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- EMDB-0822: Cryo-EM structure of dimeric quinol dependent Nitric Oxide Reduct... -

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Basic information

Entry
Database: EMDB / ID: EMD-0822
TitleCryo-EM structure of dimeric quinol dependent Nitric Oxide Reductase (qNOR) from the pathogen Neisseria meninigitidis
Map data
Sample
  • Complex: Dimeric quinol dependent Nitric Oxide Reductase
    • Protein or peptide: Nitric-oxide reductase
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: FE (III) ION
  • Ligand: CALCIUM ION
KeywordsNeisseria meningitidis / quinol-dependent electrogenic Nitric Oxide Reductase (qNOR) / OXIDOREDUCTASE
Function / homology
Function and homology information


nitric-oxide reductase / cytochrome bo3 ubiquinol oxidase activity / cytochrome-c oxidase activity / electron transport coupled proton transport / aerobic respiration / respiratory electron transport chain / heme binding / membrane / metal ion binding
Similarity search - Function
: / Nitric oxide reductase subunit B, cytochrome c-like domain / 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
Similarity search - Component
Biological speciesNeisseria meningitidis alpha14 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsJamali MMA / Gopalasingam CC
Funding support Japan, United Kingdom, 6 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP15K07029 Japan
Japan Society for the Promotion of Science (JSPS)JP18K06162 Japan
Biotechnology and Biological Sciences Research Council (BBSRC)BB/L006960/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N013972/1 United Kingdom
Wellcome Trust108466/Z/15/Z United Kingdom
Wellcome Trust109158/B/15/Z United Kingdom
CitationJournal: IUCrJ / Year: 2020
Title: The active form of quinol-dependent nitric oxide reductase from is a dimer.
Authors: M Arif M Jamali / Chai C Gopalasingam / Rachel M Johnson / Takehiko Tosha / Kazumasa Muramoto / Stephen P Muench / Svetlana V Antonyuk / Yoshitsugu Shiro / Samar S Hasnain /
Abstract: is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of ... is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of the bacterium in the human host. X-ray crystallographic analyses of qNOR, including that from (qNOR) reported here at 3.15 Å resolution, show monomeric assemblies, despite the more active dimeric sample being used for crystallization. Cryo-electron microscopic analysis of the same chromatographic fraction of qNOR, however, revealed a dimeric assembly at 3.06 Å resolution. It is shown that zinc (which is used in crystallization) binding near the dimer-stabilizing TMII region contributes to the disruption of the dimer. A similar destabilization is observed in the monomeric (∼85 kDa) cryo-EM structure of a mutant (Glu494Ala) qNOR from the opportunistic pathogen () , which primarily migrates as a monomer. The monomer-dimer transition of qNORs seen in the cryo-EM and crystallographic structures has wider implications for structural studies of multimeric membrane proteins. X-ray crystallographic and cryo-EM structural analyses have been performed on the same chromatographic fraction of qNOR to high resolution. This represents one of the first examples in which the two approaches have been used to reveal a monomeric assembly and a dimeric assembly in vitrified cryo-EM grids. A number of factors have been identified that may trigger the destabilization of helices that are necessary to preserve the integrity of the dimer. These include zinc binding near the entry of the putative proton-transfer channel and the preservation of the conformational integrity of the active site. The mutation near the active site results in disruption of the active site, causing an additional destabilization of helices (TMIX and TMX) that flank the proton-transfer channel helices, creating an inert monomeric enzyme.
History
DepositionOct 11, 2019-
Header (metadata) releaseApr 1, 2020-
Map releaseApr 1, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6l3h
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0822.png

Downloads & links

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Map

FileDownload / File: emd_0822.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.)
1.07 Å/pix.
x 200 pix.
= 214. Å		1.07 Å/pix.
x 200 pix.
= 214. Å		1.07 Å/pix.
x 200 pix.
= 214. 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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0268 / Movie #1: 0.03
Minimum - Maximum-0.12489361 - 0.19886318
Average (Standard dev.)0.0000462917 (±0.0079073785)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 214.00002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z214.000214.000214.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1250.1990.000

