[ccp4bb] Question: Linux distributions people use
Dear BB, I hesitate to ask this, but here goes. If you are a Linux admin user (i.e. the person who installs CCP4) please could you drop me a line (personally, rather than the BB) about which version of Linux you are using. This will allow me to figure out the most popular systems people have, which I will collate and send back to the list, anonymized of course. The reason for this is to allow me to set up test (virtual) machines with the most popular distributions to test the forthcoming 6.1 release. Including the version number would be a real help, i.e. SuSE 10.1, Ubuntu 8.04. If you have a number of systems, a rough idea of how many would be also helpful. Many thanks, Graeme
Re: [ccp4bb] Troubleshooting protein purification cation IEX
Hi, Many things can lead to your observation. Please outline all steps of your purification procedure as it is not clear what is done before and after the Ion Exchange steps. I am not sure if IEF in your emails refers to Isoelectric focusing, as the acronym is usually used?? Couple of suggestions: 1) Instead of contamination, you might just be seeing multiple bands due to 'aggregation' of your protein! Make sure you boil the sample prior to loading on gel and also that your loading dye contains SDS, bME/DTT. 2) We used to do entire purifications with inclusion body preps under denaturing conditions to prevent unwanted aggregation of partially folded or misfolded species. Not sure if denaturant is present all along. 3) If the problem is of contamination, try making the gradient shallow for the 35 –80% gradient step in Ion Exchange (increase to say, 20 cv or more). 4) If the problem is of contamination, try to add more steps to purification -- e.g., affinity step (if possible), anion exchange as well etc. 5) If IEF (in the sense I mean it) is what you did and it shows only 1-2 bands, the problem is likely (#1) outlined above. 6) If all else fails, cut out one or two of the bands from your gel and run a mass spec. An expensive way to find out that it is aggregation. Nevertheless Hope that helps. Raji On Sep 23, 2008, at 3:51 AM, Meg wrote: Dear All, This is with reference to the purification of our recombinant protein sample expressed in E.coli as inclusion bodies. After Solubilization refolding we perform the cation exchange chromatography of our protein sample using SP sepharose fast flow resin. Attached herewith find the SDS PAGE and IEF results of the collected fractions. In addition to our protein of interest we are also getting high molecular weigh contaminants, which we cannot get rid of in IEX. Can anyone please guide me on a technique to get rid of these bands as even after gel filtration of samples few high mol wt contaminant bands are not separated from main proteins and sample gets diluted too. In cation IEX procedure is Column Sp Sepharose Fast flow packed in fineline 35 column packed bed volume 100 ml System AKTA FPLC Equilibration 10 C.V. protein loading 3-4 C.V. [approx 1mg/ml protein conc], washing to remove unbound materials 2 C.V. step elution 0-35% gradient – 1 C.V., 35 –80% gradient – 10 C.V. and 60 –100% gradient 1 C.V. Protein elutes at 40-50% gradient. Protein details: Our protein is stable at acidic pH and has a pI of 5.8 –6.3 and buffer is Na Acetate buffer pH 4.5 and elution buffer is starting buffer containing 0.4 M NaCl. We get only one peak on AKTA but on running SDS page we get so many bands even IEF shows 1-2 bands at the most. How can we modify the method or what can be done to get rid of extra high mol wt bands. Any help will be deeply appreciated. SDS PAGE.JPGIEF.doc
Re: [ccp4bb] Troubleshooting protein purification cation IEX
Hello Meg, Since your protein is quite acidic, your next step could be e.g. anion exchange - provided that you are able to get the protein into a suitable buffer w/o losing it (since you will pass through the pI). If you can, the simplest way to do so is to add TRIS to the pH4.5 buffer until you get the desired pH (7-8) and dilute the sample a bit with pure water. An alternative, or a follow-up step could be hydrophobic column - these are very useful for removing aggregates, partially folded stuff and similar entities. Again, nothing is completely safe - HIC may cause problems since you are starting at fairly high salt (1-2 M NaCl or KCl typically). But at least you won't have to pass the pI. Cheers, Artem Dear All, This is with reference to the purification