Re: [ccp4bb] molecular replacement problem.
Thank you for the quick reply. After molecular replacement , i have done only few cycle of refinement in refmac. I have not done any solvent modification or NCS averaging. I have initially indexed the data in C2221 but Rfree was not decreasing so i reindexed the data in data in P121 space group keeping the Rfree flag of C2221. While analysing the symmetry mates , i found large space but no density. structure of Ligand binding domain is almost identical with 90% identity in sequence. I am stuck with this problem and don't know how to process further. Please give me your valuable suggestion. I will appreciate your effort. Thank you Appu On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote: Dear Appu, I am not sure that I have a complete sense of the issue at hand since some of the information needed to think your issue through is missing in your email. For example, to what high resolution cut-off were the data measured? What resolution limits were used for the MR search? How do the unit cell dimensions and space group in the two cases compare? I am guessing the ligand binding domain in your protein has the identical sequence to that of the published ligand binding domain that you use as a template in your MR search. In any case, here are a couple of my thoughts: (1) It might be worth setting up different runs of MR with different numbers for expected copies (not just two copies but also one copy and three copies just in case you have one of the extreme cases of solvent content)? (2) If the MR solution is correct and there is physical room for a DNA binding domain in your lattice (check by displaying symmetry mates), perhaps the DNA binding domain is disordered. In that case (and if all attempts with current data fail), you may have to crystallize the protein in presence of DNA. Good luck! Raji On Sat, Mar 23, 2013 at 2:26 PM, Appu kumar appu.kum...@gmail.com wrote: Dear members, I am doing a molecular replacement of a transcription factor whose ligand binding structure(24000 Da) is available in PDB but not for the DNA binding(13000 Da). When i am searching for the two copies from ligand binding domain as a template model, i am getting very good solution but i am not getting any density for the DNA binding domain to build up in density. The space gorup is P 1 21 1 (4) and unit cell parameters are Unit Cell: 57.43 69.36 105.99 90.00 90.00 90.00. Please guide me how to get the complete model structure. Table below show the matthews statistics For estimated molecular weight 37000. Nmol/asym Matthews Coeff %solvent P(2.20) P(tot) _ 1 5.7178.46 0.00 0.01 2 2.8556.91 0.62 0.70 3 1.9035.37 0.37 0.29 4 1.4313.82 0.00 0.00 _ The phaser molecular replacement gives the following table. istogram of relative frequencies of VM values -- Frequency of most common VM value normalized to 1 VM values plotted in increments of 1/VM (0.02) --- relative frequency --- 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 ||||||||||| 10.00 - 8.33 - 7.14 - 6.25 - 5.56 - 5.00 - 4.55 - 4.17 - 3.85 -- 3.57 --- 3.33 -- 3.12 -- 2.94 (COMPOSITION*1) 2.78 --- 2.63 2.50 - 2.38 2.27 -- 2.17 --- 2.08 -- 2.00 -- 1.92 --- 1.85 --- 1.79 --- 1.72 - 1.67 - 1.61 - 1.56 - 1.52 - 1.47 * (COMPOSITION*2) 1.43 - 1.39 - 1.35 - 1.32 - 1.28 - 1.25 - $TABLE : Cell Content Analysis: $SCATTER :N*Composition vs Probability:0|3x0|1:1,2: $$ N*Composition Probability $$ loggraph $$ 1 0.306066 2 0.00141804 $$ Most probable VM for resolution = 2.27817 Most probable MW of protein in asu for resolution = 92664.2 Thank a lot in advance -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Question about DNA/RNA crystal annealing
Dear ccp4bb members, I would be most happy, if you could point me to any publications, in which results of crystal annealing by warming up a DNA/RNA/oligonuculeotide crystal and again cooling it down to low temperatures were reported. I am aware of 2 publications that discuss this for DNA/protein crystals (ActaCrystD 1998, 622; JStructBiol 202, 55) and general publications about this topic (e.g. ActaD, 2005, 1173 ). Thanks a lot for your help! Best regards Bernhard
Re: [ccp4bb] molecular replacement problem.
