Re: [ccp4bb] Twinning
this can screw up your statistics on twinning and this can be due to the NCS rotation axis (or will be based on what you say) parallel to a crystallographic symmetry axis. just check that. it should still refine though but this will have opposite effect to twinning, so it can be problematic. although its not clear to me what your problem is stucture solution actually was. just as a general comment, tommi NCS axis is not parallel to any crystallographic axis. Although initially had some problem in refinement because of twinning. I did not have any problem in obtaining the solution. So my doubt was that even though there was an indication of pseudotranslation, why was there no problem in obtaining the solution? So I wanted to know whether this pseudotranslation peak was due to the presence of heavy atoms in the structure. Tommi Kajander, Ph.D., Docent Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940 -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
Re: [ccp4bb] Tev Cleavage issue !!
Hi Anita, We have had success setting up drops with TEV present. We simply added TEV at a 50:1 molar ratio and then set up the drops a couple of hours later. We went from having twinned crystals at 3A to untwinned at 2A, the crystal form also changed from orthorhombic to monoclinic, all in the same drop condition. We might have just got lucky, but it is an easy experiment to try. Cheers Peter ___ Dr Peter Czabotar Structural Biology Division The Walter and Eliza Hall Institute of Medical Research 1G Royal Parade, Parkville, Victoria, Australia Ph: (+61 3) 9345 2689 ___ On Apr 8, 2011, at 12:37 PM, anita p wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. Is it probable that they dont diffract because of the extra his tag and the tev site? I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. Is there a possible way to approach this problem? Suggestions awaited Anita __ The information in this email is confidential and intended solely for the addressee. You must not disclose, forward, print or use it without the permission of the sender. __
Re: [ccp4bb] Tev Cleavage issue !!
Dear Anita, Sometimes the protein of interest has a relatively strong inherent binding affinity to the IMAC column. Have you tried to bind the cleavage reaction to an IMAC column and then elute using a shallow imidazole gradient? In fact, Porath developed IMAC chromatography as a tool for protein fractionation long before molecular biology provided the tools necessary to add poly-histidine tags to target proteins (Nature. 1975 Dec 18;258(5536):598-9). Best regards, Martin On Apr 8, 2011, at 4:37 AM, anita p wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. Is it probable that they dont diffract because of the extra his tag and the tev site? I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. Is there a possible way to approach this problem? Suggestions awaited Anita
Re: [ccp4bb] Tev Cleavage issue !!
Thanks everyone for your suggestions ! Artem has pointed out that low diffraction of the crystal might be because of other problems .. If you could* highlight a bit more on this issue it would be helpful for me.* I have tried to seperate the cleaved, uncleaved and TEV over MonoQ column but there was a single peak and all of them came out together, there was no seperation. I even tried to cleave the protein at 30 degress and it starts precipitating. I have also tried binding it to the IMAC as Martin has suggested but then I get a single peak while running the imidazole gradient and its tev, cleaved and uncleaved together. And I also get the flowthrough while loading unto the column which should be theoritically the cleaved one but it is a combination of cleaved uncleaved and tev. awaiting for bit more suggestions Anita On Fri, Apr 8, 2011 at 10:48 AM, Artem Evdokimov artem.evdoki...@gmail.comwrote: For starters, you could re-clone the protein with e.g. just a His tag or move the tag to another end, or put some distance between the end of TEV site and the protein; or perhaps use no tag at all -- or a different one? Is it possible that the tag is messing you up - yes. Is it 'probable' - I can't say that I know because I've crystallized literally dozens of proteins with His-tags attached, and more than a few with His-tag and cleavage site. I prefer a plain His-tag or a cleaved one, to be sure - but I am not quick to blame the tag (since there are so many other possible things to blame). Based on the behavior of your protein after cleavage, it may be that you have oligomer(s) forming in solution such that cleaved and uncleaved proteins do not segregate. You may wish to explore other kinds of chromatographic separation e.g. ion exchange of HIC - they may or may not work out. You can also consider cleaving your protein at lower concentration, in the presence of detergents or polyols, etc. Cheers, Artem On Thu, Apr 7, 2011 at 9:37 PM, anita p crystals...@gmail.com wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. *Is it probable that they dont diffract because of the extra his tag and the tev site?* I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. * Is there a possible way to approach this problem?* Suggestions awaited Anita
[ccp4bb] St Andrews Protein Crystallography Summer School 2011
We are pleased to announce the 18th Summer School in Protein Crystallography to be held this year at St Andrews from 3rd – 9th September. Further details and an on-line application form can be found at: http://www.st-andrews.ac.uk/~glt2/PX2011 Best wishes Garry Taylor Jim Naismith Biomedical Sciences Research Complex University of St Andrews St Andrews FifePhone: 01334 467301 Scotland Fax : 01334 462595 KY16 9ST Mobile: 07595 088 784 WWW My lab www.st-andrews.ac.uk/~glt2/My_Lab WWW BSRC: www.st-andrews.ac.uk/bsrc The University of St Andrews is a charity registered in Scotland : No SC013532
Re: [ccp4bb] what is NCS operators of parrot
First three numbers are the Euler angles. The next 3 are the (approximate) centre of mass of the source molecule. The last 3 are the corresponding point in the target molecule. (This is a little more complex than the form used in 'dm', but unfortunately the simpler form was missing important information about where the NCS applies. That was OK when every program required that you provide a mask, but became a problem when we started generating masks automatically. Unfortunately we may have to go to yet another form for a fully general solution to the cross-crystal problem.) Kevin Wang wrote: Hi everyone: I used parrot to do density modification, and it found ncs operators. The operators found are: Non-crystallographic operators: -ncs-operator -144.47,179.729,-144.321,18.7789,16.4179,19.809,16.4383,16.4507,19.809 -ncs-operator -10.0955,179.846,169.903,27.2216,37.7585,38.88,27.0706,40.5231,38.88 What does the operators mean? Is that Eular angle? But I changed it to ratation matrix, the result is not so good. Thanks Best wishes Wang -- EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm
[ccp4bb] Script / program to change chain ID 's in symmetry mates
Dear CCP4BB members, We are using a script written in python to generate symmetry mates for a given pdb file using PYMOL. After generating symmetry mates we want to combine all the symmetry molecules in a single PDB file with all the chains having unique chain IDs. Since all the symmetry mates have same chain ID's I was wondering if some one knows a script that can give unique chain ID for each symmetry mate. We are interested in script because that dataset that we are handling is large. I thank you all in advance for your help. Best, Krishan
Re: [ccp4bb] Script / program to change chain ID 's in symmetry mates - MOLEMAN
MOLEMAN from the Uppsala Software Factory by Gerard Kleywegt of course It is easy as to say Do it! FF Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Apr 8, 2011, at 14:17 , krishan wrote: Dear CCP4BB members, We are using a script written in python to generate symmetry mates for a given pdb file using PYMOL. After generating symmetry mates we want to combine all the symmetry molecules in a single PDB file with all the chains having unique chain IDs. Since all the symmetry mates have same chain ID's I was wondering if some one knows a script that can give unique chain ID for each symmetry mate. We are interested in script because that dataset that we are handling is large. I thank you all in advance for your help. Best, Krishan
Re: [ccp4bb] Script / program to change chain ID 's in symmetry mates
I wrote a little utility called pdb_merge that is in CCP4. With the nomerge option, it checks for duplicate chain IDs, and renames chains if necessary. The main limitation is that it can only merge 2 files, but you should be able to script a loop to call it multiple times. What happens if you have 10 chains in the asu and a cubic spacegroup, I am not sure ... HTH Martyn On Fri, 2011-04-08 at 16:47 +0530, krishan wrote: Dear CCP4BB members, We are using a script written in python to generate symmetry mates for a given pdb file using PYMOL. After generating symmetry mates we want to combine all the symmetry molecules in a single PDB file with all the chains having unique chain IDs. Since all the symmetry mates have same chain ID's I was wondering if some one knows a script that can give unique chain ID for each symmetry mate. We are interested in script because that dataset that we are handling is large. I thank you all in advance for your help. Best, Krishan -- *** * * * Dr. Martyn Winn * * * * STFC Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, U.K. * * Tel: +44 1925 603455E-mail: martyn.w...@stfc.ac.uk* * Fax: +44 1925 603634Skype name: martyn.winn * * URL: http://www.ccp4.ac.uk/martyn/ * ***
Re: [ccp4bb] anisotropy vs TLS
Hi Kenneth, I know that TLS is a group B factor for regions of proteins that are moving the same. You have to be a bit careful here: first B factors do not necessarily imply motion, they imply displacement (i.e. it could mean static displacements which just vary between unit cells). That's why they are called 'displacement parameters' (as in 'ADP' if it's anisotropic). Second, 'moving the same', or even 'being displaced the same' carries the connotation that you are getting information about correlated _motion_ or displacement, which is not correct either, though it may well be one interpretation of the results. Strictly TLS only gives you information about correlated _dispersion_ (in the strict sense of dispersion as 'variance'), not correlated _displacement_, though you may well interpret correlated dispersion as correlated displacement (i.e. other interpretations of the results may fit the data equally well). The difference between displacement and dispersion is illlustrated by considering a group of atoms in a molecule: if these atoms all have identical instantaneous displacements at all times, as though they were rigidly connected, that's correlated displacement. If the atoms are moving completely independently (i.e. their displacement vectors are in general all different both in direction and magnitude), but they happen to all have the same ADPs (which are a time/lattice average of the squared displacements), that's correlated dispersion. Of course in practice you will see all shades between these two extremes. It is used in low res structures. But at what resolution does one begin anisotropic, i.e individual aniso for each atom, and leave TLS out. Depends what you mean by 'low'. You can use individual ADPs when you have a sufficient data/parameter ratio, which as a rough guide I would say is around 1.5 Ang., though you would still need to use ADP restraints (as in Shel-X) at resolutions between 1.5 and say around 1 Ang (at what point you can safely drop ADP restraints beyond 1 Ang is a