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- EMDB-15617: Four subunit cytochrome b-c1 complex from Rhodobacter sphaeroides... -

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Entry
Database: EMDB / ID: EMD-15617
TitleFour subunit cytochrome b-c1 complex from Rhodobacter sphaeroides in native nanodiscs - focussed refinement in the b-c conformation
Map dataPostprocessed masked map from RELION 3.1
Sample
  • Complex: Four subunit cytochrome b-c1 complex from Rhodobacter sphaeroides
    • Protein or peptide: Ubiquinol-cytochrome c reductase iron-sulfur subunit
    • Protein or peptide: Cytochrome b
    • Protein or peptide: Cytochrome c1
    • Protein or peptide: Cytochrome b-c1 subunit IV
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: HEME C
  • Ligand: UBIQUINONE-10
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
Ubiquinol-cytochrome c reductase iron-sulfur subunit / Cytochrome b / Cytochrome c1 / Cytochrome b-c1 subunit IV
Similarity search - Component
Biological speciesCereibacter sphaeroides (bacteria) / Cereibacter sphaeroides 2.4.1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.75 Å
AuthorsSwainsbury DJK / Hawkings FR / Martin EC / Musial S / Salisbury JH / Jackson PJ / Farmer DA / Johnson MP / Siebert CA / Hitchcock A / Hunter CN
Funding supportEuropean Union, United Kingdom, 5 items
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, 2022-
Header (metadata) releaseMar 15, 2023-
Map releaseMar 15, 2023-
UpdateMar 22, 2023-
Current statusMar 22, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15617.png

Downloads & links

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Map

FileDownload / File: emd_15617.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed masked map from RELION 3.1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.17 Å/pix.
x 220 pix.
= 257.796 Å		1.17 Å/pix.
x 220 pix.
= 257.796 Å		1.17 Å/pix.
x 220 pix.
= 257.796 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1718 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02
Minimum - Maximum-0.13379344 - 0.19981617
Average (Standard dev.)0.000116942116 (±0.0050184187)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 257.796 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Locally sharpened map using LocScale

Fileemd_15617_additional_1.map
AnnotationLocally sharpened map using LocScale
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_15617_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_15617_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Four subunit cytochrome b-c1 complex from Rhodobacter sphaeroides

EntireName: Four subunit cytochrome b-c1 complex from Rhodobacter sphaeroides
Components
  • Complex: Four subunit cytochrome b-c1 complex from Rhodobacter sphaeroides
    • Protein or peptide: Ubiquinol-cytochrome c reductase iron-sulfur subunit
    • Protein or peptide: Cytochrome b
    • Protein or peptide: Cytochrome c1
    • Protein or peptide: Cytochrome b-c1 subunit IV
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: HEME C
  • Ligand: UBIQUINONE-10

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Supramolecule #1: Four subunit cytochrome b-c1 complex from Rhodobacter sphaeroides

SupramoleculeName: Four subunit cytochrome b-c1 complex from Rhodobacter sphaeroides
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#4
Details: Four subunit cytochrome b-c1 complex from Rhodobacter sphaeroides purified in SMA copolymer lipid nanodiscs
Source (natural)Organism: Cereibacter sphaeroides (bacteria) / Strain: 2.4.1

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

MacromoleculeName: Ubiquinol-cytochrome c reductase iron-sulfur subunit / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: quinol-cytochrome-c reductase
Source (natural)Organism: 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.
Molecular weightTheoretical: 19.928375 KDa
SequenceString:
MSNAEDHAGT RRDFLYYATA GAGAVATGAA VWPLINQMNP SADVQALASI FVDVSSVEPG VQLTVKFLGK PIFIRRRTEA DIELGRSVQ LGQLVDTNAR NANIDAGAEA TDQNRTLDEA GEWLVMWGVC THLGCVPIGG VSGDFGGWFC PCHGSHYDSA G RIRKGPAP ENLPIPLAKF IDETTIQLG

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Macromolecule #2: Cytochrome b

MacromoleculeName: Cytochrome b / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: 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.
Molecular weightTheoretical: 50.087422 KDa
SequenceString: MSGIPHDHYE PRTGIEKWLH SRLPIVALAY DTIMIPTPRN LNWMWIWGVV LAFCLVLQIV TGIVLAMHYT PHVDLAFASV EHIMRNVNG GFMLRYLHAN GASLFFIAVY LHIFRGLYYG SYKAPREVTW IVGMLIYLAM MATAFMGYVL PWGQMSFWGA T VITGLFGA ...String:
MSGIPHDHYE PRTGIEKWLH SRLPIVALAY DTIMIPTPRN LNWMWIWGVV LAFCLVLQIV TGIVLAMHYT PHVDLAFASV EHIMRNVNG GFMLRYLHAN GASLFFIAVY LHIFRGLYYG SYKAPREVTW IVGMLIYLAM MATAFMGYVL PWGQMSFWGA T VITGLFGA IPGIGHSIQT WLLGGPAVDN ATLNRFFSLH YLLPFVIAAL VAIHIWAFHS TGNNNPTGVE VRRTSKAEAQ KD TVPFWPY FIIKDVFALA VVLLVFFAIV GFMPNYLGHP DNYIEANPLS TPAHIVPEWY FLPFYAILRA FTADVWVVQI ANF ISFGII DAKFFGVLAM FGAILVMALV PWLDTSPVRS GRYRPMFKIY FWLLAADFVI LTWVGAQQTT FPYDWISLIA SAYW FAYFL VILPILGAIE KPVAPPATIE EDFNAHYSPA TGGTKTVVAE

