Re: [ccp4bb] P321 space group reindex problem
Hi, it is therefore likely that your spacegroup is really P321... hopefully, your data set is not twinned, did you check that ? You are left with 2 possible indexing schemes, as already mentionned. Chek scaling derivative / native scaling for each indexation of the derivative : the lowest Rfactor will likely indicate the right one. It appears that you end up with Rfactor of about 29 %, which suggest that your derivatives are not isomorphous to your native dataset. How do cell parameters compare for each data set ? Check also how the Rfactor varies with resolution, you might still have usefull phasing info at low resolution. hope this helps laurent Le 30/05/2012 07:50, Qixu Cai a écrit : At first, I processed the data at P3 space group. But after phenix.xtriage analysis, the Xtriage told me the space group must be P321, so I used P321 to process my data, and got an acceptable Rmerge. Qixu Cai 2012/5/29 Phil Evans p...@mrc-lmb.cam.ac.uk mailto:p...@mrc-lmb.cam.ac.uk How do you know the point group is 321? What does Pointless tell you if you put in the unmerged data? Despite some of the things said earlier (by me!), the possible indexing schemes in 321 are h,k,l and -h,-k,l If that doesn't work, it suggests that the point group is a lower symmetry eg P3 Phil On 29 May 2012, at 16:29, Qixu Cai wrote: Dear all, thank you for your help. I think I must describe my case in detail. I collected a native dataset and two heavy atom derivant datasets （in fact, i don not know whether these two kind of heavy atom have soked into the crystal, i just collect the data to check it）. i processed all of three datasets with automar, and merged them by CAD. I used scaleit to get the Rfactor between datasets and got a strange result. the R factor between derivant1 and native is 26% and the R factor between derivant2 and native is 59%! so I think it may be the problem of index （space group is P321）. so i exchange the h and k of derivant2 by the some awk script and merged to native data by CAD. After scaleit analysis, I got the R factor 29% between derivant2 and native. Here is my questions, 1, at my case, is that right to invert the hand? is that the special problem of the P3 or p321 space group? 2, I have carryed out some suggestion of yours, such as use pointless （use native data as reference for derivant2 reindex）, or reindex the derivant2 dataset by （k, h, -l）, and I always got the high R factor 59% between derivant2 and native. Any suggestion? thanks a lot! Qixu Cai 在 2012-5-29，下午10:36，Laurent Maveyraud laurent.maveyr...@ipbs.fr mailto:laurent.maveyr...@ipbs.fr 写道： Hi... and apologies ! I was a little quick in my answer... in P321, h k l and -h -k l are valid indexing schemes... It is in P3 that you can have h k l and k h -l as Ian and Phil agreed on the BB ! sorry, laurent Hi, you might have several possible spacegroups possible when processing your data (at the indexation step). These will be based on the metrics of your cell (vector length and angles). If you happen to have something like a = b, and alpha=beta90° and gamma=120°, then it is likely that your crystal is trigonal or hexagonal. You will have to wait until the scaling step (or pointless after integration) in order to decide which spacegroup is the right one, based on the symmetry operations in your dataset and on systematic absences. There you have to choose between P3, P31, P32, P312, P321 in trigonal. When comparing two datasets from trigonal crystals, even for identical crystals and hence identical spacegroups, you have different ways to index your dataset... In P321, one dataset might be indexed one way (eg. h k l), the other might be index the other way (k h -l). When you compared this two dataset, they will appear to be different, because both indexing schemes, although valid, are not equivalent. Take one reflection; e.g. 3 2 1 from your crystal A. The very same reflection will be indexed 2 3 -1 for your crystal B, and the one indexed 3 2 1 for crytal B will not be equivalent to the 3 2 1 reflection from crystal A. If you try to merge your two datasets, you will have huge Rmerge, because you are trying to average non equivalent reflections. You will have to ensure that the same indexing scheme is used for both datasets, eg reindex B using the reindex k h -l command in reindex, before being able to merge A and B. hope this helps... please feel free to as if I am not clear... best regards laurent Le 29/05/2012 16:03, Qixu Cai a écrit :
Re: [ccp4bb] MAD data process problem
Thank you very much for your reply. In my own understanding, We collect the peak dataset, because of the large F'', and we can get strong anomalous signal. We collect the edge dataset, because of the large F', and combined with the remote dataset, we can use the method just like SIR to get some information about the phase. so I think for peak dataset, anomalous processing is necessary, and for edge and remote dataset, anomalous processing is not necessary. Is my understanding correct? Thank you very much for your help. Best wish, Qixu Cai 2012/5/29 Tim Gruene t...@shelx.uni-ac.gwdg.de -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Qixu Cai, MAD phasing is based on the comparison of Bijvoet-pairs, i.e. I(hkl) with I(-h-k-l), both within one data set and between data sets. Therefore you might get better results if your integration program does not assume Friedel-pairs to have identical intensities, even though the difference is probably only marginal (integration programs do not merge data). So it is safest click on 'anomalous' for all data sets involved . Tim On 05/29/12 11:11, Qixu Cai wrote: Dear all, Sorry for the question from MAD beginner. When we process the MAD datasets, including the peak-data, edge-data and remote-data, which datasets need to be process with anomalous? I know peak-data obviously need data processing with anomalous, but what about edge-data and remote-data when we want to use MAD method? Thank you very much! Best wishes, Qixu Cai - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPxJfaUxlJ7aRr7hoRAm3EAKCKkyvT8z0wg6MFjflkHkiq8RR5GQCgyvF3 lOIOLypSzCcN3N6OR/3NcC8= =m6ax -END PGP SIGNATURE-
Re: [ccp4bb] MAD data process problem
Hi, you are right, the peak dataset corresponds to the highest f'' value. However, this does not mean that f'' is null for the other wavelengthes... you still have significant anomalous signal at the edge and for the high energy remote wavelength... this will help your phasing, so use it ! As Tim mentionned it : process all wavelength with anomalous switched on ! laurent Le 30/05/2012 07:59, Qixu Cai a écrit : Thank you very much for your reply. In my own understanding, We collect the peak dataset, because of the large F'', and we can get strong anomalous signal. We collect the edge dataset, because of the large F', and combined with the remote dataset, we can use the method just like SIR to get some information about the phase. so I think for peak dataset, anomalous processing is necessary, and for edge and remote dataset, anomalous processing is not necessary. Is my understanding correct? Thank you very much for your help. Best wish, Qixu Cai 2012/5/29 Tim Gruene t...@shelx.uni-ac.gwdg.de mailto:t...@shelx.uni-ac.gwdg.de -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Qixu Cai, MAD phasing is based on the comparison of Bijvoet-pairs, i.e. I(hkl) with I(-h-k-l), both within one data set and between data sets. Therefore you might get better results if your integration program does not assume Friedel-pairs to have identical intensities, even though the difference is probably only marginal (integration programs do not merge data). So it is safest click on 'anomalous' for all data sets involved . Tim On 05/29/12 11:11, Qixu Cai wrote: Dear all, Sorry for the question from MAD beginner. When we process the MAD datasets, including the peak-data, edge-data and remote-data, which datasets need to be process with anomalous? I know peak-data obviously need data processing with anomalous, but what about edge-data and remote-data when we want to use MAD method? Thank you very much! Best wishes, Qixu Cai - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPxJfaUxlJ7aRr7hoRAm3EAKCKkyvT8z0wg6MFjflkHkiq8RR5GQCgyvF3 lOIOLypSzCcN3N6OR/3NcC8= =m6ax -END PGP SIGNATURE- -- -- Laurent Maveyraud laurent.maveyraud AT ipbs DOT fr Université Paul Sabatier / CNRS / I.P.B.S. UMR 5089 PICT -- Plateforme Intégrée de Criblage de Toulouse Département BiologieStructurale et Biophysique BP 64182 - 205 rte de Narbonne - 31077 TOULOUSE FRANCE Tél: +33 (0)561 175 435 Fax : +33 (0)561 175 994 --
Re: [ccp4bb] P321 space group reindex problem
Why the 29% Rfactor indicate the derivatives are not isomorphous to native dataset? Native dataset cell constant: 181.39 181.39 110.217 90 90 120 derivative1 cell constant: 181.909 181.909 109.62 90 90 120Rfactor to native: 26% derivative2 cell constant: 181.527 181.527 109.32 90 90 120Rfactor to native: 29% The Rfactor at low resolution is larger than in high resolution. Could you please to help me figure out where the heavy atoms had been soaked into the crystal? Thank you very much. Best wishe, Qixu Cai 2012/5/30 Laurent Maveyraud laurent.maveyr...@ipbs.fr Hi, it is therefore likely that your spacegroup is really P321... hopefully, your data set is not twinned, did you check that ? You are left with 2 possible indexing schemes, as already mentionned. Chek scaling derivative / native scaling for each indexation of the derivative : the lowest Rfactor will likely indicate the right one. It appears that you end up with Rfactor of about 29 %, which suggest that your derivatives are not isomorphous to your native dataset. How do cell parameters compare for each data set ? Check also how the Rfactor varies with resolution, you might still have usefull phasing info at low resolution. hope this helps laurent Le 30/05/2012 07:50, Qixu Cai a écrit : At first, I processed the data at P3 space group. But after phenix.xtriage analysis, the Xtriage told me the space group must be P321, so I used P321 to process my data, and got an acceptable Rmerge. Qixu Cai 2012/5/29 Phil Evans p...@mrc-lmb.cam.ac.uk mailto:p...@mrc-lmb.cam.ac.uk ** How do you know the point group is 321? What does Pointless tell you if you put in the unmerged data? Despite some of the things said earlier (by me!), the possible indexing schemes in 321 are h,k,l and -h,-k,l If that doesn't work, it suggests that the point group is a lower symmetry eg P3 Phil On 29 May 2012, at 16:29, Qixu Cai wrote: Dear all, thank you for your help. I think I must describe my case in detail. I collected a native dataset and two heavy atom derivant datasets （in fact, i don not know whether these two kind of heavy atom have soked into the crystal, i just collect the data to check it）. i processed all of three datasets with automar, and merged them by CAD. I used scaleit to get the Rfactor between datasets and got a strange result. the R factor between derivant1 and native is 26% and the R factor between derivant2 and native is 59%! so I think it may be the problem of index （space group is P321）. so i exchange the h and k of derivant2 by the some awk script and merged to native data by CAD. After scaleit analysis, I got the R factor 29% between derivant2 and native. Here is my questions, 1, at my case, is that right to invert the hand? is that the special problem of the P3 or p321 space group? 2, I have carryed out some suggestion of yours, such as use pointless （use native data as reference for derivant2 reindex）, or reindex the derivant2 dataset by （k, h, -l）, and I always got the high R factor 59% between derivant2 and native. Any suggestion? thanks a lot! Qixu Cai 在 2012-5-29，下午10:36，Laurent Maveyraud laurent.maveyr...@ipbs.fr mailto:laurent.maveyraud@**ipbs.frlaurent.maveyr...@ipbs.fr 写道： Hi... and apologies ! I was a little quick in my answer... in P321, h k l and -h -k l are valid indexing schemes... It is in P3 that you can have h k l and k h -l as Ian and Phil agreed on the BB ! sorry, laurent Hi, you might have several possible spacegroups possible when processing your data (at the indexation step). These will be based on the metrics of your cell (vector length and angles). If you happen to have something like a = b, and alpha=beta90° and gamma=120°, then it is likely that your crystal is trigonal or hexagonal. You will have to wait until the scaling step (or pointless after integration) in order to decide which spacegroup is the right one, based on the symmetry operations in your dataset and on systematic absences. There you have to choose between P3, P31, P32, P312, P321 in trigonal. When comparing two datasets from trigonal crystals, even for identical crystals and hence identical spacegroups, you have different ways to index your dataset... In P321, one dataset might be indexed one way (eg. h k l), the other might be index the other way (k h -l). When you compared this two dataset, they will appear to be different, because both indexing schemes, although valid, are not equivalent. Take one reflection; e.g. 3 2 1 from your crystal A. The very same
Re: [ccp4bb] P321 space group reindex problem
Thank you for your remind of the twin problem. I checked all of the datasets by Xtriage, and found that the native is not twinned, but the derivant1 and derivant2 are both twinned. So is the Rfactor between derivants and native useful for the judgement of the success of the heavy atom soaking? Thanks. Best wishes, Qixu Cai 2012/5/30 Laurent Maveyraud laurent.maveyr...@ipbs.fr Hi, it is therefore likely that your spacegroup is really P321... hopefully, your data set is not twinned, did you check that ? You are left with 2 possible indexing schemes, as already mentionned. Chek scaling derivative / native scaling for each indexation of the derivative : the lowest Rfactor will likely indicate the right one. It appears that you end up with Rfactor of about 29 %, which suggest that your derivatives are not isomorphous to your native dataset. How do cell parameters compare for each data set ? Check also how the Rfactor varies with resolution, you might still have usefull phasing info at low resolution. hope this helps laurent Le 30/05/2012 07:50, Qixu Cai a écrit : At first, I processed the data at P3 space group. But after phenix.xtriage analysis, the Xtriage told me the space group must be P321, so I used P321 to process my data, and got an acceptable Rmerge. Qixu Cai 2012/5/29 Phil Evans p...@mrc-lmb.cam.ac.uk mailto:p...