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- PDB-6cf3: Ethylene forming enzyme Y306A variant in complex with manganese a... -

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Basic information

Entry
Database: PDB / ID: 6cf3
TitleEthylene forming enzyme Y306A variant in complex with manganese and 2-oxoglutarate
Components2-oxoglutarate-dependent ethylene/succinate-forming enzyme
KeywordsOXIDOREDUCTASE / manganese / 2-oxoglutarate / L-arginine / split occupancy / partially occupied / tyrosine to alanine / ethylene glycol / variant induced fold changes
Function / homology
Function and homology information


2-oxoglutarate oxygenase/decarboxylase (ethylene-forming) activity / 2-oxoglutarate dioxygenase (ethene-forming) / 2-oxoglutarate/L-arginine monooxygenase/decarboxylase (succinate-forming) / ethylene biosynthetic process / dioxygenase activity / metal ion binding
Similarity search - Function
: / Non-haem dioxygenase N-terminal domain / non-haem dioxygenase in morphine synthesis N-terminal / Isopenicillin N synthase-like, Fe(2+) 2OG dioxygenase domain / 2OG-Fe(II) oxygenase superfamily / Isopenicillin N synthase-like superfamily / Oxoglutarate/iron-dependent dioxygenase / Fe(2+) 2-oxoglutarate dioxygenase domain profile.
Similarity search - Domain/homology
2-OXOGLUTARIC ACID / : / 2-oxoglutarate-dependent ethylene/succinate-forming enzyme
Similarity search - Component
Biological speciesPseudomonas savastanoi pv. phaseolicola (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.12 Å
AuthorsFellner, M. / Martinez, S. / Hu, J. / Hausinger, R.P.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM063584 United States
CitationJournal: Biochemistry / Year: 2024
Title: Structural, Spectroscopic, and Computational Insights from Canavanine-Bound and Two Catalytically Compromised Variants of the Ethylene-Forming Enzyme.
Authors: Chatterjee, S. / Fellner, M. / Rankin, J. / Thomas, M.G. / J S Rifayee, S.B. / Christov, C.Z. / Hu, J. / Hausinger, R.P.
History
DepositionFeb 13, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 13, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 10, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Jan 1, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Oct 4, 2023Group: 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 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_alt_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_alt_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_ptnr2_label_alt_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
Revision 1.4Apr 17, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: 2-oxoglutarate-dependent ethylene/succinate-forming enzyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,8884
Polymers39,6251
Non-polymers2633
Water6,323351
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)44.370, 85.410, 87.170
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP22121

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Components

#1: Protein 2-oxoglutarate-dependent ethylene/succinate-forming enzyme / Ethylene-forming enzyme / 2-oxoglutarate dioxygenase (ethylene-forming) / 2-oxoglutarate/L-arginine ...Ethylene-forming enzyme / 2-oxoglutarate dioxygenase (ethylene-forming) / 2-oxoglutarate/L-arginine monooxygenase/decarboxylase (succinate-forming)


Mass: 39624.668 Da / Num. of mol.: 1 / Mutation: N-terminal fusion to SH, Y306A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas savastanoi pv. phaseolicola (bacteria)
Gene: efe / Plasmid: pET28 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21 Gold (DE3)
References: UniProt: P32021, 2-oxoglutarate dioxygenase (ethene-forming), EC: 1.14.11.34
#2: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn
#3: Chemical ChemComp-AKG / 2-OXOGLUTARIC ACID


Mass: 146.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H6O5
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 351 / Source method: isolated from a natural source / Formula: H2O

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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: 2.08 Å3/Da / Density % sol: 40.98 % / Mosaicity: 0.2 °
Crystal growTemperature: 277.15 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 0.5 ul 64 mg/ml EFE-Y306A (1 mM manganese chloride, 25 mM HEPES pH 8.0, 1 mM TCEP, 3 mM L-Arginine, 3 mM 2-oxoglutarate) was mixed with 0.5 ul reservoir solution. The sitting drop reservoir ...Details: 0.5 ul 64 mg/ml EFE-Y306A (1 mM manganese chloride, 25 mM HEPES pH 8.0, 1 mM TCEP, 3 mM L-Arginine, 3 mM 2-oxoglutarate) was mixed with 0.5 ul reservoir solution. The sitting drop reservoir of 200 ul contained 25% w/v Polyethylene glycol 3,350, 0.1 M Bis-Tris pH 6.5, 0.2 M sodium chloride. The crystal was soaked for five minutes in 25% w/v Ethylene glycol, 75% reservoir solution before freezing it.

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-D / Wavelength: 0.9762 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Jun 3, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9762 Å / Relative weight: 1
ReflectionResolution: 1.12→44.37 Å / Num. obs: 125336 / % possible obs: 98.2 % / Redundancy: 5.9 % / Biso Wilson estimate: 8.62 Å2 / CC1/2: 0.992 / Rmerge(I) obs: 0.121 / Rpim(I) all: 0.053 / Rrim(I) all: 0.133 / Net I/σ(I): 7.9
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) all% possible all
1.12-1.144.60.64158840.7070.3290.72594
6.13-44.375.70.0958990.9820.0440.10599.1

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation5.87 Å44.37 Å
Translation5.87 Å44.37 Å

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Processing

Software
NameVersionClassification
MOSFLMdata reduction
Aimless0.5.32data scaling
PHASER2.7.16phasing
PHENIX1.13_2998refinement
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5V2Y

5v2y


Resolution: 1.12→44.37 Å / SU ML: 0.1 / Cross valid method: FREE R-VALUE / σ(F): 1.91 / Phase error: 14.97
RfactorNum. reflection% reflection
Rfree0.1673 11462 4.77 %
Rwork0.1478 --
obs0.1488 120087 97.53 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 49.16 Å2 / Biso mean: 14.2202 Å2 / Biso min: 4.9 Å2
Refinement stepCycle: final / Resolution: 1.12→44.37 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2620 0 35 351 3006
Biso mean--15.43 24.99 -
Num. residues----332
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 30

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.12-1.13270.28464390.26517046748591
1.1327-1.14610.26513560.2417464782095
1.1461-1.160.23753690.21447720808998
1.16-1.17470.21853430.20757662800598
1.1747-1.19020.23763700.19937709807999
1.1902-1.20650.224510.19797701815298
1.2065-1.22370.19044150.18287599801498
1.2237-1.2420.19423770.17617646802398
1.242-1.26140.18714210.17357682810398
1.2614-1.28210.20872950.16997698799398
1.2821-1.30420.19633480.16297753810198
1.3042-1.32790.17713640.15757564792897
1.3279-1.35340.18923170.15767672798997
1.3534-1.38110.18233510.15747662801397
1.3811-1.41110.17393770.14827578795597
1.4111-1.44390.14984010.1477361776295
1.4439-1.480.15843990.13187711811098
1.48-1.52010.14554230.12097684810799
1.5201-1.56480.14343440.11947729807399
1.5648-1.61530.14474490.11587657810699
1.6153-1.6730.13333730.11827733810699
1.673-1.740.13693870.11957649803698
1.74-1.81920.13993710.12577696806798
1.8192-1.91510.15823610.11857712807398
1.9151-2.03510.13163960.12057651804798
2.0351-2.19230.13273890.12357588797798
2.1923-2.41290.14943780.13037421779995
2.4129-2.7620.14993470.14397439778694
2.762-3.47960.17664380.152877548192100
3.4796-44.40450.18444130.164477728185100

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