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- EMDB-1893: EcoKI Type I DNA restriction-modification enzyme complex in close... -

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Basic information

Entry
Database: EMDB / ID: 1893
TitleEcoKI Type I DNA restriction-modification enzyme complex in closed state with bound 75bp cognate DNA fragment. 3D reconstruction by single particle analysis from negative stain EM.
KeywordsEcoKI / endonuclease / methyltransferase / type I restriction / DNA / HsdS / HsdM / HsdR / electron microscopy / negative stain / translocase / DEAD-box / ATPase
SampleEcoKI R2 M2 S1 complex with 75 bp cognate dsDNA fragment
SourceEscherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Synthetic construct
Map data3D reconstruction of EcoKI Type I restriction-modification enzyme with DNA fragment, from negative stain EM data.
Methodsingle particle reconstruction, at 20 A resolution
AuthorsKennaway CK / Taylor JE / Song CF / Potrzebowski W / White JH / Swiderska A / Obarska-Kosinska A / Callow P / Cooper LP / Roberts GA / Bujnicki JM / Trinick J / Kneale GG / Dryden DTF
CitationGenes Dev., 2012, 26, 92-104

Genes Dev., 2012, 26, 92-104 StrPapers
Structure and operation of the DNA-translocating type I DNA restriction enzymes.
Christopher K Kennaway / James E Taylor / Chun Feng Song / Wojciech Potrzebowski / William Nicholson / John H White / Anna Swiderska / Agnieszka Obarska-Kosinska / Philip Callow / Laurie P Cooper / Gareth A Roberts / Jean-Baptiste Artero / Janusz M Bujnicki / John Trinick / G Geoff Kneale / David T F Dryden

DateDeposition: Apr 6, 2011 / Header (metadata) release: Feb 15, 2012 / Map release: Feb 15, 2012 / Last update: Apr 6, 2011

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.1
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by radius
  • Surface level: 2.1
  • Imaged by UCSF CHIMERA
  • Download
3D viewer


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Supplemental images

Downloads & links

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Map

Fileemd_1893.map.gz (map file in CCP4 format, 433 KB)
Projections & slicesSize of images:
AxesZ (Sec.)Y (Row.)X (Col.)
48 pix
5.27 A/pix
= 252.96 A
48 pix
5.27 A/pix
= 252.96 A
48 pix
5.27 A/pix
= 252.96 A

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 5.27 A
Density
Contour Level:2.1 (by author), 2.1 (movie #1):
Minimum - Maximum-4.74770546 - 9.4093132
Average (Standard dev.)0E-8 (1)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions484848
Origin-24-24-24
Limit232323
Spacing484848
CellA=B=C: 252.95999 A
Alpha=beta=gamma: 90 deg.

CCP4 map header:

modeImage stored as Reals
A/pix X/Y/Z5.275.275.27
M x/y/z484848
origin x/y/z0.0000.0000.000
length x/y/z252.960252.960252.960
alpha/beta/gamma90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS-24-24-24
NC/NR/NS484848
D min/max/mean-4.7489.409-0.000

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Supplemental data

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Sample components

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Entire EcoKI R2 M2 S1 complex with 75 bp cognate dsDNA fragment

EntireName: EcoKI R2 M2 S1 complex with 75 bp cognate dsDNA fragment
Details: Stained with uranyl acetate / Number of components: 4 / Oligomeric State: 1x HsdS, 2x HsdM, 2x HsdR, 1x dsDNA
MassTheoretical: 440 kDa / Experimental: 420 kDa / Measured by: Small angle X-ray scattering (SAXS)

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Component #1: protein, EcoKI HsdS specificity subunit

ProteinName: EcoKI HsdS specificity subunit / a.k.a: HsdS / Oligomeric Details: Monomer / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 50 kDa
SourceSpecies: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Strain: R
Source (natural)Location in cell: Cytoplasm
External referencesGene Ontology: GO: 0005515 / InterPro: InterPro: 000055

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Component #2: protein, EcoKI HsdM methyltransferase subunit

ProteinName: EcoKI HsdM methyltransferase subunit / a.k.a: HsdM / Oligomeric Details: Dimer / Recombinant expression: No / Number of Copies: 2
MassTheoretical: 59 kDa
SourceSpecies: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Strain: R
Source (natural)Location in cell: cytoplasm
External referencesInterPro: InterPro: 003356 / Gene Ontology: GO: 0005515

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Component #3: protein, EcoKI HsdR endonuclease subunit

ProteinName: EcoKI HsdR endonuclease subunit / a.k.a: HsdR / Oligomeric Details: Monomer / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 120 kDa
SourceSpecies: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Strain: R
External referencesInterPro: InterPro: 004473 / Gene Ontology: GO: 0009307

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Component #4: nucleic-acid, Deoxyribonucleic acid

Nucleic-acidName: Deoxyribonucleic acid / a.k.a: DNA / Class: DNA / Structure: DOUBLE HELIX / Synthetic: Yes
Sequence:
AACNNNNNNG TGC
MassTheoretical: 15 kDa
SourceSpecies: Synthetic construct

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Experimental details

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Sample preparation

Specimen stateparticle
Sample solutionSpecimen conc.: 0.05 mg/ml / Buffer solution: 20mM Tris-Cl, 100 mM NaCl / pH: 4.7
Support film400 mesh copper with continuous carbon
StainingProtein was adsorbed onto UV treated carbon for 1 minute, blotted, then 1% uranyl acetate solution was applied for 1 min then blotted, three times.
VitrificationInstrument: NONE / Cryogen name: NONE / Details: Negative stain

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Electron microscopy imaging

ImagingMicroscope: JEOL 1200EXII / Date: Jul 1, 2004 / Details: Customised JEOL 1200 EX microscope, low dose mode.
Electron gunElectron source: TUNGSTEN HAIRPIN / Accelerating voltage: 80 kV / Electron dose: 40 e/A2 / Illumination mode: FLOOD BEAM
LensMagnification: 40000 X (nominal) / Astigmatism: Corrected at 80,000x / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 400 - 900 nm
Specimen HolderHolder: Side entry / Model: JEOL / Temperature: 294 K
CameraDetector: KODAK SO-163 FILM

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Image acquisition

Image acquisitionNumber of digital images: 50 / Scanner: OTHER / Sampling size: 20 microns / Bit depth: 8 / Details: Scanned on Imacon scanner

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Image processing

ProcessingMethod: single particle reconstruction / Number of class averages: 100 / Number of projections: 5786 / Details: The particles were manually selected using boxer. / Applied symmetry: C2 (2 fold cyclic)
3D reconstructionAlgorithm: Consensus starting model created from a combination of methods.
Euler angles: full range, C2 symmetry / Software: EMAN, IMAGIC, Spider, XMIPP / CTF correction: Filtered at 1st zero
Details: Final rounds of refinement done in EMAN using selected input classes.
Resolution: 20 A / Resolution method: FSC 0.5

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Atomic model buiding

Modeling #1Software: Chimera / Refinement protocol: rigid body / Target criteria: cross-correlation / Refinement space: RECIPROCAL
Details: Protocol: rigid body. Homology model based on HsdR from EcoR124 (2W00) was fitted into the density after the core methylase (HsdS and 2x HsdM) was fitted.
Input PDB model: 2W00
Chain ID: 2W00_A

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