I run the phenix.xtriage to evaluate the twining but it suggest no twining. When i reindex from C2221 to P21, the completeness of data reduced from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31 for 2.2 resolution. I do not understand why the completeness of data reduced so much on reindexing. please Can anyone explain this phenomenon. Thank you On 24 March 2013 13:30, Matthias Zebisch matthias.zebi...@bbz.uni-leipzig.de wrote: the p21 c2221 ambivalence can mean severe twinning (i had a similar case just now - try several crystals from the same condition) ! What do the twinning statistics suggest? cheers, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 3/24/2013 7:46 AM, Appu kumar wrote: Thank you for the quick reply. After molecular replacement , i have done only few cycle of refinement in refmac. I have not done any solvent modification or NCS averaging. I have initially indexed the data in C2221 but Rfree was not decreasing so i reindexed the data in data in P121 space group keeping the Rfree flag of C2221. While analysing the symmetry mates , i found large space but no density. structure of Ligand binding domain is almost identical with 90% identity in sequence. I am stuck with this problem and don't know how to process further. Please give me your valuable suggestion. I will appreciate your effort. Thank you Appu On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote: Dear Appu, I am not sure that I have a complete sense of the issue at hand since some of the information needed to think your issue through is missing in your email. For example, to what high resolution cut-off were the data measured? What resolution limits were used for the MR search? How do the unit cell dimensions and space group in the two cases compare? I am guessing the ligand binding domain in your protein has the identical sequence to that of the published ligand binding domain that you use as a template in your MR search. In any case, here are a couple of my thoughts: (1) It might be worth setting up different runs of MR with different numbers for expected copies (not just two copies but also one copy and three copies just in case you have one of the extreme cases of solvent content)? (2) If the MR solution is correct and there is physical room for a DNA binding domain in your lattice (check by displaying symmetry mates), perhaps the DNA binding domain is disordered. In that case (and if all attempts with current data fail), you may have to crystallize the protein in presence of DNA. Good luck! Raji On Sat, Mar 23, 2013 at 2:26 PM, Appu kumar appu.kum...@gmail.comwrote: Dear members, I am doing a molecular replacement of a transcription factor whose ligand binding structure(24000 Da) is available in PDB but not for the DNA binding(13000 Da). When i am searching for the two copies from ligand binding domain as a template model, i am getting very good solution but i am not getting any density for the DNA binding domain to build up in density. The space gorup is P 1 21 1 (4) and unit cell parameters are Unit Cell: 57.43 69.36 105.99 90.00 90.00 90.00. Please guide me how to get the complete model structure. Table below show the matthews statistics For estimated molecular weight 37000. Nmol/asym Matthews Coeff %solvent P(2.20) P(tot) _ 1 5.7178.46 0.00 0.01 2 2.8556.91 0.62 0.70 3 1.9035.37 0.37 0.29 4 1.4313.82 0.00 0.00 _ The phaser molecular replacement gives the following table. istogram of relative frequencies of VM values -- Frequency of most common VM value normalized to 1 VM values plotted in increments of 1/VM (0.02) --- relative frequency --- 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 ||||||||||| 10.00 - 8.33 - 7.14 - 6.25 - 5.56 - 5.00 - 4.55 - 4.17 - 3.85 -- 3.57 --- 3.33 -- 3.12 -- 2.94 (COMPOSITION*1) 2.78 --- 2.63 2.50 - 2.38 2.27
Re: [ccp4bb] Isothermal titration calorimetry
Chris, indeed nanoITC instrument analysis software is very robust and user friendly (probable more friendly than microcal, GE). Although when you need to subtract Q (heat) values (from 2 or 3 blank experiments) from your experimental data you cannot. NanoITC software can subtract Q values only from 1 blank experiment. Also if you want to present your data in a form of heat/mol in Y (vertical) axes again you cannot. It presents data in Y axes only in form of heat/injection. If you have found a way to extract 2 or 3 blank experiments from experimental data or present data in form of heat/mol, please let me know it will be very useful. The main problem in the output files from nanoITC come with an extension .nitc, by default. Unfortunately Origin (that can do all the above) can read only, filenames with an extension .itc Cheers, George -Original Message- From: Colbert, Christopher [mailto:christopher.colb...