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- EMDB-0731: CryoEM map and model of Nitrite Reductase at pH 8.1 -

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Basic information

Entry
Database: EMDB / ID: EMD-0731
TitleCryoEM map and model of Nitrite Reductase at pH 8.1
Map data
Sample
  • Organelle or cellular component: Nitrite reductase
    • Protein or peptide: Copper-containing nitrite reductase
  • Ligand: COPPER (II) ION
KeywordsCu containing nitrite reductase / OXIDOREDUCTASE
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-containing nitrite reductase
Similarity search - Component
Biological speciesAchromobacter cycloclastes (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsAdachi N / Yamaguchi T / Moriya T / Kawasaki M / Koiwai K / Shinoda A / Yamada Y / Yumoto F / Kohzuma T / Senda T
Funding support Japan, 1 items
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, 2019-
Header (metadata) releaseAug 12, 2020-
Map releaseAug 12, 2020-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0405
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0405
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6kng
  • Surface level: 0.0405
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0731.png

Downloads & links

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Map

FileDownload / File: emd_0731.map.gz / Format: CCP4 / Size: 437.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.88 Å/pix.
x 486 pix.
= 427.68 Å		0.88 Å/pix.
x 486 pix.
= 427.68 Å		0.88 Å/pix.
x 486 pix.
= 427.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.88 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0405 / Movie #1: 0.0405
Minimum - Maximum-0.07121556 - 0.16267285
Average (Standard dev.)-0.0000037978973 (±0.0022694988)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions486486486
Spacing486486486
CellA=B=C: 427.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.880.880.88
M x/y/z486486486
origin x/y/z0.0000.0000.000
length x/y/z427.680427.680427.680
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS486486486
D min/max/mean-0.0710.163-0.000

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Supplemental data

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Sample components

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Entire : Nitrite reductase

EntireName: Nitrite reductase
Components
  • Organelle or cellular component: Nitrite reductase
    • Protein or peptide: Copper-containing nitrite reductase
  • Ligand: COPPER (II) ION

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Supramolecule #1: Nitrite reductase

SupramoleculeName: Nitrite reductase / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Achromobacter cycloclastes (bacteria)
Molecular weightTheoretical: 110 KDa

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Macromolecule #1: Copper-containing nitrite reductase

MacromoleculeName: Copper-containing nitrite reductase / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO / EC number: nitrite reductase (NO-forming)
Source (natural)Organism: Achromobacter cycloclastes (bacteria)
Molecular weightTheoretical: 36.621332 KDa
SequenceString: VDISTLPRVK VDLVKPPFVH AHDQVAKTGP RVVEFTMTIE EKKLVIDREG TEIHAMTFNG SVPGPLMVVH ENDYVELRLI NPDTNTLLH NIDFHAATGA LGGGALTQVN PGEETTLRFK ATKPGVFVYH CAPEGMVPWH VTSGMNGAIM VLPRDGLKDE K GQPLTYDK ...String:
VDISTLPRVK VDLVKPPFVH AHDQVAKTGP RVVEFTMTIE EKKLVIDREG TEIHAMTFNG SVPGPLMVVH ENDYVELRLI NPDTNTLLH NIDFHAATGA LGGGALTQVN PGEETTLRFK ATKPGVFVYH CAPEGMVPWH VTSGMNGAIM VLPRDGLKDE K GQPLTYDK IYYVGEQDFY VPKDEAGNYK KYETPGEAYE DAVKAMRTLT PTHIVFNGAV GALTGDHALT AAVGERVLVV HS QANRDTR PHLIGGHGDY VWATGKFRNP PDLDQETWLI PGGTAGAAFY TFRQPGVYAY VNHNLIEAFE LGAAGHFKVT GEW NDDLMT SVVKPASM

UniProtKB: Copper-containing nitrite reductase

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Macromolecule #2: COPPER (II) ION

MacromoleculeName: COPPER (II) ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: CU
Molecular weightTheoretical: 63.546 Da
Chemical component information

ChemComp-CU:
COPPER (II) ION

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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

Concentration1.1 mg/mL
BufferpH: 8.1 / Component - Concentration: 100.0 mM / Component - Name: pottasium phosphate
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: The grid was washed by acetone prior to use.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time was 15 seconds..
DetailsThis sample was mono-disperse.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 694 / Average exposure time: 56.37 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 120000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Details: An ab initio model was generated using RELION3's own implementation of Stochastic Gradient Descent (SGD) algorithm and low-pass filtered to 25 A for use as an initial model for 3D refinement.
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Number images used: 89513
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)

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