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- EMDB-33294: Cryo-EM structure of Cytochrome bo3 from Escherichia coli, the st... -

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Basic information

Entry
Database: EMDB / ID: EMD-33294
TitleCryo-EM structure of Cytochrome bo3 from Escherichia coli, the structure complexed with an allosteric inhibitor N4
Map data
Sample
  • Complex: Cytochrome bo3 ubiquinol oxidase
    • Protein or peptide: Ubiquinol oxidase subunit 2
    • Protein or peptide: Cytochrome bo(3) ubiquinol oxidase subunit 3
    • Protein or peptide: Cytochrome bo(3) ubiquinol oxidase subunit 4
  • Protein or peptide: Cytochrome bo(3) ubiquinol oxidase subunit 1
  • Ligand: HEME O
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: COPPER (II) ION
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: methyl 3-oxidanyl-5-[oxidanyl(oxidanylidene)-$l^{4}-azanyl]-1-benzothiophene-2-carboxylate
  • Ligand: UNKNOWN ATOM OR ION
Keywordsrespiratory enzyme / membrane protein / heme protein / allosteric inhibitor / OXIDOREDUCTASE
Function / homology
Function and homology information


oxidoreduction-driven active transmembrane transporter activity / cytochrome o ubiquinol oxidase complex / ubiquinol oxidase (H+-transporting) / cytochrome bo3 ubiquinol oxidase activity / aerobic electron transport chain / oxidoreductase activity, acting on diphenols and related substances as donors, oxygen as acceptor / cytochrome-c oxidase activity / ubiquinone binding / electron transport coupled proton transport / proton transmembrane transporter activity ...oxidoreduction-driven active transmembrane transporter activity / cytochrome o ubiquinol oxidase complex / ubiquinol oxidase (H+-transporting) / cytochrome bo3 ubiquinol oxidase activity / aerobic electron transport chain / oxidoreductase activity, acting on diphenols and related substances as donors, oxygen as acceptor / cytochrome-c oxidase activity / ubiquinone binding / electron transport coupled proton transport / proton transmembrane transporter activity / ATP synthesis coupled electron transport / aerobic respiration / respiratory electron transport chain / electron transfer activity / copper ion binding / heme binding / plasma membrane
Similarity search - Function
: / Cytochrome o ubiquinol oxidase, subunit III / Cytochrome o ubiquinol oxidase subunit IV / Cytochrome o ubiquinol oxidase, subunit I / Ubiquinol oxidase subunit III domain / Cytochrome C oxidase subunit IV, prokaryotes / COX aromatic rich motif / Prokaryotic Cytochrome C oxidase subunit IV / COX Aromatic Rich Motif / Cytochrome o ubiquinol oxidase subunit II ...: / Cytochrome o ubiquinol oxidase, subunit III / Cytochrome o ubiquinol oxidase subunit IV / Cytochrome o ubiquinol oxidase, subunit I / Ubiquinol oxidase subunit III domain / Cytochrome C oxidase subunit IV, prokaryotes / COX aromatic rich motif / Prokaryotic Cytochrome C oxidase subunit IV / COX Aromatic Rich Motif / Cytochrome o ubiquinol oxidase subunit II / Ubiquinol oxidase subunit 2, cupredoxin domain / Cytochrome C oxidase subunit II, transmembrane domain / Cytochrome oxidase subunit II transmembrane region profile. / Cytochrome c oxidase subunit III / Cytochrome c oxidase subunit III-like / Cytochrome c oxidase, subunit III, 4-helical bundle / Cytochrome c oxidase subunit III / Heme-copper oxidase subunit III family profile. / Cytochrome c oxidase subunit III-like superfamily / Cytochrome c/quinol oxidase subunit II / Cytochrome C oxidase subunit II, transmembrane domain superfamily / Cytochrome c oxidase, subunit I, copper-binding site / Heme-copper oxidase catalytic subunit, copper B binding region signature. / Cytochrome c oxidase-like, subunit I domain / Cytochrome oxidase subunit I profile. / Cytochrome C oxidase subunit II, periplasmic domain / Cytochrome c oxidase subunit I / Cytochrome c oxidase-like, subunit I superfamily / Cytochrome C and Quinol oxidase polypeptide I / Cytochrome c oxidase subunit II-like C-terminal / Cytochrome oxidase subunit II copper A binding domain profile. / Cupredoxin / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Ubiquinol oxidase subunit 2 / Cytochrome bo(3) ubiquinol oxidase subunit 1 / Cytochrome bo(3) ubiquinol oxidase subunit 3 / Cytochrome bo(3) ubiquinol oxidase subunit 4
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsNishida Y / Shigematsu H / Iwamoto T / Takashima S / Shintani Y
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJCR14M2 Japan
Japan Agency for Medical Research and Development (AMED)JP19im0210617 Japan
CitationJournal: Nat Commun / Year: 2022
Title: Identifying antibiotics based on structural differences in the conserved allostery from mitochondrial heme-copper oxidases.
Authors: Yuya Nishida / Sachiko Yanagisawa / Rikuri Morita / Hideki Shigematsu / Kyoko Shinzawa-Itoh / Hitomi Yuki / Satoshi Ogasawara / Ken Shimuta / Takashi Iwamoto / Chisa Nakabayashi / Waka ...Authors: Yuya Nishida / Sachiko Yanagisawa / Rikuri Morita / Hideki Shigematsu / Kyoko Shinzawa-Itoh / Hitomi Yuki / Satoshi Ogasawara / Ken Shimuta / Takashi Iwamoto / Chisa Nakabayashi / Waka Matsumura / Hisakazu Kato / Chai Gopalasingam / Takemasa Nagao / Tasneem Qaqorh / Yusuke Takahashi / Satoru Yamazaki / Katsumasa Kamiya / Ryuhei Harada / Nobuhiro Mizuno / Hideyuki Takahashi / Yukihiro Akeda / Makoto Ohnishi / Yoshikazu Ishii / Takashi Kumasaka / Takeshi Murata / Kazumasa Muramoto / Takehiko Tosha / Yoshitsugu Shiro / Teruki Honma / Yasuteru Shigeta / Minoru Kubo / Seiji Takashima / Yasunori Shintani /
Abstract: Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to ...Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.
History
DepositionApr 25, 2022-
Header (metadata) releaseDec 21, 2022-
Map releaseDec 21, 2022-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33294.png

