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- PDB-8epx: Type IIS Restriction Endonuclease PaqCI, DNA bound -

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Basic information

Entry
Database: PDB / ID: 8epx
TitleType IIS Restriction Endonuclease PaqCI, DNA bound
Components
  • DNA 1a
  • DNA 1b
  • DNA 2a
  • DNA 2b
  • DNA 3a
  • DNA 3b
  • DNA 4a
  • DNA 4b
  • Type IIS Restriction Endonuclease PaqCI
KeywordsDNA BINDING PROTEIN / homotetramer / restriction endonuclease / DNA binding / DNA cleavage
Function / homologyDNA / DNA (> 10) / Uncharacterized protein
Function and homology information
Biological speciesPaucibacter aquatile (bacteria)
DNA molecule (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsKennedy, M.A. / Stoddard, B.L.
Funding support United States, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DGE-1762114 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1 GM105691 United States
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structures, activity and mechanism of the Type IIS restriction endonuclease PaqCI.
Authors: Madison A Kennedy / Christopher J Hosford / Caleigh M Azumaya / Yvette A Luyten / Minyong Chen / Richard D Morgan / Barry L Stoddard /
Abstract: Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in ...Type IIS restriction endonucleases contain separate DNA recognition and catalytic domains and cleave their substrates at well-defined distances outside their target sequences. They are employed in biotechnology for a variety of purposes, including the creation of gene-targeting zinc finger and TAL effector nucleases and DNA synthesis applications such as Golden Gate assembly. The most thoroughly studied Type IIS enzyme, FokI, has been shown to require multimerization and engagement with multiple DNA targets for optimal cleavage activity; however, details of how it or similar enzymes forms a DNA-bound reaction complex have not been described at atomic resolution. Here we describe biochemical analyses of DNA cleavage by the Type IIS PaqCI restriction endonuclease and a series of molecular structures in the presence and absence of multiple bound DNA targets. The enzyme displays a similar tetrameric organization of target recognition domains in the absence or presence of bound substrate, with a significant repositioning of endonuclease domains in a trapped DNA-bound complex that is poised to deliver the first of a series of double-strand breaks. PaqCI and FokI share similar structural mechanisms of DNA cleavage, but considerable differences in their domain organization and quaternary architecture, facilitating comparisons between distinct Type IIS enzymes.
History
DepositionOct 6, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 22, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jun 7, 2023Group: 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 1.3Jun 19, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Type IIS Restriction Endonuclease PaqCI
B: Type IIS Restriction Endonuclease PaqCI
C: Type IIS Restriction Endonuclease PaqCI
D: Type IIS Restriction Endonuclease PaqCI
E: DNA 4a
F: DNA 4b
H: DNA 2a
G: DNA 2b
I: DNA 3a
J: DNA 3b
K: DNA 1a
L: DNA 1b
hetero molecules


Theoretical massNumber of molelcules
Total (without water)269,54014
Polymers269,46012
Non-polymers802
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization, Size Exclusion Chromatography
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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DNA chain , 8 types, 8 molecules EFHGIJKL

#2: DNA chain DNA 4a


Mass: 4740.072 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#3: DNA chain DNA 4b


Mass: 4441.875 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#4: DNA chain DNA 2a


Mass: 4746.068 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#5: DNA chain DNA 2b


Mass: 5053.279 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#6: DNA chain DNA 3a


Mass: 5670.679 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#7: DNA chain DNA 3b


Mass: 5363.468 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#8: DNA chain DNA 1a


Mass: 7163.597 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)
#9: DNA chain DNA 1b


Mass: 6963.476 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) DNA molecule (others)

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Protein / Non-polymers , 2 types, 6 molecules ABCD

#10: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#1: Protein
Type IIS Restriction Endonuclease PaqCI


Mass: 56329.355 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Paucibacter aquatile (bacteria) / Gene: C1O66_13805 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A2N8KYF9

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Details

Has ligand of interestN

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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: Homotetramer PaqCI with associated DNA duplexes / Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Paucibacter aquatile (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.5
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaCl1
230 mMBIS-TRISC8H19NO51
310 mMcalcium chlorideCaCl1
SpecimenConc.: 0.9 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The DNA-bound PaqCI complex was generated by incubating enzyme + DNA at a 1:2 molar ratio.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 36000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1897

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2SerialEM4.0.6image acquisition
4cryoSPARC3.3.2CTF correction
7UCSF ChimeraX1.3model fitting
8Coot0.9.6model fitting
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
12cryoSPARC3.3.2classification
13cryoSPARC3.3.23D reconstruction
14PHENIX1.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 299211
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 166130 / Symmetry type: POINT
Atomic model buildingB value: 139.1 / Protocol: OTHER / Space: REAL

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