@mrc-lmb.cam.ac.uk ** How do you know the point group is 321? What does Pointless tell you if you put in the unmerged data? Despite some of the things said earlier (by me!), the possible indexing schemes in 321 are h,k,l and -h,-k,l If that doesn't work, it suggests that the point group is a lower symmetry eg P3 Phil On 29 May 2012, at 16:29, Qixu Cai wrote: Dear all, thank you for your help. I think I must describe my case in detail. I collected a native dataset and two heavy atom derivant datasets （in fact, i don not know whether these two kind of heavy atom have soked into the crystal, i just collect the data to check it）. i processed all of three datasets with automar, and merged them by CAD. I used scaleit to get the Rfactor between datasets and got a strange result. the R factor between derivant1 and native is 26% and the R factor between derivant2 and native is 59%! so I think it may be the problem of index （space group is P321）. so i exchange the h and k of derivant2 by the some awk script and merged to native data by CAD. After scaleit analysis, I got the R factor 29% between derivant2 and native. Here is my questions, 1, at my case, is that right to invert the hand? is that the special problem of the P3 or p321 space group? 2, I have carryed out some suggestion of yours, such as use pointless （use native data as reference for derivant2 reindex）, or reindex the derivant2 dataset by （k, h, -l）, and I always got the high R factor 59% between derivant2 and native. Any suggestion? thanks a lot! Qixu Cai 在 2012-5-29，下午10:36，Laurent Maveyraud laurent.maveyr...@ipbs.fr mailto:laurent.maveyraud@**ipbs.frlaurent.maveyr...@ipbs.fr 写道： Hi... and apologies ! I was a little quick in my answer... in P321, h k l and -h -k l are valid indexing schemes... It is in P3 that you can have h k l and k h -l as Ian and Phil agreed on the BB ! sorry, laurent Hi, you might have several possible spacegroups possible when processing your data (at the indexation step). These will be based on the metrics of your cell (vector length and angles). If you happen to have something like a = b, and alpha=beta90° and gamma=120°, then it is likely that your crystal is trigonal or hexagonal. You will have to wait until the scaling step (or pointless after integration) in order to decide which spacegroup is the right one, based on the symmetry operations in your dataset and on systematic absences. There you have to choose between P3, P31, P32, P312, P321 in trigonal. When comparing two datasets from trigonal crystals, even for identical crystals and hence identical spacegroups, you have different ways to index your dataset... In P321, one dataset might be indexed one way (eg. h k l), the other might be index the other way (k h -l). When you compared this two dataset, they will appear to be different, because both indexing schemes, although valid, are not equivalent. Take one reflection; e.g. 3 2 1 from your crystal A. The very same reflection will be indexed 2 3 -1 for your crystal B, and the one indexed 3 2 1 for crytal B will not be equivalent to the 3 2 1 reflection from crystal A. If you try to merge your two datasets, you will have huge
Re: [ccp4bb] P321 space group reindex problem
Hi, Le 30/05/2012 08:29, Qixu Cai a écrit : Thank you for your remind of the twin problem. It is always a pleasure to be helpful ;-) By the way, you stated the spacegoup is P321... did you check systematic absences ? could it be P3121 / P3221 ? I checked all of the datasets by Xtriage, and found that the native is not twinned, but the derivant1 and derivant2 are both twinned. So is the Rfactor between derivants and native useful for the judgement of the success of the heavy atom soaking? Well, the unhelpful answer will be : it depends what is your twin fraction ? how does the scaling derivative/native perfom (in details... not only global Rfactors) Did you try to calculate Patterson maps (isomorphous and anomalous) ? I would try to find a good MIR tutorial (CCP4 website might be a good place to look at, but have a look at phenix website...), and try to adapt it to your specific case... good luck ! laurent Thanks. Best wishes, Qixu Cai 2012/5/30 Laurent Maveyraud laurent.maveyr...@ipbs.fr mailto:laurent.maveyr...@ipbs.fr Hi, it is therefore likely that your spacegroup is really P321... hopefully, your data set is not twinned, did you check that ? You are left with 2 possible indexing schemes, as already mentionned. Chek scaling derivative / native scaling for each indexation of the derivative : the lowest Rfactor will likely indicate the right one. It appears that you end up with Rfactor of about 29 %, which suggest that your derivatives are not isomorphous to your native dataset. How do cell parameters compare for each data set ? Check also how the Rfactor varies with resolution, you might still have usefull phasing info at low resolution. hope this helps laurent Le 30/05/2012 07:50, Qixu Cai a écrit : At first, I processed the data at P3 space group. But after phenix.xtriage analysis, the Xtriage told me the space group must be P321, so I used P321 to process my data, and got an acceptable Rmerge. Qixu Cai 2012/5/29 Phil Evans p...@mrc-lmb.cam.ac.uk mailto:p...@mrc-lmb.cam.ac.uk mailto:p...@mrc-lmb.cam.ac.uk mailto:p...@mrc-lmb.cam.ac.uk__ How do you know the point group is 321? What does Pointless tell you if you put in the unmerged data? Despite some of the things said earlier (by me!), the possible indexing schemes in 321 are h,k,l and -h,-k,l If that doesn't work, it suggests that the point group is a lower symmetry eg P3 Phil On 29 May 2012, at 16:29, Qixu Cai wrote: Dear all, thank you for your help. I think I must describe my case in detail. I collected a native dataset and two heavy atom derivant datasets （in fact, i don not know whether these two kind of heavy atom have soked into the crystal, i just collect the data to check it）. i processed all of three datasets with automar, and merged them by CAD. I used scaleit to get the Rfactor between datasets and got a strange result. the R factor between derivant1 and native is 26% and the R factor between derivant2 and native is 59%! so I think it may be the problem of index （space group is P321）. so i exchange the h and k of derivant2 by the some awk script and merged to native data by CAD. After scaleit analysis, I got the R factor 29% between derivant2 and native. Here is my questions, 1, at my case, is that right to invert the hand? is that the special problem of the P3 or p321 space group? 2, I have carryed out some suggestion of yours, such as use pointless （use native data as reference for derivant2 reindex）, or reindex the derivant2 dataset by （k, h, -l）, and I always got the high R factor 59% between derivant2 and native. Any suggestion? thanks a lot! Qixu Cai 在 2012-5-29，下午10:36，Laurent Maveyraud laurent.maveyr...@ipbs.fr mailto:laurent.maveyr...@ipbs.fr mailto:laurent.maveyraud@__ipbs.fr mailto:laurent.maveyr...@ipbs.fr 写道： Hi... and apologies ! I was a little quick in my answer... in P321, h k l and -h -k l are valid indexing schemes... It is in P3 that you can have h k l and k h -l as Ian and Phil agreed on the BB ! sorry, laurent Hi, you might have several possible spacegroups possible when processing your data (at the indexation step).
