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- EMDB-10387: Glu-494-Ala inactive monomer of a quinol dependent Nitric Oxide R... -

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Basic information

Entry
Database: EMDB / ID: EMD-10387
TitleGlu-494-Ala inactive monomer of a quinol dependent Nitric Oxide Reductase (qNOR) from Alcaligenes xylosoxidans
Map data
Sample
  • Complex: Glu-494-Ala mutant of quinol dependent Nitric Oxide Reductase
    • Protein or peptide: Nitric oxide reductase subunit B
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: CALCIUM ION
KeywordsMonomer / Proton Transfer / Nitric Oxide / 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: 4.5 Å
AuthorsGopalasingam CC / Johnson RM / Antonyuk SV
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: 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 19, 2019-
Header (metadata) releaseApr 1, 2020-
Map releaseApr 1, 2020-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6t6v
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10387.png

Downloads & links

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Map

FileDownload / File: emd_10387.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 150 pix.
= 156.45 Å		1.04 Å/pix.
x 150 pix.
= 156.45 Å		1.04 Å/pix.
x 150 pix.
= 156.45 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.043 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.059758537 - 0.14851397
Average (Standard dev.)0.00022671334 (±0.0050465064)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 156.45 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0431.0431.043
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z156.450156.450156.450
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.0600.1490.000

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Supplemental data

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Sample components

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Entire : Glu-494-Ala mutant of quinol dependent Nitric Oxide Reductase

EntireName: Glu-494-Ala mutant of quinol dependent Nitric Oxide Reductase
Components
  • Complex: Glu-494-Ala mutant of quinol dependent Nitric Oxide Reductase
    • Protein or peptide: Nitric oxide reductase subunit B
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: CALCIUM ION

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Supramolecule #1: Glu-494-Ala mutant of quinol dependent Nitric Oxide Reductase

SupramoleculeName: Glu-494-Ala mutant of quinol dependent Nitric Oxide Reductase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Achromobacter xylosoxidans (bacteria)
Molecular weightTheoretical: 95 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: 1 / Enantiomer: LEVO / EC number: nitric oxide reductase (cytochrome c)
Source (natural)Organism: Alcaligenes xylosoxydans xylosoxydans (bacteria)
Molecular weightTheoretical: 84.765961 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 VHLWVEGFFA 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 L

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: 2 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 1 / 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

Concentration3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMTris-HCL
150.0 mMNaCl
0.05 % (v/v)DTMn-decyl-D-thiomaltoside
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: 98 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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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: 2239 / Average electron dose: 50.0 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: -1.5 µm / Nominal magnification: 48000
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: 684000
Startup modelType of model: NONE
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 144424
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6qq6


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

PDB-6t6v:
Glu-494-Ala inactive monomer of a quinol dependent Nitric Oxide Reductase (qNOR) from Alcaligenes xylosoxidans

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