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- PDB-8bgw: CryoEM structure of quinol-dependent Nitric Oxide Reductase (qNOR... -

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Basic information

Entry
Database: PDB / ID: 8bgw
TitleCryoEM structure of quinol-dependent Nitric Oxide Reductase (qNOR) from Alcaligenes xylosoxidans at 2.2 A resolution
ComponentsNitric oxide reductase subunit B
KeywordsMEMBRANE PROTEIN / quinol-dependent Nitric Oxide Reductase / proton transfer / quinol binding / ubiquinol oxidase
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
: / PROTOPORPHYRIN IX CONTAINING FE / Chem-LOP / UBIQUINONE-1 / Nitric oxide reductase subunit B
Similarity search - Component
Biological speciesAchromobacter xylosoxidans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsFlynn, A. / Antonyuk, S.V. / Eady, R.R. / Muench, S.P. / Hasnain, S.S.
Funding support United Kingdom, 2items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 16, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nitric oxide reductase subunit B
B: Nitric oxide reductase subunit B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)182,21027
Polymers169,4502
Non-polymers12,76025
Water8,089449
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area21530 Å2
ΔGint-236 kcal/mol
Surface area42610 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS ensembles : (Details: Local NCS retraints between domains: 1 2)

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Components

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Protein / Sugars , 2 types, 4 molecules AB

#1: Protein Nitric oxide reductase subunit B / Nitric-oxide reductase large subunit


Mass: 84724.867 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Achromobacter xylosoxidans (bacteria) / Gene: norB_1, ERS451415_02175, I6G72_25885 / Plasmid: PET-26B (+) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): C41
References: UniProt: A0A0D6H8R3, nitric oxide reductase (cytochrome c)
#5: Sugar ChemComp-10M / decyl 4-O-alpha-D-glucopyranosyl-1-thio-beta-D-glucopyranoside / (2R,3R,4S,5S,6R)-2-((2R,3S,4R,5R,6S)-6-Decylsulfanyl-4,5-dihydroxy-2-hydroxymethyl-tetrahydro-pyran-3-yloxy)-6-hydroxymethyl-tetrahydro-pyran-3,4,5-triol, n-Decyl-beta-D-thiomaltoside


Type: D-saccharide / Mass: 498.628 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C22H42O10S / Comment: detergent*YM

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Non-polymers , 6 types, 472 molecules

#2: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#6: Chemical ChemComp-UQ1 / UBIQUINONE-1


Mass: 250.290 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H18O4
#7: Chemical
ChemComp-LOP / (1R)-2-{[(R)-(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(DODECANOYLOXY)METHYL]ETHYL (9Z)-OCTADEC-9-ENOATE / LAURYL OLEYL PHOSPHATIDYL ETHANOLAMINE


Mass: 661.890 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: C35H68NO8P / Comment: phospholipid*YM
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 449 / Source method: isolated from a natural source / Formula: H2O

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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: quinol-dependent Nitric Oxide Reductase / Type: CELL / Details: quinol-dependent Nitric Oxide Reductase / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.84651 MDa / Experimental value: NO
Source (natural)Organism: Achromobacter xylosoxidans (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7 / Details: 50mM Tris-HCl, 150mM NaCl, 0.05% DTM
Buffer componentConc.: 50 mM / Name: Tris-HCLTris / Formula: Tris-HCLTris
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Sample was monodisperse
Specimen supportGrid material: GOLD / 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: 277 K
Details: Sample at 3 mg/mL was applied to glow-discharged Quantifoil Au R1.2/1.3 holey carbo grids

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2700 nm / Nominal defocus min: 900 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 / Temperature (max): 114 K / Temperature (min): 80 K
Image recordingAverage exposure time: 6.11 sec. / Electron dose: 34.9 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5466
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
EM software
IDNameVersionCategoryDetails
1crYOLO1.6.1particle selectionUsed general model for picking
2EPU2.14image acquisition
4CTFFIND4.1CTF correction
7MOLREP11.06.04model fitting
9REFMAC5.8.0267model refinement
10PHENIX1.2model refinement
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3000000
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 404950 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingB value: 47 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Cross-correlation coefficient
RefinementHighest resolution: 2.2 Å
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0120.01312941
ELECTRON MICROSCOPYr_bond_other_d00.01512333
ELECTRON MICROSCOPYr_angle_refined_deg1.6721.66817575
ELECTRON MICROSCOPYr_angle_other_deg1.3811.58228192
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.94151509
ELECTRON MICROSCOPYr_dihedral_angle_2_deg33.7120.306621
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.396151799
ELECTRON MICROSCOPYr_dihedral_angle_4_deg16.1571576
ELECTRON MICROSCOPYr_chiral_restr0.0920.21533
ELECTRON MICROSCOPYr_gen_planes_refined0.0110.0214373
ELECTRON MICROSCOPYr_gen_planes_other0.0060.023281
ELECTRON MICROSCOPYr_nbd_refined0.2120.210186
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1870.234038
ELECTRON MICROSCOPYr_nbtor_refined0.1910.213358
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0840.213892
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.1960.22018
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_other0.0410.216
ELECTRON MICROSCOPYr_mcbond_it6.1875.9936022
ELECTRON MICROSCOPYr_mcbond_other6.1875.9926020
ELECTRON MICROSCOPYr_mcangle_it8.4269.0487521
ELECTRON MICROSCOPYr_mcangle_other8.4259.057522
ELECTRON MICROSCOPYr_scbond_it6.6016.7796919
ELECTRON MICROSCOPYr_scbond_other6.6016.7796920
ELECTRON MICROSCOPYr_scangle_it9.639.82610050
ELECTRON MICROSCOPYr_scangle_other9.6299.82610051
ELECTRON MICROSCOPYr_lrange_it13.38875.64618167
ELECTRON MICROSCOPYr_lrange_other13.38875.64718168
ELECTRON MICROSCOPYr_ncsr_local_group_10.0450.0559674
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)Weight position
11AELECTRON MICROSCOPYLocal ncs0.044590.05014
12BELECTRON MICROSCOPYLocal ncs0.044590.05014
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: 0 / Total num. of bins used: 20 / % reflection obs: 100 %

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
2.24-2.2980.738139360.738139360.5990.738
2.298-2.3610.519135910.519135910.6720.519
2.361-2.430.494131450.494131450.7310.494
2.43-2.5040.446128170.446128170.7920.446
2.504-2.5860.394124860.394124860.8340.394
2.586-2.6770.356120060.356120060.8690.356
2.677-2.7780.33115900.33115900.8970.33
2.778-2.8920.275112450.275112450.9240.275
2.892-3.020.241106720.241106720.9390.241
3.02-3.1670.208101750.208101750.9520.208
3.167-3.3390.18797430.18797430.960.187
3.339-3.5410.292550.292550.9650.2
3.541-3.7850.23185960.23185960.9580.231
3.785-4.0880.2781180.2781180.9530.27
4.088-4.4780.27973940.27973940.9440.279
4.478-5.0050.27966920.27966920.9380.279
5.005-5.7780.30559460.30559460.8990.305
5.778-7.0720.37749730.37749730.860.377
7.072-9.9840.32938580.32938580.8590.329
9.984-118.30.53221100.53221100.9620.532

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