@ndsu.edu] Sent: Saturday, March 23, 2013 5:56 PM To: George Kontopidis; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Isothermal titration calorimetry George, would you please explain your comments? We've found the TA Instruments analysis software very robust and user friendly. We have the low volume nanoITC from TA instruments and get equivalent #'s in our comparison tests to the Microcal instrument. Cheers, Chris -- Christopher L. Colbert, Ph.D. Assistant Professor Department of Chemistry and Biochemistry North Dakota State University P.O. Box 6050 Dept. 2710 Fargo, ND 58108-6050 PH: (701) 231-7946 FAX: (701) 231-8324 On 3/23/13 8:47 AM, George Kontopidis gkontopi...@vet.uth.gr wrote: Keep in mind that output files from nanoITC, TA instrument cannot be red by Origin. At some point you will need to analyse your data further. George -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anastassis Perrakis Sent: Saturday, March 23, 2013 12:46 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Isothermal titration calorimetry It might be worth to consider the question more in detail. Do you want to study thermodynamics of the interaction, or a KD would do? If the former, you need ITC. If the latter, and you want to study things at the level of KD only, maybe investing on a plate reader, thermophoresis, or some biosensor technology (spr or interferometry based systems) should be considered. Then, what interactions will you study with the ITC? In general, I would agree that the lower sample volume is worth the nano options, but depending on the typical systems under study, sometimes the gain on sample quantity is not worth the money - while many times its worth it. John is if course right that for studying specific systems as the one he describes the 200 is great. A. Sent from my iPhone On 23 Mar 2013, at 11:00, John Fisher johncfishe...@gmail.com wrote: I would recommend the Microcal ITC 200, hands down. Not only is it an amazing instrument with the optional automated sample loader (which is worth every penny), but we were able to do experiments (multiple) using FULL-LENGTH p53 binding to a weak cognate protein. I believe this was the first time ITC was ever used with full length p53, as it is so labile and just loves immediately to oligomerize. Sample sizes pay for the instrument. Best, John John Fisher, M.D./PhD St. Jude Children's Research Hospital Department of Oncology Department of Structural Biology W: 901-595-6193 C: 901-409-5699 On Mar 23, 2013, at 4:45 AM, Sameh Soror shamd...@googlemail.com wrote: Dear All, I am sorry for the off topic question. I am going to buy ITC to study protein-protein protein-ligand interactions I am comparing microcal, GE and nanoITC, TA instrument.. any suggestions, recommendations, good experiences or bad experiences. is there a better system. Thank in advance for the help. Regards Sameh -- Sameh Soror Postdoc. fellow
Re: [ccp4bb] molecular replacement problem.
Hello, Here we deal with symmetry and the unique part of reciprocal space (the reciprocal space asymmetric unit so to speak). C222(1) has eight asymmetric units (international tables, space group 20); P2(1) only has two. Assuming that Friedel's law does apply, then the minimum rotation range to collect a non-redundant data set (one observation per reflection) is 90 degrees, provided that the crystal is correctly and perfectly aligned. Normally with our current data collection methods where the crystal is randomly oriented, we would collect more than 90 degrees (180 degrees, or 360 degrees with the Pilatus detectors on an intense SR beamline where you cannot really check during data collection how well the crystal fares during exposure to the X-rays - shoot first, think later. The reciprocal space asymmetric unit in C222(1) is smaller. I assume that what you are doing is to take the reduced data set file (an MTZ file probably) and reduce the symmetry from C222(1) to P2(1). You will not cover the monoclinic reciprocal space asymmetric unit by doing so. The way to do it is to take the file from processing, before (crystallographic symmetry) merging of the equivalents, and perform the scaling and merging in the P2(1) space group. Or reprocess the data frames in P2(1) if you have lost the unmerged data file. Now of course this will still give you a poor completeness if you have used a strategy to optimize data collection in the orthorhombic space group (you won't have collected enough data then for good completeness in the monoclinic space group). I hope this is clear ! HTH, Fred. On 24/03/13 11:20, Appu kumar wrote: I run the phenix.xtriage to evaluate the twining but it suggest no twining. When i reindex from C2221 to P21, the completeness of data reduced from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31 for 2.2 resolution. I do not understand why the completeness of data reduced so much on reindexing. please Can anyone explain this phenomenon. Thank you On 24 March 2013 13:30, Matthias Zebisch matthias.zebi...