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- PDB-7r0w: 2.8 Angstrom cryo-EM structure of the dimeric cytochrome b6f-PetP... -

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Entry
Database: PDB / ID: 7r0w
Title2.8 Angstrom cryo-EM structure of the dimeric cytochrome b6f-PetP complex from Synechocystis sp. PCC 6803 with natively bound lipids and plastoquinone molecules
Components
  • (Cytochrome b6-f complex subunit ...) x 4
  • Cytochrome B6Cytochrome b
  • Cytochrome b6Cytochrome b
  • Cytochrome f
  • Rieske domain, PetC
  • Uncharacterized protein Cp097, conserved in cyanobacteria
KeywordsPHOTOSYNTHESIS / Complex / cytochrome / membrane protein / electron transport
Function / homology
Function and homology information


: / cytochrome b6f complex / electron transporter, transferring electrons within cytochrome b6/f complex of photosystem II activity / cytochrome complex assembly / photosynthetic electron transport chain / plasma membrane-derived thylakoid membrane / electron transporter, transferring electrons within the cyclic electron transport pathway of photosynthesis activity / photosynthesis / respiratory electron transport chain / membrane => GO:0016020 ...: / cytochrome b6f complex / electron transporter, transferring electrons within cytochrome b6/f complex of photosystem II activity / cytochrome complex assembly / photosynthetic electron transport chain / plasma membrane-derived thylakoid membrane / electron transporter, transferring electrons within the cyclic electron transport pathway of photosynthesis activity / photosynthesis / respiratory electron transport chain / membrane => GO:0016020 / electron transfer activity / iron ion binding / heme binding / metal ion binding
Similarity search - Function
Cytochrome b6f, subunit Tsr0524-like / Cytochrome b6f, subunit Tsr0524-like / 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 ...Cytochrome b6f, subunit Tsr0524-like / Cytochrome b6f, subunit Tsr0524-like / 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 / 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
Similarity search - Domain/homology
Chem-2WA / Chem-6PL / CHLOROPHYLL A / beta,beta-caroten-4-one / FE2/S2 (INORGANIC) CLUSTER / HEME C / PROTOPORPHYRIN IX CONTAINING FE / EICOSANE / 1,2-DISTEAROYL-MONOGALACTOSYL-DIGLYCERIDE / Chem-PGV ...Chem-2WA / Chem-6PL / CHLOROPHYLL A / beta,beta-caroten-4-one / FE2/S2 (INORGANIC) CLUSTER / HEME C / PROTOPORPHYRIN IX CONTAINING FE / EICOSANE / 1,2-DISTEAROYL-MONOGALACTOSYL-DIGLYCERIDE / Chem-PGV / Chem-PL9 / Chem-SQD / Uncharacterized protein Cp097, conserved in cyanobacteria / Cytochrome B6 / Cytochrome f / Cytochrome b6-f complex subunit 4 / Cytochrome b6-f complex subunit 8 / Cytochrome b6-f complex subunit 5 / Cytochrome b6-f complex subunit 7 / Cytochrome b6
Similarity search - Component
Biological speciesSynechocystis (bacteria)
Synechocystis sp. PCC 6803 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsFarmer, D.F. / Proctor, M.S. / Malone, L.A. / Swainsbury, D.P.K. / Hawkings, F.R. / Hitchcock, A. / Johnson, M.P.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M011151/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/V006630/1 United Kingdom
Leverhulme TrustRPG-2019-045 United Kingdom
Wellcome Trustnr21005 United Kingdom
CitationJournal: Biochem J / Year: 2022
Title: Cryo-EM structures of the Synechocystis sp. PCC 6803 cytochrome b6f complex with and without the regulatory PetP subunit.
Authors: Matthew S Proctor / Lorna A Malone / David A Farmer / David J K Swainsbury / Frederick R Hawkings / Federica Pastorelli / Thomas Z Emrich-Mills / C Alistair Siebert / C Neil Hunter / Matthew ...Authors: Matthew S Proctor / Lorna A Malone / David A Farmer / David J K Swainsbury / Frederick R Hawkings / Federica Pastorelli / Thomas Z Emrich-Mills / C Alistair Siebert / C Neil Hunter / Matthew P Johnson / Andrew Hitchcock /
Abstract: In oxygenic photosynthesis, the cytochrome b6f (cytb6f) complex links the linear electron transfer (LET) reactions occurring at photosystems I and II and generates a transmembrane proton gradient via ...In oxygenic photosynthesis, the cytochrome b6f (cytb6f) complex links the linear electron transfer (LET) reactions occurring at photosystems I and II and generates a transmembrane proton gradient via the Q-cycle. In addition to this central role in LET, cytb6f also participates in a range of processes including cyclic electron transfer (CET), state transitions and photosynthetic control. Many of the regulatory roles of cytb6f are facilitated by auxiliary proteins that differ depending upon the species, yet because of their weak and transient nature the structural details of these interactions remain unknown. An apparent key player in the regulatory balance between LET and CET in cyanobacteria is PetP, a ∼10 kDa protein that is also found in red algae but not in green algae and plants. Here, we used cryogenic electron microscopy to determine the structure of the Synechocystis sp. PCC 6803 cytb6f complex in the presence and absence of PetP. Our structures show that PetP interacts with the cytoplasmic side of cytb6f, displacing the C-terminus of the PetG subunit and shielding the C-terminus of cytochrome b6, which binds the heme cn cofactor that is suggested to mediate CET. The structures also highlight key differences in the mode of plastoquinone binding between cyanobacterial and plant cytb6f complexes, which we suggest may reflect the unique combination of photosynthetic and respiratory electron transfer in cyanobacterial thylakoid membranes. The structure of cytb6f from a model cyanobacterial species amenable to genetic engineering will enhance future site-directed mutagenesis studies of structure-function relationships in this crucial ET complex.
History
DepositionFeb 2, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 6, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 27, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
X: Uncharacterized protein Cp097, conserved in cyanobacteria
I: Cytochrome b6
J: Cytochrome b6-f complex subunit 4
K: Cytochrome f
M: Cytochrome B6
N: Cytochrome b6-f complex subunit 7
O: Cytochrome b6-f complex subunit 5
P: Cytochrome b6-f complex subunit 8
A: Cytochrome b6
B: Cytochrome b6-f complex subunit 4
C: Cytochrome f
D: Rieske domain, PetC
E: Cytochrome B6
F: Cytochrome b6-f complex subunit 7
G: Cytochrome b6-f complex subunit 5
H: Cytochrome b6-f complex subunit 8
L: Rieske domain, PetC
V: Uncharacterized protein Cp097, conserved in cyanobacteria
hetero molecules


