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- EMDB-1892: EcoR124 Type I DNA restriction-modification enzyme complex (in cl... -

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Basic information

Entry
Database: EMDB / ID: EMD-1892
TitleEcoR124 Type I DNA restriction-modification enzyme complex (in closed state) with bound DNA mimic protein Ocr from phage T7. 3D reconstruction by single particle analysis from negative stain EM.
Map data3D reconstruction of EcoR124I Type I restriction enzyme complex with a bound antirestriction protein Ocr.
Sample
  • Sample: EcoR124I R2 M2 S1 complex with dimeric Ocr bound at target recognition domains
  • Protein or peptide: EcoR124I HsdS specificity subunit
  • Protein or peptide: EcoR124I HsdM methyltransferase subunit
  • Protein or peptide: EcoR124I HsdR endonuclease subunit
  • Protein or peptide: ORF 0.3 Ocr antirestriction protein
KeywordsEcoR124 / endonuclease / methyltransferase / type I restriction / DNA mimic / HsdS / HsdM / HsdR / electron microscopy / negative stain / translocase / DEAD-box / ATPase / antirestriction / phage / T7
Function / homologyProtein Ocr / Type I restriction modification DNA specificity domain / Restriction endonuclease, type I, HsdR / protein binding / DNA methylase, adenine-specific / DNA restriction-modification system
Function and homology information
Biological speciesEscherichia coli (E. coli) / Enterobacteria phage T7 (virus)
Methodsingle particle reconstruction / negative staining / Resolution: 24.0 Å
AuthorsKennaway CK / Taylor JE / Song CF / Potrzebowski W / White JH / Swiderska A / Obarska-Kosinska A / Callow P / Cooper LP / Roberts GA ...Kennaway 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
CitationJournal: Genes Dev / Year: 2012
Title: Structure and operation of the DNA-translocating type I DNA restriction enzymes.
Authors: Christopher K Kennaway / James E N Taylor / Chun Feng Song / Wojciech Potrzebowski / William Nicholson / John H White / Anna Swiderska / Agnieszka Obarska-Kosinska / Philip Callow / Laurie P ...Authors: Christopher K Kennaway / James E N 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 /
Abstract: Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. ...Type I DNA restriction/modification (RM) enzymes are molecular machines found in the majority of bacterial species. Their early discovery paved the way for the development of genetic engineering. They control (restrict) the influx of foreign DNA via horizontal gene transfer into the bacterium while maintaining sequence-specific methylation (modification) of host DNA. The endonuclease reaction of these enzymes on unmethylated DNA is preceded by bidirectional translocation of thousands of base pairs of DNA toward the enzyme. We present the structures of two type I RM enzymes, EcoKI and EcoR124I, derived using electron microscopy (EM), small-angle scattering (neutron and X-ray), and detailed molecular modeling. DNA binding triggers a large contraction of the open form of the enzyme to a compact form. The path followed by DNA through the complexes is revealed by using a DNA mimic anti-restriction protein. The structures reveal an evolutionary link between type I RM enzymes and type II RM enzymes.
History
DepositionApr 6, 2011-
Header (metadata) releaseFeb 17, 2012-
Map releaseFeb 17, 2012-
UpdateFeb 17, 2012-
Current statusFeb 17, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 3.2
  • 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_1892.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of EcoR124I Type I restriction enzyme complex with a bound antirestriction protein Ocr.
Voxel sizeX=Y=Z: 3.96 Å
Density
Contour LevelBy AUTHOR: 3.2 / Movie #1: 3.2
Minimum - Maximum-5.1513052 - 9.923256869999999
Average (Standard dev.)-0.00000001 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-40-40
Dimensions808080
Spacing808080
CellA=B=C: 316.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.963.963.96
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z316.800316.800316.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS-40-40-40
NC/NR/NS808080
D min/max/mean-5.1519.923-0.000

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Supplemental data

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Sample components

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Entire : EcoR124I R2 M2 S1 complex with dimeric Ocr bound at target recogn...