@bbz.uni-leipzig.de mailto:matthias.zebi...@bbz.uni-leipzig.de wrote: the p21 c2221 ambivalence can mean severe twinning (i had a similar case just now - try several crystals from the same condition) ! What do the twinning statistics suggest? cheers, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 emailmatth...@strubi.ox.ac.uk mailto:matth...@strubi.ox.ac.uk Websitehttp://www.strubi.ox.ac.uk - On 3/24/2013 7:46 AM, Appu kumar wrote: Thank you for the quick reply. After molecular replacement , i have done only few cycle of refinement in refmac. I have not done any solvent modification or NCS averaging. I have initially indexed the data in C2221 but Rfree was not decreasing so i reindexed the data in data in P121 space group keeping the Rfree flag of C2221. While analysing the symmetry mates , i found large space but no density. structure of Ligand binding domain is almost identical with 90% identity in sequence. I am stuck with this problem and don't know how to process further. Please give me your valuable suggestion. I will appreciate your effort. Thank you Appu On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu mailto:r...@brandeis.edu wrote: Dear Appu, I am not sure that I have a complete sense of the issue at hand since some of the information needed to think your issue through is missing in your email. For example, to what high resolution cut-off were the data measured? What resolution limits were used for the MR search? How do the unit cell dimensions and space group in the two cases compare? I am guessing the ligand binding domain in your protein has the identical sequence to that of the published ligand binding domain that you use as a template in your MR search. In any case, here are a couple of my thoughts: (1) It might be worth setting up different runs of MR with different numbers for expected copies (not just two copies but also one copy and three copies just in case you have one of the extreme cases of solvent content)? (2) If the MR solution is correct and there is physical room for a DNA binding domain in your lattice (check by displaying symmetry mates), perhaps the DNA binding domain is disordered. In that case (and if all attempts with current data fail), you may have to crystallize the protein in presence of DNA. Good luck! Raji
[ccp4bb] How to add base-pair restraint between DNA strands?
Hi guys, I need to manually build a model in Coot without a map. I want to link to 2 loops from 2 DNA strands together by base-pairings while keep the rest part as freezing as possible. The sequences of the loops are complimentary but the current orientations of the loops is not correct. Is there a way to add restraint between the loops to force them base-pairing while keep the rest part of the 2 DNA strands fixed? Another question is how to measure the angles between two DNA helices? I have seen a command to measure protein helices in PYMOL but did not find one for DNA helices. Thanks for your great help! Cheers, Frank
Re: [ccp4bb] molecular replacement problem.
Dear Appu, You want to be sure you have good reason to drop the space group from C222(1) to P2(1). There may be many reasons why your Rfree may not drop following refinement, especially if you only have one domain in your protein located and just in case there are more molecules to locate in the MR search. For C222(1) data, did Xtriage suggest any alternate space groups? Also, what program did you use for MR? If you used Phaser with your C222(1) data, did you ask to search for alternate space groups? Did you check for translational NCS? If you want me to take a look at your data, I'd be happy to look at your scaled data and MR results and try to help you out. If so, please email me off the bulletin board. Good luck! Raji On Sun, Mar 24, 2013 at 6:45 AM, vellieux frederic.velli...@ibs.fr wrote: Hello, Here we deal with symmetry and the unique part of reciprocal space (the reciprocal space asymmetric unit so to speak). C222(1) has eight asymmetric units (international tables, space group 20); P2(1) only has two. Assuming that Friedel's law does apply, then the minimum rotation range to collect a non-redundant data set (one observation per reflection) is 90 degrees, provided that the crystal is correctly and perfectly aligned. Normally with our current data collection methods where the crystal is randomly oriented, we would collect more than 90 degrees (180 degrees, or 360 degrees with the Pilatus detectors on an intense SR beamline where you cannot really check during data collection how well the crystal fares during