of our recombinant protein sample expressed in E.coli as inclusion bodies. After Solubilization refolding we perform the cation exchange chromatography of our protein sample using SP sepharose fast flow resin. Attached herewith find the SDS PAGE and IEF results of the collected fractions. In addition to our protein of interest we are also getting high molecular weigh contaminants, which we cannot get rid of in IEX. Can anyone please guide me on a technique to get rid of these bands as even after gel filtration of samples few high mol wt contaminant bands are not separated from main proteins and sample gets diluted too. In cation IEX procedure is Column Sp Sepharose Fast flow packed in fineline 35 column packed bed volume 100 ml System AKTA FPLC Equilibration 10 C.V. protein loading 3-4 C.V. [approx 1mg/ml protein conc], washing to remove unbound materials 2 C.V. step elution 0-35% gradient 1 C.V., 35 80% gradient 10 C.V. and 60 100% gradient 1 C.V. Protein elutes at 40-50% gradient. Protein details: Our protein is stable at acidic pH and has a pI of 5.8 6.3 and buffer is Na Acetate buffer pH 4.5 and elution buffer is starting buffer containing 0.4 M NaCl. We get only one peak on AKTA but on running SDS page we get so many bands even IEF shows 1-2 bands at the most. How can we modify the method or what can be done to get rid of extra high mol wt bands. Any help will be deeply appreciated.
Re: [ccp4bb] Troubleshooting protein purification cation IEX
Meg, I might add that if you have boiled your samples, you might disregard this: 1) Instead of contamination, you might just be seeing multiple bands due to 'aggregation' of your protein! Make sure you boil the sample prior to loading on gel and also that your loading dye contains SDS, bME/DTT. But multiple bands might be due to aggregation of your protein BECAUSE of boiling the samples. This is common with some membrane and somewhat hydrophobic proteins. Not boiling or not heating your samples may reduce it. By the way, how do you know that you have refolded your protein specifically? Do you have an activity assay? Refolding can yield aggregates like protein micelles: soluble in the sense that they don't precipitate, but still aggregates that can include other proteins. Can you purify your target protein while denatured? That may solve part of your problem. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: [EMAIL PROTECTED] On Sep 23, 2008, at 7:54 AM, Raji Edayathumangalam wrote: Hi, Many things can lead to your observation. Please outline all steps of your purification procedure as it is not clear what is done before and after the Ion Exchange steps. I am not sure if IEF in your emails refers to Isoelectric focusing, as the acronym is usually used?? Couple of suggestions: 1) Instead of contamination, you might just be seeing multiple bands due to 'aggregation' of your protein! Make sure you boil the sample prior to loading on gel and also that your loading dye contains SDS, bME/DTT. 2) We used to do entire purifications with inclusion body preps under denaturing conditions to prevent unwanted aggregation of partially folded or misfolded species. Not sure if denaturant is present all along. 3) If the problem is of contamination, try making the gradient shallow for the 35 –80% gradient step in Ion Exchange (increase to say, 20 cv or more). 4) If the problem is of contamination, try to add more steps to purification -- e.g., affinity step (if possible), anion exchange as well etc. 5) If IEF (in the sense I mean it) is what you did and it shows only 1-2 bands, the problem is likely (#1) outlined above. 6) If all else fails, cut out one or two of the bands from your gel and run a mass spec. An expensive way to find out that it is aggregation. Nevertheless Hope that helps. Raji On Sep 23, 2008, at 3:51 AM, Meg wrote: Dear All, This is with reference to the purification of our recombinant protein sample expressed in E.coli as inclusion bodies. After Solubilization refolding we perform the cation exchange chromatography of our protein sample using SP sepharose fast flow resin. Attached herewith find the SDS PAGE and IEF results of the collected fractions. In addition to our protein of interest we are also getting high molecular weigh contaminants, which we cannot get rid of in IEX. Can anyone please guide me on a technique to get rid of these bands as even after gel filtration of samples few high mol wt contaminant bands are not separated from main proteins