EntireName: EcoR124I R2 M2 S1 complex with dimeric Ocr bound at target recognition domains
Components
  • Sample: EcoR124I R2 M2 S1 complex with dimeric Ocr bound at target recognition domains
  • Protein or peptide: EcoR124I HsdS specificity subunit
  • Protein or peptide: EcoR124I HsdM methyltransferase subunit
  • Protein or peptide: EcoR124I HsdR endonuclease subunit
  • Protein or peptide: ORF 0.3 Ocr antirestriction protein

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Supramolecule #1000: EcoR124I R2 M2 S1 complex with dimeric Ocr bound at target recogn...

SupramoleculeName: EcoR124I R2 M2 S1 complex with dimeric Ocr bound at target recognition domains
type: sample / ID: 1000 / Details: Stained with uranyl acetate / Oligomeric state: 1x HsdS, 2x HsdM, 2x HsdR, 2x Ocr / Number unique components: 4
Molecular weightTheoretical: 428 KDa

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Macromolecule #1: EcoR124I HsdS specificity subunit

MacromoleculeName: EcoR124I HsdS specificity subunit / type: protein_or_peptide / ID: 1 / Name.synonym: HsdS / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli) / Strain: R / Location in cell: Cytoplasmic
Molecular weightTheoretical: 50 KDa
SequenceGO: protein binding
InterPro: Type I restriction modification DNA specificity domain

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Macromolecule #2: EcoR124I HsdM methyltransferase subunit

MacromoleculeName: EcoR124I HsdM methyltransferase subunit / type: protein_or_peptide / ID: 2 / Name.synonym: HsdM / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli) / Strain: R / Location in cell: cytoplasm
Molecular weightTheoretical: 59 KDa
SequenceGO: protein binding / InterPro: DNA methylase, adenine-specific

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Macromolecule #3: EcoR124I HsdR endonuclease subunit

MacromoleculeName: EcoR124I HsdR endonuclease subunit / type: protein_or_peptide / ID: 3 / Name.synonym: HsdR / Number of copies: 2 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli) / Strain: R
Molecular weightTheoretical: 120 KDa
SequenceGO: DNA restriction-modification system / InterPro: Restriction endonuclease, type I, HsdR

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Macromolecule #4: ORF 0.3 Ocr antirestriction protein

MacromoleculeName: ORF 0.3 Ocr antirestriction protein / type: protein_or_peptide / ID: 4 / Name.synonym: Ocr
Details: Ocr forms a dimer that mimics B-form DNA in shape and charge distrubution.
Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Enterobacteria phage T7 (virus) / synonym: T7
Molecular weightTheoretical: 27 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 1 minute, blotted, then 1% uranyl acetate solution was applied for 1 min then blotted, three times.
GridDetails: 400 mesh copper, continuous carbon
VitrificationCryogen name: NONE / Instrument: OTHER / Details: Negative stain

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Electron microscopy

MicroscopeJEOL 1200EXII
Electron beamAcceleration voltage: 80 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsCalibrated magnification: 37833 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.357 µm / Nominal defocus min: 0.391 µm / Nominal magnification: 40000
Sample stageSpecimen holder: Side entry / Specimen holder model: JEOL
TemperatureAverage: 294 K
Alignment procedureLegacy - Astigmatism: Corrected at 80,000x
DetailsCustomised JEOL 1200 EX microscope, low dose mode.
DateJan 13, 2011
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 15 µm / Number real images: 7 / Average electron dose: 40 e/Å2 / Details: Scanned on Imacon scanner / Bits/pixel: 8

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Image processing

CTF correctionDetails: Filtered at 1st zero
Final two d classificationNumber classes: 48
Final angle assignmentDetails: Euler angle range limited to plus minus 30 degrees from single axis of rotation
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, IMAGIC
Details: Refined in Imagic using anchor set. C2 symmetry imposed.
Number images used: 748
Details1.5 Ocr (dimer) to EcoR124 molar ratio. The particles were manually selected using boxer. Smaller R1 complexes were removed by alignment and MSA classification.

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Chain ID: A
SoftwareName: Chimera
DetailsPDBEntryID_givenInChain. Protocol: rigid body. HsdR (2W00) was fitted into the density after the core methylase (HsdS and 2x HsdM) was fitted. Ocr docked into DNA binding sites (target recognition domains) in the methylase.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation

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