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- PDB-8asj: Four subunit cytochrome b-c1 complex from Rhodobacter sphaeroides... -

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Basic information

Entry
Database: PDB / ID: 8asj
TitleFour subunit cytochrome b-c1 complex from Rhodobacter sphaeroides in native nanodiscs - focussed refinement in the b-c conformation
Components
  • Cytochrome b
  • Cytochrome b-c1 subunit IV
  • Cytochrome c1
  • Ubiquinol-cytochrome c reductase iron-sulfur subunit
KeywordsOXIDOREDUCTASE / cyt bc1 complex III membrane protein electron transport quinone cytochrome
Function / homology
Function and homology information


respiratory chain complex III / quinol-cytochrome-c reductase / ubiquinol-cytochrome-c reductase activity / respiratory electron transport chain / 2 iron, 2 sulfur cluster binding / electron transfer activity / heme binding / membrane / metal ion binding / plasma membrane
Similarity search - Function
Ubiquitinol-cytochrome C reductase, Fe-S subunit, TAT signal / Ubiquitinol-cytochrome C reductase Fe-S subunit TAT signal / Cytochrome b / Twin-arginine translocation pathway, signal sequence, bacterial/archaeal / : / Ubiquinol-cytochrome c reductase, iron-sulphur subunit / Cytochrome c1 / Cytochrome C1 family / : / Cytochrome b/b6, C-terminal ...Ubiquitinol-cytochrome C reductase, Fe-S subunit, TAT signal / Ubiquitinol-cytochrome C reductase Fe-S subunit TAT signal / Cytochrome b / Twin-arginine translocation pathway, signal sequence, bacterial/archaeal / : / Ubiquinol-cytochrome c reductase, iron-sulphur subunit / Cytochrome c1 / Cytochrome C1 family / : / 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 / 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 / Cytochrome c family profile. / Cytochrome c-like domain / Cytochrome c-like domain superfamily / Twin arginine translocation (Tat) signal profile. / Twin-arginine translocation pathway, signal sequence
Similarity search - Domain/homology
FE2/S2 (INORGANIC) CLUSTER / HEME C / PROTOPORPHYRIN IX CONTAINING FE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / UBIQUINONE-10 / Ubiquinol-cytochrome c reductase iron-sulfur subunit / Cytochrome b / Cytochrome c1 / Cytochrome b-c1 subunit IV
Similarity search - Component
Biological speciesCereibacter sphaeroides 2.4.1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsSwainsbury, D.J.K. / Hawkings, F.R. / Martin, E.C. / Musial, S. / Salisbury, J.H. / Jackson, P.J. / Farmer, D.A. / Johnson, M.P. / Siebert, C.A. / Hitchcock, A. / Hunter, C.N.
Funding supportEuropean Union, United Kingdom, 5items
OrganizationGrant numberCountry
European Research Council (ERC)854126European Union
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M000265/1 United Kingdom
Wellcome Trustnr29785 United Kingdom
Royal SocietyURF/R1/19154 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/V006630/1 United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: Cryo-EM structure of the four-subunit cytochrome complex in styrene maleic acid nanodiscs.
Authors: David J K Swainsbury / Frederick R Hawkings / Elizabeth C Martin / Sabina Musiał / Jack H Salisbury / Philip J Jackson / David A Farmer / Matthew P Johnson / C Alistair Siebert / Andrew ...Authors: David J K Swainsbury / Frederick R Hawkings / Elizabeth C Martin / Sabina Musiał / Jack H Salisbury / Philip J Jackson / David A Farmer / Matthew P Johnson / C Alistair Siebert / Andrew Hitchcock / C Neil Hunter /
Abstract: Cytochrome complexes are ubiquinol:cytochrome oxidoreductases, and as such, they are centrally important components of respiratory and photosynthetic electron transfer chains in many species of ...Cytochrome complexes are ubiquinol:cytochrome oxidoreductases, and as such, they are centrally important components of respiratory and photosynthetic electron transfer chains in many species of bacteria and in mitochondria. The minimal complex has three catalytic components, which are cytochrome , cytochrome , and the Rieske iron-sulfur subunit, but the function of mitochondrial cytochrome complexes is modified by up to eight supernumerary subunits. The cytochrome complex from the purple phototrophic bacterium   has a single supernumerary subunit called subunit IV, which is absent from current structures of the complex. In this work we use the styrene-maleic acid copolymer to purify the cytochrome complex in native lipid nanodiscs, which retains the labile subunit IV, annular lipids, and natively bound quinones. The catalytic activity of the four-subunit cytochrome complex is threefold higher than that of the complex lacking subunit IV. To understand the role of subunit IV, we determined the structure of the four-subunit complex at 2.9 Å using single particle cryogenic electron microscopy. The structure shows the position of the transmembrane domain of subunit IV, which lies across the transmembrane helices of the Rieske and cytochrome subunits. We observe a quinone at the Q quinone-binding site and show that occupancy of this site is linked to conformational changes in the Rieske head domain during catalysis. Twelve lipids were structurally resolved, making contacts with the Rieske and cytochrome subunits, with some spanning both of the two monomers that make up the dimeric complex.
History
DepositionAug 19, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 15, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ubiquinol-cytochrome c reductase iron-sulfur subunit
B: Cytochrome b
C: Cytochrome c1
D: Cytochrome b-c1 subunit IV
E: Ubiquinol-cytochrome c reductase iron-sulfur subunit
F: Cytochrome b
G: Cytochrome c1
H: Cytochrome b-c1 subunit IV
hetero molecules


