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- EMDB-0989: Structure of Dimethylformamidase, dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-0989
TitleStructure of Dimethylformamidase, dimer
Map dataOne of the primary map from the refinement in Relion
Sample
  • Complex: Dimethylformamidase
    • Protein or peptide: N,N-dimethylformamidase large subunit
    • Protein or peptide: N,N-dimethylformamidase small subunit
  • Ligand: FE (III) ION
  • Ligand: water
Keywordsab polypeptide / mononuclear iron / amidohydrolase / tetramer / HYDROLASE
Function / homologyN,N-dimethylformamidase / N,N-dimethylformamidase activity / N,N-dimethylformamidase beta subunit-like, C-terminal / N,N-dimethylformamidase beta subunit-like, C-terminal / Concanavalin A-like lectin/glucanases superfamily / Concanavalin A-like lectin/glucanase domain superfamily / N,N-dimethylformamidase large subunit / N,N-dimethylformamidase small subunit
Function and homology information
Biological speciesParacoccus sp. SSG05 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsArya CA / Yadav S
Funding support India, 3 items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
Department of Biotechnology (DBT, India)BT/PR5081/INF/22/156/2012 India
Science and Engineering Research Board (SERB)SB/S2/RJN-094/2017 India
CitationJournal: Angew Chem Int Ed Engl / Year: 2020
Title: A 2-Tyr-1-carboxylate Mononuclear Iron Center Forms the Active Site of a Paracoccus Dimethylformamidase.
Authors: Chetan Kumar Arya / Swati Yadav / Jonathan Fine / Ana Casanal / Gaurav Chopra / Gurunath Ramanathan / Kutti R Vinothkumar / Ramaswamy Subramanian /
Abstract: N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved ...N,N-dimethyl formamide (DMF) is an extensively used organic solvent but is also a potent pollutant. Certain bacterial species from genera such as Paracoccus, Pseudomonas, and Alcaligenes have evolved to use DMF as a sole carbon and nitrogen source for growth via degradation by a dimethylformamidase (DMFase). We show that DMFase from Paracoccus sp. strain DMF is a halophilic and thermostable enzyme comprising a multimeric complex of the α β or (α β ) type. One of the three domains of the large subunit and the small subunit are hitherto undescribed protein folds of unknown evolutionary origin. The active site consists of a mononuclear iron coordinated by two Tyr side-chain phenolates and one carboxylate from Glu. The Fe ion in the active site catalyzes the hydrolytic cleavage of the amide bond in DMF. Kinetic characterization reveals that the enzyme shows cooperativity between subunits, and mutagenesis and structural data provide clues to the catalytic mechanism.
History
DepositionFeb 2, 2020-
Header (metadata) releaseJun 3, 2020-
Map releaseJun 3, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.066
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.066
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6lvc
  • Surface level: 0.066
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0989.png

Downloads & links

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Map

FileDownload / File: emd_0989.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOne of the primary map from the refinement in Relion
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.066 / Movie #1: 0.066
Minimum - Maximum-0.20070964 - 0.3651514
Average (Standard dev.)-0.000010341393 (±0.009318245)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 342.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z342.400342.400342.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-31-35-52
NX/NY/NZ11798209
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.2010.365-0.000

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Supplemental data

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Half map: One of the half map from the refinement in Relion

Fileemd_0989_half_map_1.map
AnnotationOne of the half map from the refinement in Relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: One of the half map from the refinement in Relion

Fileemd_0989_half_map_2.map
AnnotationOne of the half map from the refinement in Relion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimethylformamidase

EntireName: Dimethylformamidase
Components
  • Complex: Dimethylformamidase
    • Protein or peptide: N,N-dimethylformamidase large subunit
    • Protein or peptide: N,N-dimethylformamidase small subunit
  • Ligand: FE (III) ION
  • Ligand: water

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Supramolecule #1: Dimethylformamidase

SupramoleculeName: Dimethylformamidase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: Dimer, (a2b2)
Source (natural)Organism: Paracoccus sp. SSG05 (bacteria)
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: N,N-dimethylformamidase large subunit

