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Yorodumi- EMDB-1892: EcoR124 Type I DNA restriction-modification enzyme complex (in cl... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1892 | |||||||||
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Title | EcoR124 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 data | 3D reconstruction of EcoR124I Type I restriction enzyme complex with a bound antirestriction protein Ocr. | |||||||||
Sample |
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Keywords | EcoR124 / endonuclease / methyltransferase / type I restriction / DNA mimic / HsdS / HsdM / HsdR / electron microscopy / negative stain / translocase / DEAD-box / ATPase / antirestriction / phage / T7 | |||||||||
Function / homology | Protein 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 species | Escherichia coli (E. coli) / Enterobacteria phage T7 (virus) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 24.0 Å | |||||||||
Authors | Kennaway 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 | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_1892.map.gz | 253.7 KB | EMDB map data format | |
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Header (meta data) | emd-1892-v30.xml emd-1892.xml | 15.1 KB 15.1 KB | Display Display | EMDB header |
Images | emd_1892.png | 217.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1892 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1892 | HTTPS FTP |
-Validation report
Summary document | emd_1892_validation.pdf.gz | 194.7 KB | Display | EMDB validaton report |
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Full document | emd_1892_full_validation.pdf.gz | 193.8 KB | Display | |
Data in XML | emd_1892_validation.xml.gz | 5.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1892 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1892 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_1892.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | 3D reconstruction of EcoR124I Type I restriction enzyme complex with a bound antirestriction protein Ocr. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 3.96 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : EcoR124I R2 M2 S1 complex with dimeric Ocr bound at target recogn...
Entire | Name: EcoR124I R2 M2 S1 complex with dimeric Ocr bound at target recognition domains |
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Components |
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-Supramolecule #1000: EcoR124I R2 M2 S1 complex with dimeric Ocr bound at target recogn...
Supramolecule | Name: 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 |
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Molecular weight | Theoretical: 428 KDa |
-Macromolecule #1: EcoR124I HsdS specificity subunit
Macromolecule | Name: EcoR124I HsdS specificity subunit / type: protein_or_peptide / ID: 1 / Name.synonym: HsdS / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: R / Location in cell: Cytoplasmic |
Molecular weight | Theoretical: 50 KDa |
Sequence | GO: protein binding InterPro: Type I restriction modification DNA specificity domain |
-Macromolecule #2: EcoR124I HsdM methyltransferase subunit
Macromolecule | Name: EcoR124I HsdM methyltransferase subunit / type: protein_or_peptide / ID: 2 / Name.synonym: HsdM / Number of copies: 2 / Oligomeric state: Dimer / Recombinant expression: No |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: R / Location in cell: cytoplasm |
Molecular weight | Theoretical: 59 KDa |
Sequence | GO: protein binding / InterPro: DNA methylase, adenine-specific |
-Macromolecule #3: EcoR124I HsdR endonuclease subunit
Macromolecule | Name: EcoR124I HsdR endonuclease subunit / type: protein_or_peptide / ID: 3 / Name.synonym: HsdR / Number of copies: 2 / Oligomeric state: Monomer / Recombinant expression: No |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: R |
Molecular weight | Theoretical: 120 KDa |
Sequence | GO: DNA restriction-modification system / InterPro: Restriction endonuclease, type I, HsdR |
-Macromolecule #4: ORF 0.3 Ocr antirestriction protein
Macromolecule | Name: 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 |
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Source (natural) | Organism: Enterobacteria phage T7 (virus) / synonym: T7 |
Molecular weight | Theoretical: 27 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | InterPro: Protein Ocr |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.050 mg/mL |
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Buffer | pH: 4.7 / Details: 20mM Tris-Cl, 100 mM NaCl |
Staining | Type: 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. |
Grid | Details: 400 mesh copper, continuous carbon |
Vitrification | Cryogen name: NONE / Instrument: OTHER / Details: Negative stain |
-Electron microscopy
Microscope | JEOL 1200EXII |
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Temperature | Average: 294 K |
Alignment procedure | Legacy - Astigmatism: Corrected at 80,000x |
Details | Customised JEOL 1200 EX microscope, low dose mode. |
Date | Jan 13, 2011 |
Image recording | Category: 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 |
Electron beam | Acceleration voltage: 80 kV / Electron source: TUNGSTEN HAIRPIN |
Electron optics | Calibrated magnification: 37833 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 1.357 µm / Nominal defocus min: 0.391 µm / Nominal magnification: 40000 |
Sample stage | Specimen holder: Side entry / Specimen holder model: JEOL |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera |
Details | PDBEntryID_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. |
Refinement | Space: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation |