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| | ==Crystal structure of the Acidaminococcus sp. BV3L6 Cpf1 RVR variant in complex with crRNA and target DNA (TATA PAM)== | | ==Crystal structure of the Acidaminococcus sp. BV3L6 Cpf1 RVR variant in complex with crRNA and target DNA (TATA PAM)== |
| - | <StructureSection load='5xh6' size='340' side='right' caption='[[5xh6]], [[Resolution|resolution]] 2.00Å' scene=''> | + | <StructureSection load='5xh6' size='340' side='right'caption='[[5xh6]], [[Resolution|resolution]] 2.00Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[5xh6]] is a 4 chain structure with sequence from [http://en.wikipedia.org/wiki/Acisb Acisb]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5XH6 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5XH6 FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[5xh6]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Acidaminococcus_sp._BV3L6 Acidaminococcus sp. BV3L6]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5XH6 OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5XH6 FirstGlance]. <br> |
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><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></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]] 2Å</td></tr> |
| - | <tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[5xh7|5xh7]]</td></tr> | + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><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></td></tr> |
| - | <tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">cpf1, HMPREF1246_0236 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=1111120 ACISB])</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=5xh6 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5xh6 OCA], [https://pdbe.org/5xh6 PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5xh6 RCSB], [https://www.ebi.ac.uk/pdbsum/5xh6 PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5xh6 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=5xh6 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5xh6 OCA], [http://pdbe.org/5xh6 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5xh6 RCSB], [http://www.ebi.ac.uk/pdbsum/5xh6 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5xh6 ProSAT]</span></td></tr> | + | |
| | </table> | | </table> |
| | == Function == | | == Function == |
| - | [[http://www.uniprot.org/uniprot/CPF1_ACISB CPF1_ACISB]] 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 sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). Has dsDNA endonuclease activity, results in a 4-base 5' overhang 19 and 22 bases downstream of the PAM on the non-targeted and targeted strand respectively (PubMed:26422227). In this CRISPR system correct processing of pre-crRNA requires only this protein and the CRISPR locus (By similarity).[UniProtKB:A0Q7Q2]<ref>PMID:26422227</ref> | + | [https://www.uniprot.org/uniprot/CS12A_ACISB CS12A_ACISB] 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 sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). Recognizes a short motif in the CRISPR repeat sequences (the 5' PAM or protospacer adjacent motif, TTTN in this organism) to help distinguish self versus nonself, as targets within the bacterial CRISPR locus do not have PAMs (PubMed:26422227). Has dsDNA endonuclease activity, results in staggered 4-base 5' overhangs 19 and 22 bases downstream of the PAM on the non-targeted and targeted strand respectively (PubMed:26422227). Non-target strand cleavage by the RuvC domain is probably a prerequisite of target strand cleavage by the Nuc domain (PubMed:27114038). In this CRISPR system correct processing of pre-crRNA requires only this protein and the CRISPR locus (By similarity).[UniProtKB:A0Q7Q2]<ref>PMID:26422227</ref> <ref>PMID:27114038</ref> |
| | <div style="background-color:#fffaf0;"> | | <div style="background-color:#fffaf0;"> |
| | == Publication Abstract from PubMed == | | == Publication Abstract from PubMed == |
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| | </div> | | </div> |
| | <div class="pdbe-citations 5xh6" style="background-color:#fffaf0;"></div> | | <div class="pdbe-citations 5xh6" style="background-color:#fffaf0;"></div> |
| | + | |
| | + | ==See Also== |
| | + | *[[Endonuclease 3D structures|Endonuclease 3D structures]] |
| | == References == | | == References == |
| | <references/> | | <references/> |
| | __TOC__ | | __TOC__ |
| | </StructureSection> | | </StructureSection> |
| - | [[Category: Acisb]] | + | [[Category: Acidaminococcus sp. BV3L6]] |
| - | [[Category: Ishitani, R]] | + | [[Category: Large Structures]] |
| - | [[Category: Nishimasu, H]] | + | [[Category: Ishitani R]] |
| - | [[Category: Nureki, O]] | + | [[Category: Nishimasu H]] |
| - | [[Category: Yamano, T]] | + | [[Category: Nureki O]] |
| - | [[Category: Hydrolase-rna-dna complex]]
| + | [[Category: Yamano T]] |
| - | [[Category: Nuclease]]
| + | |
| Structural highlights
Function
CS12A_ACISB 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 sequences complementary to antecedent mobile elements and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). Recognizes a short motif in the CRISPR repeat sequences (the 5' PAM or protospacer adjacent motif, TTTN in this organism) to help distinguish self versus nonself, as targets within the bacterial CRISPR locus do not have PAMs (PubMed:26422227). Has dsDNA endonuclease activity, results in staggered 4-base 5' overhangs 19 and 22 bases downstream of the PAM on the non-targeted and targeted strand respectively (PubMed:26422227). Non-target strand cleavage by the RuvC domain is probably a prerequisite of target strand cleavage by the Nuc domain (PubMed:27114038). In this CRISPR system correct processing of pre-crRNA requires only this protein and the CRISPR locus (By similarity).[UniProtKB:A0Q7Q2][1] [2]
Publication Abstract from PubMed
The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 A resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1.
Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1.,Nishimasu H, Yamano T, Gao L, Zhang F, Ishitani R, Nureki O Mol Cell. 2017 Jul 6;67(1):139-147.e2. doi: 10.1016/j.molcel.2017.04.019. Epub, 2017 Jun 6. PMID:28595896[3]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
See Also
References
- ↑ Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015 Oct 22;163(3):759-71. doi: 10.1016/j.cell.2015.09.038. Epub 2015 Sep, 25. PMID:26422227 doi:http://dx.doi.org/10.1016/j.cell.2015.09.038
- ↑ Yamano T, Nishimasu H, Zetsche B, Hirano H, Slaymaker IM, Li Y, Fedorova I, Nakane T, Makarova KS, Koonin EV, Ishitani R, Zhang F, Nureki O. Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA. Cell. 2016 May 5;165(4):949-62. doi: 10.1016/j.cell.2016.04.003. Epub 2016 Apr, 21. PMID:27114038 doi:http://dx.doi.org/10.1016/j.cell.2016.04.003
- ↑ Nishimasu H, Yamano T, Gao L, Zhang F, Ishitani R, Nureki O. Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1. Mol Cell. 2017 Jul 6;67(1):139-147.e2. doi: 10.1016/j.molcel.2017.04.019. Epub, 2017 Jun 6. PMID:28595896 doi:http://dx.doi.org/10.1016/j.molcel.2017.04.019
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