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- EMDB-30180: EcoR124I-ArdA in the Translocation State -

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Basic information

Entry
Database: EMDB / ID: EMD-30180
TitleEcoR124I-ArdA in the Translocation State
Map data
Sample
  • Complex: EcoR124I-ArdA
    • Protein or peptide: Type I restriction enzyme EcoR124II M protein
    • Protein or peptide: Antirestriction protein ArdA
    • Protein or peptide: Type I restriction enzyme R Protein
    • Protein or peptide: Type-1 restriction enzyme EcoR124II specificity protein
KeywordsCryoelectron microscopy / Innate immune mechanism / Complex / IMMUNE SYSTEM
Function / homology
Function and homology information


type I site-specific deoxyribonuclease / type I site-specific deoxyribonuclease activity / N-methyltransferase activity / site-specific DNA-methyltransferase (adenine-specific) / site-specific DNA-methyltransferase (adenine-specific) activity / DNA restriction-modification system / methylation / DNA binding / ATP binding
Similarity search - Function
Antirestriction / Antirestriction protein ArdA, domain 3 / Antirestriction protein ArdA, domain 1 / Antirestriction protein ArdA, domain 2 / Antirestriction protein (ArdA) / Restriction endonuclease, type I, methylase subunit / Restriction endonuclease, type I, HsdR, N-terminal / Type I restriction enzyme R protein, C-terminal / SWI2/SNF2 ATPase / : ...Antirestriction / Antirestriction protein ArdA, domain 3 / Antirestriction protein ArdA, domain 1 / Antirestriction protein ArdA, domain 2 / Antirestriction protein (ArdA) / Restriction endonuclease, type I, methylase subunit / Restriction endonuclease, type I, HsdR, N-terminal / Type I restriction enzyme R protein, C-terminal / SWI2/SNF2 ATPase / : / Type I restriction enzyme R protein N terminus (HSDR_N) / Type I restriction and modification enzyme - subunit R C terminal / SWI2/SNF2 ATPase / UvrB domain 3 / N6 adenine-specific DNA methyltransferase, N-terminal domain / : / Type I restriction enzyme EcoKI-like, methylase subunit, N-terminal domain superfamily / : / HsdM N-terminal domain / Restriction endonuclease, type I, HsdR / Type I restriction modification DNA specificity domain superfamily / Type I restriction modification DNA specificity domain / Type I restriction modification DNA specificity domain / N-6 DNA Methylase / DNA methylase, adenine-specific / N-6 Adenine-specific DNA methylases signature. / DNA methylase, N-6 adenine-specific, conserved site / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase superfamily 1/2, ATP-binding domain / S-adenosyl-L-methionine-dependent methyltransferase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Antirestriction protein ArdA / Type I restriction enzyme EcoR124I/EcoR124II methylase subunit / Type I restriction enzyme EcoR124I/EcoR124II specificity subunit / Type I restriction enzyme EcoR124I/EcoR124II endonuclease subunit / Type I restriction enzyme EcoR124I/EcoR124II endonuclease subunit
Similarity search - Component
Biological speciesEscherichia coli (E. coli) / Enterococcus faecalis EnGen0302 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.97 Å
AuthorsGao Y / Gao P
CitationJournal: Nat Microbiol / Year: 2020
Title: Structural insights into assembly, operation and inhibition of a type I restriction-modification system.
Authors: Yina Gao / Duanfang Cao / Jingpeng Zhu / Han Feng / Xiu Luo / Songqing Liu / Xiao-Xue Yan / Xinzheng Zhang / Pu Gao /
Abstract: Type I restriction-modification (R-M) systems are widespread in prokaryotic genomes and provide robust protection against foreign DNA. They are multisubunit enzymes with methyltransferase, ...Type I restriction-modification (R-M) systems are widespread in prokaryotic genomes and provide robust protection against foreign DNA. They are multisubunit enzymes with methyltransferase, endonuclease and translocase activities. Despite extensive studies over the past five decades, little is known about the molecular mechanisms of these sophisticated machines. Here, we report the cryo-electron microscopy structures of the representative EcoR124I R-M system in different assemblies (RMS, RMS and MS) bound to target DNA and the phage and mobile genetic element-encoded anti-restriction proteins Ocr and ArdA. EcoR124I can precisely regulate different enzymatic activities by adopting distinct conformations. The marked conformational transitions of EcoR124I are dependent on the intrinsic flexibility at both the individual-subunit and assembled-complex levels. Moreover, Ocr and ArdA use a DNA-mimicry strategy to inhibit multiple activities, but do not block the conformational transitions of the complexes. These structural findings, complemented by mutational studies of key intermolecular contacts, provide insights into assembly, operation and inhibition mechanisms of type I R-M systems.
History
DepositionApr 2, 2020-
Header (metadata) releaseMay 27, 2020-
Map releaseMay 27, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0455
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0455
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7bto
  • Surface level: 0.0455
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30180.png

