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- EMDB-16041: CryoEM structure of quinol-dependent Nitric Oxide Reductase (qNOR... -

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Basic information

Entry
Database: EMDB / ID: EMD-16041
TitleCryoEM structure of quinol-dependent Nitric Oxide Reductase (qNOR) from Alcaligenes xylosoxidans at 2.2 A resolution
Map datamain map used for model building
Sample
  • Cell: quinol-dependent Nitric Oxide Reductase
    • Protein or peptide: Nitric oxide reductase subunit B
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: FE (III) ION
  • Ligand: CALCIUM IONCalcium
  • Ligand: decyl 4-O-alpha-D-glucopyranosyl-1-thio-beta-D-glucopyranoside
  • Ligand: UBIQUINONE-1Coenzyme Q10
  • Ligand: (1R)-2-{[(R)-(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(DODECANOYLOXY)METHYL]ETHYL (9Z)-OCTADEC-9-ENOATE
  • Ligand: water
Keywordsquinol-dependent Nitric Oxide Reductase / proton transfer / quinol binding / ubiquinol oxidase / MEMBRANE PROTEIN
Function / homology
Function and homology information


nitric oxide reductase (cytochrome c) / nitric oxide reductase activity / cytochrome-c oxidase activity / aerobic respiration / heme binding / membrane
Similarity search - Function
Cytochrome c oxidase subunit I / Cytochrome c oxidase-like, subunit I superfamily / Cytochrome C and Quinol oxidase polypeptide I
Similarity search - Domain/homology
Nitric oxide reductase subunit B
Similarity search - Component
Biological speciesAchromobacter xylosoxidans (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsFlynn A / Antonyuk SV / Eady RR / Muench SP / Hasnain SS
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust108466/Z/15/Z United Kingdom
Wellcome Trust221524/Z/20/Z United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: A 2.2 Å cryoEM structure of a quinol-dependent NO Reductase shows close similarity to respiratory oxidases.
Authors: Alex J Flynn / Svetlana V Antonyuk / Robert R Eady / Stephen P Muench / S Samar Hasnain /
Abstract: Quinol-dependent nitric oxide reductases (qNORs) are considered members of the respiratory heme-copper oxidase superfamily, are unique to bacteria, and are commonly found in pathogenic bacteria where ...Quinol-dependent nitric oxide reductases (qNORs) are considered members of the respiratory heme-copper oxidase superfamily, are unique to bacteria, and are commonly found in pathogenic bacteria where they play a role in combating the host immune response. qNORs are also essential enzymes in the denitrification pathway, catalysing the reduction of nitric oxide to nitrous oxide. Here, we determine a 2.2 Å cryoEM structure of qNOR from Alcaligenes xylosoxidans, an opportunistic pathogen and a denitrifying bacterium of importance in the nitrogen cycle. This high-resolution structure provides insight into electron, substrate, and proton pathways, and provides evidence that the quinol binding site not only contains the conserved His and Asp residues but also possesses a critical Arg (Arg720) observed in cytochrome bo, a respiratory quinol oxidase.
History
DepositionOct 28, 2022-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 2023-
UpdateAug 16, 2023-
Current statusAug 16, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16041.png

Downloads & links

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Map

FileDownload / File: emd_16041.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map used for model building
Voxel sizeX=Y=Z: 0.91 Å
Density
Contour LevelBy AUTHOR: 0.015
Minimum - Maximum-0.06622768 - 0.17982407
Average (Standard dev.)0.00004599626 (±0.0017005954)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 364.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16041_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map used in refinement

Fileemd_16041_half_map_1.map
Annotationhalf map used in refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map used for cross validation

Fileemd_16041_half_map_2.map
Annotationhalf map used for cross validation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : quinol-dependent Nitric Oxide Reductase

EntireName: quinol-dependent Nitric Oxide Reductase
Components
  • Cell: quinol-dependent Nitric Oxide Reductase
    • Protein or peptide: Nitric oxide reductase subunit B
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: FE (III) ION
  • Ligand: CALCIUM IONCalcium
  • Ligand: decyl 4-O-alpha-D-glucopyranosyl-1-thio-beta-D-glucopyranoside
  • Ligand: UBIQUINONE-1Coenzyme Q10
  • Ligand: (1R)-2-{[(R)-(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(DODECANOYLOXY)METHYL]ETHYL (9Z)-OCTADEC-9-ENOATE
  • Ligand: water

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Supramolecule #1: quinol-dependent Nitric Oxide Reductase

SupramoleculeName: quinol-dependent Nitric Oxide Reductase / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: quinol-dependent Nitric Oxide Reductase
Source (natural)Organism: Achromobacter xylosoxidans (bacteria)

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Macromolecule #1: Nitric oxide reductase subunit B

