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Structure paper

TitleType IV-A CRISPR-Csf complex: Assembly, dsDNA targeting, and CasDinG recruitment.
Journal, issue, pagesMol Cell, Vol. 83, Issue 14, Page 2493-22508.e5, Year 2023
Publish dateJul 20, 2023
AuthorsNing Cui / Jun-Tao Zhang / Yongrui Liu / Yanhong Liu / Xiao-Yu Liu / Chongyuan Wang / Hongda Huang / Ning Jia /
PubMed AbstractType 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.
External linksMol Cell / PubMed:37343553
MethodsEM (single particle) / X-ray diffraction
Resolution2.6 - 3.7 Å
Structure data

33180

7xfz

EMDB-33180, PDB-7xfz:
CryoEM structure of type IV-A Csf-crRNAsp14-dsDNA ternary complex
Method: EM (single particle) / Resolution: 3.0 Å

33181

7xg0

EMDB-33181, PDB-7xg0:
CryoEM structure of type IV-A Csf-crRNA-dsDNA ternary complex
Method: EM (single particle) / Resolution: 2.6 Å

33183

7xg2

EMDB-33183, PDB-7xg2:
CryoEM structure of type IV-A NTS-nicked dsDNA bound Csf-crRNA ternary complex
Method: EM (single particle) / Resolution: 2.8 Å

33184

7xg3

EMDB-33184, PDB-7xg3:
CryoEM structure of type IV-A CasDinG bound NTS-nicked Csf-crRNA-dsDNA quaternary complex
Method: EM (single particle) / Resolution: 3.0 Å

33185

7xg4

EMDB-33185, PDB-7xg4:
CryoEM structure of type IV-A CasDinG bound NTS-nicked Csf-crRNA-dsDNA quaternary complex in a second state
Method: EM (single particle) / Resolution: 3.7 Å

PDB-7xex:
Crystal strucutre of apoCasDinG
Method: X-RAY DIFFRACTION / Resolution: 3.0 Å

PDB-7xf0:
Crystal strucutre of CasDinG in complex with ATP
Method: X-RAY DIFFRACTION / Resolution: 3.1 Å

PDB-7xf1:
Crystal strucutre of apoCasDinG in complex with ssDNA
Method: X-RAY DIFFRACTION / Resolution: 3.2 Å

Chemicals

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

ChemComp-ZN:
Unknown entry

Source
  • pseudomonas aeruginosa (bacteria)
  • synthetic construct (others)
KeywordsSTRUCTURAL PROTEIN / Nuclease / STRUCTURAL PROTEIN/DNA / STRUCTURAL PROTEIN-DNA complex / STRUCTURAL PROTEIN/RNA/DNA / STRUCTURAL PROTEIN-RNA-DNA COMPLEX

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