Downloads & links

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Map

FileDownload / File: emd_33294.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.19 Å/pix.
x 300 pix.
= 355.599 Å		1.19 Å/pix.
x 300 pix.
= 355.599 Å		1.19 Å/pix.
x 300 pix.
= 355.599 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.18533 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.017
Minimum - Maximum-0.044421144 - 0.09902562
Average (Standard dev.)-0.0000033511142 (±0.002196367)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 355.599 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_33294_msk_1.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_33294_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33294_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Cytochrome bo3 ubiquinol oxidase

EntireName: Cytochrome bo3 ubiquinol oxidase
Components
  • Complex: Cytochrome bo3 ubiquinol oxidase
    • Protein or peptide: Ubiquinol oxidase subunit 2
    • Protein or peptide: Cytochrome bo(3) ubiquinol oxidase subunit 3
    • Protein or peptide: Cytochrome bo(3) ubiquinol oxidase subunit 4
  • Protein or peptide: Cytochrome bo(3) ubiquinol oxidase subunit 1
  • Ligand: HEME O
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: COPPER (II) ION
  • Ligand: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • Ligand: methyl 3-oxidanyl-5-[oxidanyl(oxidanylidene)-$l^{4}-azanyl]-1-benzothiophene-2-carboxylate
  • Ligand: UNKNOWN ATOM OR ION