Re: [ccp4bb] XDSAPP - A new GUI for data processing using XDS
Very neat. Is there some way to set parameters not listed under 'Settings'? Data from the Australian Synchrotron uses ROTATION_AXIS=-1.0 0.0 0.0 but I haven't figured out how to change this. Editing the generated XDS.INP between runs doesn't seem to do it. Cheers, /Daniel -- Daniel Ericsson Postdoctoral Research Fellow Kobe group School of Chemistry and Molecular Bioscience University of Queensland d.erics...@uq.edu.au +61 44 99 23 036 On Tue, May 29, 2012 at 10:39 PM, Müller, Uwe u...@helmholtz-berlin.dewrote: Dear CCP4BB users, we would hereby like to introduce XDSAPP, a GUI for the easy and convenient processing of diffraction data sets using XDS (see also Krug et al. (2012). J. Appl. Cryst. 45, 568-572). XDSAPP automates the data processing, generates plots of various data set statistics and produces intensity files suitable for further analysis using CCP4, SHELX and CNS. XDSAPP has the following features: - detector support for Rayonics MX-225, MARCCD-165 mm, PILATUS 6M and ADSC Quantum Q315r - Successfully tested on various Linux platforms - Full XDS-multiprocessor support - Space group determination using pointless - Twinning and pseudo-translation analysis using phenix.xtriage and sfcheck (CCP4 and PHENIX programs must be installed separately) - Life graphics during Integration - Graphical output of statistics of CORRECT and XDSSTAT (XDSSTAT must be installed separately) - All XDS output files (.pck, .cbf, .LP) available for viewing within the GUI - Automatic multi-crystal processing - Output files in mtz, hkl and cv format XDSAPP is freely available for academics. More information on XDSAPP including download instructions can be found at: http://www.helmholtz-berlin.de/bessy-mx Uwe Mueller, Manfred S. Weiss and Michael Krug Dr. Uwe Mueller Soft Matter and Functional Materials Macromolecular Crystallography (BESSY-MX) | Group leader Elektronenspeicherring BESSY II Albert-Einstein-Str. 15, D-12489 Berlin, Germany Fon: +49 30 8062 14974 Fax: +49 30 8062 14975 url: www.helmholtz-berlin.de/bessy-mx email:u...@helmholtz-berlin.de* * -- Helmholtz-Zentrum Berlin für Materialien und Energie GmbH Mitglied der Hermann von Helmholtz-Gemeinschaft Deutscher Forschungszentren e.V. Aufsichtsrat: Vorsitzender Prof. Dr. Dr. h.c. mult. Joachim Treusch, stv. Vorsitzende Dr. Beatrix Vierkorn-Rudolph Geschäftsführung: Prof. Dr. Anke Rita Kaysser-Pyzalla, Thomas Frederking Sitz Berlin, AG Charlottenburg, 89 HRB 5583 Postadresse: Hahn-Meitner-Platz 1 D-14109 Berlin http://www.helmholtz-berlin.de
Re: [ccp4bb] P321 space group reindex problem
Hi there, I am not certain that the thread is P321 space group reindex problem any more. But: trigonal (and hexagonal) space groups are (usually?) polar. The cell axis c can go up or can go down, and in order to get a consistent indexing you need to check both indexing systems when you scale additional data to your native (the indexing chosen by your first crystals defines the standard indexing - I must say that I haven't checked in the drawings of the international tables if having c going up or going down leads to a difference in that particular space group, P321, I'd need to draw both possibilities and check but I'm sorry I do not have the time right now - in fact it's too bad that the International Tables do not indicate Polar or Non-polar). For practical purposes, a derivative is considered non isomorphous when the differences in unit cell parameters exceed ca. 1% (this is because if you take 2 crystals from the same crystallisation drop and collect and process diffraction crystals from these 2 crystals, you will never get exactly the very same values for the unit cell parameters; non-isomorphism effects start at ca. 1% change and you'll never get 2 perfectly isomorphous crystals - even if you collect diffraction data twice from the same crystals you will not get perfect isomorphism). From the values mentioned, 1% of the cell parameters of the native for a and b is 1.81 Angstroem and for c 1.1 Angstroem (the angles do not matter for a trigonal space group). Had you obtained a value for a, b larger than ca. 183 Angstroem, or below ca. 109.2 Angstroem (only in the direction indicated by the changes mentioned in your mail - I ignored changes in the opposite direction) then you would have been able to say that the crystals were non-isomorphous to each other. For me they are isomorphous to each other and I ignore these small differences in unit cell parameters. The difference of 3% in Rfactor (I suppose this is |FP - FPH| / |FP|, no Sigma character on my keyboard to indicate the summations over h k l) is I think due to 2 different chemicals (heavy-atom compounds) in derivative 1 and derivative 2. Differences in R-factors at low resolutions are often associated with solvent effects, and I think you will have 2 different mother liquors and hence 2 different solvents in derivative 1 and in derivative 2. That is assuming that derivative 1 and derivative 2 were prepared using 2 different chemicals. And typically low-resolution data (below 15 Angstroem resolution or so) is kept out during phasing by the MIR method. To locate the heavy atom constellations in the 2 derivatives, you could compute and interpret difference Patterson maps - including automated interpretation, vector search and the likes -, you could use direct methods (the heavy atom constellation is similar to a small molecule because there are far fewer atoms there than in the full macromolecule, and direct methods work extremely well for small molecules - you would need to use the isomorphous differences in order to use direct methods; no mention is made of any anomalous signal so I do not know if you could this as well). HTH, Fred. Qixu Cai wrote: Why the 29% Rfactor indicate the derivatives are not isomorphous to native dataset? Native dataset cell constant: 181.39 181.39 110.217 90 90 120 derivative1 cell constant: 181.909 181.909 109.62 90 90 120Rfactor to native: 26% derivative2 cell constant: 181.527 181.527 109.32 90 90 120Rfactor to native: 29% The Rfactor at low resolution is larger than in high resolution. Could you please to help me figure out where the heavy atoms had been soaked into the crystal? Thank you very much. Best wishe, Qixu Cai 2012/5/30 Laurent Maveyraud laurent.maveyr...@ipbs.fr mailto:laurent.maveyr...@ipbs.fr Hi, it is therefore likely that your spacegroup is really P321... hopefully, your data set is not twinned, did you check that ? You are left with 2 possible indexing schemes, as already mentionned. Chek scaling derivative / native scaling for each indexation of the derivative : the lowest Rfactor will likely indicate the right one. It appears that you end up with Rfactor of about 29 %, which suggest that your derivatives are not isomorphous to your native dataset. How do cell parameters compare for each data set ? Check also how the Rfactor varies with resolution, you might still have usefull phasing info at low resolution. hope this helps laurent Le 30/05/2012 07:50, Qixu Cai a écrit : At first, I processed the data at P3 space group. But after phenix.xtriage analysis, the Xtriage told me the space group must be P321, so I used P321 to process my data, and got an acceptable Rmerge. Qixu Cai 2012/5/29 Phil Evans p...@mrc-lmb.cam.ac.uk mailto:p...@mrc-lmb.cam.ac.uk
Re: [ccp4bb] P321 space group reindex problem