Downloads & links

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Map

FileDownload / File: emd_30180.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 256 pix.
= 345.6 Å		1.35 Å/pix.
x 256 pix.
= 345.6 Å		1.35 Å/pix.
x 256 pix.
= 345.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0455 / Movie #1: 0.0455
Minimum - Maximum-0.13530184 - 0.30656937
Average (Standard dev.)0.0005737086 (±0.0072190813)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 345.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z345.600345.600345.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ280280280
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-0.1350.3070.001

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Supplemental data

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Sample components

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Entire : EcoR124I-ArdA

EntireName: EcoR124I-ArdA
Components
  • Complex: EcoR124I-ArdA
    • Protein or peptide: Type I restriction enzyme EcoR124II M protein
    • Protein or peptide: Antirestriction protein ArdA
    • Protein or peptide: Type I restriction enzyme R Protein
    • Protein or peptide: Type-1 restriction enzyme EcoR124II specificity protein

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Supramolecule #1: EcoR124I-ArdA

SupramoleculeName: EcoR124I-ArdA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Type I restriction enzyme EcoR124II M protein

MacromoleculeName: Type I restriction enzyme EcoR124II M protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: site-specific DNA-methyltransferase (adenine-specific)
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 58.07709 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKMTSIQQRA ELHRQIWQIA NDVRGSVDGW DFKQYVLGAL FYRFISENFS SYIEAGDDSI CYAKLDDSVI TDDIKDDAIK TKGYFIYPS QLFCNVAAKA NTNDRLNADL NSIFVAIESS AYGYPSEADI KGLFADFDTT SNRLGNTVKD KNARLAAVLK G VEGLKLGD ...String:
MKMTSIQQRA ELHRQIWQIA NDVRGSVDGW DFKQYVLGAL FYRFISENFS SYIEAGDDSI CYAKLDDSVI TDDIKDDAIK TKGYFIYPS QLFCNVAAKA NTNDRLNADL NSIFVAIESS AYGYPSEADI KGLFADFDTT SNRLGNTVKD KNARLAAVLK G VEGLKLGD FNEHQIDLFG DAYEFLISNY AANAGKSGGE FFTPQHVSKL IAQLAMHGQT HVNKIYDPAA GSGSLLLQAK KQ FDNHIIE EGFFGQEINH TTYNLARMNM FLHNINYDKF DIKLGNTLTE PHFRDEKPFD AIVSNPPYSV KWIGSDDPTL IND ERFAPA GVLAPKSKAD FAFVLHALNY LSAKGRAAIV CFPGIFYRGG AEQKIRQYLV DNNYVETVIS LAPNLFFGTT IAVN ILVLS KHKTDTNVQF IDASELFKKE TNNNILTDAH IEQIMQVFAS KEDVAHLAKS VAFETVVAND YNLSVSSYVE AKDNR EIID IAELNAELKT TVSKIDQLRK DIDAIVAEIE GCEVQK

UniProtKB: Type I restriction enzyme EcoR124I/EcoR124II methylase subunit

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Macromolecule #2: Antirestriction protein ArdA

MacromoleculeName: Antirestriction protein ArdA / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Enterococcus faecalis EnGen0302 (bacteria)
Molecular weightTheoretical: 19.135883 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MDDMQVYIAN LGKYNEGELV GAWFTFPIDF EEVKEKIGLN DEYEEYAIHD YELPFTVDEY TSIGELNRLW EMVSELPEEL QSELSALLT HFSSIEELSE HQEDIIIHSD CDDMYDVARY YIEETGALGE VPASLQNYID YQAYGRDLDL SGTFISTNHG I FEIVY

UniProtKB: Antirestriction protein ArdA

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Macromolecule #3: Type I restriction enzyme R Protein

