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- PDB-7btq: EcoR124I-DNA in the Restriction-Alleviation State -

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Basic information

Entry
Database: PDB / ID: 7btq
TitleEcoR124I-DNA in the Restriction-Alleviation State
Components
  • (DNA (64-MER)) x 2
  • Type I restriction enzyme EcoR124II M protein
  • Type I restriction enzyme R Protein
  • Type-1 restriction enzyme EcoR124II specificity protein
KeywordsIMMUNE SYSTEM / Cryoelectron microscopy / Innate immune mechanism / Complex
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
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 ...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
DNA / DNA (> 10) / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.54 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, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 27, 2020Provider: repository / Type: Initial release
Revision 1.1Jun 24, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Sep 9, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Mar 27, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Type I restriction enzyme EcoR124II M protein
B: DNA (64-MER)
C: DNA (64-MER)
D: Type I restriction enzyme EcoR124II M protein
E: Type-1 restriction enzyme EcoR124II specificity protein
F: Type I restriction enzyme R Protein


Theoretical massNumber of molelcules
Total (without water)322,1196
Polymers322,1196
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16970 Å2
ΔGint-83 kcal/mol
Surface area131800 Å2

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Components

#1: Protein Type I restriction enzyme EcoR124II M protein / M.EcoR124II


Mass: 58077.090 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: hsdM, hsm / Production host: Escherichia coli (E. coli)
References: UniProt: P10484, site-specific DNA-methyltransferase (adenine-specific)
#2: DNA chain DNA (64-MER)


Mass: 19657.736 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#3: DNA chain DNA (64-MER)


Mass: 19792.592 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#4: Protein Type-1 restriction enzyme EcoR124II specificity protein / S.EcoR124II / Type I restriction enzyme EcoR124II specificity protein / S protein


Mass: 46235.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: hsdS, hss / Production host: Escherichia coli (E. coli) / References: UniProt: P10485
#5: Protein Type I restriction enzyme R Protein


Mass: 120278.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: hsdR / Production host: Escherichia coli (E. coli)
References: UniProt: Q304R3, UniProt: P10486*PLUS, type I site-specific deoxyribonuclease

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: EcoR124I-DNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDVersionCategory
7Chimeramodel fitting
9PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83797 / Symmetry type: POINT

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