If the data sets are twinned, large differences between derivatives are to be expected unless the twin fraction is very, very low (1-2%). Given the above, I think nothing can be said until the data are all detwinned - and of course the correct axial interchange done. Adrian On 30 May 2012, at 09:55, Vellieux Frederic wrote: Hi there, I am not certain that the thread is P321 space group reindex problem any more. But: trigonal (and hexagonal) space groups are (usually?) polar. The cell axis c can go up or can go down, and in order to get a consistent indexing you need to check both indexing systems when you scale additional data to your native (the indexing chosen by your first crystals defines the standard indexing - I must say that I haven't checked in the drawings of the international tables if having c going up or going down leads to a difference in that particular space group, P321, I'd need to draw both possibilities and check but I'm sorry I do not have the time right now - in fact it's too bad that the International Tables do not indicate Polar or Non-polar). For practical purposes, a derivative is considered non isomorphous when the differences in unit cell parameters exceed ca. 1% (this is because if you take 2 crystals from the same crystallisation drop and collect and process diffraction crystals from these 2 crystals, you will never get exactly the very same values for the unit cell parameters; non-isomorphism effects start at ca. 1% change and you'll never get 2 perfectly isomorphous crystals - even if you collect diffraction data twice from the same crystals you will not get perfect isomorphism). From the values mentioned, 1% of the cell parameters of the native for a and b is 1.81 Angstroem and for c 1.1 Angstroem (the angles do not matter for a trigonal space group). Had you obtained a value for a, b larger than ca. 183 Angstroem, or below ca. 109.2 Angstroem (only in the direction indicated by the changes mentioned in your mail - I ignored changes in the opposite direction) then you would have been able to say that the crystals were non-isomorphous to each other. For me they are isomorphous to each other and I ignore these small differences in unit cell parameters. The difference of 3% in Rfactor (I suppose this is |FP - FPH| / |FP|, no Sigma character on my keyboard to indicate the summations over h k l) is I think due to 2 different chemicals (heavy-atom compounds) in derivative 1 and derivative 2. Differences in R-factors at low resolutions are often associated with solvent effects, and I think you will have 2 different mother liquors and hence 2 different solvents in derivative 1 and in derivative 2. That is assuming that derivative 1 and derivative 2 were prepared using 2 different chemicals. And typically low-resolution data (below 15 Angstroem resolution or so) is kept out during phasing by the MIR method. To locate the heavy atom constellations in the 2 derivatives, you could compute and interpret difference Patterson maps - including automated interpretation, vector search and the likes -, you could use direct methods (the heavy atom constellation is similar to a small molecule because there are far fewer atoms there than in the full macromolecule, and direct methods work extremely well for small molecules - you would need to use the isomorphous differences in order to use direct methods; no mention is made of any anomalous signal so I do not know if you could this as well). HTH, Fred. Qixu Cai wrote: Why the 29% Rfactor indicate the derivatives are not isomorphous to native dataset? Native dataset cell constant: 181.39 181.39 110.217 90 90 120 derivative1 cell constant: 181.909 181.909 109.62 90 90 120 Rfactor to native: 26% derivative2 cell constant: 181.527 181.527 109.32 90 90 120 Rfactor to native: 29% The Rfactor at low resolution is larger than in high resolution. Could you please to help me figure out where the heavy atoms had been soaked into the crystal? Thank you very much. Best wishe, Qixu Cai 2012/5/30 Laurent Maveyraud laurent.maveyr...@ipbs.fr mailto:laurent.maveyr...@ipbs.fr Hi, it is therefore likely that your spacegroup is really P321... hopefully, your data set is not twinned, did you check that ? You are left with 2 possible indexing schemes, as already mentionned. Chek scaling derivative / native scaling for each indexation of the derivative : the lowest Rfactor will likely indicate the right one. It appears that you end up with Rfactor of about 29 %, which suggest that your derivatives are not isomorphous to your native dataset. How do cell parameters compare for each data set ? Check also how the Rfactor varies with resolution, you might still have usefull phasing info at low resolution. hope this helps
Re: [ccp4bb] Akta vs HPLC
if you have peek surface or titanium parts (if i recall right) there are no problems with salt solutions. tommi On May 30, 2012, at 3:39 AM, aaleshin wrote: Back in Iowa State University we used Waters HPLC for protein purification during many years without noticeable damage to the stainless steel tubings. But Dan was right about the pumps, someone in the lab forgot to flush the high salt pump with water after its use and damaged the pump... Alex On May 29, 2012, at 5:14 PM, Daniel Anderson wrote: Hi, Ho, Your question has a lot of variables. HPLC columns should not be used on the Akta within my field of view because the Akta within my field of view does not have gradual pump acceleration and deceleration. HPLC columns can be damaged by sudden changes in pressure or composition. The HPLC within my field of view has wetted stainless steel surfaces and the mobile phase should not contain chloride ion or reductant. Chloride ion would accelerate corrosion of the stainless steel (and result in metal ions in the protein). Reductant would strip off the passivation (during maintenance I soak the stainless parts in nitric acid to keep them stainless) later resulting in corrosion. The Waters sales representative once told me that the pumps have to be salt-free and methanol-flushed at the end of every working day. Good luck implementing that policy. -Dan Ho Leung Ng wrote: Hello, My Akta Purifier is being repaired, and I'm thinking about borrowing a colleague's HPLC in the interim. What makes the Aktas different from HPLCs? I've used HPLCs for purifying small molecules and peptides but not proteins. Anything I should be careful about regarding keeping the machines, columns, and proteins happy? Thank you, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu mailto:h...@hawaii.edu
Re: [ccp4bb] Moleculardimensions kits
hi i dont know about the midas but proplex is good..but if u really wanna go for some molecular dimension screen then opt for morpheus..it is damn good..if any protein is ever going to crystallize, it will also give crystal in this screen too.. u could find a hit in this screen also.. best of luck On Tue, May 29, 2012 at 9:55 PM, Barbara Medagli barbara.meda...@elettra.trieste.it wrote: Dear all, This is Barbara Medagli, working in the structural biology lab in Italy Surfing in the molecular dimensions web site I found this two screens: MIDAS and ProPlex. I was thinking in buy them as I have to order other items to this company Some of you already used those kits? Any experience with them? Thanks for the help Barbara Medagli, PhD Structural Biology Lab Sincrotrone Trieste SCpA S.S. 14, km 163.5, Loc. Basovizza 34012 Trieste/Italy phone +39040 3758807/8537 fax +39040 3758029 -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] P321 space group reindex problem
Hi Fred, On Wed, May 30, 2012 at 08:55:35AM +0200, Vellieux Frederic wrote: For practical purposes, a derivative is considered non isomorphous when the differences in unit cell parameters exceed ca. 1% (this is because if you take 2 crystals from the same crystallisation drop and collect and process diffraction crystals from these 2 crystals, you will never get exactly the very same values for the unit cell parameters; non-isomorphism effects start at ca. 1% change and you'll never get 2 perfectly isomorphous crystals - even if you collect diffraction data twice from the same crystals you will not get perfect isomorphism). From the values mentioned, 1% of the cell parameters of the native for a and b is 1.81 Angstroem and for c 1.1 Angstroem (the angles do not matter for a trigonal space group). Had you obtained a value for a, b larger than ca. 183 Angstroem, or below ca. 109.2 Angstroem (only in the direction indicated by the changes mentioned in your mail - I ignored changes in the opposite direction) then you would have been able to say that the crystals were non-isomorphous to each other. For me they are isomorphous to each other and I ignore these small differences in unit cell parameters. I would be careful with the (popular) percentage-rule here: the absolute value of cell differences is much more important. At least if we assume that the change in cell parameters roughly corresponds with a shift in actual atoms. If you have a 1000A cell then a 1% difference could mean a shift of 10A ... clearly, a helix moved 10A away results in something completely different. But with a cell of 20A you could have a 0.2A shift, which you might hardly notice. See eg. 5.2 in Garman Murray (2003): http://journals.iucr.org/d/issues/2003/11/00/ba5042/index.html which shows 5.2. Non-isomorphism One of the biggest problems of heavy-atom derivatization is that incorporation of a heavy atom into the lattice often induces a change in the unit cell away from the native crystal values, i.e. the derivatized crystal is non-isomorphous to the native crystals. The heavy atom may perturb the arrangement of protein molecules in the crystal or distort the protein molecule, causing a change in unit-cell lengths. Note, however, that it is also possible for the protein to move within the original unit cell (resulting in a different sampling of the molecular transform). The same unit cell is thus a necessary but not sufficient condition for isomorphism. Crick Magdoff (1956[Crick, F. H. C. Magdoff, B. S. (1956). Acta Cryst. 