[English] 日本語
Yorodumi
- EMDB-1890: EcoR124 Type I DNA restriction-modification enzyme complex in clo... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-1890
TitleEcoR124 Type I DNA restriction-modification enzyme complex in closed state with bound 30bp cognate DNA fragment. 3D reconstruction by single particle analysis from negative stain EM.
Map dataEcoR124I R2 M2 S1 complex with 30bp DNA fragment bound (closed state). DNA is not visible in the negative stain images or map.
Sample
  • Sample: EcoR124I R2 M2 S1 complex with 30 bp cognate dsDNA fragment
  • Protein or peptide: EcoR124I HsdS specificity subunit
  • Protein or peptide: EcoR124I HsdM methyltransferase subunit
  • Protein or peptide: EcoR124I HsdR endonuclease subunit
  • DNA: Deoxyribonucleic acidDNA
KeywordsEcoR124 / endonuclease / methyltransferase / type I restriction / DNA / HsdS / HsdM / HsdR / electron microscopy / negative stain / translocase / DEAD-box / ATPase
Function / homologyType 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) / synthetic construct (others)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 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-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.43
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.43
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

-
Map

FileDownload / File: emd_1890.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEcoR124I R2 M2 S1 complex with 30bp DNA fragment bound (closed state). DNA is not visible in the negative stain images or map.
Voxel sizeX=Y=Z: 3.96 Å
Density
Contour LevelBy AUTHOR: 0.43 / Movie #1: 0.43
Minimum - Maximum-0.54730725 - 1.30585396
Average (Standard dev.)0.01822406 (±0.11469037)
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-0.5471.3060.018

-
Supplemental data

-
Sample components

-
Entire : EcoR124I R2 M2 S1 complex with 30 bp cognate dsDNA fragment

EntireName: EcoR124I R2 M2 S1 complex with 30 bp cognate dsDNA fragment
Components
  • Sample: EcoR124I R2 M2 S1 complex with 30 bp cognate dsDNA fragment
  • Protein or peptide: EcoR124I HsdS specificity subunit
  • Protein or peptide: EcoR124I HsdM methyltransferase subunit
  • Protein or peptide: EcoR124I HsdR endonuclease subunit
  • DNA: Deoxyribonucleic acidDNA

-
Supramolecule #1000: EcoR124I R2 M2 S1 complex with 30 bp cognate dsDNA fragment

SupramoleculeName: EcoR124I R2 M2 S1 complex with 30 bp cognate dsDNA fragment
type: sample / ID: 1000 / Details: Stained with uranyl acetate / Oligomeric state: 1x HsdS, 2x HsdM, 2x HsdR, 1x dsDNA / Number unique components: 4
Molecular weightExperimental: 384 KDa / Theoretical: 415 KDa / Method: Small angle neutron scattering (SANS)

-
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

-
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

-
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

-
Macromolecule #4: Deoxyribonucleic acid

MacromoleculeName: Deoxyribonucleic acid / type: dna / ID: 4 / Name.synonym: DNA / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15 KDa
SequenceString:
CCGTGCAGAA TTCGAGGTCG ACGGATCCGG

-
Experimental details

-
Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

-
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
VitrificationCryogen name: NONE / Instrument: OTHER / Details: Negative stain

-
Electron microscopy

MicroscopeJEOL 1200EXII
Electron beamAcceleration voltage: 80 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 0.991 µm / Nominal defocus min: 0.405 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.
DateSep 1, 2010
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 15 µm / Number real images: 30 / Average electron dose: 40 e/Å2 / Details: Scanned on Imacon scanner / Bits/pixel: 8

-
Image processing

CTF correctionDetails: Filtered at 1st zero
Final two d classificationNumber classes: 100
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: 21.0 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: 3806
DetailsThe particles were manually selected using boxer.

-
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.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242):Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more