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- PDB-6l1x: Quinol-dependent nitric oxide reductase (qNOR) from Neisseria men... -

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Basic information

Entry
Database: PDB / ID: 6l1x
TitleQuinol-dependent nitric oxide reductase (qNOR) from Neisseria meningitidis in the monomeric oxidized state with zinc complex.
ComponentsNitric-oxide reductase
KeywordsOXIDOREDUCTASE / reductase / membrane-bound / nitric oxide
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
Cytochrome C Oxidase; Chain A / Cytochrome c oxidase-like, subunit I domain / Cytochrome c oxidase subunit I / Cytochrome c oxidase-like, subunit I superfamily / Cytochrome C and Quinol oxidase polypeptide I / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
: / PROTOPORPHYRIN IX CONTAINING FE / Nitric-oxide reductase
Similarity search - Component
Biological speciesNeisseria meningitidis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MIR / Resolution: 3.15 Å
AuthorsJamali, M.M.A. / Antonyuk, S.V. / Tosha, T. / Muramoto, K. / Hasnain, S.S. / Shiro, Y.
Funding support Japan, United Kingdom, 5items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP15K07029 Japan
Japan Society for the Promotion of Science (JSPS)JP18K06162 Japan
Japan Society for the Promotion of Science (JSPS)JP17H03092 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
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 1, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 1, 2020Provider: repository / Type: Initial release
Revision 1.1Jun 3, 2020Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nitric-oxide reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,9148
Polymers84,3891
Non-polymers1,5257
Water181
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2750 Å2
ΔGint-95 kcal/mol
Surface area26940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)96.120, 116.680, 123.550
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Nitric-oxide reductase


Mass: 84389.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis (strain alpha14) (bacteria)
Strain: alpha14 / Gene: norB, NMO_1451 / Production host: Escherichia coli (E. coli) / References: UniProt: C6S880, nitric-oxide reductase

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Non-polymers , 5 types, 8 molecules

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


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1 / 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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.17 Å3/Da / Density % sol: 70.5 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 50 mM HEPES pH8 26% PEG 600 (v/v) 0.5 mM zinc sulphate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL44XU / Wavelength: 0.9 Å
DetectorType: RAYONIX MX300HE / Detector: CCD / Date: May 24, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9 Å / Relative weight: 1
ReflectionResolution: 3.1→49.92 Å / Num. obs: 19034 / % possible obs: 83.3 % / Redundancy: 4.1 % / Biso Wilson estimate: 61 Å2 / CC1/2: 0.997 / Rmerge(I) obs: 0.113 / Rpim(I) all: 0.064 / Rrim(I) all: 0.141 / Net I/σ(I): 6.5
Reflection shellResolution: 3.1→3.31 Å / Redundancy: 4 % / Rmerge(I) obs: 0.556 / Mean I/σ(I) obs: 2.2 / Num. unique obs: 3849 / CC1/2: 0.284 / Rpim(I) all: 0.42 / Rrim(I) all: 0.7 / % possible all: 84.5

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
PDB_EXTRACT3.22data extraction
MOSFLMdata reduction
Aimlessdata scaling
MOLREPphasing
RefinementMethod to determine structure: MIR / Resolution: 3.15→49.92 Å / Cor.coef. Fo:Fc: 0.907 / Cor.coef. Fo:Fc free: 0.903 / SU B: 31.933 / SU ML: 0.498 / Cross valid method: THROUGHOUT / ESU R Free: 0.566 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: SF FILE CONTAINS FRIEDEL PAIRS UNDER I/F_MINUS AND I/F_PLUS COLUMNS.
RfactorNum. reflection% reflectionSelection details
Rfree0.30253 1063 5.3 %RANDOM
Rwork0.25029 ---
obs0.25313 19034 81.46 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 100.951 Å2
Baniso -1Baniso -2Baniso -3
1--5.94 Å20 Å20 Å2
2--0.18 Å20 Å2
3---5.76 Å2
Refinement stepCycle: 1 / Resolution: 3.15→49.92 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5562 0 91 1 5654
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0040.0125866
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg0.9991.6428006
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2965703
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.28521.962260
X-RAY DIFFRACTIONr_dihedral_angle_3_deg19.75515832
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.5111520
X-RAY DIFFRACTIONr_chiral_restr0.0850.2721
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.024493
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it4.88110.152828
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it8.1215.2083525
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it5.06910.2423038
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined13.7199656
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 3.15→3.231 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.455 85 -
Rwork0.44 1401 -
obs--83.62 %

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