MacromoleculeName: N,N-dimethylformamidase large subunit / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: N,N-dimethylformamidase
Source (natural)Organism: Paracoccus sp. SSG05 (bacteria)
Molecular weightTheoretical: 86.341758 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKDIAIRGYC DRPSVATGET IRFYVSANET RGTFDAELVR LIHGDSNPAG PGYKEEAIKS DLEGQYPARF QRTQFGSYVE VADPDAGLQ PDGAFSVHLF LWSTTPSRGR QGIASRWNDE RQSGWNLAIE DGRVVFTIGD GSGATSSVVS DRPLFQQIWY S ITGVYDPE ...String:
MKDIAIRGYC DRPSVATGET IRFYVSANET RGTFDAELVR LIHGDSNPAG PGYKEEAIKS DLEGQYPARF QRTQFGSYVE VADPDAGLQ PDGAFSVHLF LWSTTPSRGR QGIASRWNDE RQSGWNLAIE DGRVVFTIGD GSGATSSVVS DRPLFQQIWY S ITGVYDPE KKQLRLYQKS VVNRTNSRFG LVVPLDSDCA VSADATVKAA DSETSLLIAG LGEAAAQDGR TWCIAHYNGK VD APKIYGC ALGQDDAEKL SRGEIVRPIS RLAHWDFSAG IGLNGIPTDH VVDASGYGHH GRCMNQPSRG STGWNWDGHE ENF IHCPEQ YGALWFHEDC LDDCRWEKDF EFTVPEGLKS DFYAVKIRYE DTEDYIPFFV LPPRGTATAP ILVIASTLSY LAYA NEQIM HKADIGQAVA GHTPVLNEND VELHKNLSYY GLSTYDGHID GRGVQYTSWR RPIMNLRPKH RQGFGSIWEL PADLH LIDW LNHNGFEYDV ATEHDLNDQG AELLRRYKVV LTGSHPEYQT WANADAWEDY LADGGRGMYL AANGMYWIVE VHPEKP WVM EVRKELGVTA WEAPPGEYHY STNGRRGGRF RGRARATQKI WGTGMSSFGF DHSGYFVQMP DSQDERVAWI MEGIDPE ER IGDGGLVGGG AGGYELDRYD LALGTPPNTL LLASSVEHSV VYTVIPDDKA FPHPGMNGGE HPFVRADITY FSTANGGG M FATSSISWLG SLSWNDYDNN VSKMTKNVLN QFIKDEPAPR VKLAAALEHH HHHH

UniProtKB: N,N-dimethylformamidase large subunit

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Macromolecule #2: N,N-dimethylformamidase small subunit

MacromoleculeName: N,N-dimethylformamidase small subunit / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: N,N-dimethylformamidase
Source (natural)Organism: Paracoccus sp. SSG05 (bacteria)
Molecular weightTheoretical: 16.083823 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MTEASESCVR DPSNYRDRSA DWYAFYDERR RKEIIDIIDE HPEIVEEHAA NPFGYRKHPS PYLQRVHNYF RMQPTFGRYY IYSEREWDA YRIATIREFG ELPELGDERF KTEEEAMHAV FLRRIEDVRA ELA

UniProtKB: N,N-dimethylformamidase small subunit

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Macromolecule #3: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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: 7.2 / Component - Concentration: 50.0 mM / Component - Name: Tris-Cl
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR
Details: Glow discharge was carried out with Quorum glocube at 25 mA for 90 seconds
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV
Details: Blot force was 0 and blotting was done for 3.5 seconds.
DetailsSample at no salt exists mostly as dimer. Buffer used had no salt.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 5.148 µm / Calibrated defocus min: 1.526 µm / Calibrated magnification: 130841 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.148 µm / Nominal defocus min: 1.526 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.7 K / Max: 77.7 K
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1147 / Average exposure time: 60.0 sec. / Average electron dose: 27.0 e/Å2
Details: A total of 25 frames were saved from the 60 second exposure, resulting in ~1.08 electron/frame
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 169988
Details: Two rounds of 2D classification was performed prior to angular assignment and reconstruction
Startup modelType of model: OTHER
Details: The model was obtained from the 2D class averages of the data.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 80674
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER / Overall B value: 42
Output model

PDB-6lvc:
Structure of Dimethylformamidase, dimer

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