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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 dataNegative stain EM reconstruction of M.EcoKI-Ocr (Two different models submitted to PDB: 2Y7C, 2Y7H)
Sample
  • Sample: E. coli M.EcoKI (M2S1) bound to dimeric ocr from T7 phage
  • Protein or peptide: HsdSThe Heaven Sword and Dragon Saber
  • Protein or peptide: HsdM
  • Protein or peptide: 0.3 gene
KeywordsEcoKI / methyltransferase / type I restriction / Ocr / HsdS / HsdM / T7
Function / homology
Function and homology information


type I site-specific deoxyribonuclease complex / symbiont-mediated evasion of host restriction-modification system / protein binding / N-methyltransferase activity / site-specific DNA-methyltransferase (adenine-specific) / site-specific DNA-methyltransferase (adenine-specific) activity / DNA restriction-modification system / DNA binding / cytosol
Similarity search - Function
B-form DNA mimic Ocr / DNA mimic ocr / Protein Ocr / Protein Ocr / Type I restriction modification DNA specificity domain superfamily / Type I restriction modification DNA specificity domain / N6 adenine-specific DNA methyltransferase, N-terminal domain / Type I restriction enzyme EcoKI-like, methylase subunit, N-terminal domain superfamily / HsdM N-terminal domain / Type I restriction modification DNA specificity domain ...B-form DNA mimic Ocr / DNA mimic ocr / Protein Ocr / Protein Ocr / Type I restriction modification DNA specificity domain superfamily / Type I restriction modification DNA specificity domain / N6 adenine-specific DNA methyltransferase, N-terminal domain / Type I restriction enzyme EcoKI-like, methylase subunit, N-terminal domain superfamily / HsdM N-terminal domain / 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 superfamily
Similarity search - Domain/homology
Protein Ocr / Type I restriction enzyme EcoKI specificity subunit / Type I restriction enzyme EcoKI methylase subunit
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Enterobacteria phage T7 (virus)
Methodsingle particle reconstruction / negative staining / Resolution: 18.0 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
  • Download
  • 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
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-2y7h
  • Surface level: 4.05
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1534.gif

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)
AnnotationNegative stain EM reconstruction of M.EcoKI-Ocr (Two different models submitted to PDB: 2Y7C, 2Y7H)
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

Histogram

Histogram (log scale)
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
Components
  • Sample: E. coli M.EcoKI (M2S1) bound to dimeric ocr from T7 phage
  • Protein or peptide: HsdSThe Heaven Sword and Dragon Saber
  • Protein or peptide: HsdM
  • Protein or peptide: 0.3 gene

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Supramolecule #1000: E. coli M.EcoKI (M2S1) bound to dimeric ocr from T7 phage

SupramoleculeName: E. coli M.EcoKI (M2S1) bound to dimeric ocr from T7 phage
type: sample / ID: 1000 / Details: stained with 1pc uranyl acetate / Oligomeric state: HsdS-(HsdM)2-(ocr)2 / Number unique components: 5
Molecular weightTheoretical: 200 KDa

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Macromolecule #1: HsdS

MacromoleculeName: HsdS / type: protein_or_peptide / ID: 1 / Name.synonym: EcoKI specificity subunit / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12 / Location in cell: Cytoplasmic
Molecular weightTheoretical: 51.4 KDa
SequenceGO: protein binding
InterPro: Type I restriction modification DNA specificity domain

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Macromolecule #2: HsdM

MacromoleculeName: HsdM / type: protein_or_peptide / ID: 2 / Name.synonym: EcoKI methylase subunit / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K-12 / Location in cell: cytoplasm
Molecular weightTheoretical: 59.3 KDa
SequenceGO: protein binding / InterPro: DNA methylase, adenine-specific

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Macromolecule #3: 0.3 gene

MacromoleculeName: 0.3 gene / type: protein_or_peptide / ID: 3 / Name.synonym: ocr, 0.3 gene, from T7 phage / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: No
Source (natural)Organism: Enterobacteria phage T7 (virus) / synonym: phage T7
Molecular weightTheoretical: 13.8 KDa
SequenceInterPro: Protein Ocr

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.050 mg/mL
BufferpH: 4.7 / Details: 20mM Tris-Cl, 100 mM NaCl,
StainingType: NEGATIVE
Details: Protein was adsorbed onto UV treated carbon for 2 mins, blotted, then 1% uranyl acetate solution was applied for 1 min then blotted, twice.
GridDetails: 400 mesh copper
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy #1

MicroscopeJEOL 1200EX
Electron beamAcceleration voltage: 80 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.87 µm / Nominal defocus min: 0.275 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry / Specimen holder model: OTHER
TemperatureAverage: 294 K
Microscopy ID1
Alignment procedureLegacy - Astigmatism: Corrected at 80,000x
DetailsCustomised JEOL 1200 EX microscope, low dose mode.
DateFeb 1, 2008
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.25 µm / Number real images: 12 / Average electron dose: 25 e/Å2 / Bits/pixel: 14

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Electron microscopy #2

MicroscopeJEOL 1200EX
Electron beamAcceleration voltage: 80 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.87 µm / Nominal defocus min: 0.275 µm / Nominal magnification: 40000
Sample stageSpecimen holder: Side entry / Specimen holder model: OTHER
TemperatureAverage: 294 K
Microscopy ID2
Alignment procedureLegacy - Astigmatism: Corrected at 80,000x
DetailsCustomised JEOL 1200 EX microscope, low dose mode.
DateFeb 1, 2008
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.25 µm / Number real images: 12 / Average electron dose: 25 e/Å2 / Bits/pixel: 14

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Image processing

CTF correctionDetails: Filtered at 1st zero
Final two d classificationNumber classes: 600
Final angle assignmentDetails: EMAN
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, IMAGIC
Details: Final map calculated from combined datasets taken at 50,000x and 40,000.
Number images used: 17807
DetailsThe particles were selected using boxer in autobox mode.

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Atomic model buiding 1

Initial modelPDB ID:

1s7z


Chain - Chain ID: A
SoftwareName: Situs
DetailsProtocol: Rigid body. atomic model of whole complex (M2,S1,ocr2) fitted as single rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-2y7c:
Atomic model of the Ocr-bound methylase complex from the Type I restriction-modification enzyme EcoKI (M2S1). Based on fitting into EM map 1534.

PDB-2y7h:
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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Atomic model buiding 2

Initial modelPDB ID:

1yf2


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B
SoftwareName: Situs
DetailsProtocol: Rigid body. HsdS homology model based on 1yf2 used. Atomic model of whole complex (M2,S1,ocr2) fitted as single rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-2y7c:
Atomic model of the Ocr-bound methylase complex from the Type I restriction-modification enzyme EcoKI (M2S1). Based on fitting into EM map 1534.

PDB-2y7h:
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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Atomic model buiding 3

Initial modelPDB ID:

2ar0


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: D / Chain - #4 - Chain ID: E / Chain - #5 - Chain ID: F / Chain - #6 - Chain ID: G / Chain - #7 - Chain ID: H
SoftwareName: Situs
DetailsProtocol: 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
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-2y7c:
Atomic model of the Ocr-bound methylase complex from the Type I restriction-modification enzyme EcoKI (M2S1). Based on fitting into EM map 1534.

PDB-2y7h:
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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