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- PDB-7xmd: Cryo-EM structure of Cytochrome bo3 from Escherichia coli, the st... -

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Basic information

Entry
Database: PDB / ID: 7xmd
TitleCryo-EM structure of Cytochrome bo3 from Escherichia coli, the structure complexed with an allosteric inhibitor N4
Components
  • (Cytochrome bo(3) ubiquinol oxidase subunit ...) x 3
  • Ubiquinol oxidase subunit 2
KeywordsOXIDOREDUCTASE / respiratory enzyme / membrane protein / heme protein / allosteric inhibitor
Function / homology
Function and homology information


cytochrome o ubiquinol oxidase complex / oxidoreduction-driven active transmembrane transporter activity / 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 ...cytochrome o ubiquinol oxidase complex / oxidoreduction-driven active transmembrane transporter activity / 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 / respirasome / 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 / Ubiquinol oxidase subunit 2, cupredoxin domain ...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 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 oxidase subunit II, transmembrane domain / Cytochrome oxidase subunit II transmembrane region profile. / 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 I / Cytochrome c oxidase-like, subunit I superfamily / Cytochrome C and Quinol oxidase polypeptide I / Cytochrome C oxidase subunit II, periplasmic domain / 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
COPPER (II) ION / PROTOPORPHYRIN IX CONTAINING FE / HEME O / Chem-JYR / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsNishida, Y. / Shigematsu, H. / Iwamoto, T. / Takashima, S. / Shintani, Y.
Funding support Japan, 2items
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, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 21, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cytochrome bo(3) ubiquinol oxidase subunit 1
B: Ubiquinol oxidase subunit 2
C: Cytochrome bo(3) ubiquinol oxidase subunit 3
D: Cytochrome bo(3) ubiquinol oxidase subunit 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,57411
Polymers145,3144
Non-polymers3,2607
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Cytochrome bo(3) ubiquinol oxidase subunit ... , 3 types, 3 molecules ACD

#1: Protein Cytochrome bo(3) ubiquinol oxidase subunit 1 / Cytochrome b562-o complex subunit I / Cytochrome o ubiquinol oxidase subunit 1 / Cytochrome o ...Cytochrome b562-o complex subunit I / Cytochrome o ubiquinol oxidase subunit 1 / Cytochrome o subunit 1 / Oxidase bo(3) subunit 1 / Ubiquinol oxidase chain A / Ubiquinol oxidase polypeptide I / Ubiquinol oxidase subunit 1


Mass: 74424.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
References: UniProt: P0ABI8, ubiquinol oxidase (H+-transporting)
#3: Protein Cytochrome bo(3) ubiquinol oxidase subunit 3 / Cytochrome o ubiquinol oxidase subunit 3 / Cytochrome o subunit 3 / Oxidase bo(3) subunit 3 / ...Cytochrome o ubiquinol oxidase subunit 3 / Cytochrome o subunit 3 / Oxidase bo(3) subunit 3 / Ubiquinol oxidase chain C / Ubiquinol oxidase polypeptide III / Ubiquinol oxidase subunit 3


Mass: 22642.566 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: P0ABJ3
#4: Protein Cytochrome bo(3) ubiquinol oxidase subunit 4 / Cytochrome o ubiquinol oxidase subunit 4 / Cytochrome o subunit 4 / Oxidase bo(3) subunit 4 / ...Cytochrome o ubiquinol oxidase subunit 4 / Cytochrome o subunit 4 / Oxidase bo(3) subunit 4 / Ubiquinol oxidase chain D / Ubiquinol oxidase polypeptide IV / Ubiquinol oxidase subunit 4


Mass: 12037.402 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: P0ABJ6

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Protein , 1 types, 1 molecules B