matter of taste). Or can one still use TLS to first compensate for large motions and then dampen down the individual atoms with aniso ADP? Not sure what you mean by 'dampen down': do you mean 'restrained'? For the purposes of refinement it would be tricky to do this in practice because TLS and ADP parameters are not independent. The first thing the refinement program does with the TLS parameters is calculate the equivalent ADPs and use those in the SF calculation, though of course the results are ultimately still expressed in terms of changes in the TLS parameters. A better way of doing this would be to use only ADPs in refinement, and interpret the results post-refinement as TLS + 'residual' ADPs. This is how TLS was used years ago for small molecules before refinement of TLS parameters directly was coded (though the residual ADPs were usually viewed as just 'random error'). The problem here is what you call 'residual', i.e. because they are not independent, you can interpret the results as either correlated displacement or correlated dispersion, and the data aren't going to help you to decide which is the more correct interpretation. If both the aniso and TLS are used, how does a person interpret the results? What programs are there to see just what is large body motions and what is atoms. I've never tried this, so I can't say if it would be a useful exercise or not. I think I would stick to the TLS + individual _Biso_ model: that is already hard enough to interpret! Cheers -- Ian
Re: [ccp4bb] Tev Cleavage issue !!
Hi Anita, Admittedly, there are proteins that naturally bind to Ni-NTA so tightly that they co-elute with our His6-tagged proteins even on an imidazole gradient. However, we do need some luck to come across a protein with such property. For most proteins, they would just flow through the Ni-NTA in the presence of 10-20mM imidazole. Are you sure that what you saw as a lower molecular weight band on SDS gel was not really a clipped form of your protein that failed to be cleaved by TEV and still carried a His tag when undenatured? I guess that your TEV is also His-tagged, so seeing it in the Ni-NTA fraction is reasonable. But I would expect some sort of separation from your protein on the imidazole gradient even if the peaks are overlapping. Also, on Q column, TEV, being an extremely basic protein, simply won't bind. If you saw the TEV band in the Q fractions, then that suggests that you may have incorrectly identified your protein bands on the SDS gel. It would be interesting to see your SDS gel. Also providing more specific details of your chromatographies may help a lot. For example, what was the approximate concentration of imidazole when your peak came out from the Ni-NTA when eluted with a gradient? What was the condition you used for Q column and what is your protein's PI? There are just too many factors that could effect the performance of the ion-exchange chromatography. On the other hand, if it is true that your protein binds to Ni-NTA so well even without a His tag, then why not try expressing it alone without a His tag? Shouldn't you be able to purify it easily with Ni-NTA? Finally, the difficulty in TEV cleavage could indicate a construction problem. I assume that your protein is N-terminally His-tagged. To my experience, TEV wants one or two more amino acids between the G/S in ENLYFQ^G/S and the folded protein domain, i.e., it wants some space on the right hand side of the cleavage site. Adding one or two amino acids after the current cleavage site may help. Zhijie From: anita p Sent: Friday, April 08, 2011 5:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Tev Cleavage issue !! Thanks everyone for your suggestions ! Artem has pointed out that low diffraction of the crystal might be because of other problems .. If you could highlight a bit more on this issue it would be helpful for me. I have tried to seperate the cleaved, uncleaved and TEV over MonoQ column but there was a single peak and all of them came out together, there was no seperation. I even tried to cleave the protein at 30 degress and it starts precipitating. I have also tried binding it to the IMAC as Martin has suggested but then I get a single peak while running the imidazole gradient and its tev, cleaved and uncleaved together. And I also get the flowthrough while loading unto the column which should be theoritically the cleaved one but it is a combination of cleaved uncleaved and tev. awaiting for bit more suggestions Anita On Fri, Apr 8, 2011 at 10:48 AM, Artem Evdokimov artem.evdoki...@gmail.com wrote: For starters, you could re-clone the protein with e.g. just a His tag or move the tag to another end, or put some distance between the end of TEV site and the protein; or perhaps use no tag at all -- or a different one? Is it possible that the tag is messing you up - yes. Is it 'probable' - I can't say that I know because I've crystallized literally dozens of proteins with His-tags attached, and more than a few with His-tag and cleavage site. I prefer a plain His-tag or a cleaved one, to be sure - but I am not quick to blame the tag (since there are so many other possible things to blame). Based on the behavior of your protein after cleavage, it may be that you have oligomer(s) forming in solution such that cleaved and uncleaved proteins do not segregate. You may wish to explore other kinds of chromatographic separation e.g. ion exchange of HIC - they may or may not work out. You can also consider cleaving your protein at lower concentration, in the presence of detergents or polyols, etc. Cheers, Artem On Thu, Apr 7, 2011 at 9:37 PM, anita p crystals...@gmail.com wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. Is it probable that they dont diffract because of the extra his tag and the tev site? I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. Is there a
Re: [ccp4bb] Tev Cleavage issue !!
