https://wiki.uni-konstanz.de/ccp4/index.php?title=Crystal_growth:_Protein-DNA_complexes&feed=atom&action=historyCrystal growth: Protein-DNA complexes - Revision history2024-03-28T20:27:33ZRevision history for this page on the wikiMediaWiki 1.39.6https://wiki.uni-konstanz.de/ccp4/index.php?title=Crystal_growth:_Protein-DNA_complexes&diff=2342&oldid=prevKay at 08:54, 9 September 20152015-09-09T08:54:11Z<p></p>
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<td colspan="2" style="background-color: #fff; color: #202122; text-align: center;">Revision as of 09:54, 9 September 2015</td>
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<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>7. Other useful tips?</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>7. Other useful tips?</div></td></tr>
<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Protein DNA crystals can be very fragile so good idea to screen with cryoprotectant in the screen to save manipulations</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;">* </ins>Protein DNA crystals can be very fragile so good idea to screen with cryoprotectant in the screen to save manipulations</div></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Test crystals with a gel to see if DNA is present</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;">* </ins>Test crystals with a gel to see if DNA is present</div></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div>Use of SAXS to get molecular envelop of complex</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;">* </ins>Use of SAXS to get molecular envelop of complex</div></td></tr>
<tr><td colspan="2" class="diff-side-deleted"></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;">* general guidelines to prepare protein/DNA complexes suitable for crystallization trials: [http://dx.doi.org/10.1007/978-1-59745-209-0_11 Hollis T (2007) Crystallization of protein-DNA complexes. Methods Mol. Biol. 363: 225–237]</ins></div></td></tr>
<tr><td colspan="2" class="diff-side-deleted"></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;">* design and composition of a specialized screen for protein/DNA complexes: [http://dx.doi.org/10.1107/S1744309112025316 Pryor EE Jr, Wozniak DJ & Hollis T (2012) Crystallization of Pseudomonas aeruginosa AmrZ protein: development of a comprehensive method for obtaining and optimization of protein-DNA crystals. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 68: 985–993]</ins></div></td></tr>
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<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><br/></td></tr>
<tr><td class="diff-marker" data-marker="−"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #ffe49c; vertical-align: top; white-space: pre-wrap;"><div><del style="font-weight: bold; text-decoration: none;">Several </del>references <del style="font-weight: bold; text-decoration: none;">below</del>:</div></td><td class="diff-marker" data-marker="+"></td><td style="color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #a3d3ff; vertical-align: top; white-space: pre-wrap;"><div><ins style="font-weight: bold; text-decoration: none;">== Some </ins>references: <ins style="font-weight: bold; text-decoration: none;">==</ins></div></td></tr>
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<tr><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>Schultz, S. C., Shields, G. C. & Steitz, T. A. (1990).</div></td><td class="diff-marker"></td><td style="background-color: #f8f9fa; color: #202122; font-size: 88%; border-style: solid; border-width: 1px 1px 1px 4px; border-radius: 0.33em; border-color: #eaecf0; vertical-align: top; white-space: pre-wrap;"><div>Schultz, S. C., Shields, G. C. & Steitz, T. A. (1990).</div></td></tr>
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<p><b>New page</b></p><div>Tips suggested by the CCP4 community as the best way to crystallize a protein/DNA complex <br />
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1. One of the biggest variables is to screen different lengths of DNA<br />
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2. Good to try both blunt and sticky ends. Perhaps as a first attempt try sticky ends with complementary ends<br />
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3. Could try purification by gel filtration if the complex is tight. Probably easier, quicker, cheaper and just as successful just to mix and pray!<br />
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4. Screen for crystals using a wide variety of crystal screens<br />
Natrix, Nucleix, PEGS, Index, JCSG core, MPD screen, PEG ion screen, home made PEG salt screen (+/- Mg++)<br />
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5. DNA can be ordered from a wide range of suppliers. Could try using un-purified DNA for screening, purify your own or order HPLC purified DNA<br />
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6. Good to try a wide range of ratio’s for protein/DNA. Most popular seems to be a ratio of 1/1.2<br />
<br />
7. Other useful tips?<br />
<br />
Protein DNA crystals can be very fragile so good idea to screen with cryoprotectant in the screen to save manipulations<br />
Test crystals with a gel to see if DNA is present<br />
Use of SAXS to get molecular envelop of complex<br />
<br />
<br />
Several references below:<br />
<br />
Schultz, S. C., Shields, G. C. & Steitz, T. A. (1990).<br />
Crystallization of Escherichia coli catabolite gene activator protein with its DNA binding site. J. Mol.Biol. 213, 159–166.<br />
<br />
Tan et al, Crystallization of the Yeast MATa2/MCM1/DNA Ternary Complex: General Methods and Principles for Protein/DNA Cocrystallization J. Mol. Biol. (2000) 297, 947-959<br />
<br />
Cannone et al, Crystallization of bFGF-DNA aptamer complexes using a<br />
Sparse Matrix designed for protein–nucleic acid complexes Journal of Crystal Growth, 2001 232 (2001) 409–417<br />
<br />
Book chapter 8 from Crystallization of nucleic acids and proteins: a practical approach.<br />
Edited by A.Ducruix and R. Giege (Oxford University Press)</div>Cwilliam