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Macromolecule #3: Cytochrome c1

MacromoleculeName: Cytochrome c1 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: 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.
Molecular weightTheoretical: 30.661664 KDa
SequenceString: MIRKLTLTAA TALALSGGAA MAAGGGHVED VPFSFEGPFG TFDQHQLQRG LQVYTEVCAA CHGMKFVPIR SLSEPGGPEL PEDQVRAYA TQFTVTDEET GEDREGKPTD HFPHSALENA PDLSLMAKAR AGFHGPMGTG ISQLFNGIGG PEYIYSVLTG F PEEPPKCA ...String:
MIRKLTLTAA TALALSGGAA MAAGGGHVED VPFSFEGPFG TFDQHQLQRG LQVYTEVCAA CHGMKFVPIR SLSEPGGPEL PEDQVRAYA TQFTVTDEET GEDREGKPTD HFPHSALENA PDLSLMAKAR AGFHGPMGTG ISQLFNGIGG PEYIYSVLTG F PEEPPKCA EGHEPDGFYY NRAFQNGSVP DTCKDANGVK TTAGSWIAMP PPLMDDLVEY ADGHDASVHA MAEDVSAFLM WA AEPKLMA RKQAGFTAVM FLTVLSVLLY LTNKRLWAGV KGKKKTNV

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Macromolecule #4: Cytochrome b-c1 subunit IV

MacromoleculeName: Cytochrome b-c1 subunit IV / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: 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.
Molecular weightTheoretical: 14.415301 KDa
SequenceString:
MFSFIDDIPS FEQIKARVRD DLRKHGWEKR WNDSRLVQKS RELLNDEELK IDPATWIWKR MPSREEVAAR RQRDFETVWK YRYRLGGFA SGALLALALA GIFSTGNFGG SSDAGNRPSV VYPIE

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Macromolecule #5: FE2/S2 (INORGANIC) CLUSTER

MacromoleculeName: FE2/S2 (INORGANIC) CLUSTER / type: ligand / ID: 5 / Number of copies: 2 / Formula: FES
Molecular weightTheoretical: 175.82 Da
Chemical component information

ChemComp-FES:
FE2/S2 (INORGANIC) CLUSTER

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Macromolecule #6: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 6 / Number of copies: 12 / Formula: PEE
Molecular weightTheoretical: 744.034 Da
Chemical component information

ChemComp-PEE:
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE, phospholipid*YM

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Macromolecule #7: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 7 / Number of copies: 4 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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Macromolecule #8: HEME C

MacromoleculeName: HEME C / type: ligand / ID: 8 / Number of copies: 2 / Formula: HEC
Molecular weightTheoretical: 618.503 Da
Chemical component information

ChemComp-HEC:
HEME C

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Macromolecule #9: UBIQUINONE-10

MacromoleculeName: UBIQUINONE-10 / type: ligand / ID: 9 / Number of copies: 1 / Formula: U10
Molecular weightTheoretical: 863.343 Da
Chemical component information

ChemComp-U10:
UBIQUINONE-10

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mM2-Amino-2-(hydroxymethyl)-1,3-propanediolTris
200.0 mMNaClNaCl

Details: Solutions were freshly prepared from concentrated stock solutions.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 298 K / Instrument: LEICA EM GP
Details: 15 ul of sample was applied to the grid, incubated for 30 s, blotted for 4 s then plunged in liquid ethane..
DetailsMonodisperse particles consisting of four-subunit cyt b-c1 solubilised in styrene maleic acid nanodiscs

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 15867 / Average exposure time: 1.13 sec. / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsImages were motion corrected using Motiocorr 2 within RELION using 5 x 5 patches.
Particle selectionNumber selected: 4060135
Details: Particles were picked using crYOLO 1.7.5 using a model trained on this dataset
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.75 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: Refinement performed without alignment from particles within a single class after focussed refinement
Number images used: 72118
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 3.1)
Software - details: Alignment free masked classification on previous consensus reconstruction
Details: One high quality class was selected for final refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-8asj:
Four subunit cytochrome b-c1 complex from Rhodobacter sphaeroides in native nanodiscs - focussed refinement in the b-c conformation

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