You could also try upping the tev:prot ratio, such that the protein is ~100% cleaved, then do IMAC or simply some other, non-IMAC chromatography step, such as ion exchange or SEC, depending on the size and charge of your protein relative to TEV. JPK On Fri, Apr 8, 2011 at 8:17 AM, Zhijie Li zhijie...@utoronto.ca wrote: Hi Anita, Admittedly, there are proteins that naturally bind to Ni-NTA so tightly that they co-elute with our His6-tagged proteins even on an imidazole gradient. However, we do need some luck to come across a protein with such property. For most proteins, they would just flow through the Ni-NTA in the presence of 10-20mM imidazole. Are you sure that what you saw as a lower molecular weight band on SDS gel was not really a clipped form of your protein that failed to be cleaved by TEV and still carried a His tag when undenatured? I guess that your TEV is also His-tagged, so seeing it in the Ni-NTA fraction is reasonable. But I would expect some sort of separation from your protein on the imidazole gradient even if the peaks are overlapping. Also, on Q column, TEV, being an extremely basic protein, simply won't bind. If you saw the TEV band in the Q fractions, then that suggests that you may have incorrectly identified your protein bands on the SDS gel. It would be interesting to see your SDS gel. Also providing more specific details of your chromatographies may help a lot. For example, what was the approximate concentration of imidazole when your peak came out from the Ni-NTA when eluted with a gradient? What was the condition you used for Q column and what is your protein's PI? There are just too many factors that could effect the performance of the ion-exchange chromatography. On the other hand, if it is true that your protein binds to Ni-NTA so well even without a His tag, then why not try expressing it alone without a His tag? Shouldn't you be able to purify it easily with Ni-NTA? Finally, the difficulty in TEV cleavage could indicate a construction problem. I assume that your protein is N-terminally His-tagged. To my experience, TEV wants one or two more amino acids between the G/S in ENLYFQ^G/S and the folded protein domain, i.e., it wants some space on the right hand side of the cleavage site. Adding one or two amino acids after the current cleavage site may help. Zhijie From: anita p Sent: Friday, April 08, 2011 5:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Tev Cleavage issue !! Thanks everyone for your suggestions ! Artem has pointed out that low diffraction of the crystal might be because of other problems .. If you could highlight a bit more on this issue it would be helpful for me. I have tried to seperate the cleaved, uncleaved and TEV over MonoQ column but there was a single peak and all of them came out together, there was no seperation. I even tried to cleave the protein at 30 degress and it starts precipitating. I have also tried binding it to the IMAC as Martin has suggested but then I get a single peak while running the imidazole gradient and its tev, cleaved and uncleaved together. And I also get the flowthrough while loading unto the column which should be theoritically the cleaved one but it is a combination of cleaved uncleaved and tev. awaiting for bit more suggestions Anita On Fri, Apr 8, 2011 at 10:48 AM, Artem Evdokimov artem.evdoki...@gmail.com wrote: For starters, you could re-clone the protein with e.g. just a His tag or move the tag to another end, or put some distance between the end of TEV site and the protein; or perhaps use no tag at all -- or a different one? Is it possible that the tag is messing you up - yes. Is it 'probable' - I can't say that I know because I've crystallized literally dozens of proteins with His-tags attached, and more than a few with His-tag and cleavage site. I prefer a plain His-tag or a cleaved one, to be sure - but I am not quick to blame the tag (since there are so many other possible things to blame). Based on the behavior of your protein after cleavage, it may be that you have oligomer(s) forming in solution such that cleaved and uncleaved proteins do not segregate. You may wish to explore other kinds of chromatographic separation e.g. ion exchange of HIC - they may or may not work out. You can also consider cleaving your protein at lower concentration, in the presence of detergents or polyols, etc. Cheers, Artem On Thu, Apr 7, 2011 at 9:37 PM, anita p crystals...@gmail.com wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. Is it probable that they dont diffract because of the extra his tag and the tev site? I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on
Re: [ccp4bb] anisotropy vs TLS