MacromoleculeName: Type I restriction enzyme R Protein / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO / EC number: type I site-specific deoxyribonuclease
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 120.278859 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTHQTHTIAE SNNFIVLDKY IKAEPTGDSY QSESDLEREL IQDLRNQGYE FISVKSQSAM LANVREQLQN LNGVVFNDSE WRRFTEQYL DNPSDGILDK TRKIHIDYIC DFIFDDERLE NIYLIDKKNL MRNKVQIIQQ FEQAGSHANR YDVTILVNGL P LVQIELKK ...String:
MTHQTHTIAE SNNFIVLDKY IKAEPTGDSY QSESDLEREL IQDLRNQGYE FISVKSQSAM LANVREQLQN LNGVVFNDSE WRRFTEQYL DNPSDGILDK TRKIHIDYIC DFIFDDERLE NIYLIDKKNL MRNKVQIIQQ FEQAGSHANR YDVTILVNGL P LVQIELKK RGVAIREAFN QIHRYSKESF NSENSLFKYL QLFVISNGTD TRYFANTTKR DKNSFDFTMN WAKSDNTLIK DL KDFTATC FQKHTLLNVL VNYSVFDSSQ TLLVMRPYQI AATERILWKI KSSFTAKNWS KPESGGYIWH TTGSGKTLTS FKA ARLATE LDFIDKVFFV VDRKDLDYQT MKEYQRFSPD SVNGSENTAG LKRNLDKDDN KIIVTTIQKL NNLMKAESDL PVYN QQVVF IFDECHRSQF GEAQKNLKKK FKRYYQFGFT GTPIFPENAL GSETTASVFG RELHSYVITD AIRDEKVLKF KVDYN DVRP QFKSLETETD EKKLSAAENQ QAFLHPMRIQ EITQYILNNF RQKTHRTFPG SKGFNAMLAV SSVDAAKAYY ATFKRL QEE AANKSATYKP LRIATIFSFA ANEEQNAIGE ISDETFDTSA MDSSAKEFLD AAIREYNSHF KTNFSTDSNG FQNYYRD LA QRVKNQDIDL LIVVGMFLTG FDAPTLNTLF VDKNLRYHGL MQAFSRTNRI YDATKTFGNI VTFRDLERST IDAITLFG D KNTKNVVLEK SYTEYMEGFT DAATGEAKRG FMTVVSELEQ RFPDPTSIES EKEKKDFVKL FGEYLRAENI LQNYDEFAT LKALQQIDLS DPVAVEKFKA EHYVDDEKFA ELQTIRLPAD RKIQDYRSAY NDIRDWQRRE KEAEKKEKST TDWDDVVFEV DLLKSQEIN LDYILGLIFE HNRQNKGKGE MIEEVKRLIR SSLGNRAKEG LVVDFIQQTN LDDLPDKASI IDAFFTFAQR E QQREAEAL IKEENLNEDA AKRYIRTSLK REYATENGTE LNETLPKLSP LNPQYKTKKQ AVFQKIVSFI EKFKGVGGKI

UniProtKB: Type I restriction enzyme EcoR124I/EcoR124II endonuclease subunit

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Macromolecule #4: Type-1 restriction enzyme EcoR124II specificity protein

MacromoleculeName: Type-1 restriction enzyme EcoR124II specificity protein
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 46.235773 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSEMSYLEKL LDGVEVEWLP LGEITKYEQP TKYLVKAKDY HDTYTIPVLT AGKTFILGYT NETHGIYQAS KAPVIIFDDF TTANKWVDF DFKAKSSAMK MVTSCDDNKT LLKYVYYWLN TLPSEFAEGD HKRQWISNYS QKKIPIPCPD NPEKSLAIQS E IVRILDKF ...String:
MSEMSYLEKL LDGVEVEWLP LGEITKYEQP TKYLVKAKDY HDTYTIPVLT AGKTFILGYT NETHGIYQAS KAPVIIFDDF TTANKWVDF DFKAKSSAMK MVTSCDDNKT LLKYVYYWLN TLPSEFAEGD HKRQWISNYS QKKIPIPCPD NPEKSLAIQS E IVRILDKF TALTAELTAE LNMRKKQYNY YRDQLLSFKE GEVEWKTLGE IGKWYGGGTP SKNKIEFWEN GSIPWISPKD MG RTLVDSS EDYITEEAVL HSSTKLIPAN SIAIVVRSSI LDKVLPSALI KVPATLNQDM KAVIPHENIL VKYIYHMIGS RGS DILRAA KKTGGSVASI DSKKLFSFKI PVPNINEQQR IVEILDKFDT LTNSITEGLP REIELRQKQY EYYRDLLFSF PKPE TVSN

UniProtKB: Type I restriction enzyme EcoR124I/EcoR124II specificity subunit

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.97 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 268962
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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