and sample gets diluted too. In cation IEX procedure is Column Sp Sepharose Fast flow packed in fineline 35 column packed bed volume 100 ml System AKTA FPLC Equilibration 10 C.V. protein loading 3-4 C.V. [approx 1mg/ml protein conc], washing to remove unbound materials 2 C.V. step elution 0-35% gradient – 1 C.V., 35 –80% gradient – 10 C.V. and 60 –100% gradient 1 C.V. Protein elutes at 40-50% gradient. Protein details: Our protein is stable at acidic pH and has a pI of 5.8 –6.3 and buffer is Na Acetate buffer pH 4.5 and elution buffer is starting buffer containing 0.4 M NaCl. We get only one peak on AKTA but on running SDS page we get so many bands even IEF shows 1-2 bands at the most. How can we modify the method or what can be done to get rid of extra high mol wt bands. Any help will be deeply appreciated. SDS PAGE.JPGIEF.doc
[ccp4bb] recipees for prep of test derivatives
Hi, Could anyone give a more or less exact recipee for preparing a derivate for lysozyme or proteinase K? like what used, concentration and soak time. we'd need some data sets for a lab course only thing that really worked so far was lysozyem KI SIRAS (which however doesnt seem to be good enough for demonstration purposes, so we need more) Thanks very much! best, Tommi Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 [EMAIL PROTECTED]
[ccp4bb] test data sets part II...
Also, i think that would be nice if this type of info could be put on the web, part of the wiki for instance.. if there is some consensus to what works + the typical proteins easily available. Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 [EMAIL PROTECTED]
Re: [ccp4bb] Crystallogrphy today
Perhaps you could translate and annotate it, then send it to the CCP4BB? JPK ps seriously, why do you say no need for review--is it boring, not well written, obsolete, or what? James is still pretty useful, I think, and that was put out only two years later *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: Marius Schmidt [EMAIL PROTECTED] To: Jacob Keller [EMAIL PROTECTED]; CCP4BB@JISCMAIL.AC.UK Sent: Monday, September 22, 2008 8:42 PM Subject: Re: [ccp4bb] Crystallogrphy today i have a suggestion for a nice book for you, you will love it. it is in German, great!, has over 400 pages and it IS THE SOURCE. M. von Laue Roentgenstrahlinterferenzen Physik und Chemie und Ihre Anwendungen, Band VI 2. Auflage (1st edition burnt down by cannonizing at WWII) 1948 Akademische Verlagsgesellschaft, Geest Portig K.-G., Leipzig everything is covered, even protein crystallography, however in a very skeptic way, no need for a review ever. Cheers Marius To understand the fundamentals of any discipline, I have always found it completely worthwhile to go back to the original source, where the idea was first discovered or presented. This is really, really valuable, although not always possible. I wonder whether others agree with me about this...but I feel pretty strongly about this matter. Often one can read many reviews on some subject, which never really get to the gist of the matter, but when one reads the original source, the subject is usually laid out clearly because guess what: nobody knew it yet, so it had to be explained clearly. Furthermore, one gets a sense of the excitement of discovery, and the unsurety about some new proposed hypothesis which has not yet become cannonized into fact. For this reason, it is sometimes even worthwhile to saunter down to the...library! Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] ***
Re: [ccp4bb] Troubleshooting protein purification cation IEX
Another suggestion that comes to mind is crosslinking via Cys, are you purifying under reducing conditions ? Or since you have inclusion bodies they're in general not correctly folded hence expose hydrophobic regions and can interact with other proteins, to avoid unspecific interaction add 20% glycerol to your lysis buffer. Jürgen On 23 Sep 2008, at 00:51, Meg wrote: - Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
[ccp4bb] The mystery of the S-tensor WAS: re-indexing, re-orienting and TLS-tensors
