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</o:shapelayout></xml><![endif]--></head><body lang=EN-US link=blue vlink=purple><div class=WordSection1><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>Just a quick note from somebody who does this for a living....<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>-If the compound in question retains its bromine it will practically scream at you when you look at the fofc maps. No need for elaborate anomalous difference maps, unless you like to go through the motions, which is commendable.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>-Beware that if the part of the compound were the bromine is bound is dangling, i.e. not well ordered, you will lose the anomalous signal, so use common sense to decide if the compound is bound or not. <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>-Always good to collect an intrinsic control to make sure that you have the procedure under control or experiment with a high redundancy (>10x) lysozyme dataset collected at home to get comfortable with the procedure. The sulfurs in the lysozyme dataset light up in an anomalous map, when the collection was done right.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>-I usually only use anomalous maps in the context of sulfur containing compounds, since it allows me to determine the orientation of thiazole rings and such. Everything with more electrons is usually easily detected in the fofc maps. <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>-Make sure you collect a high redundancy dataset (> x6) on or a bit beyond the Br edge. Don’t overkill it, radiation damage is you enemy. <o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>Good luck.<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'>Carsten<o:p></o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D'><o:p> </o:p></span></p><div style='border:none;border-left:solid blue 1.5pt;padding:0in 0in 0in 4.0pt'><div><div style='border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'>From:</span></b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'> phenixbb-bounces@phenix-online.org [mailto:phenixbb-bounces@phenix-online.org] <b>On Behalf Of </b>Jason<br><b>Sent:</b> Tuesday, February 08, 2011 1:14 PM<br><b>To:</b> phenixbb@phenix-online.org<br><b>Subject:</b> [phenixbb] How to locate and refine a ligand using anomalousscattering<o:p></o:p></span></p></div></div><p class=MsoNormal><o:p> </o:p></p><div><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'>Hello everyone,<o:p></o:p></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><o:p> </o:p></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'>I have a few crystals to be x-rayed next week. Before that I hope to get a clear idea about what I am doing (I am new to anomalous scattering). <o:p></o:p></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><o:p> </o:p></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'>Facts:<o:p></o:p></p><ol start=1 type=1><li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;mso-list:l2 level1 lfo1'><span style='font-family:"'Times New Roman'","serif"'>The crystal is a protein co-crystallized with a ligand<o:p></o:p></span></li><li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;mso-list:l2 level1 lfo1'><span style='font-family:"'Times New Roman'","serif"'>The protein structure is known.<o:p></o:p></span></li><li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;mso-list:l2 level1 lfo1'><span style='font-family:"'Times New Roman'","serif"'>The ligand has a heavy atom bromide (absorption K-egde=13.47Kev)<o:p></o:p></span></li><li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;mso-list:l2 level1 lfo1'><span style='font-family:"'Times New Roman'","serif"'>Data resolution is ~3 angstrom<o:p></o:p></span></li></ol><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><o:p> </o:p></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'>Goals:<o:p></o:p></p><ol start=1 type=1><li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;mso-list:l0 level1 lfo2'><span style='font-family:"'Times New Roman'","serif"'>Locate the bromide position<o:p></o:p></span></li><li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;mso-list:l0 level1 lfo2'><span style='font-family:"'Times New Roman'","serif"'>Locate and refine the ligand<o:p></o:p></span></li></ol><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><o:p> </o:p></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'>Questions:<o:p></o:p></p><ol start=1 type=1><li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;mso-list:l1 level1 lfo3'><span style='font-family:"'Times New Roman'","serif"'>Do I need to carry out MAD experiment at 3 wavelengths or there is some other easier way since the protein structure is known (I am not expecting big change of the protein structure itself)?<o:p></o:p></span></li><li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;mso-list:l1 level1 lfo3'><span style='font-family:"'Times New Roman'","serif"'>Assuming I have the MAD data, what should I do next using phenix to achieve the two goals listed above? Here are some thoughts:<o:p></o:p></span></li></ol><ol start=2 type=1><ul type=circle><li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;mso-list:l1 level2 lfo3'><span style='font-family:"'Times New Roman'","serif"'>Using phenix.hyss to locate the anomalous scatterers <o:p></o:p></span></li><li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;mso-list:l1 level2 lfo3'><span style='font-family:"'Times New Roman'","serif"'>Using phenix.autobuild to build the protein model (which data set to use?)<o:p></o:p></span></li><li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;mso-list:l1 level2 lfo3'><span style='font-family:"'Times New Roman'","serif"'>Using coot to add ligands to the protein structure (Is the relative position between the protein and the ligand known based on phenix.hyss and phenix.autobuild?)<o:p></o:p></span></li><li class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;mso-list:l1 level2 lfo3'><span style='font-family:"'Times New Roman'","serif"'>Using phenix.refine to refine the ligand+protein complex<o:p></o:p></span></li></ul></ol><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><o:p> </o:p></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'>Thank you all for reading this.<o:p></o:p></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><o:p> </o:p></p></div><div><p class=MsoNormal>======================<o:p></o:p></p></div><p class=MsoNormal>Jason<o:p></o:p></p><div><p class=MsoNormal>Structural Biology Department<o:p></o:p></p></div><div><p class=MsoNormal>University of Pittsburgh<o:p></o:p></p></div><div><p class=MsoNormal>======================<o:p></o:p></p></div><p class=MsoNormal><o:p> </o:p></p></div></div></body></html>