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- PDB-6l3h: Cryo-EM structure of dimeric quinol dependent Nitric Oxide Reduct... -

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Basic information

Entry
Database: PDB / ID: 6l3h
TitleCryo-EM structure of dimeric quinol dependent Nitric Oxide Reductase (qNOR) from the pathogen Neisseria meninigitidis
ComponentsNitric-oxide reductase
KeywordsOXIDOREDUCTASE / Neisseria meningitidis / quinol-dependent electrogenic Nitric Oxide Reductase (qNOR)
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 alpha14 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsJamali, M.M.A. / Gopalasingam, C.C. / Johnson, R.M. / Tosha, T. / Muench, S.P. / Muramoto, K. / Antonyuk, S.V. / Shiro, Y. / Hasnain, S.S.
Funding support Japan, United Kingdom, 6items
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, 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 ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id

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Structure visualization

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Assembly

Deposited unit
A: Nitric-oxide reductase
B: Nitric-oxide reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,43610
Polymers168,7782
Non-polymers2,6588
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10090 Å2
ΔGint-200 kcal/mol
Surface area52060 Å2

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Components

#1: Protein Nitric-oxide reductase


Mass: 84389.211 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neisseria meningitidis alpha14 (bacteria)
Strain: alpha14 / Gene: norB, NMO_1451 / Plasmid: pRSET-C / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): C43 / References: UniProt: C6S880, nitric-oxide reductase
#2: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Dimeric quinol dependent Nitric Oxide Reductase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)Organism: Neisseria meningitidis alpha14 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C43 / Plasmid: pRSET-C
Buffer solutionpH: 8
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Blot time of 6 seconds, with accompanying blot force of 6.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: -3500 nm / Nominal defocus min: -1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 69.44 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3182
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1.10CTF correction
7UCSF Chimera1.13.1model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
13PHENIX1.1.15model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles 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.
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 233556 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6L1X

6l1x


Accession code: 6L1X / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00712368
ELECTRON MICROSCOPYf_angle_d0.71716916
ELECTRON MICROSCOPYf_dihedral_angle_d6.1976878
ELECTRON MICROSCOPYf_chiral_restr0.0461808
ELECTRON MICROSCOPYf_plane_restr0.0042080

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