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- PDB-5eeh: Crystal structure of carminomycin-4-O-methyltransferase DnrK in c... -

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Basic information

Entry
Database: PDB / ID: 5eeh
TitleCrystal structure of carminomycin-4-O-methyltransferase DnrK in complex with SAH and 2-chloro-4-nitrophenol
ComponentsCarminomycin 4-O-methyltransferase DnrK
KeywordsTRANSFERASE / unnatural substrate / Structural Genomics / PSI-Biology / Protein Structure Initiative / Enzyme Discovery for Natural Product Biosynthesis / NatPro
Function / homology
Function and homology information


carminomycin 4-O-methyltransferase / daunorubicin biosynthetic process / O-methyltransferase activity / methyltransferase activity / methylation
Similarity search - Function
Helix Hairpins - #1350 / O-methyltransferase domain / O-methyltransferase domain / SAM-dependent O-methyltransferase class II-type profile. / O-methyltransferase COMT-type / Vaccinia Virus protein VP39 / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / Helix Hairpins / Arc Repressor Mutant, subunit A / Winged helix DNA-binding domain superfamily ...Helix Hairpins - #1350 / O-methyltransferase domain / O-methyltransferase domain / SAM-dependent O-methyltransferase class II-type profile. / O-methyltransferase COMT-type / Vaccinia Virus protein VP39 / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / Helix Hairpins / Arc Repressor Mutant, subunit A / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
2-chloranyl-4-nitro-phenol / S-ADENOSYL-L-HOMOCYSTEINE / Carminomycin 4-O-methyltransferase DnrK
Similarity search - Component
Biological speciesStreptomyces peucetius (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.82 Å
AuthorsWang, F. / Singh, S. / Thorson, J.S. / Phillips Jr., G.N. / Enzyme Discovery for Natural Product Biosynthesis (NatPro)
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U01 GM098248 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R37 AI52188 United States
CitationJournal: Acs Chem.Biol. / Year: 2016
Title: Functional AdoMet Isosteres Resistant to Classical AdoMet Degradation Pathways.
Authors: Huber, T.D. / Wang, F. / Singh, S. / Johnson, B.R. / Zhang, J. / Sunkara, M. / Van Lanen, S.G. / Morris, A.J. / Phillips, G.N. / Thorson, J.S.
History
DepositionOct 22, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 16, 2015Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2016Group: Database references
Revision 1.2Sep 28, 2016Group: Database references
Revision 1.3Sep 20, 2017Group: Author supporting evidence / Derived calculations / Category: pdbx_audit_support / pdbx_struct_oper_list
Item: _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation
Revision 1.4Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Sep 27, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Carminomycin 4-O-methyltransferase DnrK
B: Carminomycin 4-O-methyltransferase DnrK
C: Carminomycin 4-O-methyltransferase DnrK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)127,62230
Polymers123,0013
Non-polymers4,62127
Water16,448913
1
A: Carminomycin 4-O-methyltransferase DnrK
hetero molecules

A: Carminomycin 4-O-methyltransferase DnrK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,62124
Polymers82,0012
Non-polymers3,62022
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_757-x+2,y,-z+21
Buried area8830 Å2
ΔGint-165 kcal/mol
Surface area25950 Å2
MethodPISA
2
B: Carminomycin 4-O-methyltransferase DnrK
hetero molecules

C: Carminomycin 4-O-methyltransferase DnrK
hetero molecules


Theoretical massNumber of molelcules
Total (without water)84,81218
Polymers82,0012
Non-polymers2,81116
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_858-x+3,y,-z+31
Buried area7620 Å2
ΔGint-115 kcal/mol
Surface area26370 Å2
MethodPISA
Unit cell
Length a, b, c (Å)123.347, 110.678, 116.869
Angle α, β, γ (deg.)90.000, 120.240, 90.000
Int Tables number5
Space group name H-MC121
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21chain B
31chain C

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111chain AA11 - 1006
211chain BB13 - 1006
311chain CC11 - 1003

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Components

#1: Protein Carminomycin 4-O-methyltransferase DnrK / COMT / Anthracycline 4-O-methyltransferase


Mass: 41000.406 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptomyces peucetius (bacteria) / Gene: dnrK / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q06528, carminomycin 4-O-methyltransferase
#2: Chemical ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE


Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C14H20N6O5S
#3: Chemical
ChemComp-P9P / 2-chloranyl-4-nitro-phenol


