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- PDB-8c59: CpG specific M.MpeI methyltransferase crystallized in the presenc... -

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Basic information

Entry
Database: PDB / ID: 8c59
TitleCpG specific M.MpeI methyltransferase crystallized in the presence of 5-bromocytosine (converted to 5mC) and 5-methylcytosine containing dsDNA
Components
  • Cytosine-specific methyltransferase
  • DNA (5'-D(*CP*CP*AP*CP*AP*TP*GP*(5CM)P*GP*CP*TP*GP*AP*A)-3')
  • DNA (5'-D(*GP*TP*TP*CP*AP*GP*(5CM)P*GP*CP*AP*TP*GP*TP*G)-3')
KeywordsTRANSFERASE / M.MpeI / DNA methyltransferase / DNA / 5-methylcytosine / 5-bromocytosine / 5BrC / 5mC / CpG
Function / homology
Function and homology information


DNA (cytosine-5-)-methyltransferase / DNA (cytosine-5-)-methyltransferase activity / DNA restriction-modification system
Similarity search - Function
DNA methylase, C-5 cytosine-specific, active site / C-5 cytosine-specific DNA methylases active site. / C-5 cytosine-specific DNA methylase (Dnmt) domain profile. / C-5 cytosine methyltransferase / C-5 cytosine-specific DNA methylase / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
CITRIC ACID / CARBONATE ION / S-ADENOSYLMETHIONINE / DNA / DNA (> 10) / Cytosine-specific methyltransferase
Similarity search - Component
Biological speciesMalacoplasma penetrans HF-2 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.7 Å
AuthorsWojciechowski, M. / Czapinska, H. / Krwawicz, J. / Rafalski, D. / Bochtler, M.
Funding support Poland, European Union, 8items
OrganizationGrant numberCountry
Foundation for Polish ScienceTEAM/2010-6/1 Poland
European Union (EU)TEAM/2010-6/1European Union
Foundation for Polish SciencePOIR.04.04.00-00-31DF/17-00 Poland
European Union (EU)POIR.04.04.00-00-31DF/17-00European Union
Polish National Science CentreUMO-2014/13/B/NZ1/03991 Poland
Polish National Science CentreUMO-2014/14/M/NZ5/00558 Poland
Polish National Science CentreUMO-2018/30/Q/NZ2/00669 Poland
European Union (EU)POIG.02.02.00-14-024/08-00European Union
Citation
Journal: To Be Published
Title: Cytosine analogues as DNA methyltransferase substrates and inhibitors
Authors: Wojciechowski, M. / Czapinska, H. / Krwawicz, J. / Rafalski, D. / Bochtler, M.
#1: Journal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2013
Title: CpG underrepresentation and the bacterial CpG-specific DNA methyltransferase M.MpeI.
Authors: Wojciechowski, M. / Czapinska, H. / Bochtler, M.
#2: Journal: Science / Year: 2012
Title: Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation.
Authors: Song, J. / Teplova, M. / Ishibe-Murakami, S. / Patel, D.J.
History
DepositionJan 6, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cytosine-specific methyltransferase
B: DNA (5'-D(*CP*CP*AP*CP*AP*TP*GP*(5CM)P*GP*CP*TP*GP*AP*A)-3')
C: DNA (5'-D(*GP*TP*TP*CP*AP*GP*(5CM)P*GP*CP*AP*TP*GP*TP*G)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,33116
Polymers54,8293
Non-polymers1,50213
Water10,647591
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)84.511, 84.511, 173.115
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number92
Space group name H-MP41212
Components on special symmetry positions
IDModelComponents
11A-408-

GOL

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Cytosine-specific methyltransferase


Mass: 46238.941 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Malacoplasma penetrans HF-2 (bacteria) / Strain: HF-2 / Gene: MYPE4940 / Plasmid: pET-28a / Production host: Escherichia coli (E. coli) / Strain (production host): 2566 / References: UniProt: Q8EVR5

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DNA chain , 2 types, 2 molecules BC

#2: DNA chain DNA (5'-D(*CP*CP*AP*CP*AP*TP*GP*(5CM)P*GP*CP*TP*GP*AP*A)-3')


Mass: 4263.809 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#3: DNA chain DNA (5'-D(*GP*TP*TP*CP*AP*GP*(5CM)P*GP*CP*AP*TP*GP*TP*G)-3')


Mass: 4325.829 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)

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Non-polymers , 6 types, 604 molecules

#4: Chemical ChemComp-SAM / S-ADENOSYLMETHIONINE / S-Adenosyl methionine


Mass: 398.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H22N6O5S
#5: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#6: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol


Mass: 92.094 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C3H8O3
#7: Chemical ChemComp-CIT / CITRIC ACID / Citric acid


Mass: 192.124 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H8O7
#8: Chemical ChemComp-CO3 / CARBONATE ION / Carbonate


Mass: 60.009 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: CO3
#9: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 591 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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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: 56.11 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 5.6
Details: 10% PEG 3350, 150 mM NaCl, 50 mM sodium citrate (final pH, 5.6). For cryoprotection glycerol was added to 25% v/v

