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- EMDB-1534: EcoKI type I RM methyltransferase with DNA mimic Ocr. Negative st... -

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Basic information

Entry
Database: EMDB / ID: EMD-1534
TitleEcoKI type I RM methyltransferase with DNA mimic Ocr. Negative stain 3D.
Map data
SampleE. coli M.EcoKI (M2S1) bound to dimeric ocr from T7 phage:
HsdSThe Heaven Sword and Dragon Saber / HsdM / 0.3 gene
KeywordsEcoKI / methyltransferase / type I restriction / Ocr / HsdS / HsdM / T7
Function / homology
Function and homology information


restriction-modification system evasion by virus / protein binding / site-specific DNA-methyltransferase (adenine-specific) / site-specific DNA-methyltransferase (adenine-specific) activity / N-methyltransferase activity / DNA restriction-modification system / DNA binding / cytosol
Similarity search - Function
DNA mimic ocr / B-form DNA mimic Ocr / Protein Ocr / Protein Ocr / Type I restriction modification DNA specificity domain / N6 adenine-specific DNA methyltransferase, N-terminal domain superfamily / Type I restriction modification DNA specificity domain / Type I restriction modification DNA specificity domain / N-6 DNA Methylase / DNA methylase, adenine-specific ...DNA mimic ocr / B-form DNA mimic Ocr / Protein Ocr / Protein Ocr / Type I restriction modification DNA specificity domain / N6 adenine-specific DNA methyltransferase, N-terminal domain superfamily / Type I restriction modification DNA specificity domain / Type I restriction modification DNA specificity domain / N-6 DNA Methylase / DNA methylase, adenine-specific / DNA methylase, adenine-specific / N-6 Adenine-specific DNA methylases signature. / DNA methylase, N-6 adenine-specific, conserved site / S-adenosyl-L-methionine-dependent methyltransferase
Similarity search - Domain/homology
Protein Ocr / Type-1 restriction enzyme EcoKI specificity protein / Type I restriction enzyme EcoKI M protein
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Enterobacteria phage T7 (bacteriophage)
Methodsingle particle reconstruction / negative staining / Resolution: 18 Å
AuthorsKennaway CK / Obarska-Kosinska A / White JH / Tuszynska I / Cooper LP / Bujnicki JM / Trinick J / Dryden DTF
CitationJournal: Nucleic Acids Res / Year: 2009
Title: The structure of M.EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein.
Authors: Christopher K Kennaway / Agnieszka Obarska-Kosinska / John H White / Irina Tuszynska / Laurie P Cooper / Janusz M Bujnicki / John Trinick / David T F Dryden /
Abstract: Type-I DNA restriction-modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a ...Type-I DNA restriction-modification (R/M) systems are important agents in limiting the transmission of mobile genetic elements responsible for spreading bacterial resistance to antibiotics. EcoKI, a Type I R/M enzyme from Escherichia coli, acts by methylation- and sequence-specific recognition, leading to either methylation of DNA or translocation and cutting at a random site, often hundreds of base pairs away. Consisting of one specificity subunit, two modification subunits, and two DNA translocase/endonuclease subunits, EcoKI is inhibited by the T7 phage antirestriction protein ocr, a DNA mimic. We present a 3D density map generated by negative-stain electron microscopy and single particle analysis of the central core of the restriction complex, the M.EcoKI M(2)S(1) methyltransferase, bound to ocr. We also present complete atomic models of M.EcoKI in complex with ocr and its cognate DNA giving a clear picture of the overall clamp-like operation of the enzyme. The model is consistent with a large body of experimental data on EcoKI published over 40 years.
History
DepositionJul 8, 2008-
Header (metadata) releaseJul 8, 2008-
Map releaseApr 1, 2009-
UpdateFeb 4, 2011-
Current statusFeb 4, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 4.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2y7c
  • Surface level: 4.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2y7h
  • Surface level: 4.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1534.map.gz / Format: CCP4 / Size: 422.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.13 Å/pix.
x 48 pix.
= 150. Å
3.13 Å/pix.
x 48 pix.
= 150. Å
3.13 Å/pix.
x 48 pix.
= 150. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.125 Å
Density
Contour LevelBy AUTHOR: 6.41 / Movie #1: 4.05
Minimum - Maximum-3.13662 - 13.363099999999999
Average (Standard dev.)-0.104744 (±2.51747)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-24-24-24
Dimensions484848
Spacing484848
CellA=B=C: 150 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.1253.1253.125
M x/y/z484848
origin x/y/z0.0000.0000.000
length x/y/z150.000150.000150.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-75-75-74
NX/NY/NZ150150150
MAP C/R/S123
start NC/NR/NS-24-24-24
NC/NR/NS484848
D min/max/mean-3.13713.363-0.105