Dear all, a few weeks ago I was wondering how to apply a rotation matrix to TLS-tensors and Ian was so kind to supply us with the corresponding formulas. After looking a bit into it, however, the whole matter seems to be complicated by the fact that one value of S is undefined, however, after the transformation it appears within S' at several places. Did we miss something obvious? Or is this actually more complicated than it looked like in the first place? Thanks Clemens Ian Tickle schrieb: Clemens, One thing I should have pointed out, though you may have realised it already: you will most likely want to keep the same relative origins for the new TLS groups so this means that the translational component of the transformation (vector p or matrix P) will always be zero. This is true even if your desired transformation of the co-ordinates contains a non-zero translation component, because this translation is then also applied to the local origin of the TLS group, hence there is no resultant translation *relative* to that origin. In other words the translational component p is only relevant if you want to change the relative origins of the TLS groups (e.g. from COG to COR or vice versa), which is unlikely if all you are doing is re-indexing. Having p=0 (and P=O) of course considerably simplifies the equations, i.e.: T' = RTR~ and the same transformation is applied separately to L and S. Cheers -- Ian -Original Message- From: Clemens Grimm [mailto:[EMAIL PROTECTED] Sent: 09 July 2008 14:20 To: Ian Tickle Subject: RE: [ccp4bb] re-indexing, re-orienting and TLS-tensors Hi Ian, thanks for the formulas! I'll probably give Mthematica a try. Clemens Quoting Ian Tickle [EMAIL PROTECTED]: Hi Clemens The relevant matrix algebra is all there in TLSANL, i.e. you could put some code together based on that to do what you want, however there isn't an option (even an undocumented one!) to do exactly what you want and I don't know of any program which will do that. The math isn't actually all that difficult, all you need is a routine for 3x3 matrix multiplication, or you could use something like R or Mathematica. -- Ian -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Clemens Grimm Sent: 09 July 2008 12:24 To: [EMAIL PROTECTED]; Eleanor Dodson Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] re-indexing, re-orienting and TLS-tensors this was a difficult case due to poor convergence behaviour during the TLS refinement. At the moment I'm not sure what would be more complicated, re-creating the tensors from scratch or writing a script. What about TLSANL? Is there an (undocumented ?) feature that could be used for things like that? Clemens Quoting Eleanor Dodson [EMAIL PROTECTED]: I would run the TLS again! Eleanor Clemens Grimm wrote: Dear all, after re-indexing a dataset I had to re-orient my coordinates accordingly. The model contains some 24 TLS-tensors. Now my question is how to apply the rotation matrix also to the TLS-tensors. What is the mathematical operation and is there a program that can do the job for me? Thanks, Clemens Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing [EMAIL PROTECTED] and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674 Disclaimer This communication is
Re: [ccp4bb] The mystery of the S-tensor WAS: re-indexing, re-orienting and TLS-tensors
Hi Clemens The element of S you are referring to is not undefined: the value of one of the diagonal elements of S can be chosen arbitrarily because it makes no difference as far as the individual atomic motions are concerned. The software normally chooses the value for the arbitrary element so that the sum of the diagonal elements of S (i.e. the trace) is zero, but it wouldn't make any difference if any other value were chosen. HTH -- Ian -Original Message- From: Clemens Grimm [mailto:[EMAIL PROTECTED] Sent: 23 September 2008 16:41 To: CCP4BB@JISCMAIL.AC.UK Cc: Ian Tickle Subject: The mystery of the S-tensor WAS: re-indexing, re-orienting and TLS-tensors Dear all, a few weeks ago I was wondering how to apply a rotation matrix to TLS-tensors and Ian was so kind to supply us with the corresponding formulas. After looking a bit into it, however, the whole matter seems to be complicated by the fact that one value of S is undefined, however, after the transformation it appears within S' at several places. Did we miss something obvious? Or is this actually more complicated than it looked like in the first place? Thanks Clemens Ian Tickle schrieb: Clemens, One thing I should have pointed out, though you may have realised it already: you will most likely want to keep the same relative origins for the new TLS groups so this means that the translational component of the transformation (vector p or matrix P) will always be zero. This is true even if your desired transformation of the co-ordinates contains a non-zero translation