exposure to the X-rays - shoot first, think later. The reciprocal space asymmetric unit in C222(1) is smaller. I assume that what you are doing is to take the reduced data set file (an MTZ file probably) and reduce the symmetry from C222(1) to P2(1). You will not cover the monoclinic reciprocal space asymmetric unit by doing so. The way to do it is to take the file from processing, before (crystallographic symmetry) merging of the equivalents, and perform the scaling and merging in the P2(1) space group. Or reprocess the data frames in P2(1) if you have lost the unmerged data file. Now of course this will still give you a poor completeness if you have used a strategy to optimize data collection in the orthorhombic space group (you won't have collected enough data then for good completeness in the monoclinic space group). I hope this is clear ! HTH, Fred. On 24/03/13 11:20, Appu kumar wrote: I run the phenix.xtriage to evaluate the twining but it suggest no twining. When i reindex from C2221 to P21, the completeness of data reduced from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31 for 2.2 resolution. I do not understand why the completeness of data reduced so much on reindexing. please Can anyone explain this phenomenon. Thank you On 24 March 2013 13:30, Matthias Zebisch matthias.zebi...@bbz.uni-leipzig.de wrote: the p21 c2221 ambivalence can mean severe twinning (i had a similar case just now - try several crystals from the same condition) ! What do the twinning statistics suggest? cheers, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 3/24/2013 7:46 AM, Appu kumar wrote: Thank you for the quick reply. After molecular replacement , i have done only few cycle of refinement in refmac. I have not done any solvent modification or NCS averaging. I have initially indexed the data in C2221 but Rfree was not decreasing so i reindexed the data in data in P121 space group keeping the Rfree flag of C2221. While analysing the symmetry mates , i found large space but no density. structure of Ligand binding domain is almost identical with 90% identity in sequence. I am stuck with this problem and don't know how to process further. Please give me your valuable suggestion. I will appreciate your effort. Thank you Appu On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote: Dear Appu, I am not sure that I have a complete sense of the issue at hand since some of the information needed to think your issue through is missing in your email. For example, to what high resolution cut-off were the data measured? What resolution limits were used for the MR search? How do the unit cell dimensions and space group in the two cases compare? I am guessing the ligand binding domain in your protein has the identical sequence to that of the published ligand binding domain that you use as a template in your MR search. In any case, here are a couple of my thoughts: (1) It might be worth setting up different runs of MR with different numbers for expected copies
Re: [ccp4bb] molecular replacement problem.
Sorry for the misconception. Yes i am expanding the space group from merged mtz file. Actually i have enough number of images collected. when i indexed, integrate, and scale the data in either C2221 or P 21, it fetches the overall 98% completeness. But when i am trying to reindex the data from C2221 to P21 keeping the Rfree flag of C2221, the completeness of data reduced drastically to 40%. This is what i am not getting. I am a beginner so i have to read a lot which i am doing also, but i had few practical confusion which i shared and off course i am getting good response. Thank you all for your kind response and educating me on the problem i faced. Thank you all for your valuable response. On 24 March 2013 15:15, vellieux frederic.velli...@ibs.fr wrote: Hello, Here we deal with symmetry and the unique part of reciprocal space (the reciprocal space asymmetric unit so to speak). C222(1) has eight asymmetric units (international tables, space group 20); P2(1) only has two. Assuming that Friedel's law does apply, then the minimum rotation range to collect a non-redundant data set (one observation per reflection) is 90 degrees, provided that the crystal is correctly and perfectly aligned. Normally with our current data collection methods where the crystal is randomly oriented, we would collect more than 90 degrees (180 degrees, or 360 degrees with the Pilatus detectors on an intense SR beamline where you cannot really check during data collection how well the crystal fares during exposure to the X-rays - shoot first, think later. The reciprocal space asymmetric unit in C222(1) is smaller. I assume that what you are doing is to take the reduced data set file (an MTZ file probably) and reduce the symmetry from C222(1) to