Mass: 173.554 Da / Num. of mol.: 15 / Source method: obtained synthetically / Formula: C6H4ClNO3
#4: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: SO4
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 913 / 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: 2.8 Å3/Da / Density % sol: 56.1 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8 / Details: 1.26M (NH4)SO4, 0.1M Tris pH 8.0, 0.2M Li2SO4

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-F / Wavelength: 0.98 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Jun 20, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 1.82→48.5 Å / Num. all: 578485 / Num. obs: 235152 / % possible obs: 98.6 % / Observed criterion σ(I): -3 / Redundancy: 2.46 % / Biso Wilson estimate: 21.17 Å2 / Rmerge F obs: 0.996 / Rmerge(I) obs: 0.075 / Rrim(I) all: 0.08 / Rsym value: 0.071 / Χ2: 1.002 / Net I/σ(I): 10.53 / Num. measured all: 695966
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Highest resolution (Å)Redundancy (%)Rmerge F obsRmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsRrim(I) all% possible all
1.82-1.8652.330.520.4132.784467456152311981.00692.3
1.71-1.820.7560.4631.497310452782433520.6182.1
1.82-1.970.8960.2913.0711347149252485210.37698.5
1.97-2.160.9610.1595.6611087445092448310.20499.4
2.16-2.410.9820.0968.910088840919405990.12399.2
2.41-2.780.990.06712.318914036113357590.08699
2.78-3.410.9930.0516.727491530476300690.06498.7
3.41-4.80.9940.04121.585746323592230610.05397.7
4.80.9940.03922.863143713080126430.0596.7

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Processing

Software
NameVersionClassification
XSCALEdata scaling
PHENIX(phenix.refine: 1.9_1692)refinement
PDB_EXTRACT3.15data extraction
XDSdata reduction
PHENIX(phenix.refine: 1.9_1692)phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5EEG

5eeg


Resolution: 1.82→48.5 Å / SU ML: 0.17 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 17.99 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.1855 1974 1.65 %Random selection
Rwork0.1544 117392 --
obs0.1549 119366 98.49 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 133.9 Å2 / Biso mean: 32.4476 Å2 / Biso min: 8.23 Å2
Refinement stepCycle: final / Resolution: 1.82→48.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7918 0 288 913 9119
Biso mean--33.59 41.69 -
Num. residues----1025
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0078527
X-RAY DIFFRACTIONf_angle_d1.11811652
X-RAY DIFFRACTIONf_chiral_restr0.0461310
X-RAY DIFFRACTIONf_plane_restr0.0051528
X-RAY DIFFRACTIONf_dihedral_angle_d12.2593092
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A4805X-RAY DIFFRACTION3.88TORSIONAL
12B4805X-RAY DIFFRACTION3.88TORSIONAL
13C4805X-RAY DIFFRACTION3.88TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.82-1.86550.24741330.22197809794292
1.8655-1.9160.25051420.19538255839797
1.916-1.97230.24111220.18038256837897
1.9723-2.0360.2081470.16818317846498
2.036-2.10880.19431410.16318371851299
2.1088-2.19320.18731440.16078376852099
2.1932-2.2930.19921400.15358453859399
2.293-2.41390.19131450.15668430857599
2.4139-2.56510.20541420.154684808622100
2.5651-2.76320.18321470.159284798626100
2.7632-3.04120.19881430.160984798622100
3.0412-3.48120.19641430.155585278670100
3.4812-4.38550.13821440.130185448688100
4.3855-48.54480.16831410.143886168757100
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.61740.06230.44730.3031-0.05011.18980.036-0.09140.00620.0685-0.0226-0.0633-0.10980.1177-0.00840.1088-0.01970.01690.1112-0.01240.132377.145228.7529115.0895
20.73250.32820.45050.6501-0.03041.12580.031-0.007-0.0996-0.04530.05010.02420.1041-0.0725-0.07740.1323-0.04080.01520.13940.02970.1828104.121151.2115118.2218
30.49630.2660.11581.27561.23231.4491-0.00990.2256-0.0416-0.6107-0.1790.225-0.442-0.22940.10970.41450.1648-0.09630.3368-0.08070.230971.628859.4978152.3874
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1(chain 'A' and resid 11 through 407)A0
2X-RAY DIFFRACTION2(chain 'B' and resid 13 through 407)B0
3X-RAY DIFFRACTION3(chain 'C' and resid 11 through 404)C0

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