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Supramolecule #1: Cytochrome bo3 ubiquinol oxidase

SupramoleculeName: Cytochrome bo3 ubiquinol oxidase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2-#4
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 144 KDa

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Macromolecule #1: Cytochrome bo(3) ubiquinol oxidase subunit 1

MacromoleculeName: Cytochrome bo(3) ubiquinol oxidase subunit 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: ubiquinol oxidase (H+-transporting)
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 74.424469 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MFGKLSLDAV PFHEPIVMVT IAGIILGGLA LVGLITYFGK WTYLWKEWLT SVDHKRLGIM YIIVAIVMLL RGFADAIMMR SQQALASAG EAGFLPPHHY DQIFTAHGVI MIFFVAMPFV IGLMNLVVPL QIGARDVAFP FLNNLSFWFT VVGVILVNVS L GVGEFAQT ...String:
MFGKLSLDAV PFHEPIVMVT IAGIILGGLA LVGLITYFGK WTYLWKEWLT SVDHKRLGIM YIIVAIVMLL RGFADAIMMR SQQALASAG EAGFLPPHHY DQIFTAHGVI MIFFVAMPFV IGLMNLVVPL QIGARDVAFP FLNNLSFWFT VVGVILVNVS L GVGEFAQT GWLAYPPLSG IEYSPGVGVD YWIWSLQLSG IGTTLTGINF FVTILKMRAP GMTMFKMPVF TWASLCANVL II ASFPILT VTVALLTLDR YLGTHFFTND MGGNMMMYIN LIWAWGHPEV YILILPVFGV FSEIAATFSR KRLFGYTSLV WAT VCITVL SFIVWLHHFF TMGAGANVNA FFGITTMIIA IPTGVKIFNW LFTMYQGRIV FHSAMLWTIG FIVTFSVGGM TGVL LAVPG ADFVLHNSLF LIAHFHNVII GGVVFGCFAG MTYWWPKAFG FKLNETWGKR AFWFWIIGFF VAFMPLYALG FMGMT RRLS QQIDPQFHTM LMIAASGAVL IALGILCLVI QMYVSIRDRD QNRDLTGDPW GGRTLEWATS SPPPFYNFAV VPHVHE RDA FWEMKEKGEA YKKPDHYEEI HMPKNSGAGI VIAAFSTIFG FAMIWHIWWL AIVGFAGMII TWIVKSFDED VDYYVPV AE IEKLENQHFD EITKAGLKNG N

UniProtKB: Cytochrome bo(3) ubiquinol oxidase subunit 1

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Macromolecule #2: Ubiquinol oxidase subunit 2

MacromoleculeName: Ubiquinol oxidase subunit 2 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 36.209559 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRLRKYNKSL GWLSLFAGTV LLSGCNSALL DPKGQIGLEQ RSLILTAFGL MLIVVIPAIL MAVGFAWKYR ASNKDAKYSP NWSHSNKVE AVVWTVPILI IIFLAVLTWK TTHALEPSKP LAHDEKPITI EVVSMDWKWF FIYPEQGIAT VNEIAFPANT P VYFKVTSN ...String:
MRLRKYNKSL GWLSLFAGTV LLSGCNSALL DPKGQIGLEQ RSLILTAFGL MLIVVIPAIL MAVGFAWKYR ASNKDAKYSP NWSHSNKVE AVVWTVPILI IIFLAVLTWK TTHALEPSKP LAHDEKPITI EVVSMDWKWF FIYPEQGIAT VNEIAFPANT P VYFKVTSN SVMNSFFIPR LGSQIYAMAG MQTRLHLIAN EPGTYDGISA SYSGPGFSGM KFKAIATPDR AAFDQWVAKA KQ SPNTMSD MAAFEKLAAP SEYNQVEYFS NVKPDLFADV INKFMAHGKS MDMTQPEGEH SAHEGMEGMD MSHAESAHHH HHH HHHR