9, 901-908.]) calculated that a 0.5 Å change in all three unit-cell edges of a 100 Å cubed unit cell would change the diffraction intensities by an average of 15% in a 3 Å resolution sphere. The predicted intensity changes induced by non-isomorphism increase at higher resolution. When faced with a non-isomorphous derivative, it is the absolute change in the cell which should be considered compared with the working resolution, rather than the relative change, i.e. a change of 1.0% in a 100 Å unit cell edge has a similar effect to that of a 0.5% change in a 200 Å unit cell edge, if compared at similar resolutions. As a general rule of thumb, a change in cell dimensions of dmin/4 is tolerable, where dmin is the resolution limit (Drenth, 1999[Drenth, J. (1999). Principles of Protein X-ray Crystallography, 2nd ed. Berlin: Springer-Verlag.]). For instance, for 2.5 Å data, a 0.6 Å change in the unit cell might be acceptable, whereas at 3.5 Å this could rise to 0.8 Å. Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
[ccp4bb] TCEP resistant disulfide
Has anyone worked with, or seen an example of, a solvent accessible disulfide that cannot be reduced with TCEP? Alternatively solvent accessible disulfides that resist reduction with DTT but can be reduced with TCEP? Thanks in advance, Jose. Jose Antonio Cuesta-Seijo, PhD Carlsberg Laboratory Gamle Carlsberg Vej 10 DK-1799 Copenhagen V Denmark Tlf +45 3327 5332 Email josea.cuesta.se...@carlsberglab.dk
Re: [ccp4bb] P321 space group reindex problem
Hello Fred On 30 May 2012 07:55, Vellieux Frederic frederic.velli...@ibs.fr wrote: Hi there, But: trigonal (and hexagonal) space groups are (usually?) polar. The cell axis c can go up or can go down, and in order to get a consistent indexing you need to check both indexing systems when you scale additional data to your native (the indexing chosen by your first crystals defines the standard indexing - I must say that I haven't checked in the drawings of the international tables if having c going up or going down leads to a difference in that particular space group, P321, I'd need to draw both possibilities and check but I'm sorry I do not have the time right now - in fact it's too bad that the International Tables do not indicate Polar or Non-polar). It does, at least my edition (Vol. A: 5th ed., 2002, Table 10.2.1.2, p.806) does - ITC has everything you need to know about space groups (and a lot more besides)! See also this table that I made where all polar non-polar SGs are listed individually: http://www.ccp4.ac.uk/dist/html/alternate_origins.html Counting only the non-enantiomorphic ones, PG3 (4 SGs) and PG6 (6 SGs) are polar, whereas PG321 (3 SGs), PG312 (3 SGs), PG32 (1 SG) and PG622 (6 SGs) are non-polar. So in all 10 are polar and 13 are non-polar. A 2-fold axis perp to another axis always implies that there's no preferred direction along the other axis, so it's non-polar. Cheers -- Ian
Re: [ccp4bb] P321 space group reindex problem
Hi Ian, You're right the information is there... but not where I was expecting it (on the page corresponding to an individual space group). It had never occurred to me that it could be somewhere else. So thanks, and regards to Jasmine. Fred. Ian Tickle wrote: Hello Fred On 30 May 2012 07:55, Vellieux Frederic frederic.velli...@ibs.fr wrote: Hi there, But: trigonal (and hexagonal) space groups are (usually?) polar. The cell axis c can go up or can go down, and in order to get a consistent indexing you need to check both indexing systems when you scale additional data to your native (the indexing chosen by your first crystals defines the standard indexing - I must say that I haven't checked in the drawings of the international tables if having c going up or going down leads to a difference in that particular space group, P321, I'd need to draw both possibilities and check but I'm sorry I do not have the time right now - in fact it's too bad that the International Tables do not indicate Polar or Non-polar). It does, at least my edition (Vol. A: 5th ed., 2002, Table 10.2.1.2, p.806) does - ITC has everything you need to know about space groups (and a lot more besides)! See also this table that I made where all polar non-polar SGs are listed individually: http://www.ccp4.ac.uk/dist/html/alternate_origins.html Counting only the non-enantiomorphic ones, PG3 (4 SGs) and PG6 (6 SGs) are polar, whereas PG321 (3 SGs), PG312 (3 SGs), PG32 (1 SG) and PG622 (6 SGs) are non-polar. So in all 10 are polar and 13 are non-polar. A 2-fold axis perp to another axis always implies that there's no preferred direction along the other axis, so it's non-polar. Cheers -- Ian
[ccp4bb] to determine missing atoms and residues in a PDB file
Dear All, I have a PDB file which does not have the REMARKS cards 465 (for missing residues) and 470 (for missing atoms). This is not a deposited PDB file. Is there any program to figure out the missing residues and atoms (some programs complain about missing atoms) ? Or do I have to check in any particular file generated during the processing of the diffraction data? The S2C program from Prof. Dunbrack's Lab does not show any option of uploading pdb files (this solution was mentioned to a previous query on the BB). Thanks in advance, sreetama
Re: [ccp4bb] P321 space group reindex problem
It does, at least my edition (Vol. A: 5th ed., 2002, Table 10.2.1.2, p.806) does - ITC has everything you need to know about space groups (and a lot more besides)! Actually, as the aforementioned table indicates, it's not correct to talk about polar and non-polar space groups, but only about polar and non-polar directions in space groups. Many space-groups have both polar and non-polar directions, which would seem to imply that these space groups are both polar and non-polar at the same time!!! For example, P3 has a polar direction parallel to the 3-fold and no non-polar directions, whereas P321, even though it's classed as a non-polar space group, nevertheless has 3 polar directions [100], [010], [-1-10] parallel to the 3 2-folds in the xy plane, plus 4 non-polar directions (including the 3-fold). Note that any direction perpendicular to an even-fold rotation axis is always non-polar: these 'trivial' cases are not shown explicitly in the above table. From the point of view of deciding which are the alternate settings I don't think it's helpful to consider polar directions anyway. What matters is which symmetry axes of the lattice are not present in the point group. So in the case of P321 the hexagonal lattice has a 6-fold parallel to c which can be thought of as the product of a 3- and a 2-fold both || c, and also 2-folds perp to the 6-fold. The 3-fold and the perp 2-folds are all present in P321 but the 2-fold || c is not, so that is what gives rise to the 2 alternate settings (h,k,l) and (-h,-k,l). In P3 all 2-folds are missing so you get 4 alternate settings. The same missing symmetry can also give rise to merohedral twinning, so it's nice to kill 2 birds with 1 stone! Cheers -- Ian
Re: [ccp4bb] P321 space group reindex problem