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Supplemental data

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Sample components

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Entire E. coli M.EcoKI (M2S1) bound to dimeric ocr from T7 phage

EntireName: E. coli M.EcoKI (M2S1) bound to dimeric ocr from T7 phage
Details: stained with 1pc uranyl acetate / Number of Components: 5 / Oligomeric State: HsdS-(HsdM)2-(ocr)2
MassTheoretical: 200 kDa

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Component #1: protein, HsdS

ProteinName: HsdSThe Heaven Sword and Dragon Saber / a.k.a: EcoKI specificity subunit / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 51.4 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: K-12
Source (natural)Location in cell: Cytoplasmic
External referencesGene Ontology: protein binding
InterPro: Type I restriction modification DNA specificity domain

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Component #2: protein, HsdM

ProteinName: HsdM / a.k.a: EcoKI methylase subunit / Oligomeric Details: Dimer / Recombinant expression: No / Number of Copies: 2
MassTheoretical: 59.3 kDa
SourceSpecies: Escherichia coli (E. coli) / Strain: K-12
Source (natural)Location in cell: cytoplasm
External referencesInterPro: DNA methylase, adenine-specific / Gene Ontology: protein binding

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Component #3: protein, 0.3 gene

ProteinName: 0.3 gene / a.k.a: ocr, 0.3 gene, from T7 phage / Oligomeric Details: Dimer / Recombinant expression: No / Number of Copies: 2
MassTheoretical: 13.8 kDa
SourceSpecies: Enterobacteria phage T7 (bacteriophage)
External referencesInterPro: Protein Ocr

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: negative staining
Sample solutionSpecimen conc.: 0.05 mg/mL / Buffer solution: 20mM Tris-Cl, 100 mM NaCl, / pH: 4.7
Support film400 mesh copper
StainingProtein was adsorbed onto UV treated carbon for 2 mins, blotted, then 1% uranyl acetate solution was applied for 1 min then blotted, twice.
VitrificationCryogen Name: NONE

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Electron microscopy imaging #1

ImagingMicroscope: JEOL 1200EX / Date: Feb 1, 2008 / Details: Customised JEOL 1200 EX microscope, low dose mode.
Electron gunElectron Source: LAB6 / Accelerating Voltage: 80 kV / Electron Dose: 25 e/Å2 / Illumination Mode: FLOOD BEAM
LensMagnification: 50000 X (nominal) / Astigmatism: Corrected at 80,000x / Cs: 2 mm / Imaging Mode: BRIGHT FIELD / Defocus: 275 - 870 nm
Specimen HolderHolder: Side entry / Model: OTHER / Temperature: 294
CameraDetector: KODAK SO-163 FILM

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Electron microscopy imaging #2

ImagingMicroscope: JEOL 1200EX / Date: Feb 1, 2008 / Details: Customised JEOL 1200 EX microscope, low dose mode.
Electron gunElectron Source: LAB6 / Accelerating Voltage: 80 kV / Electron Dose: 25 e/Å2 / Illumination Mode: FLOOD BEAM
LensMagnification: 40000 X (nominal) / Astigmatism: Corrected at 80,000x / Cs: 2 mm / Imaging Mode: BRIGHT FIELD / Defocus: 275 - 870 nm
Specimen HolderHolder: Side entry / Model: OTHER / Temperature: 294
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of Digital Images: 12 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling Size: 6.25 µm / Bit depth: 14

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Image processing

ProcessingMethod: single particle reconstruction / Number of Class Averages: 600 / Number of Projections: 17807
Details: The particles were selected using boxer in autobox mode.
Applied Symmetry: C2 (2 fold cyclic)
3D reconstructionAlgorithm: Projection matching from random blobs / Euler angles: EMAN / Software: EMAN, IMAGIC / CTF correction: Filtered at 1st zero
Details: Final map calculated from combined datasets taken at 50,000x and 40,000.
Resolution: 18 Å / Resolution Method: FSC 0.5

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Atomic model buiding

Modeling #1Software: Situs / Refinement protocol: rigid body / Refinement space: REAL
Details: Protocol: Rigid body. atomic model of whole complex (M2,S1,ocr2) fitted as single rigid body
Input PDB model: 1S7Z
Chain ID: A
Modeling #2Software: Situs / Refinement protocol: rigid body / Refinement space: REAL
Details: Protocol: Rigid body. HsdS homology model based on 1yf2 used. Atomic model of whole complex (M2,S1,ocr2) fitted as single rigid body
Input PDB model: 1YF2
Chain ID: A, B
Modeling #3Software: Situs / Refinement protocol: rigid body / Refinement space: REAL
Details: Protocol: Rigid body. Modified version of hsdM pdb used with alternative chain trace. Atomic model of whole complex (M2,S1,ocr2) fitted as single rigid body
Input PDB model: 2AR0
Chain ID: A, B, C, D, E, F, G, H
Output model

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