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| [[Image:2b0d.gif|left|200px]]
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| {{Structure
| | ==EcoRV Restriction Endonuclease/GAATTC/Ca2+== |
| |PDB= 2b0d |SIZE=350|CAPTION= <scene name='initialview01'>2b0d</scene>, resolution 2.0Å
| | <StructureSection load='2b0d' size='340' side='right'caption='[[2b0d]], [[Resolution|resolution]] 2.00Å' scene=''> |
| |SITE= | | == Structural highlights == |
| |LIGAND= <scene name='pdbligand=CA:CALCIUM ION'>CA</scene>
| | <table><tr><td colspan='2'>[[2b0d]] is a 4 chain structure with sequence from [https://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2B0D OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=2B0D FirstGlance]. <br> |
| |ACTIVITY= [http://en.wikipedia.org/wiki/Type_II_site-specific_deoxyribonuclease Type II site-specific deoxyribonuclease], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=3.1.21.4 3.1.21.4]
| | </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> |
| |GENE= ecoRVR ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=562 Escherichia coli])
| | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CA:CALCIUM+ION'>CA</scene></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=2b0d FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=2b0d OCA], [https://pdbe.org/2b0d PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=2b0d RCSB], [https://www.ebi.ac.uk/pdbsum/2b0d PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=2b0d ProSAT]</span></td></tr> |
| | </table> |
| | == Function == |
| | [https://www.uniprot.org/uniprot/T2E5_ECOLX T2E5_ECOLX] Recognizes the double-stranded sequence GATATC and cleaves after T-3. |
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| '''EcoRV Restriction Endonuclease/GAATTC/Ca2+'''
| | ==See Also== |
| | | *[[Endonuclease 3D structures|Endonuclease 3D structures]] |
| | | __TOC__ |
| ==Overview== | | </StructureSection> |
| The crystal structure of EcoRV endonuclease bound to non-cognate DNA at 2.0 angstroms resolution shows that very small structural adaptations are sufficient to ensure the extreme sequence specificity characteristic of restriction enzymes. EcoRV bends its specific GATATC site sharply by 50 degrees into the major groove at the center TA step, generating unusual base-base interactions along each individual DNA strand. In the symmetric non-cognate complex bound to GAATTC, the center step bend is relaxed to avoid steric hindrance caused by the different placement of the exocyclic thymine methyl groups. The decreased base-pair unstacking in turn leads to small conformational rearrangements in the sugar-phosphate backbone, sufficient to destabilize binding of crucial divalent metal ions in the active site. A second crystal structure of EcoRV bound to the base-analog GAAUTC site shows that the 50 degrees center-step bend of the DNA is restored. However, while divalent metals bind at high occupancy in this structure, one metal ion shifts away from binding at the scissile DNA phosphate to a position near the 3'-adjacent phosphate group. This may explain why the 10(4)-fold attenuated cleavage efficiency toward GAATTC is reconstituted by less than tenfold toward GAAUTC. Examination of DNA binding and bending by equilibrium and stopped-flow florescence quenching and fluorescence resonance energy transfer (FRET) methods demonstrates that the capacity of EcoRV to bend the GAATTC non-cognate site is severely limited, but that full bending of GAAUTC is achieved at only a threefold reduced rate compared with the cognate complex. Together, the structural and biochemical data demonstrate the existence of distinct mechanisms for ensuring specificity at the bending and catalytic steps, respectively. The limited conformational rearrangements observed in the EcoRV non-cognate complex provide a sharp contrast to the extensive structural changes found in a non-cognate BamHI-DNA crystal structure, thus demonstrating a diversity of mechanisms by which restriction enzymes are able to achieve specificity.
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| ==About this Structure==
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| 2B0D is a [[Single protein]] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=2B0D OCA].
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| ==Reference==
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| Non-cognate enzyme-DNA complex: structural and kinetic analysis of EcoRV endonuclease bound to the EcoRI recognition site GAATTC., Hiller DA, Rodriguez AM, Perona JJ, J Mol Biol. 2005 Nov 18;354(1):121-36. Epub 2005 Oct 3. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16236314 16236314]
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| [[Category: Escherichia coli]] | | [[Category: Escherichia coli]] |
| [[Category: Single protein]] | | [[Category: Large Structures]] |
| [[Category: Type II site-specific deoxyribonuclease]]
| | [[Category: Hiller DA]] |
| [[Category: Hiller, D A.]] | | [[Category: Perona JJ]] |
| [[Category: Perona, J J.]] | | [[Category: Rodriguez AM]] |
| [[Category: Rodriguez, A M.]] | |
| [[Category: CA]]
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| [[Category: indirect readout]]
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| [[Category: noncognate]]
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| [[Category: protein-nucleic acid recognition]]
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| [[Category: restriction enzyme]]
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| [[Category: substrate specificity]]
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| ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Mar 20 15:56:04 2008''
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