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- PDB-7xg3: CryoEM structure of type IV-A CasDinG bound NTS-nicked Csf-crRNA-... -

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Basic information

Entry
Database: PDB / ID: 7xg3
TitleCryoEM structure of type IV-A CasDinG bound NTS-nicked Csf-crRNA-dsDNA quaternary complex
Components
  • Csf1
  • Csf2
  • Csf3
  • Csf4
  • Csf5
  • NTS
  • TS
  • crRNA
KeywordsSTRUCTURAL PROTEIN/RNA/DNA / Nuclease / STRUCTURAL PROTEIN-RNA-DNA complex
Function / homologyP-loop containing nucleotide triphosphate hydrolases / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta / DNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesPseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsZhang, J.T. / Cui, N. / Huang, H.D. / Jia, N.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2023
Title: Type IV-A CRISPR-Csf complex: Assembly, dsDNA targeting, and CasDinG recruitment.
Authors: Ning Cui / Jun-Tao Zhang / Yongrui Liu / Yanhong Liu / Xiao-Yu Liu / Chongyuan Wang / Hongda Huang / Ning Jia /
Abstract: Type IV CRISPR-Cas systems, which are primarily found on plasmids and exhibit a strong plasmid-targeting preference, are the only one of the six known CRISPR-Cas types for which the mechanistic ...Type IV CRISPR-Cas systems, which are primarily found on plasmids and exhibit a strong plasmid-targeting preference, are the only one of the six known CRISPR-Cas types for which the mechanistic details of their function remain unknown. Here, we provide high-resolution functional snapshots of type IV-A Csf complexes before and after target dsDNA binding, either in the absence or presence of CasDinG, revealing the mechanisms underlying Csf complex assembly, "DWN" PAM-dependent dsDNA targeting, R-loop formation, and CasDinG recruitment. Furthermore, we establish that CasDinG, a signature DinG family helicase, harbors ssDNA-stimulated ATPase activity and ATP-dependent 5'-3' DNA helicase activity. In addition, we show that CasDinG unwinds the non-target strand (NTS) and target strand (TS) of target dsDNA from the Csf complex. These molecular details advance our mechanistic understanding of type IV-A CRISPR-Csf function and should enable Csf complexes to be harnessed as genome-engineering tools for biotechnological applications.
History
DepositionApr 2, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 9, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Csf1
B: Csf3
C: Csf2
D: Csf2
E: Csf2
F: Csf2
G: Csf2
H: Csf5
I: crRNA
J: NTS
K: TS
L: Csf4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)383,29513
Polymers383,23012
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 5 types, 9 molecules ABCDEFGHL

#1: Protein Csf1


Mass: 28675.768 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)
#2: Protein Csf3


Mass: 24421.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)
#3: Protein
Csf2


Mass: 37322.234 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)
#4: Protein Csf5


Mass: 29152.414 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)
#8: Protein Csf4


Mass: 67939.883 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)

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DNA chain , 2 types, 2 molecules JK

#6: DNA chain NTS


Mass: 9981.448 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas aeruginosa (bacteria)
#7: DNA chain TS


Mass: 16547.568 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Pseudomonas aeruginosa (bacteria)

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RNA chain / Non-polymers , 2 types, 2 molecules I

#5: RNA chain crRNA


Mass: 19899.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Production host: Escherichia coli (E. coli)
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Type IV-A CasDinG bound NTS-nicked Csf-crRNA-dsDNA quaternary complexCOMPLEX#1-#80MULTIPLE SOURCES
2CsfCOMPLEX#1-#4, #81RECOMBINANT
3crRNACOMPLEX#51RECOMBINANT
4dsDNACOMPLEX#6-#71SYNTHETIC
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Pseudomonas aeruginosa (bacteria)287
32Pseudomonas aeruginosa (bacteria)287
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli (E. coli)562
32Escherichia coli (E. coli)562
Buffer solutionpH: 8
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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59086 / Symmetry type: POINT
RefinementCross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006625107
ELECTRON MICROSCOPYf_angle_d1.146634633
ELECTRON MICROSCOPYf_chiral_restr0.06323949
ELECTRON MICROSCOPYf_plane_restr0.00994034
ELECTRON MICROSCOPYf_dihedral_angle_d18.8374430

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