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- PDB-9es7: Cryo-EM structure of Spinacia oleracea cytochrome b6f complex wit... -

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Basic information

Entry
Database: PDB / ID: 9es7
TitleCryo-EM structure of Spinacia oleracea cytochrome b6f complex with water molecules at 1.94 A resolution
Components
  • (Cytochrome b6-f complex subunit ...) x 5
  • Cytochrome b6
  • Cytochrome b6-f complex iron-sulfur subunit, chloroplastic
  • Cytochrome f
  • Thylakoid soluble phosphoprotein
KeywordsOXIDOREDUCTASE / b6f complex / photosynthesis / membrane protein / electron transport / proton transport / quinone catalysis / water channels
Function / homology
Function and homology information


PSII associated light-harvesting complex II binding / chloroplast photosystem I binding / chloroplast photosystem II binding / cytochrome b6f complex / plastoquinol-plastocyanin reductase / plastoquinol--plastocyanin reductase activity / : / thylakoid membrane / cytochrome complex assembly / photosynthetic electron transport chain ...PSII associated light-harvesting complex II binding / chloroplast photosystem I binding / chloroplast photosystem II binding / cytochrome b6f complex / plastoquinol-plastocyanin reductase / plastoquinol--plastocyanin reductase activity / : / thylakoid membrane / cytochrome complex assembly / photosynthetic electron transport chain / chloroplast thylakoid membrane / : / response to light stimulus / photosynthesis / chloroplast / respiratory electron transport chain / 2 iron, 2 sulfur cluster binding / electron transfer activity / oxidoreductase activity / iron ion binding / lipid binding / heme binding / metal ion binding / membrane / plasma membrane
Similarity search - Function
Thylakoid soluble phosphoprotein TSP9 / Thylakoid soluble phosphoprotein TSP9 superfamily / Thylakoid soluble phosphoprotein TSP9 / : / Cytochrome B6-F complex subunit VI (PetL) / Cytochrome b6-f complex subunit 6 / Cytochrome b6-f complex iron-sulfur subunit / Cytochrome b6/f complex, subunit 5 / Cytochrome b6-f complex, subunit 8 / Cytochrome b6/f complex, subunit IV ...Thylakoid soluble phosphoprotein TSP9 / Thylakoid soluble phosphoprotein TSP9 superfamily / Thylakoid soluble phosphoprotein TSP9 / : / Cytochrome B6-F complex subunit VI (PetL) / Cytochrome b6-f complex subunit 6 / Cytochrome b6-f complex iron-sulfur subunit / Cytochrome b6/f complex, subunit 5 / Cytochrome b6-f complex, subunit 8 / Cytochrome b6/f complex, subunit IV / PetM of cytochrome b6/f complex subunit 7 / Cytochrome b6, PetB / Cytochrome b6/f complex, subunit 5 superfamily / Cytochrome b6-f complex, subunit 8 superfamily / Cytochrome B6-F complex subunit 5 / PetN / PetM family of cytochrome b6f complex subunit 7 / Cytochrome f transmembrane anchor / Cytochrome f / Cytochrome f large domain / Cytochrome f large domain superfamily / Apocytochrome F, C-terminal / Apocytochrome F, N-terminal / Cytochrome f family profile. / : / : / Cytochrome b/b6, C-terminal / Cytochrome b(C-terminal)/b6/petD / Cytochrome b/b6 C-terminal region profile. / Cytochrome b/b6, C-terminal domain superfamily / Cytochrome b/b6/petB / Rieske iron-sulphur protein, C-terminal / Rudiment single hybrid motif / Cytochrome b/b6, N-terminal / Cytochrome b/b6-like domain superfamily / Cytochrome b/b6 N-terminal region profile. / Di-haem cytochrome, transmembrane / Rieske iron-sulphur protein / Rieske [2Fe-2S] domain / Rieske [2Fe-2S] iron-sulphur domain / Rieske [2Fe-2S] iron-sulfur domain profile. / Rieske [2Fe-2S] iron-sulphur domain superfamily
Similarity search - Domain/homology
BETA-CAROTENE / CHLOROPHYLL A / FE2/S2 (INORGANIC) CLUSTER / HEME C / PROTOPORPHYRIN IX CONTAINING FE / 1,2-DISTEAROYL-MONOGALACTOSYL-DIGLYCERIDE / Chem-SQD / Uncharacterized protein LOC110795548 / Cytochrome b6 / Cytochrome b6-f complex subunit 4 ...BETA-CAROTENE / CHLOROPHYLL A / FE2/S2 (INORGANIC) CLUSTER / HEME C / PROTOPORPHYRIN IX CONTAINING FE / 1,2-DISTEAROYL-MONOGALACTOSYL-DIGLYCERIDE / Chem-SQD / Uncharacterized protein LOC110795548 / Cytochrome b6 / Cytochrome b6-f complex subunit 4 / Cytochrome b6-f complex iron-sulfur subunit, chloroplastic / Cytochrome f / Cytochrome b6-f complex subunit 8 / Cytochrome b6-f complex subunit 5 / Thylakoid soluble phosphoprotein / Cytochrome b6-f complex subunit 6
