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- PDB-7zyv: Cryo-EM structure of catalytically active Spinacia oleracea cytoc... -

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Basic information

Entry
Database: PDB / ID: 7zyv
TitleCryo-EM structure of catalytically active Spinacia oleracea cytochrome b6f in complex with endogenous plastoquinones at 2.13 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
KeywordsPHOTOSYNTHESIS / cytochrome / Spinacia oleracea / plastoquinones / cryo-EM
Function / homology
Function and homology information


PSII associated light-harvesting complex II binding / chloroplast photosystem I binding / chloroplast photosystem II binding / plastoquinol--plastocyanin reductase activity / cytochrome b6f complex / plastoquinol-plastocyanin reductase / electron transporter, transferring electrons within cytochrome b6/f complex of photosystem II activity / thylakoid membrane / cytochrome complex assembly / : ...PSII associated light-harvesting complex II binding / chloroplast photosystem I binding / chloroplast photosystem II binding / plastoquinol--plastocyanin reductase activity / cytochrome b6f complex / plastoquinol-plastocyanin reductase / electron transporter, transferring electrons within cytochrome b6/f complex of photosystem II activity / thylakoid membrane / cytochrome complex assembly / : / photosynthetic electron transport chain / ubiquinol-cytochrome-c reductase activity / mitochondrial electron transport, ubiquinol to cytochrome c / electron transporter, transferring electrons within the cyclic electron transport pathway of photosynthesis activity / chloroplast thylakoid membrane / response to light stimulus / localization / photosynthesis / chloroplast / 2 iron, 2 sulfur cluster binding / electron transfer activity / iron ion binding / lipid binding / heme binding / membrane / metal ion binding / 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 / PetM of cytochrome b6/f complex subunit 7 ...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 / Cytochrome b/b6, N-terminal / Cytochrome b/b6-like domain superfamily / Cytochrome b/b6 N-terminal region profile. / Di-haem cytochrome, transmembrane / Rudiment single hybrid motif / Rieske iron-sulphur protein / Rieske [2Fe-2S] iron-sulphur domain / Rieske [2Fe-2S] 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 / Chem-PL9 / Chem-SQD / Cytochrome b6-f complex subunit 7 / Cytochrome b6 / Cytochrome b6-f complex subunit 4 ...BETA-CAROTENE / CHLOROPHYLL A / FE2/S2 (INORGANIC) CLUSTER / HEME C / PROTOPORPHYRIN IX CONTAINING FE / Chem-PL9 / Chem-SQD / Cytochrome b6-f complex subunit 7 / 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: 2.13 Å
AuthorsSarewicz, M. / Szwalec, M. / Pintscher, S. / Indyka, P. / Rawski, M. / Pietras, R. / Mielecki, B. / Koziej, L. / Jaciuk, M. / Glatt, S. / Osyczka, A.
Funding support Poland, 2items
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
CitationJournal: Sci Adv / Year: 2023
Title: High-resolution cryo-EM structures of plant cytochrome bf at work.
Authors: Marcin Sarewicz / Mateusz Szwalec / Sebastian Pintscher / Paulina Indyka / Michał Rawski / Rafał Pietras / Bohun Mielecki / Łukasz Koziej / Marcin Jaciuk / Sebastian Glatt / Artur Osyczka /
Abstract: Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome bf (Cyt bf) is known as one of the key steps of photosynthesis, but the ...Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome bf (Cyt bf) is known as one of the key steps of photosynthesis, but the catalytic mechanism in the plastoquinone oxidation site (Q) remains elusive. Here, we describe two high-resolution cryo-EM structures of the spinach Cyt bf homodimer with endogenous plastoquinones and in complex with plastocyanin. Three plastoquinones are visible and line up one after another head to tail near Q in both monomers, indicating the existence of a channel in each monomer. Therefore, quinones appear to flow through Cyt bf in one direction, transiently exposing the redox-active ring of quinone during catalysis. Our work proposes an unprecedented one-way traffic model that explains efficient quinol oxidation during photosynthesis and respiration.
History
DepositionMay 25, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 25, 2023Provider: repository / Type: Initial release

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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
R: Thylakoid soluble phosphoprotein
Q: Thylakoid soluble phosphoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)290,61150
Polymers271,40718
Non-polymers19,20432
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 8 molecules AICKDLRQ

#1: Protein Cytochrome b6


Mass: 24186.504 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / 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) / 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)
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) / 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) / 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) / References: UniProt: Q9M3L0
#6: Protein Cytochrome b6-f complex subunit 7


Mass: 12953.800 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Spinacia oleracea (spinach) / References: UniProt: A0A0K9RMM1
#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) / 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) / References: UniProt: P61045

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Non-polymers , 8 types, 32 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-CLA / CHLOROPHYLL A


Mass: 893.489 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C55H72MgN4O5 / Feature type: SUBJECT OF INVESTIGATION
#13: Chemical
ChemComp-UMQ / UNDECYL-MALTOSIDE / UNDECYL-BETA-D-MALTOPYRANOSIDE


Mass: 496.589 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C23H44O11 / Feature type: SUBJECT OF INVESTIGATION / Comment: detergent*YM
#14: Chemical
ChemComp-PL9 / 2,3-DIMETHYL-5-(3,7,11,15,19,23,27,31,35-NONAMETHYL-2,6,10,14,18,22,26,30,34-HEXATRIACONTANONAENYL-2,5-CYCLOHEXADIENE-1,4-DIONE-2,3-DIMETHYL-5-SOLANESYL-1,4-BENZOQUINONE / PLASTOQUINONE 9


Mass: 749.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C53H80O2 / 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

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Details

Has ligand of interestY

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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 / Type: COMPLEX / Entity ID: #1-#9 / Source: NATURAL
Molecular weightValue: 0.22 MDa / Experimental value: NO
Source (natural)Organism: Spinacia oleracea (spinach) / Cellular location: thylakoids / Organ: Leaves / Organelle: Chloroplasts
Buffer solutionpH: 8
Details: Buffer and the diluted protein sample were filtered through a 0.22 um syringe filter. Prior to freezing, protein sample was concentrated on centrifugal filter units with 50K MWCO.
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris2-Amino-2-(hydroxymethyl)propane-1,3-diol1
215 mMsodium chlorideNaCl1
30.8 mMundecyl maltoside(2R,3R,4S,5S,6R)-2-[(2R,3S,4R,5R,6R)-4,5-dihydroxy-2-(hydroxymethyl)-6-undecoxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol1
SpecimenConc.: 6.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 / Details: blot time 2 s, wait time 0 s, blot force -1

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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 / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.82 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 7651
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv3.3.1particle selectionblob, template and Topaz picker
2EPU2.1image acquisition
4cryoSPARCv3.3.1CTF correctionpatch CTF, global and local CTF refinement
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10cryoSPARCv3.3.1initial Euler assignmentAb-Initio Reconstruction
11cryoSPARCv3.3.1final Euler assignmentNon-uniform Refinement
12cryoSPARCv3.3.1classificationAb-Initio Reconstruction
13cryoSPARCv3.3.13D reconstructionNon-uniform Refinement
Image processingDetails: 20 eV slit, fully tuned before the experiment
CTF correctionDetails: Non-uniform Refinement with iterative global CTF refinement and anisotropic magnification fitting
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 42951
Details: given number of particles is after blob picker and 2D cleaning
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 685979 / Details: Non-uniform Refinement / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 7QRM

7qrm

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