component, because this translation is then also applied to the local origin of the TLS group, hence there is no resultant translation *relative* to that origin. In other words the translational component p is only relevant if you want to change the relative origins of the TLS groups (e.g. from COG to COR or vice versa), which is unlikely if all you are doing is re-indexing. Having p=0 (and P=O) of course considerably simplifies the equations, i.e.: T' = RTR~ and the same transformation is applied separately to L and S. Cheers -- Ian -Original Message- From: Clemens Grimm [mailto:[EMAIL PROTECTED] Sent: 09 July 2008 14:20 To: Ian Tickle Subject: RE: [ccp4bb] re-indexing, re-orienting and TLS-tensors Hi Ian, thanks for the formulas! I'll probably give Mthematica a try. Clemens Quoting Ian Tickle [EMAIL PROTECTED]: Hi Clemens The relevant matrix algebra is all there in TLSANL, i.e. you could put some code together based on that to do what you want, however there isn't an option (even an undocumented one!) to do exactly what you want and I don't know of any program which will do that. The math isn't actually all that difficult, all you need is a routine for 3x3 matrix multiplication, or you could use something like R or Mathematica. -- Ian -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Clemens Grimm Sent: 09 July 2008 12:24 To: [EMAIL PROTECTED]; Eleanor Dodson Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] re-indexing, re-orienting and TLS-tensors this was a difficult case due to poor convergence behaviour during the TLS refinement. At the moment I'm not sure what would be more complicated, re-creating the tensors from scratch or writing a script. What about TLSANL? Is there an (undocumented ?) feature that could be used for things like that? Clemens Quoting Eleanor Dodson [EMAIL PROTECTED]: I would run the TLS again! Eleanor Clemens Grimm wrote: Dear all, after re-indexing a dataset I had to re-orient my coordinates accordingly. The model contains some 24 TLS-tensors. Now my question is how to apply the rotation matrix also to the TLS-tensors. What is the mathematical operation and is there a program that can do the job for me? Thanks, Clemens Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing [EMAIL PROTECTED] and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email
Re: [ccp4bb] The mystery of the S-tensor WAS: re-indexing, re-orienting and TLS-tensors
Clemens Sorry my last 2 e-mails probably weren't so clear, because the software may not actually write out S11 S22 explicitly. Unfortunately there's no consistency on what the programs do write out, so you may need to solve some linear equations for the unknowns S11, S22 S33 needed to construct the complete S tensor. For example Refmac writes out (see http://www.ccp4.ac.uk/dist/html/refmac5/files/tls.html) S22-S11 and S11-S33 as the first 2 elements of the S matrix (8 elements in all), so let: x = S22-S11 y = S11-S33 and we also have: S11+S22+S33 = 0 Therefore the solution is: S11 = (y-x)/3 S22 = (y+2x)/3 S33 = -(2y+x)/3 You can satisfy yourself that this is the solution by substitution in the original equations. The reason that Refmac writes out the differences between the diagonal elements, and not the elements themselves, is simply that only the differences are involved in the calculation of the atomic Uaniso tensors, and the third difference needed is expressible in terms of the other two, i.e. S33-S22 = -(x+y). Hope this is clearer now! -- Ian -Original Message- From: Clemens Grimm [mailto:[EMAIL PROTECTED] Sent: 23 September 2008 16:41 To: CCP4BB@JISCMAIL.AC.UK Cc: Ian Tickle Subject: The mystery of the S-tensor WAS: re-indexing, re-orienting and TLS-tensors Dear all, a few weeks ago I was wondering how to apply a rotation matrix to TLS-tensors and Ian was so kind to supply us with the corresponding formulas. After looking a bit into it, however, the whole matter seems to be complicated by the fact that one value of S is undefined, however, after the transformation it appears within S' at several places. Did we miss something obvious? Or is this actually more complicated than it looked like in the first place? Thanks Clemens Ian Tickle schrieb: Clemens, One thing I should have pointed out, though you may have realised it already: you will most likely want to keep the same relative origins for the new TLS groups so this means that the translational component of the transformation (vector p or matrix P) will always