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.9116 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Sep 5, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9116 Å / Relative weight: 1
ReflectionResolution: 1.7→20 Å / Num. obs: 69838 / % possible obs: 99.8 % / Redundancy: 8.78 % / Biso Wilson estimate: 38.44 Å2 / CC1/2: 1 / Rrim(I) all: 0.041 / Rsym value: 0.039 / Net I/σ(I): 27.87
Reflection shellResolution: 1.7→1.8 Å / Redundancy: 8.52 % / Mean I/σ(I) obs: 2.15 / Num. unique obs: 11063 / CC1/2: 0.822 / Rrim(I) all: 0.98 / Rsym value: 0.919 / % possible all: 99.8

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Processing

Software
NameVersionClassification
XDSdata reduction
XDSdata scaling
REFMACphasing
ARP/wARPmodel building
REFMAC5.8.0258refinement
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4dkj

4dkj


Resolution: 1.7→19.95 Å / Cor.coef. Fo:Fc: 0.973 / Cor.coef. Fo:Fc free: 0.966 / SU B: 4.723 / SU ML: 0.068 / Cross valid method: THROUGHOUT / ESU R: 0.091 / ESU R Free: 0.089 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGEN ATOMS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES : WITH TLS ADDED. IONS AND SOLVENT MOLECULES HAVE BEEN MODELLED TENTATIVELY. S-ADENOSYLMETHIONINE (SAM) HAS BEEN MODELLED IN ...Details: HYDROGEN ATOMS HAVE BEEN ADDED IN THE RIDING POSITIONS. U VALUES : WITH TLS ADDED. IONS AND SOLVENT MOLECULES HAVE BEEN MODELLED TENTATIVELY. S-ADENOSYLMETHIONINE (SAM) HAS BEEN MODELLED IN THE CO-SUBSTRATE BINDING POCKET BUT ONLY PARTS OF THE MOLECULE CORRESPONDING TO ITS BASE AND AMINO ACID END ARE RESOLVED IN THE ELECTRON DENSITY. THE CENTRAL PART OF THE CO-SUBSTRATE COULD NOT BE UNAMBIGUOUSLY TRACED, WHICH MAY BE DUE TO ITS DECOMPOSITION TO HOMOSERINE LACTONE (HSL) AND METHYLTHIOADENOSINE (MTA). 5-BROMOCYTOSINE CONTAINING DNA HAS BEEN USED FOR CRYSTALLIZATION BUT 5-METHYLCYTOSINE IS OBSERVED IN THE CRYSTALS IN THE M.MPEI SUBSTRATE BINDING POCKET WHICH INDICATES THAT THE REACTION MUST HAVE TAKEN PLACE EITHER IN SOLUTION PRIOR TO CRYSTALLIZATION OR IN THE CRYSTAL. THE RESIDUAL DIFFERENCE DENSITY NEXT TO THE SUBSTRATE BASE LIKELY RESULTS FROM THE RESIDUAL PRESENCE OF THE ACTIVE SITE LOOP IN THE "IN" CONFORMATION BUT THE OCCUPANCY OF THIS CONFORMER WAS TOO LOW TO BE INCLUDED IN THE MODEL. Since the pH of the crystallization drop (reservoir buffer pH 5.6) was close to the pK for the HCO3-/CO32-+H+ equilibrium (pKa=6.1), a mixture of carbonate and hydrogencarbonate is likely present in the crystal.
RfactorNum. reflection% reflectionSelection details
Rfree0.19278 3498 5 %RANDOM
Rwork0.16652 ---
obs0.16782 66340 99.83 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 38.536 Å2
Baniso -1Baniso -2Baniso -3
1-0.38 Å2-0 Å2-0 Å2
2--0.38 Å2-0 Å2
3----0.77 Å2
Refinement stepCycle: 1 / Resolution: 1.7→19.95 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3107 570 99 591 4367
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0060.0184279
X-RAY DIFFRACTIONr_bond_other_d0.0020.023864
X-RAY DIFFRACTIONr_angle_refined_deg1.141.8645907
X-RAY DIFFRACTIONr_angle_other_deg0.81838994
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.5955452
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.65325.273165
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.03715726
X-RAY DIFFRACTIONr_dihedral_angle_4_deg14.0681514
X-RAY DIFFRACTIONr_chiral_restr0.0710.2609
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.024513
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02978
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.6792.2681709
X-RAY DIFFRACTIONr_mcbond_other0.6792.2681709
X-RAY DIFFRACTIONr_mcangle_it1.193.392194
X-RAY DIFFRACTIONr_mcangle_other1.193.3912195
X-RAY DIFFRACTIONr_scbond_it0.7782.512570
X-RAY DIFFRACTIONr_scbond_other0.7782.5072565
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other1.3423.7233710
X-RAY DIFFRACTIONr_long_range_B_refined5.93829.9415704
X-RAY DIFFRACTIONr_long_range_B_other5.08327.7615340
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.7→1.74 Å
RfactorNum. reflection% reflection
Rfree0.272 252 -
Rwork0.281 4770 -
obs--99.62 %
Refinement TLS params.Method: refined / Origin x: 22.761 Å / Origin y: 22.44 Å / Origin z: 46.066 Å
111213212223313233
T0.0387 Å2-0.0238 Å20.0006 Å2-0.1266 Å20.0071 Å2--0.005 Å2
L0.9903 °20.8856 °2-0.0374 °2-1.9251 °20.3458 °2--0.9666 °2
S0.0909 Å °-0.1558 Å °0.0272 Å °0.0029 Å °-0.1216 Å °-0.01 Å °-0.0614 Å °0.0753 Å °0.0307 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A7 - 393
2X-RAY DIFFRACTION1B1 - 7
3X-RAY DIFFRACTION1C1 - 14

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