5B2O

Crystal structure of Francisella novicida Cas9 in complex with sgRNA and target DNA (TGG PAM)


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.70 Å
  • R-Value Free: 0.207 
  • R-Value Work: 0.184 
  • R-Value Observed: 0.186 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Structure and Engineering of Francisella novicida Cas9

Hirano, H.Gootenberg, J.S.Horii, T.Abudayyeh, O.O.Kimura, M.Hsu, P.D.Nakane, T.Ishitani, R.Hatada, I.Zhang, F.Nishimasu, H.Nureki, O.

(2016) Cell 164: 950-961

  • DOI: https://doi.org/10.1016/j.cell.2016.01.039
  • Primary Citation of Related Structures:  
    5B2O, 5B2P, 5B2Q

  • PubMed Abstract: 

    The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox.


  • Organizational Affiliation

    Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.


Macromolecules

Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
CRISPR-associated endonuclease Cas91,632Francisella tularensis subsp. novicida U112Mutation(s): 1 
Gene Names: cas9FTN_0757
EC: 3.1
UniProt
Find proteins for A0Q5Y3 (Francisella tularensis subsp. novicida (strain U112))
Explore A0Q5Y3 
Go to UniProtKB:  A0Q5Y3
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupA0Q5Y3
Sequence Annotations
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  • Reference Sequence
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Entity ID: 2
MoleculeChains LengthOrganismImage
Guide RNA94synthetic construct
Sequence Annotations
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  • Reference Sequence
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Entity ID: 3
MoleculeChains LengthOrganismImage
Target DNA30Francisella tularensis subsp. novicida
Sequence Annotations
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  • Reference Sequence

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Entity ID: 4
MoleculeChains LengthOrganismImage
DNA (5'-D(*TP*GP*GP*TP*AP*TP*CP*GP*G)-3')9Francisella tularensis subsp. novicida
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 6 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
ZN
Query on ZN

Download Ideal Coordinates CCD File 
E [auth A]ZINC ION
Zn
PTFCDOFLOPIGGS-UHFFFAOYSA-N
EDO
Query on EDO

Download Ideal Coordinates CCD File 
AA [auth A]
AB [auth C]
BA [auth A]
CA [auth A]
DA [auth A]
AA [auth A],
AB [auth C],
BA [auth A],
CA [auth A],
DA [auth A],
EA [auth A],
PA [auth B],
QA [auth B],
RA [auth B],
SA [auth B],
T [auth A],
TA [auth B],
U [auth A],
UA [auth B],
V [auth A],
VA [auth B],
W [auth A],
WA [auth B],
X [auth A],
XA [auth B],
Y [auth A],
YA [auth B],
Z [auth A]
1,2-ETHANEDIOL
C2 H6 O2
LYCAIKOWRPUZTN-UHFFFAOYSA-N
ACT
Query on ACT

Download Ideal Coordinates CCD File 
FA [auth A],
ZA [auth B]
ACETATE ION
C2 H3 O2
QTBSBXVTEAMEQO-UHFFFAOYSA-M
CA
Query on CA

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IA [auth B]
J [auth A]
JA [auth B]
K [auth A]
KA [auth B]
IA [auth B],
J [auth A],
JA [auth B],
K [auth A],
KA [auth B],
L [auth A],
LA [auth B],
M [auth A],
MA [auth B],
N [auth A],
NA [auth B],
O [auth A],
OA [auth B],
P [auth A],
Q [auth A],
R [auth A],
S [auth A]
CALCIUM ION
Ca
BHPQYMZQTOCNFJ-UHFFFAOYSA-N
CL
Query on CL

Download Ideal Coordinates CCD File 
H [auth A],
I [auth A]
CHLORIDE ION
Cl
VEXZGXHMUGYJMC-UHFFFAOYSA-M
NA
Query on NA

Download Ideal Coordinates CCD File 
F [auth A],
G [auth A],
GA [auth B],
HA [auth B]
SODIUM ION
Na
FKNQFGJONOIPTF-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.70 Å
  • R-Value Free: 0.207 
  • R-Value Work: 0.184 
  • R-Value Observed: 0.186 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 81.922α = 90
b = 159.105β = 107.04
c = 96.808γ = 90
Software Package:
Software NamePurpose
Aimlessdata scaling
PHENIXrefinement
PDB_EXTRACTdata extraction

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2016-03-02
    Type: Initial release
  • Version 1.1: 2016-03-09
    Changes: Database references
  • Version 1.2: 2017-10-04
    Changes: Advisory, Data collection, Derived calculations
  • Version 1.3: 2024-03-20
    Changes: Data collection, Database references, Derived calculations