Theoretical massNumber of molelcules
Total (without water)258,01645
Polymers240,58818
Non-polymers17,42827
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis, Blue native PAGE
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 8 molecules XVIAKCDL

#1: Protein Uncharacterized protein Cp097, conserved in cyanobacteria


Mass: 7231.374 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Synechocystis (bacteria) / Strain: PCC 6714 / Gene: cp097, D082_20950 / Plasmid: pET28a / Details (production host): 5' 6xHis tag / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0A068MZK2
#2: Protein Cytochrome b6 / Cytochrome b


Mass: 25075.533 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / References: UniProt: Q57038
#4: Protein Cytochrome f /


Mass: 35259.340 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / References: UniProt: P26287
#9: Protein Rieske domain, PetC


Mass: 20562.344 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Synechocystis sp. PCC 6803 (bacteria)

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Cytochrome b6-f complex subunit ... , 4 types, 8 molecules JBNFOGPH

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


Mass: 17455.783 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / References: UniProt: P27589
#6: Protein/peptide Cytochrome b6-f complex subunit 7 / Cytochrome b6-f complex subunit PetM / Cytochrome b6-f complex subunit VII


Mass: 3827.555 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / References: UniProt: P74810
#7: Protein/peptide Cytochrome b6-f complex subunit 5 / Cytochrome b6-f complex subunit PetG / Cytochrome b6-f complex subunit V


