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- PDB-2y7h: Atomic model of the DNA-bound methylase complex from the Type I r... -
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Basic information
Entry | Database: PDB / ID: 2y7h | ||||||
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Title | Atomic model of the DNA-bound methylase complex from the Type I restriction-modification enzyme EcoKI (M2S1). Based on fitting into EM map 1534. | ||||||
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![]() | TRANSFERASE/DNA / TRANSFERASE-DNA COMPLEX | ||||||
Function / homology | ![]() type I site-specific deoxyribonuclease complex / N-methyltransferase activity / site-specific DNA-methyltransferase (adenine-specific) / site-specific DNA-methyltransferase (adenine-specific) activity / DNA restriction-modification system / methylation / DNA binding / cytosol Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 18 Å | ||||||
![]() | Kennaway, C.K. / Obarska-Kosinska, A. / White, J.H. / Tuszynska, I. / Cooper, L.P. / Bujnicki, J.M. / Trinick, J. / Dryden, D.T.F. | ||||||
![]() | ![]() 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. | ||||||
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 299.2 KB | Display | ![]() |
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PDB format | ![]() | 233.7 KB | Display | ![]() |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 1534MC ![]() 2y7cC C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 51468.191 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() References: UniProt: P05719, type I site-specific deoxyribonuclease | ||||||
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#2: Protein | Mass: 59378.324 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() References: UniProt: P08957, type I site-specific deoxyribonuclease, site-specific DNA-methyltransferase (adenine-specific) #3: DNA chain | | Mass: 6118.968 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: FLIPPED OUT ADENINE / Source: (synth.) ![]() ![]() #4: DNA chain | | Mass: 6149.978 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: FLIPPED OUT ADENINE BASE / Source: (synth.) ![]() ![]() #5: Chemical | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: M.ECOKI WITH DNA / Type: COMPLEX |
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Buffer solution | Name: 20MM TRIS-CL, 100 MM NACL / pH: 4.7 / Details: 20MM TRIS-CL, 100 MM NACL |
Specimen | Conc.: 0.05 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO |
EM staining | Type: NEGATIVE / Material: Uranyl Acetate |
Specimen support | Details: CARBON |
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Electron microscopy imaging
Microscopy | Model: JEOL 1200EX / Date: Feb 1, 2008 |
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Electron gun | Electron source: TUNGSTEN HAIRPIN / Accelerating voltage: 80 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 40000 X / Calibrated magnification: 39500 X / Nominal defocus max: 870 nm / Nominal defocus min: 275 nm / Cs: 2 mm |
Specimen holder | Temperature: 294 K |
Image recording | Electron dose: 25 e/Å2 / Film or detector model: KODAK SO-163 FILM |
Radiation wavelength | Relative weight: 1 |
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Processing
EM software |
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CTF correction | Details: FILTERED AT FIRST ZERO | ||||||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||||||
3D reconstruction | Method: RANDOM SPHERES STARTING MODEL / Resolution: 18 Å / Num. of particles: 17807 / Nominal pixel size: 3.12 Å / Actual pixel size: 3.12 Å / Magnification calibration: TMV Details: HSDM N-TERMINAL DOMAIN RETRACED FROM PDB ENTRY 2AR0. DISORDERED C-TERMINUS OF HSDM MODELLED INTO DENSITY. Symmetry type: POINT | ||||||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT / Space: REAL / Details: METHOD--UROX REFINEMENT PROTOCOL--RIGID BODY | ||||||||||||||||||||||||||||
Atomic model building |
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Refinement | Highest resolution: 18 Å | ||||||||||||||||||||||||||||
Refinement step | Cycle: LAST / Highest resolution: 18 Å
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