Similarity search - Component
Biological speciesSpinacia oleracea (spinach)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.94 Å
AuthorsPietras, R. / Pintscher, S. / Mielecki, B. / Szwalec, M. / Wojcik-Augustyn, A. / Indyka, P. / Rawski, M. / Koziej, L. / Jaciuk, M. / Wazny, G. ...Pietras, R. / Pintscher, S. / Mielecki, B. / Szwalec, M. / Wojcik-Augustyn, A. / Indyka, P. / Rawski, M. / Koziej, L. / Jaciuk, M. / Wazny, G. / Glatt, S. / Osyczka, A.
Funding support Poland, 3items
OrganizationGrant numberCountry
Foundation for Polish SciencePOIR.04.04.00-00-5B54/17-00 Poland
Foundation for Polish ScienceTEAM TECH CORE FACILITY/2017-4/6 Poland
Polish National Science Centre2022/47/B/NZ1/03308 Poland
CitationJournal: Nat Plants / Year: 2024
Title: Molecular basis of plastoquinone reduction in plant cytochrome bf.
Authors: Sebastian Pintscher / Rafał Pietras / Bohun Mielecki / Mateusz Szwalec / Anna Wójcik-Augustyn / Paulina Indyka / Michał Rawski / Łukasz Koziej / Marcin Jaciuk / Grzegorz Ważny / ...Authors: Sebastian Pintscher / Rafał Pietras / Bohun Mielecki / Mateusz Szwalec / Anna Wójcik-Augustyn / Paulina Indyka / Michał Rawski / Łukasz Koziej / Marcin Jaciuk / Grzegorz Ważny / Sebastian Glatt / Artur Osyczka /
Abstract: A multi-subunit enzyme, cytochrome bf (cytbf), provides the crucial link between photosystems I and II in the photosynthetic membranes of higher plants, transferring electrons between plastoquinone ...A multi-subunit enzyme, cytochrome bf (cytbf), provides the crucial link between photosystems I and II in the photosynthetic membranes of higher plants, transferring electrons between plastoquinone (PQ) and plastocyanin. The atomic structure of cytbf is known, but its detailed catalytic mechanism remains elusive. Here we present cryogenic electron microscopy structures of spinach cytbf at 1.9 Å and 2.2 Å resolution, revealing an unexpected orientation of the substrate PQ in the haem ligand niche that forms the PQ reduction site (Q). PQ, unlike Q inhibitors, is not in direct contact with the haem. Instead, a water molecule is coordinated by one of the carbonyl groups of PQ and can act as the immediate proton donor for PQ. In addition, we identify water channels that connect Q with the aqueous exterior of the enzyme, suggesting that the binding of PQ in Q displaces water through these channels. The structures confirm large movements of the head domain of the iron-sulfur protein (ISP-HD) towards and away from the plastoquinol oxidation site (Q) and define the unique position of ISP-HD when a Q inhibitor (2,5-dibromo-3-methyl-6-isopropylbenzoquinone) is bound. This work identifies key conformational states of cytbf, highlights fundamental differences between substrates and inhibitors and proposes a quinone-water exchange mechanism.
History
DepositionMar 25, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 16, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 1.2Nov 27, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cytochrome b6
B: Cytochrome b6-f complex subunit 4
C: Cytochrome f
D: Cytochrome b6-f complex iron-sulfur subunit, chloroplastic
E: Cytochrome b6-f complex subunit 6
F: Cytochrome b6-f complex subunit 7
G: Cytochrome b6-f complex subunit 5
H: Cytochrome b6-f complex subunit 8
I: Cytochrome b6
J: Cytochrome b6-f complex subunit 4
K: Cytochrome f
L: Cytochrome b6-f complex iron-sulfur subunit, chloroplastic
M: Cytochrome b6-f complex subunit 6
N: Cytochrome b6-f complex subunit 7
O: Cytochrome b6-f complex subunit 5
P: Cytochrome b6-f complex subunit 8
Q: Thylakoid soluble phosphoprotein
R: Thylakoid soluble phosphoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)284,71040
Polymers271,40718
Non-polymers13,30322
Water5,927329
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 4 types, 8 molecules AICKDLQR

#1: Protein Cytochrome b6


Mass: 24186.504 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / Organ: leaf / References: UniProt: P00165
#3: Protein Cytochrome f