Does anyone know what the record is for most reflections per atom? JPK On Fri, Apr 8, 2011 at 8:06 AM, Ian Tickle ianj...@gmail.com wrote: Hi Kenneth, I know that TLS is a group B factor for regions of proteins that are moving the same. You have to be a bit careful here: first B factors do not necessarily imply motion, they imply displacement (i.e. it could mean static displacements which just vary between unit cells). That's why they are called 'displacement parameters' (as in 'ADP' if it's anisotropic). Second, 'moving the same', or even 'being displaced the same' carries the connotation that you are getting information about correlated _motion_ or displacement, which is not correct either, though it may well be one interpretation of the results. Strictly TLS only gives you information about correlated _dispersion_ (in the strict sense of dispersion as 'variance'), not correlated _displacement_, though you may well interpret correlated dispersion as correlated displacement (i.e. other interpretations of the results may fit the data equally well). The difference between displacement and dispersion is illlustrated by considering a group of atoms in a molecule: if these atoms all have identical instantaneous displacements at all times, as though they were rigidly connected, that's correlated displacement. If the atoms are moving completely independently (i.e. their displacement vectors are in general all different both in direction and magnitude), but they happen to all have the same ADPs (which are a time/lattice average of the squared displacements), that's correlated dispersion. Of course in practice you will see all shades between these two extremes. It is used in low res structures. But at what resolution does one begin anisotropic, i.e individual aniso for each atom, and leave TLS out. Depends what you mean by 'low'. You can use individual ADPs when you have a sufficient data/parameter ratio, which as a rough guide I would say is around 1.5 Ang., though you would still need to use ADP restraints (as in Shel-X) at resolutions between 1.5 and say around 1 Ang (at what point you can safely drop ADP restraints beyond 1 Ang is a matter of taste). Or can one still use TLS to first compensate for large motions and then dampen down the individual atoms with aniso ADP? Not sure what you mean by 'dampen down': do you mean 'restrained'? For the purposes of refinement it would be tricky to do this in practice because TLS and ADP parameters are not independent. The first thing the refinement program does with the TLS parameters is calculate the equivalent ADPs and use those in the SF calculation, though of course the results are ultimately still expressed in terms of changes in the TLS parameters. A better way of doing this would be to use only ADPs in refinement, and interpret the results post-refinement as TLS + 'residual' ADPs. This is how TLS was used years ago for small molecules before refinement of TLS parameters directly was coded (though the residual ADPs were usually viewed as just 'random error'). The problem here is what you call 'residual', i.e. because they are not independent, you can interpret the results as either correlated displacement or correlated dispersion, and the data aren't going to help you to decide which is the more correct interpretation. If both the aniso and TLS are used, how does a person interpret the results? What programs are there to see just what is large body motions and what is atoms. I've never tried this, so I can't say if it would be a useful exercise or not. I think I would stick to the TLS + individual _Biso_ model: that is already hard enough to interpret! Cheers -- Ian -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Tev Cleavage issue !!
I'd say since you obtained crystals with your tag it is not a disturbing factor and either disordered or making contacts. So removing the tag you might end up not getting crystals in the worst case. Now to the question why they don't diffract. Did you test the old fashioned way at RT in capillary ? Maybe your freezing is the problem. The inability to purify TEVed protein suggests that you have at least a dimer perhaps or higher multimer. Since you observe three bands on the gel, uncleaved, cleaved and TEV itself. Any idea about DLS or migration behavior on a SEC column for your uncleaved protein ? Since you have crystals what I would do is crush them up and rescreen whatever commercial screens you have available using some of the crushed crystals as seed. A second option would be to take your current condition and modify it with say the most frequent 12 cryo solutions added in maybe 5-10% effective concentration and see if you still obtain crystals. Before freezing them I would then freeze directly from the drop and a second crystal would be soaked into whatever concentration of the cryo is required to properly freeze. I bet you will find something that works. Of course you don't stop there. Once your crystals are on the gonio and diffract or don't diffract you will flash anneal them once or multiple times and report back in a nice table which condition resulted in the 2.3A dataset. So you have roughly 146 experiments to do alone from cryo- optimization. Good luck ! Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Apr 7, 2011, at 22:37, anita p crystals...@gmail.com wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. Is it probable that they dont diffract because of the extra his tag and the tev site? I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. Is there a possible way to approach this problem? Suggestions awaited Anita
Re: [ccp4bb] Script / program to change chain ID 's in symmetry mates - MOLEMAN