On Wed, 30 May 2012 13:16:12 +0100 Ian Tickle ianj...@gmail.com wrote: From the point of view of deciding which are the alternate settings I don't think it's helpful to consider polar directions anyway. What matters is which symmetry axes of the lattice are not present in the point group. Possibly relevant here is a set of tables I put together for our free-electron laser experiments, where tens or even hundreds of thousands of patterns have to be indexed independently: https://www.desy.de/~twhite/crystfel/twin-calculator.pdf Underlying these tables is the same underlying information as everything else mentioned on this thread, but these ones tell you what the apparent symmetry would be if the intensities were mixed up according to all the available indexing ambiguities. So far, no-one has been able to reliably resolve these ambiguities in our case: the intensities are just obscured by too much noise, partiality, a spiky X-ray spectrum and so on. That's why we have to have so many patterns. For the time being, we just merge according to the higher symmetry, accept that the data may be (perfectly) twinned, and handle it at the later stages. Later on when we've hopefully solved this problem, these tables will serve as a menu of options for doing the whole thing backwards. I agree that polarity isn't the right criterion. Point group 2 is polar but does not exhibit any indexing ambiguity. Point group 4/m, which is most definitely not polar, does. This paper, Le Page et al., has some similar tables but lists the actual ambiguity operators: http://scripts.iucr.org/cgi-bin/paper?S0108767384001392 Of course, all of this only covers merohedral ambiguities, not pseudo-merohedral ones which might arise by accident in special cases. Comments on and corrections to the tables are welcome! Tom
Re: [ccp4bb] TCEP resistant disulfide
Hi Jose, Has anyone worked with, or seen an example of, a solvent accessible disulfide that cannot be reduced with TCEP? Something to keep in mind is that TCEP is not very stable with phosphate buffers near neutral pH. Alternatively solvent accessible disulfides that resist reduction with DTT but can be reduced with TCEP? This could be due to the effective pH of TCEP HCl (~1.5-8.5) being wider than DTT ~7. Take Care, Sean Seaver P212121 http://store.p212121.com/
[ccp4bb] Three Senior Faculty Positions in X-ray Crystallography, Cryo-Electron Microscopy, and NMR
University of Texas Medical Branch Senior Faculty in X-ray Crystallography Sealy Center for Structural Biology Molecular Biophysics UTMB Health seeks senior faculty applicants in structural biology in the Sealy Center for Structural Biology and Molecular Biophysics (SCSBMB). The Center supports a graduate program in molecular biophysics and five well-run core laboratories in X-ray, Cryo-EM, NMR, Computation and Solution Biophysics, each with an excellent PhD-level manager. For details see: http://www.scsb.utmb.edu/ Core facility instrumentation includes: newly purchased Rigaku Ultimate Homelab Plus, comprising the FR-E+DW SuperBright x-ray source with the R-AXIS IV and the BioSAXS-1000 Kratky camera; plus a Bruker M06HF high-brilliance source with the Bruker platform-CCD; new 2200FS, 2100, 1400 JEOL Cryo-EM instruments (the 2200FS is in BSL/3 containment); newly upgraded 800-, and 600- MHz NMR spectrometers with Bruker Avance III consoles and solution biophysics instrumentation including a Biacore T-100, a Thermofluor high-throughput-screening instrument, plus spectroscopic, kinetic, calorimetric, analytical UC, and mass spectrometry instrumentation. The successful candidate must be a highly motivated individual with a PhD or MD degree, a strong publication record, and a record of independent well-funded grant support. The ideal candidate will have extensive experience in crystallography applied to the study of the structure, dynamics, novel mechanisms, and functions of bio-macromolecules, viruses, assemblies etc. Candidates for positions in either the department of Biochemistry and Molecular Biology or Pharmacology and Toxicology should also have or seek overlap with the highly collaborative established biomedical research community in UTMB’s basic science departments, and centers and programs of excellence such as the Institute for Human Infection and Immunity, the Galveston National Laboratory, the Center for Tropical Diseases, the Institute for Translational Sciences, the Sealy Center for Cancer Cell Biology, the Sealy Center for Environmental Health and Medicine, the Sealy Center on Aging, the George P. and Cynthia Woods Mitchell Center for Neurodegenerative Diseases, the Moody center for Brain and Spinal Cord Injury Research, the Sealy Center for Molecular Medicine and the Chemical Biology Program. These and other entities provide a wide variety of core services, in recombinant DNA, genomics, proteomics, high-throughput drug screening, mass spectrometry, membrane protein crystallization, and protein expression and purification. Excellent collaborative opportunities also exist through UTMB’s participation in the Gulf Coast Consortia and the Keck Center for Interdisciplinary Bioscience http://www.gulfcoastconsortia.org/home.aspx Applicants are requested to submit electronically: a cover letter expressing interest in being considered, a curriculum vitae, current funding, a summary of research accomplishments, and future goals to mailto: scsbmb.recruit...@utmb.edu Direct inquiries to Dr. B. Montgomery Pettitt, mpett...@utmb.edu, 409-772-0723. UTMB has been aggressively recruiting in a large number of research areas and is an equal opportunity, affirmative action institution that proudly values diversity. Candidates of all backgrounds are encouraged to apply. Faculty Positions * Cryo-Electron Microscopy: Announcement * NMR: Announcement * X-ray Crystallography: Announcement -- Yours sincerely, Mark A. White, Ph.D. Associate Professor of Biochemistry and Molecular Biology, Manager, Sealy Center for Structural Biology and Molecular Biophysics X-ray Crystallography Laboratory, Basic Science Building, Room 6.660 C University of Texas Medical Branch Galveston, TX 77555-0647 Tel. (409) 747-4747 Cell. (281) 734-3614 Fax. (409) 747-1404 mailto://mawh...@utmb.edu http://xray.utmb.edu http://xray.utmb.edu/~white
Re: [ccp4bb] MAD data process problem
Dear Qixu Cai, The following paper should be informative for your query:- http://dx.doi.org/10.1107/S0909049595013288 Best wishes, John Prof John R Helliwell DSc On 29 May 2012, at 10:11, Qixu Cai caiq...@gmail.com wrote: Dear all, Sorry for the question from MAD beginner. When we process the MAD datasets, including the peak-data, edge-data and remote-data, which datasets need to be process with anomalous? I know peak-data obviously need data processing with anomalous, but what about edge-data and remote-data when we want to use MAD method? Thank you very much! Best wishes, Qixu Cai
[ccp4bb] Einstein would be proud
Hi Folks, Sorry for a non-CCP4 post but I simply couldn't resist. Here's a video that's simply out of this world! This video was made by an undergraduate student at Northeastern and it just won him a trip to outer space. Yes, outer space! Not only did the student write the script and make the video but he also did all the chalk art and composed and played the music in the video. The narration is slow but totally funky! Well worth the fifteen minutes, in my opinion... http://www.northeastern.edu/insolution/other/2012/04/rocket-man/ Please DO NOT miss it if you like science or art or music or imagination or philosophy or some vague combination of the five... Enjoy! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] to determine missing atoms and residues in a PDB file
sreetama das wrote: Dear All, I have a PDB file which does not have the REMARKS cards 465 (for missing residues) and 470 (for missing atoms). This is not a deposited PDB file. Is there any program to figure out the missing residues and atoms (some programs complain about missing atoms) ? Or do I have to check in any particular file generated during the processing of the diffraction data? The S2C program from Prof. Dunbrack's Lab does not show any option of uploading pdb files (this solution was mentioned to a previous query on the BB). Thanks in advance, sreetama I think if you generate SEQRES records for the sequence of your protein (or grab them from another pdb if another structure has been deposited), add them to your raw pdb, and submit to the validation server at the PDB, the validation report will include detailed listing of missing residues and missing atom in partial residues.
