5fq5

From Proteopedia

(Difference between revisions)

Revision as of 13:04, 11 May 2016

Crystal structure of Cas9-sgRNA-DNA complex solved by native SAD phasing

Structural highlights

5fq5 is a 5 chain structure. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Ligands:	,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum

Function

[CAS9_STRP1] 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) (Probable). 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. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed by 3'-5' exonucleolytically. DNA-binding requires protein and both RNA species. Cas9 probably recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself.^[1] ^[2]

Publication Abstract from PubMed

Recent improvements in data-collection strategies have pushed the limits of native SAD (single-wavelength anomalous diffraction) phasing, a method that uses the weak anomalous signal of light elements naturally present in macromolecules. These involve the merging of multiple data sets from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose. Both approaches yield data of high multiplicity while minimizing radiation damage and systematic error, thus ensuring accurate measurements of the anomalous differences. Here, the combined use of these two strategies is described to solve cases of native SAD phasing that were particular challenges: the integral membrane diacylglycerol kinase (DgkA) with a low Bijvoet ratio of 1% and the large 200 kDa complex of the CRISPR-associated endonuclease (Cas9) bound to guide RNA and target DNA crystallized in the low-symmetry space group C2. The optimal native SAD data-collection strategy based on systematic measurements performed on the 266 kDa multiprotein/multiligand tubulin complex is discussed.

Data-collection strategy for challenging native SAD phasing.,Olieric V, Weinert T, Finke AD, Anders C, Li D, Olieric N, Borca CN, Steinmetz MO, Caffrey M, Jinek M, Wang M Acta Crystallogr D Struct Biol. 2016 Mar 1;72(Pt 3):421-9. doi:, 10.1107/S2059798315024110. Epub 2016 Mar 1. PMID:26960129^[3]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011 Mar 31;471(7340):602-7. doi: 10.1038/nature09886. PMID:21455174 doi:http://dx.doi.org/10.1038/nature09886
↑ Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012 Aug 17;337(6096):816-21. doi: 10.1126/science.1225829. Epub 2012, Jun 28. PMID:22745249 doi:http://dx.doi.org/10.1126/science.1225829
↑ Olieric V, Weinert T, Finke AD, Anders C, Li D, Olieric N, Borca CN, Steinmetz MO, Caffrey M, Jinek M, Wang M. Data-collection strategy for challenging native SAD phasing. Acta Crystallogr D Struct Biol. 2016 Mar 1;72(Pt 3):421-9. doi:, 10.1107/S2059798315024110. Epub 2016 Mar 1. PMID:26960129 doi:http://dx.doi.org/10.1107/S2059798315024110

1 Structural highlights
2 Function
3 Publication Abstract from PubMed
4 References

Proteopedia Page Contributors and Editors (what is this?)

OCA

Retrieved from "http://52.214.119.220/wiki/index.php/5fq5"

@@ Line 1: / Line 1: @@
-'''Unreleased structure'''
-The entry 5fq5 is ON HOLD  until Paper Publication
+==Crystal structure of Cas9-sgRNA-DNA complex solved by native SAD phasing==
+<StructureSection load='5fq5' size='340' side='right' caption='[[5fq5]], [[Resolution|resolution]] 2.14&Aring;' scene=''>
+== Structural highlights ==
+<table><tr><td colspan='2'>[[5fq5]] is a 5 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5FQ5 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5FQ5 FirstGlance]. <br>
+</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=K:POTASSIUM+ION'>K</scene>, <scene name='pdbligand=MG:MAGNESIUM+ION'>MG</scene></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=5fq5 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5fq5 OCA], [http://pdbe.org/5fq5 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5fq5 RCSB], [http://www.ebi.ac.uk/pdbsum/5fq5 PDBsum]</span></td></tr>
+</table>
+== Function ==
+[[http://www.uniprot.org/uniprot/CAS9_STRP1 CAS9_STRP1]] 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) (Probable). 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. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed by 3'-5' exonucleolytically. DNA-binding requires protein and both RNA species. Cas9 probably recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself.<ref>PMID:21455174</ref> <ref>PMID:22745249</ref>
+<div style="background-color:#fffaf0;">
+== Publication Abstract from PubMed ==
+Recent improvements in data-collection strategies have pushed the limits of native SAD (single-wavelength anomalous diffraction) phasing, a method that uses the weak anomalous signal of light elements naturally present in macromolecules. These involve the merging of multiple data sets from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose. Both approaches yield data of high multiplicity while minimizing radiation damage and systematic error, thus ensuring accurate measurements of the anomalous differences. Here, the combined use of these two strategies is described to solve cases of native SAD phasing that were particular challenges: the integral membrane diacylglycerol kinase (DgkA) with a low Bijvoet ratio of 1% and the large 200 kDa complex of the CRISPR-associated endonuclease (Cas9) bound to guide RNA and target DNA crystallized in the low-symmetry space group C2. The optimal native SAD data-collection strategy based on systematic measurements performed on the 266 kDa multiprotein/multiligand tubulin complex is discussed.
-Authors: Olieric, V., Weinert, T., Finke, A., Anders, C., Jinek, M., Wang, M.
+Data-collection strategy for challenging native SAD phasing.,Olieric V, Weinert T, Finke AD, Anders C, Li D, Olieric N, Borca CN, Steinmetz MO, Caffrey M, Jinek M, Wang M Acta Crystallogr D Struct Biol. 2016 Mar 1;72(Pt 3):421-9. doi:, 10.1107/S2059798315024110. Epub 2016 Mar 1. PMID:26960129<ref>PMID:26960129</ref>
-Description: Crystal structure of Cas9-sgRNA-DNA complex solved by native SAD phasing
+From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
-[[Category: Unreleased Structures]]
+</div>
-[[Category: Wang, M]]
+<div class="pdbe-citations 5fq5" style="background-color:#fffaf0;"></div>
-[[Category: Jinek, M]]
+== References ==
+<references/>
+__TOC__
+</StructureSection>
+[[Category: Anders, C]]
 [[Category: Finke, A]]
+[[Category: Jinek, M]]
 [[Category: Olieric, V]]
-[[Category: Anders, C]]
+[[Category: Wang, M]]
 [[Category: Weinert, T]]
+[[Category: Cas9]]
+[[Category: Crispr]]
+[[Category: Genome editing]]
+[[Category: Hydrolase-dna complex]]
+[[Category: Protein-rna complex]]

5fq5

From Proteopedia

Revision as of 13:04, 11 May 2016

Crystal structure of Cas9-sgRNA-DNA complex solved by native SAD phasing

Structural highlights

Function

Publication Abstract from PubMed

References

Contents

Proteopedia Page Contributors and Editors (what is this?)

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