P2(1). You will not cover the monoclinic reciprocal space asymmetric unit by doing so. The way to do it is to take the file from processing, before (crystallographic symmetry) merging of the equivalents, and perform the scaling and merging in the P2(1) space group. Or reprocess the data frames in P2(1) if you have lost the unmerged data file. Now of course this will still give you a poor completeness if you have used a strategy to optimize data collection in the orthorhombic space group (you won't have collected enough data then for good completeness in the monoclinic space group). I hope this is clear ! HTH, Fred. On 24/03/13 11:20, Appu kumar wrote: I run the phenix.xtriage to evaluate the twining but it suggest no twining. When i reindex from C2221 to P21, the completeness of data reduced from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31 for 2.2 resolution. I do not understand why the completeness of data reduced so much on reindexing. please Can anyone explain this phenomenon. Thank you On 24 March 2013 13:30, Matthias Zebisch matthias.zebi...@bbz.uni-leipzig.de wrote: the p21 c2221 ambivalence can mean severe twinning (i had a similar case just now - try several crystals from the same condition) ! What do the twinning statistics suggest? cheers, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 3/24/2013 7:46 AM, Appu kumar wrote: Thank you for the quick reply. After molecular replacement , i have done only few cycle of refinement in refmac. I have not done any solvent modification or NCS averaging. I have initially indexed the data in C2221 but Rfree was not decreasing so i reindexed the data in data in P121 space group keeping the Rfree flag of C2221. While analysing the symmetry mates , i found large space but no density. structure of Ligand binding domain is almost identical with 90% identity in sequence. I am stuck with this problem and don't know how to process further. Please give me your valuable suggestion. I will appreciate your effort. Thank you Appu On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote: Dear Appu, I am not sure that I have a complete sense of the issue at hand since some of the information needed to think your issue through is missing in your email. For example, to what high resolution cut-off were the data measured? What resolution limits were used for the MR search? How do the unit cell dimensions and space group in the two cases compare? I am guessing the ligand binding domain in your protein has the identical sequence to that of the published ligand binding domain that you use as a template in your MR search. In any case, here are a couple of my thoughts: (1) It might be worth setting up different runs of MR with different numbers for expected copies (not just two copies but also one copy and
Re: [ccp4bb] Isothermal titration calorimetry
Hi George, this is probably a very stupid suggestion and you likely have tried it, but I'll suggest the obvious nevertheless. What happens to your .nitc file when you rename it to .itc can you read it in Origin then ? Jürgen On Mar 24, 2013, at 6:39 AM, George Kontopidis wrote: Chris, indeed nanoITC instrument analysis software is very robust and user friendly (probable more friendly than microcal, GE). Although when you need to subtract Q (heat) values (from 2 or 3 blank experiments) from your experimental data you cannot. NanoITC software can subtract Q values only from 1 blank experiment. Also if you want to present your data in a form of heat/mol in Y (vertical) axes again you cannot. It presents data in Y axes only in form of heat/injection. If you have found a way to extract 2 or 3 blank experiments from experimental data or present data in form of heat/mol, please let me know it will be very useful. The main problem in the output files from nanoITC come with an extension .nitc, by default. Unfortunately Origin (that can do all the above) can read only, filenames with an extension .itc Cheers, George -Original Message- From: Colbert, Christopher [mailto:christopher.colb...@ndsu.edu] Sent: Saturday, March 23, 2013 5:56 PM To: George Kontopidis; CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Isothermal titration calorimetry George, would you please explain your comments? We've found the TA Instruments analysis software very robust and user friendly. We have the low volume nanoITC from TA instruments and get equivalent #'s in our comparison tests to the Microcal instrument. Cheers, Chris -- Christopher L. Colbert, Ph.D. Assistant Professor Department of Chemistry and Biochemistry North Dakota State University P.O. Box 6050 Dept. 2710 Fargo, ND 58108-6050 PH: (701) 231-7946 FAX: (701) 231-8324 On 3/23/13 8:47 AM, George Kontopidis gkontopi...@vet.uth.grmailto:gkontopi...