pdbset does this nicely: pdbset xyzin mono.pdb xyzout dimer.pdb eof symgen x, y, z symgen 1-x, 1-y, z !select chain A B C D E F G H I K chain symm 2 A N chain symm 2 B O chain symm 2 C P chain symm 2 D Q chain symm 2 E R chain symm 2 F S chain symm 2 G T chain symm 2 H U chain symm 2 J W chain symm 2 K X chain symm 2 L Y chain symm 2 X K eof Felix Frolow wrote: MOLEMAN from the Uppsala Software Factory by Gerard Kleywegt of course It is easy as to say Do it! FF Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Apr 8, 2011, at 14:17 , krishan wrote: Dear CCP4BB members, We are using a script written in python to generate symmetry mates for a given pdb file using PYMOL. After generating symmetry mates we want to combine all the symmetry molecules in a single PDB file with all the chains having unique chain IDs. Since all the symmetry mates have same chain ID's I was wondering if some one knows a script that can give unique chain ID for each symmetry mate. We are interested in script because that dataset that we are handling is large. I thank you all in advance for your help. Best, Krishan
Re: [ccp4bb] Script / program to change chain ID 's in symmetry mates
Krishan, Here is a simple stand-alone python script that should do what you want. Greg #!/usr/bin/python # # rechainpdb.py # usage: # python rechainpdb.py mypdbfile1.pdb mypdbfile2.pdb mypdbfile3.pdb # # This will generate a new file with all pdbfile (ATOMs and HETATMs) in a single # file, with each pdbfile having a different chain id. Max pdbs is 26. # #ATOM 1 N ASP W 175 24.971 27.228 -12.398 1.00161.17 N #12345678901234567890123456789012345678901234567890123456789012345678901234567890 # 1 2 3 4 5 6 7 8 # # chain id is char 22 import sys if (len(sys.argv)==1): # no arguments were given print +++ print YOU NEED TO SUPPLY A LIST OF PDB FILES. print e.g. print python rechainpdb.py mypdbfile1.pdb mypdbfile2.pdb print print This will generate a single new file with each pdb in print the list having a different chain id. print +++ sys.exit(\nExiting...\n) #possible chain ids CHAINLIST=ABCDEFGHIJKLMNOPQRSTUVWXYZ filelist=[] #OUTPUT file name - default is to use the name of first pdb outputfilename=sys.argv[1][:-4]+_allfiles.pdb # generate a list of lists, where each file listed after the script # is a list of strings (lines) for x in range(1,len(sys.argv)): currentfile = open(sys.argv[x],'r') crudefile=currentfile.readlines() currentfile.close() cleanfile=[] for line in crudefile: cleanfile.append(line.strip()) filelist.append(cleanfile) # set up output file outputfile = open(outputfilename,'w') # write out one large pdbfile with each having a unique chain id for x in range(0,len(filelist)): for line in filelist[x]: if ((line[0:4]==ATOM) or (line[0:6]==HETATM)): print outputfile, line[:21]+CHAINLIST[x]+line[22:] print print # # # # # # # # # # # # # # # # # # # # # # # print print The following chain ids were given to the input pdbfiles: print for i in range(1,len(sys.argv)): print chain id given:, CHAINLIST[i-1],sys.argv[i] print - - - - - - - - - - - - - - - - - - - - - - - - print print Final file created:,outputfilename print print # # # # # # # # # # # # # # # # # # # # # # # On Apr 8, 2011, at 7:17 AM, krishan wrote: Dear CCP4BB members, We are using a script written in python to generate symmetry mates for a given pdb file using PYMOL. After generating symmetry mates we want to combine all the symmetry molecules in a single PDB file with all the chains having unique chain IDs. Since all the symmetry mates have same chain ID's I was wondering if some one knows a script that can give unique chain ID for each symmetry mate. We are interested in script because that dataset that we are handling is large. I thank you all in advance for your help. Best, Krishan -- Department of Biophysics Johns Hopkins University 302 Jenkins Hall 3400 N. Charles St. Baltimore, MD 21218 Phone: (410) 516-7850 (office) Phone: (410) 516-3476 (lab) Fax: (410) 516-4118 gdbow...@jhu.edu
Re: [ccp4bb] anisotropy vs TLS
On Friday, April 08, 2011, Jacob Keller wrote: Does anyone know what the record is for most reflections per atom? It goes up as high as 300 reflections per modelled atom in some virus structures modelled with strict NCS. There's one outlier in the current PDB set with 1000 refls/atom. I forget what the story is on that one. Ethan
[ccp4bb] problems with tinker energy parameters
Hi all, I've been getting blocked running minimize/optimize from tinker. The issue is atom types and energy parameters. For small molecules, I can hand check the types and get tinker to complete successfully. However as my molecules get larger (and I'm attempting to automate a screening protocol), by hand isn't going to scale. I've tried several approaches to parsing a file and getting types set. As examples, I've used obabel to convert a PDB to a TXYZ which should correctly target the atom types for the MM2 parameter set. I've also used MolKit and Pybabel to attempt the same thing. It's frustrating that I can load the molecule into Avogadro or Ghemical and have no issues at all with energy minimization. 1. Is there an option to tinker programs to tell me exactly where the issue is? 2. Does anyone have a reliable atom type setting script? 3. Is there a force field set I should be using besides MM2? Thanks in advance for any assistance... britt
