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- EMDB-4619: Cryo-EM structure of dimeric quinol dependent nitric oxide reduct... -

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Basic information

Entry
Database: EMDB / ID: EMD-4619
TitleCryo-EM structure of dimeric quinol dependent nitric oxide reductase (qNOR) Val495Ala mutant from Alcaligenes xylosoxidans
Map data
Sample
  • Complex: Quinol Dependent Nitric Oxide Reductase
    • Protein or peptide: Nitric oxide reductase subunit B
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: FE (III) ION
  • Ligand: CALCIUM ION
  • Ligand: (1R)-2-{[(R)-(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(DODECANOYLOXY)METHYL]ETHYL (9Z)-OCTADEC-9-ENOATE
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: water
KeywordsProton Transfer / Membrane Protein / Homodimer / OXIDOREDUCTASE
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) / Alcaligenes xylosoxydans xylosoxydans (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGopalasingam CC / Johnson RM / Chiduza GN
Funding support United Kingdom, 4 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/L006960/1 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/N013972/1 United Kingdom
Wellcome Trust109158/B/15/Z 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.
History
DepositionFeb 17, 2019-
Header (metadata) releaseSep 11, 2019-
Map releaseSep 11, 2019-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing 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
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  • 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-6qq6
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4619.png

Downloads & links

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Map

FileDownload / File: emd_4619.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 200 pix.
= 210. Å		1.05 Å/pix.
x 200 pix.
= 210. Å		1.05 Å/pix.
x 200 pix.
= 210. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0394 / Movie #1: 0.06
Minimum - Maximum-0.30891758 - 0.44059387
Average (Standard dev.)0.000302113 (±0.013368749)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 209.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z210.000210.000210.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.3090.4410.000

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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
Components
  • Complex: Quinol Dependent Nitric Oxide Reductase
    • Protein or peptide: Nitric oxide reductase subunit B
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: FE (III) ION
  • Ligand: CALCIUM ION
  • Ligand: (1R)-2-{[(R)-(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(DODECANOYLOXY)METHYL]ETHYL (9Z)-OCTADEC-9-ENOATE
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: water

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Supramolecule #1: Quinol Dependent Nitric Oxide Reductase

SupramoleculeName: Quinol Dependent Nitric Oxide Reductase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Achromobacter xylosoxidans (bacteria)
Molecular weightTheoretical: 170 KDa

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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: Alcaligenes xylosoxydans xylosoxydans (bacteria)
Molecular weightTheoretical: 83.093938 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 AAGTEREGTS 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 AFATTALAFI 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 LLSGK

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

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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: (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: 5 / Number of copies: 3 / 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 #6: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 6 / Number of copies: 2 / Formula: LMT
Molecular weightTheoretical: 510.615 Da
Chemical component information

ChemComp-LMT:
DODECYL-BETA-D-MALTOSIDE / detergent*YM

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 18 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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

Concentration5 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Blot for 6 seconds with blot force of 6 prior to plunge freezing.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 1803 / Average electron dose: 49.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -3.0 µm / Nominal defocus min: -1.2 µm / Nominal magnification: 47710
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 614931
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 227359
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6qq5


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6qq6:
Cryo-EM structure of dimeric quinol dependent nitric oxide reductase (qNOR) Val495Ala mutant from Alcaligenes xylosoxidans

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