@vet.uth.gr wrote: Keep in mind that output files from nanoITC, TA instrument cannot be red by Origin. At some point you will need to analyse your data further. George -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anastassis Perrakis Sent: Saturday, March 23, 2013 12:46 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Isothermal titration calorimetry It might be worth to consider the question more in detail. Do you want to study thermodynamics of the interaction, or a KD would do? If the former, you need ITC. If the latter, and you want to study things at the level of KD only, maybe investing on a plate reader, thermophoresis, or some biosensor technology (spr or interferometry based systems) should be considered. Then, what interactions will you study with the ITC? In general, I would agree that the lower sample volume is worth the nano options, but depending on the typical systems under study, sometimes the gain on sample quantity is not worth the money - while many times its worth it. John is if course right that for studying specific systems as the one he describes the 200 is great. A. Sent from my iPhone On 23 Mar 2013, at 11:00, John Fisher johncfishe...@gmail.commailto:johncfishe...@gmail.com wrote: I would recommend the Microcal ITC 200, hands down. Not only is it an amazing instrument with the optional automated sample loader (which is worth every penny), but we were able to do experiments (multiple) using FULL-LENGTH p53 binding to a weak cognate protein. I believe this was the first time ITC was ever used with full length p53, as it is so labile and just loves immediately to oligomerize. Sample sizes pay for the instrument. Best, John John Fisher, M.D./PhD St. Jude Children's Research Hospital Department of Oncology Department of Structural Biology W: 901-595-6193 C: 901-409-5699 On Mar 23, 2013, at 4:45 AM, Sameh Soror shamd...@googlemail.commailto:shamd...@googlemail.com wrote: Dear All, I am sorry for the off topic question. I am going to buy ITC to study protein-protein protein-ligand interactions I am comparing microcal, GE and nanoITC, TA instrument.. any suggestions, recommendations, good experiences or bad experiences. is there a better system. Thank in advance for the help. Regards Sameh -- Sameh Soror Postdoc. fellow .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] How to convert file format from CNS to CCP4
Ok. you filled my mailbox the second time today - please do stop sending junk to the list. -tommi On Mar 23, 2013, at 3:59 PM, Wei Feng wrote: Dear Steffi, Thank you very much for you patient reply! I have tried to use your script to convert the map format, but no ***omit_map.coeff can be found in all outputted file. After Heavy-atom search, Heavy-atom refinement/SAD phasing and SAD Phasing - Density Modification only two map outputted , one is fourier_map, other is density_modify.map.(see the attachments). And the cell parameter of the structure is 131.598 131.598 80.788 90.00 90.00 120.00 P622(177) Can you help me to check out why these maps can not be converted by sftools? Thank you for your time! Best! Wei At 2013-03-21 04:13:41,Stefanie Becker stefanie.bec...@uni-konstanz.de wrote: Hi Wei! i am very sorry for the belated answer. I checked my notebook and found I remembered it incorrectly. Indeed you can do the conversion with SFtools. Here is a small script that should do it (you need to change the space group and cell parameters of course ) hope that helps! steffi #!/bin/csh -f rm temp.mtz sftoolsend read composite_omit_map.coeff CNS 156.773 163.390 595.757 90 90 90 22 END write temp.mtz MTZ quit end cad hklin1 temp.mtz hklout omit_map_070419.mtzeof LABI FILE 1 ALL LABO FILE 1 E1=FWT E2=PHWT end eof Am Mittwoch, 20. März 2013 02:14 CET, ccp4...@hotmail.com schrieb: Dear steffi, Can you give me a link? I did not find it in google. Thank you! Wei 在 2013-03-19 20:55:42，Stefanie Becker stefanie.bec...@uni-konstanz.de 写道： Am Dienstag, 19. März 2013 04:37 CET, Wei Feng ccp4...@hotmail.com schrieb: Hi! don't know if you already got the answer by now, but there is a small program called map2mtz that should do it (i think you can just google it) good luck! steffi Dear all, I have used CNS to calculate the experimental phase of my structure. After Heavy-atom search, Heavy-atom refinement/SAD phasing and SAD Phasing - Density Modification - Selection of Map. Some files outputted: sad_phase2.hkl sad_phase2.sdb density_modify.hkl density_modify.map density_modify.mask ... I want to use these files to do the model building, but I do not know how to do it in CNS. So I want to convert these files to CCP4 format and do the model building by ARP/Warp, but I do not know which files should be converted and which software can be used to convert the file format from CNS to CCP4. Thank you for your time! Wei