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Supplemental data

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Sample components

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Entire : Dimeric quinol dependent Nitric Oxide Reductase

EntireName: Dimeric quinol dependent Nitric Oxide Reductase
Components
  • Complex: Dimeric quinol dependent Nitric Oxide Reductase
    • Protein or peptide: Nitric-oxide reductase
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: FE (III) ION
  • Ligand: CALCIUM ION

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Supramolecule #1: Dimeric quinol dependent Nitric Oxide Reductase

SupramoleculeName: Dimeric quinol dependent Nitric Oxide Reductase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Neisseria meningitidis alpha14 (bacteria)
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: Nitric-oxide reductase

MacromoleculeName: Nitric-oxide reductase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: nitric-oxide reductase
Source (natural)Organism: Neisseria meningitidis alpha14 (bacteria) / Strain: alpha14
Molecular weightTheoretical: 84.389211 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGQYKKLWYL LFAVLAVCFT ILGYMGSEVY KKAPPYPEQV VSASGKVLMA KDDILAGQSA WQTTGGMEVG SVLGHGAYQA PDWTADWLH RELSAWLDLT AQQTYGKKFD EVSPEEQAVL KTRLADEYRN QSRIKEDGSV VISDTRVKAI ESILPYYHGV Y GDDPALQT ...String:
MGQYKKLWYL LFAVLAVCFT ILGYMGSEVY KKAPPYPEQV VSASGKVLMA KDDILAGQSA WQTTGGMEVG SVLGHGAYQA PDWTADWLH RELSAWLDLT AQQTYGKKFD EVSPEEQAVL KTRLADEYRN QSRIKEDGSV VISDTRVKAI ESILPYYHGV Y GDDPALQT TREHFAMKNN TLPSQEAREK LFDFFFWTSW SASTNRPDET FTYTNNWPHE PLINNVPTTE NYMWSFTSVV LL LMGIGLL MWGYSFLTKH EEVEVPTEDP ISKVQLTPSQ KALGKYVFLT VALFVVQVLL GGLTAHYTVE GQGFYGIDEA LGF EMSDWF PYALTRTWHI QSAIFWIATG FLTAGLFLAP IVNGGKDPKF QRAGVNFLYI ALFIVVGGSY AGNFFALTHI LPPE FNFWF GHQGYEYLDL GRFWQLLLMV GLLLWLFLML RCTVSAFKEK GVDKNLLAIF VASMVGVGVF YAPGLFYGEK SPIAV MEYW RWWVVHLWVE GFFEVFATAA FAFVFYNMGF VRRSTATAST LAAAAIFMLG GVPGTLHHLY FSGSTSASMA IGACFS ALE VVPLVLLGRE AYEHWSYQHL SEWAKRLRWP LMCFVAVAFW NMIGAGVFGF LINPPISLFY IQGLNTSAVH AHAALFG VY GFLALGFVLL VARYLKPNVQ FDDKLMTWGF WLLNGGLVGM IAISLLPVGV IQAYASITHG LWYARSEEFL QMEILDTL R WVRTAADLIF IGGAICVAIQ ATKIVFGRDK

UniProtKB: Nitric-oxide reductase

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4.0 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Blot time of 6 seconds, with accompanying blot force of 6..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 3182 / Average exposure time: 8.0 sec. / Average electron dose: 69.44 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -3.5 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 130000
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: 970000
Details: 2D templates generated from 2,000 manually picked particles. Templates (low pass filtered to 20 angstrom) then used for automated picking of micrographs.
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 233556
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6l1x


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

PDB-6l3h:
Cryo-EM structure of dimeric quinol dependent Nitric Oxide Reductase (qNOR) from the pathogen Neisseria meninigitidis

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