Re: [ccp4bb] program to calculate electron density at x,y,z (SUMMARY)
Thanks to everyone for their suggestions. The closest solution was 1. Expand dataset in P1 using SFTOOLS (keyword EXPAND) 2. Write it out in text file (WRITE data.hkl format(3i5,2f16.3) col col1 col2) 3. Use program HYDENS (Bart Hazes) It should be noted that the current version of HYDENS only works in orthorhombic spacegroups (although there may be a workaround of pre-calculating fractional coordinates with coordconv and then setting the unit cell to 1x1x1). This was the only solution that actually calculates electron density at the atomic positions. The rest of options were all to calculate the map and then interpolate it to the select (x,y,z). Several options exist for that (in the order they were received): 1. MAPMAN (PEEK keyword) (Gerard Kleywegt, James Holton, Edward Berry) 2. CCTBX (Pavel Afonin) 3. COOT (density_at_point, probably uses clipper libs) (Paul Emsley) 4. CHIMERA (Values at Atom Positions) (Eric Pettersen) On Fri, 2011-04-01 at 11:16 -0400, Ed Pozharski wrote: I need to calculate the electron density values for a list of spatial locations (e.g. atom positions in a model) using an mtz-file that already contains map coefficients. To write my own code may be easier than I think (if one can manipulate mtz columns, isn't the only problem left how to incorporate symmetry-related reflections?), but I would need an alternative at least for troubleshooting purposes. So, Does anyone know of a software tool that can calculate point electron density for every atom in a structure? If I would have to bring a dependency into this, the best choice for me would be clipper libs. Thanks in advance, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
[ccp4bb] Assigning secondary structure
Hello all, Given a PDB file of a newly solved protein structure, what is the standard procedure for assigning regions of secondary structure? And by this I mean to ask, how does one decide which residues form beta strands, which alpha helices, and so on? Is DSSP sufficient for this? Are we supposed to manually walk through the entire molecule and assign secondary structure as we deem appropriate based on hydrogen bonding behaviour? Some other procedure? And what of structures solved to ~2.7 A (or worse) where we can't be sure of H-bonding. Cheers, Cale
Re: [ccp4bb] off topic: Is deglycosylation necessary for crystallization?
First, I don't think glycosylation occurs on lysine residues. You should try to crystallize it as is, if that doesn't work, try to remove the glycosylation with some or all of the following: EndoH, PNGaseF, EndoF1, EndoF3. We have used them all to various degrees of efficiency to remove glycans from insect-expressed proteins. It may or may not crash out, that is protein dependent. You can also mutagenize the potential N-linked sites which has been very successful for me. There is no way to say if an insect signal sequence works better then the native signal sequence, the only way to know is to try both. If you have a lot of protein in the cells, that might suggest signal sequence cleavage is inefficient, and perhaps and insect signal sequence would be better. Sometimes adding a gly-gly linker after the signal sequence will improve cleavage. If you do deglycosylate I wouldn't worry about purifying the deglycosylated from glycosylated, just let the crystallization experiment select for the crystallizable material. In other words, separating different glycoforms is like any other purification, you just have to keep trying things until you get the results you want. In theory a lectin column would do it, but those aren't 'high-resolution' separations (in my hands...) In short, I would try everything you can think of until you get good crystals. Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Fri, Apr 8, 2011 at 2:06 PM, joybeiyang joybeiy...@gmail.com wrote: Dear All, I am currently working with a protein which is glycosylated on several N and K. I use insect cells for secretion expression, so the protein was supposed to be correctly glycosylated. However after several steps of purification, there are still a minor doublet under the major band, which later on was proved to be my target protein without glycosylation. My question is: 1. Should I neglect the doublet and just crystallize at this point? (The unglycosylated form compare to the glycosylated form were about 5%:95%) 2. Should I deglycosylate the protein before crystallization and if so could anyone share with me a protocol? (Is precipitation likely to occur during deglycosylation?) 3. Was the unglycosylated form arise from cell death during cell culture or was it arise from the overexpression of target protein and inadequacy of glycosylation machinery and false secretion? How could I prevent it? Will a insect specific signal peptide work better than the native signal peptide? 4. Is it possible to separate the glycosylated and unglycosylated form through either a monoQ/S or a Lectin Sepharose 4B column? Thank you very much for your input in advance and I greatly appreciate it! With all my best wishes, Bei 2011-04-08 joybeiyang
Re: [ccp4bb] off topic: Is deglycosylation necessary for crystallization?
