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==Crystal structure of Francisella novicida Cas9 RHA in complex with sgRNA and target DNA (TGG PAM)==
==Crystal structure of Francisella novicida Cas9 RHA in complex with sgRNA and target DNA (TGG PAM)==
<StructureSection load='5b2q' size='340' side='right' caption='[[5b2q]], [[Resolution|resolution]] 1.70&Aring;' scene=''>
<StructureSection load='5b2q' size='340' side='right'caption='[[5b2q]], [[Resolution|resolution]] 1.70&Aring;' scene=''>
== Structural highlights ==
== Structural highlights ==
<table><tr><td colspan='2'>[[5b2q]] is a 4 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5B2Q OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5B2Q FirstGlance]. <br>
<table><tr><td colspan='2'>[[5b2q]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Francisella_tularensis_subsp._novicida Francisella tularensis subsp. novicida], [https://en.wikipedia.org/wiki/Francisella_tularensis_subsp._novicida_U112 Francisella tularensis subsp. novicida U112] and [https://en.wikipedia.org/wiki/Synthetic_construct Synthetic construct]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5B2Q OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5B2Q FirstGlance]. <br>
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=ACT:ACETATE+ION'>ACT</scene>, <scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene>, <scene name='pdbligand=NA:SODIUM+ION'>NA</scene>, <scene name='pdbligand=ZN:ZINC+ION'>ZN</scene></td></tr>
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.7&#8491;</td></tr>
<tr id='NonStdRes'><td class="sblockLbl"><b>[[Non-Standard_Residue|NonStd Res:]]</b></td><td class="sblockDat"><scene name='pdbligand=DU:2-DEOXYURIDINE-5-MONOPHOSPHATE'>DU</scene></td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=ACT:ACETATE+ION'>ACT</scene>, <scene name='pdbligand=CA:CALCIUM+ION'>CA</scene>, <scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene>, <scene name='pdbligand=EDO:1,2-ETHANEDIOL'>EDO</scene>, <scene name='pdbligand=NA:SODIUM+ION'>NA</scene>, <scene name='pdbligand=ZN:ZINC+ION'>ZN</scene></td></tr>
<tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[5b2o|5b2o]], [[5b2p|5b2p]]</td></tr>
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=5b2q FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5b2q OCA], [https://pdbe.org/5b2q PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5b2q RCSB], [https://www.ebi.ac.uk/pdbsum/5b2q PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5b2q ProSAT]</span></td></tr>
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5b2q FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5b2q OCA], [http://pdbe.org/5b2q PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5b2q RCSB], [http://www.ebi.ac.uk/pdbsum/5b2q PDBsum]</span></td></tr>
</table>
</table>
== Function ==
== Function ==
[[http://www.uniprot.org/uniprot/CAS9_FRATN CAS9_FRATN]] CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer; Cas9 is inactive in the absence of the 2 guide RNAs (gRNA). Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself, as targets within the bacterial CRISPR locus do not have PAMs. PAM recognition is also required for catalytic activity (By similarity). Cuts target DNA when Cas9 and gRNAs are mixed.<ref>PMID:24270795</ref>
[https://www.uniprot.org/uniprot/CAS9_FRATN CAS9_FRATN] CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA. Subsequently Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer; Cas9 is inactive in the absence of the 2 guide RNAs (gRNA). Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself, as targets within the bacterial CRISPR locus do not have PAMs. PAM recognition is also required for catalytic activity (By similarity). Cuts target DNA when Cas9 and gRNAs are mixed.<ref>PMID:24270795</ref>  
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
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 A 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.


Structure and Engineering of Francisella novicida Cas9.,Hirano H, Gootenberg JS, Horii T, Abudayyeh OO, Kimura M, Hsu PD, Nakane T, Ishitani R, Hatada I, Zhang F, Nishimasu H, Nureki O Cell. 2016 Feb 25;164(5):950-61. doi: 10.1016/j.cell.2016.01.039. Epub 2016 Feb, 11. PMID:26875867<ref>PMID:26875867</ref>
==See Also==
 
*[[Endonuclease 3D structures|Endonuclease 3D structures]]
From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
</div>
<div class="pdbe-citations 5b2q" style="background-color:#fffaf0;"></div>
== References ==
== References ==
<references/>
<references/>
__TOC__
__TOC__
</StructureSection>
</StructureSection>
[[Category: Hirano, H]]
[[Category: Francisella tularensis subsp. novicida]]
[[Category: Ishitani, R]]
[[Category: Francisella tularensis subsp. novicida U112]]
[[Category: Nakane, T]]
[[Category: Large Structures]]
[[Category: Nishimasu, H]]
[[Category: Synthetic construct]]
[[Category: Nureki, O]]
[[Category: Hirano H]]
[[Category: Crispr-cas9]]
[[Category: Ishitani R]]
[[Category: Genome engineering]]
[[Category: Nakane T]]
[[Category: Hydrolase-rna-dna complex]]
[[Category: Nishimasu H]]
[[Category: Nureki O]]

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