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Structure paper

TitleStructures of Cas9 endonucleases reveal RNA-mediated conformational activation.
Journal, issue, pagesScience, Vol. 343, Issue 6176, Page 1247997, Year 2014
Publish dateMar 14, 2014
AuthorsMartin Jinek / Fuguo Jiang / David W Taylor / Samuel H Sternberg / Emine Kaya / Enbo Ma / Carolin Anders / Michael Hauer / Kaihong Zhou / Steven Lin / Matias Kaplan / Anthony T Iavarone / Emmanuelle Charpentier / Eva Nogales / Jennifer A Doudna /
PubMed AbstractType II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA ...Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.
External linksScience / PubMed:24505130 / PubMed Central
MethodsEM (single particle) / X-ray diffraction
Resolution2.201 - 21.0 Å
Structure data

EMDB-5858:
Structures of Cas9 endonucleases reveal RNA-mediated conformational activation
Method: EM (single particle) / Resolution: 19.0 Å

EMDB-5859:
Structures of Cas9 endonucleases reveal RNA-mediated conformational activation
Method: EM (single particle) / Resolution: 21.0 Å

EMDB-5860:
Structures of Cas9 endonucleases reveal RNA-mediated conformational activation
Method: EM (single particle) / Resolution: 21.0 Å

PDB-4cmp:
Crystal structure of S. pyogenes Cas9
Method: X-RAY DIFFRACTION / Resolution: 2.62 Å

PDB-4cmq:
Crystal structure of Mn-bound S.pyogenes Cas9
Method: X-RAY DIFFRACTION / Resolution: 3.09 Å

PDB-4ogc:
Crystal structure of the Type II-C Cas9 enzyme from Actinomyces naeslundii
Method: X-RAY DIFFRACTION / Resolution: 2.8 Å

PDB-4oge:
Crystal structure of the Type II-C Cas9 enzyme from Actinomyces naeslundii
Method: X-RAY DIFFRACTION / Resolution: 2.201 Å

Chemicals

ChemComp-SO4:
SULFATE ION / Sulfate

ChemComp-MG:
Unknown entry

ChemComp-HOH:
WATER / Water

ChemComp-MN:
Unknown entry

ChemComp-ZN:
Unknown entry

ChemComp-SPD:
SPERMIDINE / Spermidine

ChemComp-ACT:
ACETATE ION / Acetate

Source
  • streptococcus pyogenes (bacteria)
  • Enterobacteria phage lambda (virus)
  • actinomyces naeslundii (bacteria)
KeywordsHYDROLASE / DNASE / RNA-GUIDED / IMMUNITY / CRRNA / GENOME EDITING / ENDONUCLEASE / CRISPR-Cas / Cas9 / HNH / RuvC / RNA-guided DNA endonuclease / cytoplasmic

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