Mass: 35355.664 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / Organ: leaf / References: UniProt: P16013
#4: Protein Cytochrome b6-f complex iron-sulfur subunit, chloroplastic / Plastohydroquinone:plastocyanin oxidoreductase iron-sulfur protein / Rieske iron-sulfur protein / ISP / RISP


Mass: 24347.898 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / Organ: leaf
References: UniProt: P08980, plastoquinol-plastocyanin reductase
#9: Protein Thylakoid soluble phosphoprotein


Mass: 10608.004 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / Organ: leaf / References: UniProt: Q8GT36

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Cytochrome b6-f complex subunit ... , 5 types, 10 molecules BJEMFNGOHP

#2: Protein Cytochrome b6-f complex subunit 4 / 17 kDa polypeptide


Mass: 17456.662 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / Organ: leaf / References: UniProt: P00166
#5: Protein/peptide Cytochrome b6-f complex subunit 6 / Cytochrome b6-f complex subunit PetL / Cytochrome b6-f complex subunit VI


Mass: 3452.200 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / Organ: leaf / References: UniProt: Q9M3L0
#6: Protein Cytochrome b6-f complex subunit 7 / Cytochrome b6-f complex subunit PetM / Cytochrome b6-f complex subunit VII


Mass: 12953.800 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / Organ: leaf / References: UniProt: A0A9R0IV89
#7: Protein/peptide Cytochrome b6-f complex subunit 5 / Cytochrome b6-f complex subunit PetG / Cytochrome b6-f complex subunit V


Mass: 4171.985 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / Organ: leaf / References: UniProt: P69461
#8: Protein/peptide Cytochrome b6-f complex subunit 8 / Cytochrome b6-f complex subunit PetN / Cytochrome b6-f complex subunit VIII


Mass: 3170.809 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / Organ: leaf / References: UniProt: P61045

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Non-polymers , 9 types, 351 molecules

#10: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical
ChemComp-HEC / HEME C


Mass: 618.503 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H34FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#12: Chemical
ChemComp-UMQ / UNDECYL-MALTOSIDE / UNDECYL-BETA-D-MALTOPYRANOSIDE


Mass: 496.589 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C23H44O11 / Comment: detergent*YM
#13: Chemical ChemComp-LMG / 1,2-DISTEAROYL-MONOGALACTOSYL-DIGLYCERIDE


Mass: 787.158 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C45H86O10 / Feature type: SUBJECT OF INVESTIGATION
#14: Chemical ChemComp-CLA / CHLOROPHYLL A


Mass: 893.489 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C55H72MgN4O5 / Feature type: SUBJECT OF INVESTIGATION
#15: Chemical ChemComp-SQD / 1,2-DI-O-ACYL-3-O-[6-DEOXY-6-SULFO-ALPHA-D-GLUCOPYRANOSYL]-SN-GLYCEROL / SULFOQUINOVOSYLDIACYLGLYCEROL


Mass: 795.116 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C41H78O12S / Feature type: SUBJECT OF INVESTIGATION
#16: Chemical ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe2S2 / Feature type: SUBJECT OF INVESTIGATION
#17: Chemical ChemComp-BCR / BETA-CAROTENE


Mass: 536.873 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C40H56 / Feature type: SUBJECT OF INVESTIGATION
#18: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 329 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: cytochrome b6f complex incubated with decylplastoquinone
Type: COMPLEX / Entity ID: #1-#9 / Source: NATURAL
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)Organism: Spinacia oleracea (spinach) / Organ: leaf
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
250 mMsodium chlorideNaCl1
31 mMn-undecyl beta-maltosideC23H44O111
40.6 mMdecylplastoquinoneC18H28O21
514 mMdimethyl sulfoxideC2H6OS1
SpecimenConc.: 6.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: final concentration of cytochrome b6f dimers 30uM
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
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 / Nominal magnification: 105000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40.7 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7784
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.0particle selection
2EPU2.10.0.1941RELimage acquisition
4cryoSPARC4.4.0CTF correction
7UCSF ChimeraX1.7model fitting
9Coot0.9.8model refinement
10PHENIX1.20.1-4487model refinement
11cryoSPARC4.4.0initial Euler assignment
13cryoSPARC4.4.0classification
14cryoSPARC4.4.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6110000 / Details: template picker, TOPAZ
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 1.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1320078 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7ZYV

7zyv


Accession code: 7ZYV / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00216540
ELECTRON MICROSCOPYf_angle_d0.48322622
ELECTRON MICROSCOPYf_dihedral_angle_d11.6255904
ELECTRON MICROSCOPYf_chiral_restr0.0412530
ELECTRON MICROSCOPYf_plane_restr0.0042778

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