Re: [ccp4bb] to determine missing atoms and residues in a PDB file
On 30/05/12 12:50, sreetama das wrote: Dear All, I have a PDB file which does not have the REMARKS cards 465 (for missing residues) and 470 (for missing atoms). This is not a deposited PDB file. Is there any program to figure out the missing residues and atoms (some programs complain about missing atoms) ? Or do I have to check in any particular file generated during the processing of the diffraction data? The S2C program from Prof. Dunbrack's Lab does not show any option of uploading pdb files (this solution was mentioned to a previous query on the BB). The fill-partial-residues function or the Rotamer probability validation tool in Coot will find standard residues with missing atoms.
Re: [ccp4bb] Einstein would be proud
Thanks for sharing Raji, this is indeed a great visualization. Very talented student, I'm sure we will read more about his research in the future. Jürgen On May 30, 2012, at 12:19 PM, Raji Edayathumangalam wrote: Hi Folks, Sorry for a non-CCP4 post but I simply couldn't resist. Here's a video that's simply out of this world! This video was made by an undergraduate student at Northeastern and it just won him a trip to outer space. Yes, outer space! Not only did the student write the script and make the video but he also did all the chalk art and composed and played the music in the video. The narration is slow but totally funky! Well worth the fifteen minutes, in my opinion... http://www.northeastern.edu/insolution/other/2012/04/rocket-man/ Please DO NOT miss it if you like science or art or music or imagination or philosophy or some vague combination of the five... Enjoy! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University .. 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://web.mac.com/bosch_lab/
[ccp4bb] GFP membrane protein expression
Dear all Is there any method to check membrane protein overexpression using GFP when the C terminus is in periplasm? My reading so far all mention that for C terminus fusion to work, it has to be cytoplasm. Thank you.
Re: [ccp4bb] GFP membrane protein expression
Dear all Is there any method to check membrane protein overexpression using GFP when the C terminus is in periplasm? My reading so far all mention that for C terminus fusion to work, it has to be cytoplasm. Thank you. Dear Theresa, Superfolder GFP (sGFP) is reported to translocate and fold into the periplasm of E. coli using a secretary signal peptide (Aronson DE et al.Traffic. 2011), suggesting it would likewise be active when fused to the C-terminus of a membrane protein. Because sGFP is no longer sensitive to upstream membrane protein integration the formation of any aggregates (inclusion bodies) are likely to be fluorescent. Nonetheless, with FSEC you should be able to quickly estimate the amount of folded material. Alternatively if your membrane protein has an Nin topology you may consider the addition of an N-terminal GFP fusion. Again, you have the drawback that you can get uncoupled translation of the N-terminal fusion resulting in free GFP. However, as long as you know this then one can simply isolate membranes first before estimating overexpression levels. If you have an Nout topology the N-terminal GFP may affect folding/targeting and I would probably avoid this route. One last option it to consider the addition of an extra TM segment. Jeff Abramson's group has these vectors (Hseih JM et al. Prot. Sci. 2010). Best of luck! Cheers David. Dr. David Drew Royal Society University Research Fellow Division of Molecular Biosciences Imperial College London South Kensington Campus London SW7 2AZ d.d...@imperial.ac.uk Tel: 020-7594-9624 (ext. 49624)
Re: [ccp4bb] to determine missing atoms and residues in a PDB file
I do not seem to understand the meaning of fixing. Fixing something can mean a) repairing it, implying that something was broken or amiss. Lack of experimental information expressed as omission of atoms is not something that needs fixing. b) keeping it constant. Like in having one single energy minimized conformation and accepting it once fixed as in (a). c) injection of conscience-expanding drugs, often also hallucinogenic, as in fixing in (b). d) removal of reproductive organs, as in possible punishment for mutilating experimental structure models by fixing as in (a,b,c) A discrete ensemble of probable rotamers following a distribution with constrained occupancy probabilities according to their empirically observed frequency (optimally context sensitive) might be an approximate solution to a to this date quite contentious issue. I am sure Paul will make it happen ;-) Anyhow - beware of fixers. BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of debayan dey Sent: Wednesday, May 30, 2012 10:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] to determine missing atoms and residues in a PDB file The missing residues/atoms in the PDB file can be found out and fixed in Schrödinger program's Prime module refinement.It will automatically detect the missing atoms in a residue and will fix it. After fixing it can energy minimize it.For missing portions of residue it can build the missing portions using homology model.The other option for fixing missing atoms is to use the following server: http://lorentz.immstr.pasteur.fr/pdb/frozen_submission.php -Debayan Dey On Wed, May 30, 2012 at 5:20 PM, sreetama das somon_...@yahoo.co.in wrote: Dear All, I have a PDB file which does not have the REMARKS cards 465 (for missing residues) and 470 (for missing atoms). This is not a deposited PDB file. Is there any program to figure out the missing residues and atoms (some programs complain about missing atoms) ? Or do I have to check in any particular file generated during the processing of the diffraction data? The S2C program from Prof. Dunbrack's Lab does not show any option of uploading pdb files (this solution was mentioned to a previous query on the BB). Thanks in advance, sreetama -- Debayan Dey Research Fellow Dept. of Physics, Biocrystallography and Computational Biology Laboratory Indian Institute of Science India
