYr_lrange_other17.509116.31311210
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
2.7-2.771.05128801.05128800.8111.05
2.77-2.8460.544125840.544125840.8740.544
2.846-2.9290.477121190.477121190.9020.477
2.929-3.0190.41117770.41117770.9140.41
3.019-3.1180.316114650.316114650.9290.316
3.118-3.2270.226111290.226111290.9490.226
3.227-3.3490.203107120.203107120.9540.203
3.349-3.4850.194102050.194102050.9560.194
3.485-3.640.198100140.198100140.9640.198
3.64-3.8180.20893310.20893310.9680.208
3.818-4.0240.23790020.23790020.9650.237
4.024-4.2680.26184590.26184590.9630.261
4.268-4.5620.27180000.27180000.9630.271
4.562-4.9270.24474460.24474460.9620.244
4.927-5.3960.24168040.24168040.9410.241
5.396-6.0310.33661350.33661350.8880.336
6.031-6.9620.45555000.45555000.8210.455
6.962-8.5190.41545930.41545930.850.415
8.519-12.0170.27135750.27135750.9350.271
12.017-119.521.06519591.06519590.6121.065

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