Theoretical massNumber of molelcules
Total (without water)244,03229
Polymers230,1868
Non-polymers13,84621
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, Particle dimensions and high-resolution map consistent with a dimeric complex, native gel electrophoresis, Native PAGE shows the predominant species is a heterodimer
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area41960 Å2
ΔGint-501 kcal/mol
Surface area74720 Å2
MethodPISA

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Components

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Protein , 4 types, 8 molecules AEBFCGDH

#1: Protein Ubiquinol-cytochrome c reductase iron-sulfur subunit


Mass: 19928.375 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Cereibacter sphaeroides 2.4.1 (bacteria)
Strain: ATCC 17023 / DSM 158 / JCM 6121 / CCUG 31486 / LMG 2827 / NBRC 12203 / NCIMB 8253 / ATH 2.4.1.
References: UniProt: Q3IY09, quinol-cytochrome-c reductase
#2: Protein Cytochrome b


Mass: 50087.422 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Cereibacter sphaeroides 2.4.1 (bacteria)
Strain: ATCC 17023 / DSM 158 / JCM 6121 / CCUG 31486 / LMG 2827 / NBRC 12203 / NCIMB 8253 / ATH 2.4.1.
References: UniProt: Q3IY10
#3: Protein Cytochrome c1


Mass: 30661.664 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Cereibacter sphaeroides 2.4.1 (bacteria)
Strain: ATCC 17023 / DSM 158 / JCM 6121 / CCUG 31486 / LMG 2827 / NBRC 12203 / NCIMB 8253 / ATH 2.4.1.
References: UniProt: Q3IY11
#4: Protein Cytochrome b-c1 subunit IV


Mass: 14415.301 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Cereibacter sphaeroides 2.4.1 (bacteria)
Strain: ATCC 17023 / DSM 158 / JCM 6121 / CCUG 31486 / LMG 2827 / NBRC 12203 / NCIMB 8253 / ATH 2.4.1.
References: UniProt: Q3J2Z2

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Non-polymers , 5 types, 21 molecules

#5: 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
#6: Chemical
ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE


Mass: 744.034 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C41H78NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: DOPE, phospholipid*YM
#7: 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
#8: Chemical ChemComp-HEC / HEME C


Mass: 618.503 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H34FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#9: Chemical ChemComp-U10 / UBIQUINONE-10 / Coenzyme Q10


Mass: 863.343 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C59H90O4 / 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: Four subunit cytochrome b-c1 complex from Rhodobacter sphaeroides
Type: COMPLEX
Details: Four subunit cytochrome b-c1 complex from Rhodobacter sphaeroides purified in SMA copolymer lipid nanodiscs
Entity ID: #1-#4 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Cereibacter sphaeroides (bacteria) / Strain: 2.4.1
Buffer solutionpH: 8
Details: Solutions were freshly prepared from concentrated stock solutions.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris2-Amino-2-(hydroxymethyl)-1,3-propanediol1
2200 mMNaClNaCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Monodisperse particles consisting of four-subunit cyt b-c1 solubilised in styrene maleic acid nanodiscs
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: 80 % / Chamber temperature: 298 K
Details: 15 ul of sample was applied to the grid, incubated for 30 s, blotted for 4 s then plunged in liquid ethane.

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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 FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 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: 1.13 sec. / Electron dose: 1 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 15867
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1crYOLO1.7.5particle selectionModel trained on this dataset
2EPUimage acquisition
4CTFFIND4.1.14CTF correctionRun within RELION 3.1
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classificationAlignment free masked classification on previous consensus reconstruction
13RELION3.13D reconstruction
Image processingDetails: Images were motion corrected using Motiocorr 2 within RELION using 5 x 5 patches.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4060135
Details: Particles were picked using crYOLO 1.7.5 using a model trained on this dataset
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.75 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 72118 / Algorithm: FOURIER SPACE
Details: Refinement performed without alignment from particles within a single class after focussed refinement
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00315206
ELECTRON MICROSCOPYf_angle_d0.80320746
ELECTRON MICROSCOPYf_dihedral_angle_d20.9985327
ELECTRON MICROSCOPYf_chiral_restr0.0462147
ELECTRON MICROSCOPYf_plane_restr0.0092597

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