Mass: 4177.004 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / References: UniProt: P74149
#8: Protein/peptide Cytochrome b6-f complex subunit 8 / Cytochrome b6-f complex subunit PetN / Cytochrome b6-f complex subunit VIII


Mass: 3328.965 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Synechocystis sp. PCC 6803 (bacteria) / Strain: PCC 6803 / Kazusa / References: UniProt: P72717

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Protein/peptide , 1 types, 2 molecules ME

#5: Protein/peptide Cytochrome B6 / Cytochrome b


Mass: 3376.149 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Synechocystis sp. PCC 6803 (bacteria) / References: UniProt: A0A6P1VG96

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Non-polymers , 13 types, 29 molecules

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


Mass: 616.487 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical
ChemComp-PGV / (1R)-2-{[{[(2S)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL (11E)-OCTADEC-11-ENOATE / PHOSPHATIDYLGLYCEROL / 2-VACCENOYL-1-PALMITOYL-SN-GLYCEROL-3-PHOSPHOGLYCEROL / Phosphatidylglycerol


Mass: 749.007 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C40H77O10P / Comment: phospholipid*YM
#12: Chemical ChemComp-ECH / beta,beta-caroten-4-one / echinenone / Echinenone


Mass: 550.856 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C40H54O
#13: 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 / Plastoquinone


Mass: 749.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C53H80O2
#14: Chemical ChemComp-CLA / CHLOROPHYLL A / Chlorophyll a


Mass: 893.489 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C55H72MgN4O5 / Feature type: SUBJECT OF INVESTIGATION
#15: Chemical ChemComp-HEC / HEME C / Heme C


Mass: 618.503 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H34FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#16: Chemical ChemComp-LMG / 1,2-DISTEAROYL-MONOGALACTOSYL-DIGLYCERIDE


Mass: 787.158 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C45H86O10
#17: Chemical ChemComp-6PL / (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE / 1-PALMITOYL-2-STEAROYL-SN-GLYCERO-3-PHOSPHOCHOLINE


Mass: 763.100 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C42H85NO8P / Comment: phospholipid*YM
#18: Chemical ChemComp-2WA / (1S,8E)-1-{[(2S)-1-hydroxy-3-{[(1S)-1-hydroxypentadecyl]oxy}propan-2-yl]oxy}heptadec-8-en-1-ol


Mass: 570.927 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C35H70O5
#19: Chemical ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER / Iron–sulfur cluster


Mass: 175.820 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe2S2 / Feature type: SUBJECT OF INVESTIGATION
#20: 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
#21: Chemical ChemComp-LFA / EICOSANE / LIPID FRAGMENT / Icosane


Mass: 282.547 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H42
#22: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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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 in association with PetP / Type: COMPLEX
Details: strep-PetA used to purify cytochrome b6f complex from Synechocystis sp. PCC 6803. 6xHis-PetP recombinantly expressed in E. coli used to pull down cytochrome b6f complex and form the cytochrome b6f-PetP complex.
Entity ID: #1-#9 / Source: MULTIPLE SOURCES
Source (natural)Organism: Synechocystis sp. PCC 6803 (bacteria)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
125 mLSodium phosphateNa3PO41
210 mMMagnesium chlorideMgCl21
3150 mMSodium chlorideNaClSodium chloride1
40.02 % (w/v)glyco-diosgeninC56H92O251
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was monodisperse but not present in every hole
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 70 % / Chamber temperature: 288 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.5 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 20133
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20_4459refinement
PHENIX1.20_4459refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1.14CTF correction
7Coot0.9.2model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1169445
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 152860 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7PPW

7ppw
PDB Unreleased entry

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 93 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004517270
ELECTRON MICROSCOPYf_angle_d0.946823519
ELECTRON MICROSCOPYf_chiral_restr0.1252557
ELECTRON MICROSCOPYf_plane_restr0.00632866
ELECTRON MICROSCOPYf_dihedral_angle_d18.44122688

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