be zero. This is true even if your desired transformation of the co-ordinates contains a non-zero translation component, because this translation is then also applied to the local origin of the TLS group, hence there is no resultant translation *relative* to that origin. In other words the translational component p is only relevant if you want to change the relative origins of the TLS groups (e.g. from COG to COR or vice versa), which is unlikely if all you are doing is re-indexing. Having p=0 (and P=O) of course considerably simplifies the equations, i.e.: T' = RTR~ and the same transformation is applied separately to L and S. Cheers -- Ian -Original Message- From: Clemens Grimm [mailto:[EMAIL PROTECTED] Sent: 09 July 2008 14:20 To: Ian Tickle Subject: RE: [ccp4bb] re-indexing, re-orienting and TLS-tensors Hi Ian, thanks for the formulas! I'll probably give Mthematica a try. Clemens Quoting Ian Tickle [EMAIL PROTECTED]: Hi Clemens The relevant matrix algebra is all there in TLSANL, i.e. you could put some code together based on that to do what you want, however there isn't an option (even an undocumented one!) to do exactly what you want and I don't know of any program which will do that. The math isn't actually all that difficult, all you need is a routine for 3x3 matrix multiplication, or you could use something like R or Mathematica. -- Ian -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Clemens Grimm Sent: 09 July 2008 12:24 To: [EMAIL PROTECTED]; Eleanor Dodson Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] re-indexing, re-orienting and TLS-tensors this was a difficult case due to poor convergence behaviour during the TLS refinement. At the moment I'm not sure what would be more complicated, re-creating the tensors from scratch or writing a script. What about TLSANL? Is there an (undocumented ?) feature that could be used for things like that? Clemens Quoting Eleanor Dodson [EMAIL PROTECTED]: I would run the TLS again! Eleanor Clemens Grimm wrote: Dear all, after re-indexing a dataset I had to re-orient my coordinates accordingly. The model contains some 24 TLS-tensors. Now my question is how to apply the rotation matrix also to the TLS-tensors. What is the mathematical operation and is there a program that can do the job for me? Thanks, Clemens Disclaimer This communication is confidential and may contain
Re: [ccp4bb] Crystallogrphy today
Speaking of good material to attempt to understand crystallographic concepts with , there a video link to the talk given by Airlie McCoy based on the Liking likelyhood paper Its on the phaser publications page or at this linkhttp://erice2005.docking.org/vcourse/16mon/1145-McCoy/McCoy.wmv http://erice2005.docking.org/vcourse/16mon/1145-McCoy/McCoy.wmv There is also a talk from Randy reed at that locationhttp://erice2005.docking.org/vcourse/18wed/0945-Read/Read.wmv It would be great if other such talks could be made available as videos . , for all of us to benefit from . It will be great to have a youtube or google video channel for such videos. That way google pays for the bandwidth instead of erice2005 or ccp4. Thanks for this thread.. Hari On Tue, Sep 23, 2008 at 11:31 AM, Jacob Keller [EMAIL PROTECTED] wrote: Perhaps you could translate and annotate it, then send it to the CCP4BB? JPK ps seriously, why do you say no need for review--is it boring, not well written, obsolete, or what? James is still pretty useful, I think, and that was put out only two years later *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: Marius Schmidt [EMAIL PROTECTED] To: Jacob Keller [EMAIL PROTECTED]; CCP4BB@JISCMAIL.AC.UK Sent: Monday, September 22, 2008 8:42 PM Subject: Re: [ccp4bb] Crystallogrphy today i have a suggestion for a nice book for you, you will love it. it is in German, great!, has over 400 pages and it IS THE SOURCE. M. von Laue Roentgenstrahlinterferenzen Physik und Chemie und Ihre Anwendungen, Band VI 2. Auflage (1st edition burnt down by cannonizing at WWII) 1948 Akademische Verlagsgesellschaft, Geest Portig K.-G., Leipzig everything is covered, even protein crystallography, however in a very skeptic way, no need for a review ever. Cheers Marius To understand the fundamentals of any discipline, I have always found it completely worthwhile to go back to the original source, where the idea was first discovered or presented. This is really, really valuable, although not always possible. I wonder whether others agree with me about this...but I feel pretty strongly about this matter. Often one can read many reviews on some subject, which never really get to the gist of the matter, but when one reads the original source, the subject is usually laid out clearly because guess what: nobody knew it yet, so it had to be explained clearly. Furthermore, one gets a sense of the excitement of discovery, and the unsurety about some new proposed hypothesis which has not yet become cannonized into fact. For this reason, it is sometimes even worthwhile to saunter down to the...library! Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] ***