UniProtKB: Ubiquinol oxidase subunit 2

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Macromolecule #3: Cytochrome bo(3) ubiquinol oxidase subunit 3

MacromoleculeName: Cytochrome bo(3) ubiquinol oxidase subunit 3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 22.642566 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MATDTLTHAT AHAHEHGHHD AGGTKIFGFW IYLMSDCILF SILFATYAVL VNGTAGGPTG KDIFELPFVL VETFLLLFSS ITYGMAAIA MYKNNKSQVI SWLALTWLFG AGFIGMEIYE FHHLIVNGMG PDRSGFLSAF FALVGTHGLH VTSGLIWMAV L MVQIARRG ...String:
MATDTLTHAT AHAHEHGHHD AGGTKIFGFW IYLMSDCILF SILFATYAVL VNGTAGGPTG KDIFELPFVL VETFLLLFSS ITYGMAAIA MYKNNKSQVI SWLALTWLFG AGFIGMEIYE FHHLIVNGMG PDRSGFLSAF FALVGTHGLH VTSGLIWMAV L MVQIARRG LTSTNRTRIM CLSLFWHFLD VVWICVFTVV YLMGAM

UniProtKB: Cytochrome bo(3) ubiquinol oxidase subunit 3

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Macromolecule #4: Cytochrome bo(3) ubiquinol oxidase subunit 4

MacromoleculeName: Cytochrome bo(3) ubiquinol oxidase subunit 4 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 12.037402 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSHSTDHSGA SHGSVKTYMT GFILSIILTV IPFWMVMTGA ASPAVILGTI LAMAVVQVLV HLVCFLHMNT KSDEGWNMTA FVFTVLIIA ILVVGSIWIM WNLNYNMMMH

UniProtKB: Cytochrome bo(3) ubiquinol oxidase subunit 4

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Macromolecule #5: HEME O

MacromoleculeName: HEME O / type: ligand / ID: 5 / Number of copies: 1 / Formula: HEO
Molecular weightTheoretical: 838.854 Da
Chemical component information

ChemComp-HEO:
HEME O

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Macromolecule #6: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 6 / Number of copies: 1 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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Macromolecule #7: COPPER (II) ION

MacromoleculeName: COPPER (II) ION / type: ligand / ID: 7 / Number of copies: 1 / Formula: CU
Molecular weightTheoretical: 63.546 Da
Chemical component information

ChemComp-CU:
COPPER (II) ION

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Macromolecule #8: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine

MacromoleculeName: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / type: ligand / ID: 8 / Number of copies: 2 / 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 #9: methyl 3-oxidanyl-5-[oxidanyl(oxidanylidene)-$l^{4}-azanyl]-1-ben...

MacromoleculeName: methyl 3-oxidanyl-5-[oxidanyl(oxidanylidene)-$l^{4}-azanyl]-1-benzothiophene-2-carboxylate
type: ligand / ID: 9 / Number of copies: 1 / Formula: JYR
Molecular weightTheoretical: 253.231 Da
Chemical component information

ChemComp-JYR:
methyl 3-oxidanyl-5-[oxidanyl(oxidanylidene)-$l^{4}-azanyl]-1-benzothiophene-2-carboxylate

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Macromolecule #10: UNKNOWN ATOM OR ION

MacromoleculeName: UNKNOWN ATOM OR ION / type: ligand / ID: 10 / Number of copies: 1 / Formula: UNX

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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

Concentration13 mg/mL
BufferpH: 7.2
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
20.0 mMTristris(hydroxymethyl)aminomethane
0.01 %C12Mn-Dodecyl-beta-D-maltoside
0.5 %DMSODimethyl sulfoxid
0.25 mMN4N4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Pressure: 0.007 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 7173 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1fft

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PHENIX / Number images used: 67692
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-7xmd:
Cryo-EM structure of Cytochrome bo3 from Escherichia coli, the structure complexed with an allosteric inhibitor N4

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