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- PDB-6knf: CryoEM map and model of Nitrite Reductase at pH 6.2 -

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Basic information

Entry
Database: PDB / ID: 6knf
TitleCryoEM map and model of Nitrite Reductase at pH 6.2
ComponentsCopper-containing nitrite reductase
KeywordsOXIDOREDUCTASE / Cu containing nitrite reductase
Function / homology
Function and homology information


denitrification pathway / nitrite reductase (NO-forming) / nitrite reductase (NO-forming) activity / nitrate assimilation / periplasmic space / copper ion binding
Similarity search - Function
Nitrite reductase, copper-type / Multicopper oxidase, type 1 / Multicopper oxidase / Multicopper oxidase / Multicopper oxidase, N-terminal / Multicopper oxidase / Twin arginine translocation (Tat) signal profile. / Twin-arginine translocation pathway, signal sequence / Cupredoxin
Similarity search - Domain/homology
COPPER (II) ION / Copper-containing nitrite reductase
Similarity search - Component
Biological speciesAchromobacter cycloclastes (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsAdachi, N. / Yamaguchi, T. / Moriya, T. / Kawasaki, M. / Koiwai, K. / Shinoda, A. / Yamada, Y. / Yumoto, F. / Kohzuma, T. / Senda, T.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP19am0101071 Japan
CitationJournal: J Struct Biol / Year: 2021
Title: 2.85 and 2.99 Å resolution structures of 110 kDa nitrite reductase determined by 200 kV cryogenic electron microscopy.
Authors: Naruhiko Adachi / Takahide Yamaguchi / Toshio Moriya / Masato Kawasaki / Kotaro Koiwai / Akira Shinoda / Yusuke Yamada / Fumiaki Yumoto / Takamitsu Kohzuma / Toshiya Senda /
Abstract: Cu-containing nitrite reductases (NiRs) are 110 kDa enzymes that play central roles in denitrification. Although the NiRs have been well studied, with over 100 Protein Data Bank entries, such issues ...Cu-containing nitrite reductases (NiRs) are 110 kDa enzymes that play central roles in denitrification. Although the NiRs have been well studied, with over 100 Protein Data Bank entries, such issues as crystal packing, photoreduction, and lack of high pH cases have impeded structural analysis of their catalytic mechanisms. Here we show the cryogenic electron microscopy (cryo-EM) structures of Achromobacter cycloclastes NiR (AcNiR) at pH 6.2 and 8.1. The optimization of 3D-reconstruction parameters achieved 2.99 and 2.85 Å resolution. Comprehensive comparisons with cryo-EM and 56 AcNiR crystal structures suggested crystallographic artifacts in residues 185-215 and His255' due to packing and photoreduction, respectively. We used a newly developed map comparison method to detect structural change around the type 2 Cu site. While the theoretical estimation of coordinate errors of cryo-EM structures remains difficult, combined analysis using X-ray and cryo-EM structures will allow deeper insight into the local structural changes of proteins.
History
DepositionAug 5, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 12, 2020Provider: repository / Type: Initial release
Revision 1.0Aug 12, 2020Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 12, 2020Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 12, 2020Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Aug 12, 2020Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 12, 2020Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Aug 12, 2020Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 12, 2020Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Aug 25, 2021Group: Database references / Category: citation / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 1.3Jul 2, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1Jul 2, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: Copper-containing nitrite reductase
B: Copper-containing nitrite reductase
C: Copper-containing nitrite reductase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)110,2459
Polymers109,8643
Non-polymers3816
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12680 Å2
ΔGint-95 kcal/mol
Surface area35590 Å2

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Components

#1: Protein Copper-containing nitrite reductase / Cu-NIR


Mass: 36621.332 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Achromobacter cycloclastes (bacteria) / References: UniProt: P25006, nitrite reductase (NO-forming)
#2: Chemical
ChemComp-CU / COPPER (II) ION


Mass: 63.546 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Cu / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: Nitrite reductase / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.11 MDa / Experimental value: NO
Source (natural)Organism: Achromobacter cycloclastes (bacteria)
Buffer solutionpH: 6.2
Buffer componentConc.: 100 mM / Name: potassium phosphate
SpecimenConc.: 1.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-disperse.
Specimen supportDetails: The grid was washed by acetone prior to use. / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K / Details: Blotting time was 15 seconds

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 120000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 794

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.06CTF correction
8PHENIXmodel refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 121353 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0077953
ELECTRON MICROSCOPYf_angle_d0.63510845
ELECTRON MICROSCOPYf_dihedral_angle_d5.4891074
ELECTRON MICROSCOPYf_chiral_restr0.0481197
ELECTRON MICROSCOPYf_plane_restr0.0051422

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