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- EMDB-4618: Cryo-EM structure of dimeric quinol dependent nitric oxide reduct... -

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Basic information

Entry
Database: EMDB / ID: EMD-4618
TitleCryo-EM structure of dimeric quinol dependent nitric oxide reductase (qNOR) from Alcaligenes xylosoxidans
Map data
SampleQuinol Dependent Nitric Oxide Reductase:
Nitric oxide reductase subunit B / (ligand) x 3
Function / homology
Function and homology information


nitric oxide reductase (cytochrome c) / nitric oxide reductase activity / cytochrome-c oxidase activity / aerobic respiration / heme binding / integral component of membrane
Cytochrome c oxidase subunit I / Cytochrome c oxidase-like, subunit I superfamily / Cytochrome C and Quinol oxidase polypeptide I
Nitric oxide reductase subunit B
Biological speciesAchromobacter xylosoxidans (bacteria) / Alcaligenes xylosoxydans xylosoxydans (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsGopalasingam CC / Johnson RM / Chiduza GN / Tosha T / Yamamoto M / Shiro Y / Antonyuk SV / Muench SP / Hasnain SS
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/L006960/1 United Kingdom
Wellcome Trust109158/B/15/Z United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/N013972/1 United Kingdom
Wellcome Trust108466/Z/15/Z United Kingdom
CitationJournal: Sci Adv / Year: 2019
Title: Dimeric structures of quinol-dependent nitric oxide reductases (qNORs) revealed by cryo-electron microscopy.
Authors: Chai C Gopalasingam / Rachel M Johnson / George N Chiduza / Takehiko Tosha / Masaki Yamamoto / Yoshitsugu Shiro / Svetlana V Antonyuk / Stephen P Muench / S Samar Hasnain /
Abstract: Quinol-dependent nitric oxide reductases (qNORs) are membrane-integrated, iron-containing enzymes of the denitrification pathway, which catalyze the reduction of nitric oxide (NO) to the major ozone ...Quinol-dependent nitric oxide reductases (qNORs) are membrane-integrated, iron-containing enzymes of the denitrification pathway, which catalyze the reduction of nitric oxide (NO) to the major ozone destroying gas nitrous oxide (NO). Cryo-electron microscopy structures of active qNOR from and an activity-enhancing mutant have been determined to be at local resolutions of 3.7 and 3.2 Å, respectively. They unexpectedly reveal a dimeric conformation (also confirmed for qNOR from ) and define the active-site configuration, with a clear water channel from the cytoplasm. Structure-based mutagenesis has identified key residues involved in proton transport and substrate delivery to the active site of qNORs. The proton supply direction differs from cytochrome c-dependent NOR (cNOR), where water molecules from the cytoplasm serve as a proton source similar to those from cytochrome c oxidase.
Validation ReportPDB-ID: 6qq5

SummaryFull reportAbout validation report
History
DepositionFeb 17, 2019-
Header (metadata) releaseSep 11, 2019-
Map releaseSep 11, 2019-
UpdateSep 18, 2019-
Current statusSep 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6qq5
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4618.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 220 pix.
= 235.4 Å
1.07 Å/pix.
x 220 pix.
= 235.4 Å
1.07 Å/pix.
x 220 pix.
= 235.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.0355 / Movie #1: 0.06
Minimum - Maximum-0.30790263 - 0.44810045
Average (Standard dev.)0.00035212337 (±0.011198243)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 235.40001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z235.400235.400235.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.3080.4480.000

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Supplemental data

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Sample components

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Entire Quinol Dependent Nitric Oxide Reductase

EntireName: Quinol Dependent Nitric Oxide Reductase / Number of components: 5

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Component #1: protein, Quinol Dependent Nitric Oxide Reductase

ProteinName: Quinol Dependent Nitric Oxide Reductase / Recombinant expression: No
MassTheoretical: 170 kDa
SourceSpecies: Achromobacter xylosoxidans (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria) / Strain: C41 (DE3)

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Component #2: protein, Nitric oxide reductase subunit B

ProteinName: Nitric oxide reductase subunit B / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 83.121984 kDa
SourceSpecies: Alcaligenes xylosoxydans xylosoxydans (bacteria)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: ligand, PROTOPORPHYRIN IX CONTAINING FE

LigandName: PROTOPORPHYRIN IX CONTAINING FE / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.616487 kDa

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Component #4: ligand, FE (III) ION

LigandName: FE (III) ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 5.584505 MDa

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Component #5: ligand, CALCIUM ION

LigandName: CALCIUM IONCalcium / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 4.007805 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 3 mg/mL / pH: 7
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %
Details: Blot for 6 seconds with blot force of 6 prior to plunge freezing.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 65 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 75000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: -1500.0 - -3500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 3213

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 56134
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Input PDB model: 3AYF
Chain ID: A
Output model

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