#2: Protein Ubiquinol oxidase subunit 2 /


Mass: 36209.559 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: cyoA, ACU57_11830, AM464_19145, APX88_10780, AWP93_14560, BANRA_03634, BEA19_24285, BJI68_14080, BJJ90_20095, BK383_14750, BMC79_002872, BO068_001687, BOB65_000403, BON66_08870, BON73_06180, ...Gene: cyoA, ACU57_11830, AM464_19145, APX88_10780, AWP93_14560, BANRA_03634, BEA19_24285, BJI68_14080, BJJ90_20095, BK383_14750, BMC79_002872, BO068_001687, BOB65_000403, BON66_08870, BON73_06180, BON74_21890, BON77_14335, BON80_10695, BON86_25900, BON87_05825, BON89_00395, BON92_15695, BON93_19065, BON98_18690, BSR05_07160, BXT93_14110, BZL69_16590, C2121_000756, C5N07_12950, CA593_00980, CDC27_01080, CDL36_18475, CDL37_08970, CQP61_20675, CR539_06390, CT143_00345, CXJ73_004380, D0X26_07630, D3822_17775, D3Y67_14555, D9E34_22950, D9J03_07430, DAH17_06675, DAH34_12595, DAH36_12985, DD762_23785, DKP82_19825, DN627_04300, DNQ45_18500, DNX30_04955, DTM16_23740, DTM45_11185, DU321_07410, DXT70_02705, E2119_01520, E2122_05970, E2131_02675, E2135_01205, E4K51_12275, E5P26_04320, E5P27_05630, E5P28_11110, E5P29_09785, E5P31_06375, E5P32_04100, E5P34_04850, E5P35_04720, E5P36_01955, E5P40_03855, E5S36_14935, E5S46_14035, E5S51_13330, E5S58_14090, E5S61_17450, EC3234A_4c01010, EI021_16450, EIA08_09775, EIZ93_01035, EL79_3413, EL80_3367, ELT17_06040, ELT20_08135, ELT41_07160, ELT49_07745, ELT51_19775, ELU85_09175, ELV10_08020, ELV16_21785, ELX48_13290, ELX76_14920, ELX85_08605, EVY14_00280, ExPECSC038_03272, EYV17_02895, EYV18_09930, EYY21_23390, F2N31_13445, F3N40_15010, F9S83_01735, F9V24_00335, FA849_16035, FA868_02450, FE587_05330, FEJ01_04230, FEL34_02865, FGG80_03370, FGY90_08715, FHD44_10295, FJQ51_12155, FQF29_11475, FTV93_12140, FV293_05915, FWK02_10070, G3813_001713, G4A38_05615, G5603_08425, GIB53_01940, GKF86_04705, GKF89_04260, GLW94_08520, GQM13_16545, GQW68_14795, GQW80_03945, GRW05_22945, GRW57_14390, GRW81_04315, GSY44_02240, GTP88_03650, GUB08_11340, H0O53_01600, HIN64_001359, HJQ60_002319, HJS37_002124, HJU54_001612, HL601_03105, HLZ50_08430, HMJ82_07910, HMU48_15410, HMV41_13070, HMV95_11775, HNC36_06575, HNC59_10400, HNC66_06810, HNC99_09295, HND12_08495, HVW04_08795, HVX16_19275, HX136_19320, IA00_004007, IH772_12955, IT029_003268, J0541_001708, JNP96_06590, NCTC10082_01037, NCTC10764_05312, NCTC10974_04302, NCTC11112_03815, NCTC12950_04133, NCTC13148_06650, NCTC8008_03342, NCTC8450_01418, NCTC8500_04220, NCTC8621_03944, NCTC9037_03917, NCTC9044_03552, NCTC9117_04753, PGD_02879, SAMEA3472067_03229, SAMEA3751407_02102, WP2S18E08_34950
Production host: Escherichia coli (E. coli) / References: UniProt: A0A024L5V9

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Non-polymers , 6 types, 7 molecules

#5: Chemical ChemComp-HEO / HEME O / Heme O


Mass: 838.854 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C49H58FeN4O5
#6: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#7: Chemical ChemComp-CU / COPPER (II) ION / Copper


Mass: 63.546 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cu
#8: Chemical ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE / Discrete optimized protein energy


Mass: 744.034 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C41H78NO8P / Comment: DOPE, phospholipid*YM
#9: Chemical ChemComp-JYR / methyl 3-oxidanyl-5-[oxidanyl(oxidanylidene)-$l^{4}-azanyl]-1-benzothiophene-2-carboxylate


Mass: 253.231 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H7NO5S
#10: Chemical ChemComp-UNX / UNKNOWN ATOM OR ION


Num. of mol.: 1 / Source method: obtained synthetically

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Details

Has ligand of interestN

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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 bo3 ubiquinol oxidase / Type: COMPLEX / Entity ID: #2-#4 / Source: RECOMBINANT
Molecular weightValue: 0.144 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.2
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaClSodium chloride1
220 mMtris(hydroxymethyl)aminomethaneTris1
30.01 %n-Dodecyl-beta-D-maltosideC12M1
40.5 %Dimethyl sulfoxidDMSODimethyl sulfoxide1
50.25 mMN4N41
SpecimenConc.: 13 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 7173

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
7Cootmodel fitting
12PHENIX3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 67692 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049973
ELECTRON MICROSCOPYf_angle_d0.67513590
ELECTRON MICROSCOPYf_dihedral_angle_d12.7283454
ELECTRON MICROSCOPYf_chiral_restr0.0431501
ELECTRON MICROSCOPYf_plane_restr0.0061638

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