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- EMDB-28534: Type IIS Restriction Endonuclease PaqCI, DNA bound -

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Basic information

Entry
Database: EMDB / ID: EMD-28534
TitleType IIS Restriction Endonuclease PaqCI, DNA bound
Map data
Sample
  • Complex: Homotetramer PaqCI with associated DNA duplexes
    • Protein or peptide: Type IIS Restriction Endonuclease PaqCI
    • DNA: DNA 4a
    • DNA: DNA 4b
    • DNA: DNA 2a
    • DNA: DNA 2b
    • DNA: DNA 3a
    • DNA: DNA 3b
    • DNA: DNA 1a
    • DNA: DNA 1b
  • Ligand: CALCIUM ION
Keywordshomotetramer / restriction endonuclease / DNA binding / DNA cleavage / DNA BINDING PROTEIN
Function / homologyUncharacterized protein
Function and homology information
Biological speciesPaucibacter aquatile (bacteria) / DNA molecule (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsKennedy MA / Stoddard BL
Funding support United States, 2 items
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, 2022-
Header (metadata) releaseMar 22, 2023-
Map releaseMar 22, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28534.png

Downloads & links

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Map

FileDownload / File: emd_28534.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.12 Å/pix.
x 300 pix.
= 336.6 Å		1.12 Å/pix.
x 300 pix.
= 336.6 Å		1.12 Å/pix.
x 300 pix.
= 336.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.122 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.8
Minimum - Maximum-5.3905516 - 10.124696999999999
Average (Standard dev.)0.0034762975 (±0.20157057)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 336.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28534_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_28534_half_map_1.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_28534_half_map_2.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homotetramer PaqCI with associated DNA duplexes

EntireName: Homotetramer PaqCI with associated DNA duplexes
Components
  • Complex: Homotetramer PaqCI with associated DNA duplexes
    • Protein or peptide: Type IIS Restriction Endonuclease PaqCI
    • DNA: DNA 4a
    • DNA: DNA 4b
    • DNA: DNA 2a
    • DNA: DNA 2b
    • DNA: DNA 3a
    • DNA: DNA 3b
    • DNA: DNA 1a
    • DNA: DNA 1b
  • Ligand: CALCIUM ION

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Supramolecule #1: Homotetramer PaqCI with associated DNA duplexes

SupramoleculeName: Homotetramer PaqCI with associated DNA duplexes / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#9
Source (natural)Organism: Paucibacter aquatile (bacteria)

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Macromolecule #1: Type IIS Restriction Endonuclease PaqCI

MacromoleculeName: Type IIS Restriction Endonuclease PaqCI / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Paucibacter aquatile (bacteria)
Molecular weightTheoretical: 56.329355 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPYDHNAEAD FAASEVARML VADPGLCYDA ASLPASISAS ASYEPSAAGW PKADGLVSVL EGGTSTQRAI ALEYKRPQEG IHGLLTAIG QAHGYLHKGY SGAAIVIPGR YSSHPTPAEY VRDVLNAISG SRAIAVFSYS PPDTTSPTPF AGRIQCVRPL V FDAGRVHL ...String:
MPYDHNAEAD FAASEVARML VADPGLCYDA ASLPASISAS ASYEPSAAGW PKADGLVSVL EGGTSTQRAI ALEYKRPQEG IHGLLTAIG QAHGYLHKGY SGAAIVIPGR YSSHPTPAEY VRDVLNAISG SRAIAVFSYS PPDTTSPTPF AGRIQCVRPL V FDAGRVHL RPANQGPKTQ WVHMREGSTT RDAFFRFLQV AKRLSADPTA PRPTLRSELV AAIGRLAPGR DPIEYITNTA DN KFLTKVW QFFWLEWLAT PAVLTPWKLE AGVYSAPGAR TRILREDGTD FSQLWEGRVN SLKETIAGML NRGEISEAQG WEA FVGGIS ATGGGQDKQG VRARAHSYRE DIDSALAQLR WIEDDGLPTD QGYRFMTICE RYGGANSRAA IDYMGATLIQ TGRY ASFLH YINRLSERKF AENPLAYTKP GPGGMPVFTE ESYWEYLQDL ETKLTDELRV MRKVSGRARP RVRTTFQVEL TLLRN YGFV SSTRHRLGVG IPIDWEQVVQ ALNVDL

UniProtKB: Uncharacterized protein

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Macromolecule #2: DNA 4a

MacromoleculeName: DNA 4a / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 4.740072 KDa
SequenceString:
(DG)(DG)(DC)(DG)(DC)(DA)(DG)(DG)(DT)(DG) (DG)(DG)(DA)(DA)(DG)

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Macromolecule #3: DNA 4b

MacromoleculeName: DNA 4b / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 4.441875 KDa
SequenceString:
(DC)(DT)(DT)(DC)(DC)(DC)(DA)(DC)(DC)(DT) (DG)(DC)(DG)(DC)(DC)

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Macromolecule #4: DNA 2a

MacromoleculeName: DNA 2a / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 4.746068 KDa
SequenceString:
(DT)(DC)(DT)(DT)(DC)(DC)(DC)(DA)(DC)(DC) (DT)(DG)(DC)(DG)(DC)(DC)

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Macromolecule #5: DNA 2b

MacromoleculeName: DNA 2b / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 5.053279 KDa
SequenceString:
(DG)(DG)(DC)(DG)(DC)(DA)(DG)(DG)(DT)(DG) (DG)(DG)(DA)(DA)(DG)(DA)

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Macromolecule #6: DNA 3a

MacromoleculeName: DNA 3a / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 5.670679 KDa
SequenceString:
(DT)(DG)(DG)(DC)(DG)(DC)(DA)(DG)(DG)(DT) (DG)(DG)(DG)(DA)(DA)(DG)(DA)(DA)

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Macromolecule #7: DNA 3b

MacromoleculeName: DNA 3b / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 5.363468 KDa
SequenceString:
(DT)(DT)(DC)(DT)(DT)(DC)(DC)(DC)(DA)(DC) (DC)(DT)(DG)(DC)(DG)(DC)(DC)(DA)

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Macromolecule #8: DNA 1a

MacromoleculeName: DNA 1a / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 7.163597 KDa
SequenceString:
(DC)(DC)(DT)(DC)(DG)(DC)(DG)(DT)(DG)(DG) (DC)(DG)(DC)(DA)(DG)(DG)(DT)(DG)(DG)(DG) (DA)(DA)(DG)

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Macromolecule #9: DNA 1b

MacromoleculeName: DNA 1b / type: dna / ID: 9 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: DNA molecule (others)
Molecular weightTheoretical: 6.963476 KDa
SequenceString:
(DC)(DT)(DT)(DC)(DC)(DC)(DA)(DC)(DC)(DT) (DG)(DC)(DG)(DC)(DC)(DA)(DC)(DG)(DC)(DG) (DA)(DG)(DG)

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Macromolecule #10: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

Concentration0.9 mg/mL
BufferpH: 6.5
Component:
ConcentrationFormulaName
100.0 mMNaClsodium chloride
30.0 mMC8H19NO5BIS-TRIS
10.0 mMCaClcalcium chloride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K
DetailsThe DNA-bound PaqCI complex was generated by incubating enzyme + DNA at a 1:2 molar ratio.

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 1897 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 36000

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Image processing

Particle selectionNumber selected: 299211
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 166130
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER / Overall B value: 139.1
Output model

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

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