Re: [ccp4bb] recipees for prep of test derivatives
what is it that you want to demonstrate? If it is just for structure solution, would S-SAD phasing be an option? That way all you need is a crystal, probably diffracting to better than, say, 2A. And a little bit of multiplicity. I assume this is feasible for Lysozyme whereas I don't know the diffraction qualities of proteinase K crystals. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 23 Sep 2008, Tommi Kajander wrote: Hi, Could anyone give a more or less exact recipee for preparing a derivate for lysozyme or proteinase K? like what used, concentration and soak time. we'd need some data sets for a lab course only thing that really worked so far was lysozyem KI SIRAS (which however doesnt seem to be good enough for demonstration purposes, so we need more) Thanks very much! best, Tommi Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 [EMAIL PROTECTED]
Re: [ccp4bb] test data sets part II...
Dear Tommi, I'm not sure whether 'test data sets part I' relates to the query I mailed the other day but, in light of the recent posts regarding fundamental literature, your suggestion seems an excellent one. Perhaps I'm not looking in the right places, but finding examples of image sets exhibiting e.g., radiation damage, crystal slippage, autoindexing failures or any of the aberrations shown in Phil Evans' excellent scala tutorial seems difficult. Likewise for examples of good and bad final-refinement maps. Making primary/intermediate data available would benefit both students and algorithm comparison/validation (e.g. measuring performance on standard data sets is routine in data-visualization/image-processing papers). Tommi Kajander wrote: Also, i think that would be nice if this type of info could be put on the web, part of the wiki for instance.. if there is some consensus to what works + the typical proteins easily available. Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 [EMAIL PROTECTED] -- Alastair Fyfe Graduate Student Biomolecular Engineering Dept. University of California, Santa Cruz
Re: [ccp4bb] Troubleshooting protein purification cation IEX
Another option is to purify in denatured condition, then refold. --Chun -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Meg Sent: Tuesday, September 23, 2008 12:51 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Troubleshooting protein purification cation IEX Dear All, This is with reference to the purification of our recombinant protein sample expressed in E.coli as inclusion bodies. After Solubilization refolding we perform the cation exchange chromatography of our protein sample using SP sepharose fast flow resin. Attached herewith find the SDS PAGE and IEF results of the collected fractions. In addition to our protein of interest we are also getting high molecular weigh contaminants, which we cannot get rid of in IEX. Can anyone please guide me on a technique to get rid of these bands as even after gel filtration of samples few high mol wt contaminant bands are not separated from main proteins and sample gets diluted too. In cation IEX procedure is Column Sp Sepharose Fast flow packed in fineline 35 column packed bed volume 100 ml System AKTA FPLC Equilibration 10 C.V. protein loading 3-4 C.V. [approx 1mg/ml protein conc], washing to remove unbound materials 2 C.V. step elution 0-35% gradient - 1 C.V., 35 -80% gradient - 10 C.V. and 60 -100% gradient 1 C.V. Protein elutes at 40-50% gradient. Protein details: Our protein is stable at acidic pH and has a pI of 5.8 -6.3 and buffer is Na Acetate buffer pH 4.5 and elution buffer is starting buffer containing 0.4 M NaCl. We get only one peak on AKTA but on running SDS page we get so many bands even IEF shows 1-2 bands at the most. How can we modify the method or what can be done to get rid of extra high mol wt bands. Any help will be deeply appreciated.