MacromoleculeName: Nitric oxide reductase subunit B / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: nitric oxide reductase (cytochrome c)
Source (natural)Organism: Achromobacter xylosoxidans (bacteria)
Molecular weightTheoretical: 84.724867 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGPYRRLWFT LIAVLAVTFA LLGFYGGEVY RQAPPIPEEV ASADGTRLFG RDDILDGQTA WQSIGGMQLG SIWGHGAYQA PDWTADWLH RELMAWLDLA ARDAHGRDYG QLDAPAQAAL REQLKAEYRA NRADAAGGKL TLSPRRAQAV AQTEAYYDQL F SDAPALHR ...String:
MGPYRRLWFT LIAVLAVTFA LLGFYGGEVY RQAPPIPEEV ASADGTRLFG RDDILDGQTA WQSIGGMQLG SIWGHGAYQA PDWTADWLH RELMAWLDLA ARDAHGRDYG QLDAPAQAAL REQLKAEYRA NRADAAGGKL TLSPRRAQAV AQTEAYYDQL F SDAPALHR SRENYAMKEN TLPDANRRRQ MTHFFFWTAW AAATEREGTS VTYTNNWPHE PLIGNHPSSE NVMWSIISVV VL LAGIGLL IWAWAFLRGK EEDEPPAPAR DPLTTFALTP SQRALGKYLF LVVALFGFQV LLGGFTAHYT VEGQKFYGID LSQ WFPYSL VRTWHIQSAL FWIATGFLAA GLFLAPLING GRDPKYQKAG VDILFWALVL VVVGSFAGNY LAIAQIMPPD LNFW LGHQG YEYVDLGRLW QIGKFAGICF WLVLMLRGIV PALRTPGGDK NLLALLTASV GAIGLFYGAG FFYGERTHLT VMEYW RWWI VHLWVEGFFE VFATTALAFI FSTLGLVSRR MATTASLASA SLFMLGGIPG TFHHLYFAGT TTPVMAVGAS FSALEV VPL IVLGHEAWEN WRLKTRAPWM ENLKWPLMCF VAVAFWNMLG AGVFGFMINP PVSLYYIQGL NTTPVHAHAA LFGVYGF LA LGFTLLVLRY IRPQYALSPG LMKLAFWGLN LGLALMIFTS LLPIGLIQFH ASVSEGMWYA RSEAFMQQDI LKTLRWGR T FGDVVFLLGA LAMVVQVILG LLSGKPAAAE PVLRAEPARR

UniProtKB: Nitric oxide reductase subunit B

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Macromolecule #2: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 2 / Number of copies: 4 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE / Heme B

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Macromolecule #3: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: decyl 4-O-alpha-D-glucopyranosyl-1-thio-beta-D-glucopyranoside

MacromoleculeName: decyl 4-O-alpha-D-glucopyranosyl-1-thio-beta-D-glucopyranoside
type: ligand / ID: 5 / Number of copies: 2 / Formula: 10M
Molecular weightTheoretical: 498.628 Da
Chemical component information

ChemComp-10M:
decyl 4-O-alpha-D-glucopyranosyl-1-thio-beta-D-glucopyranoside / detergent*YM

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Macromolecule #6: UBIQUINONE-1

MacromoleculeName: UBIQUINONE-1 / type: ligand / ID: 6 / Number of copies: 2 / Formula: UQ1
Molecular weightTheoretical: 250.29 Da
Chemical component information

ChemComp-UQ1:
UBIQUINONE-1

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Macromolecule #7: (1R)-2-{[(R)-(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(DODECANO...

MacromoleculeName: (1R)-2-{[(R)-(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(DODECANOYLOXY)METHYL]ETHYL (9Z)-OCTADEC-9-ENOATE
type: ligand / ID: 7 / Number of copies: 13 / Formula: LOP
Molecular weightTheoretical: 661.89 Da
Chemical component information

ChemComp-LOP:
(1R)-2-{[(R)-(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(DODECANOYLOXY)METHYL]ETHYL (9Z)-OCTADEC-9-ENOATE / phospholipid*YM

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Macromolecule #8: water

MacromoleculeName: water / type: ligand / ID: 8 / Number of copies: 449 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration3 mg/mL
BufferpH: 7 / Component - Concentration: 50.0 mM / Component - Formula: Tris-HCLTris / Component - Name: Tris-HCLTris / Details: 50mM Tris-HCl, 150mM NaCl, 0.05% DTM
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Sample at 3 mg/mL was applied to glow-discharged Quantifoil Au R1.2/1.3 holey carbo grids.
DetailsSample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 114.0 K
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 5466 / Average exposure time: 6.11 sec. / Average electron dose: 34.9 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3000000
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6qq5


Details: Low-pass filtered to 60A for first 3D classification
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 6 / Avg.num./class: 121415 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 404950
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Overall B value: 47 / Target criteria: Cross-correlation coefficient
Output model

PDB-8bgw:
CryoEM structure of quinol-dependent Nitric Oxide Reductase (qNOR) from Alcaligenes xylosoxidans at 2.2 A resolution

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