Hi Bei, First of all, I think you meant N-linked glycans, and the following discussion are based on this assumption. I am not aware of any direct glycosylation on lysines except for the O-glycosylation of hydroxylysines, which is really rare. 1. If you have enough protein, you should screen both deglycosylated and un-deglycosylated. Some proteins do crystallize with full N-linked glycans. 2. If you do not have enough protein to try everything, or if the un-degly'ed won't crystallize, deglycosylation should be tested. Two things you want to know: 1) how easy it will be to deglycosylate the protein to nearly homogenous; 2) how much less soluble the protein becomes after certain glycosidase treatment. 3. You need to try different glycosidases. PNGase F is the one that you should try first as it removes the whole N-linked glycan. Then come the endoglycosidases: Endo F1, F3, Endo H, which leave a single GlcNAc on the Asn. If these all fail, then consider exoglycosidases: sialidase, galactosidase, hexaminidase, mannosidase, etc.. Generally speaking, the smaller the resulting N-glycan residue is, the less flexibility you have left on the protein, hence higher chance or getting highly diffractive crystals. The condition you need for a efficient cleavage with certain enzyme can vary a lot. This is caused mainly by the accessibility of the cleavage site. In the most facile cases, treatment at 4C with trace amout(1:1) of PNGase O/N can result in complete removal of the N-glycans. On the other extreme, for certain proteins you may need more enzyme than your substrate protein and cut at 37C, and still only get a partial deglycosylation. Being the enzyme that cuts the deepest on the N-glycan, one problem with PNGase is that the cleavage site (the NH2-C linkage on the Asn side chain) on certain N-linked glycosylation site could be quite inaccessible. And this actually happens a lot. In many cases, you may find you have to heat the reaction to 37C in order to get certain level of PNGase cleavage. For highly N-glycosylated proteins (having more than 4 or 5 sites), PNGase treatment is almost sure to fail. The other thing is, often, PNGase treatment results in poorly soluble proteins as the removal of the N-glcan exposes some surface patch for aggregation, or destabilized the protein itself. The Endo F1, F3, H, especially Endo H, can often result in better completeness of N-glycan removal, and better solubility of the protein, with the expense of one GlcNAc left on the protein. However, these ones require your N-glycans to be of certain type. For example, Endo H requires your N-glycans to be high-mannose type, which has to be generated from certain cell lines, or by a series of exo-glycosidase treatment. (But for insect cell produced proteins, endo H is very likely to work, see below.) 4. You can often achieve some degree of seperation of the glysylated form and un-glycosylated on ion-exchange, even when the N-glycans are not the charged type(for example, high mannose). This is likely due to a solubility difference of the differently glycosylated species. In the cases you have charged N-glycans, for example, proteins made from normal mammalian cells, the negative charge of the sialic acid on the N-glycans could be so significant, that it basically mask the protein's own surface charge. Consequently, the protein, regardless of its real PI, will bind to Q, while the deglycoylated will still behave normally. This will result in the separation of the two or more species. Actually, highly glycosylated protein with complex type glycans (with terminal sialic acid) often run as multiple peaks on ion-exchange, due to the heterogeneity of the N-glycan. HIC should separate the deglycolsylated protein too. Lectin columns should work. However the applicability of lectins depends on your N-glycan's type, and this could be a quite complicated topic to discuss. Also I am not aware of a good lectin for high-mannose. 5. Finally, to my knowledge, proteins produced from insect cells are mainly high-mannose type (I never experimentally tested this though, just took this idea from some literature). This means: 1) the N-glycans from insect cells are likely susceptible to Endo H treatment, which is great; 2) the N-glycans are not charged; 3) the N-glcans themselves are more or less homogenous as they are likely to be Man3,4,5 only, and are also smaller than the complex type glycans made from mammalian cells. This is a bless for crystallization without deglycosylation. ...OK, I found a paper right on this issue: http://www.ncbi.nlm.nih.gov/pubmed/16997012 6. The incomplete glycosylation could simply be that the N-glycosulation machinary missed a few molecules. Especially if the protein is not from the same host species and is over produced. I would say: do not worry about it. Zhijie From: joybeiyang Sent: Friday, April 08, 2011 2:06 PM To:
Re: [ccp4bb] off topic: Is deglycosylation necessary for crystallization?
You may also want to have a look at this summary of a 2006 discussion: http://www.mail-archive.com/ccp4bb@dl.ac.uk/msg01697.html From: joybeiyang Sent: Friday, April 08, 2011 2:06 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic: Is deglycosylation necessary for crystallization? Dear All, I am currently working with a protein which is glycosylated on several N and K. I use insect cells for secretion expression, so the protein was supposed to be correctly glycosylated. However after several steps of purification, there are still a minor doublet under the major band, which later on was proved to be my target protein without glycosylation. My question is: 1. Should I neglect the doublet and just crystallize at this point? (The unglycosylated form compare to the glycosylated form were about 5%:95%) 2. Should I deglycosylate the protein before crystallization and if so could anyone share with me a protocol? (Is precipitation likely to occur during deglycosylation?) 3. Was the unglycosylated form arise from cell death during cell culture or was it arise from the overexpression of target protein and inadequacy of glycosylation machinery and false secretion? How could I prevent it? Will a insect specific signal peptide work better than the native signal peptide? 4. Is it possible to separate the glycosylated and unglycosylated form through either a monoQ/S or a Lectin Sepharose 4B column? Thank you very much for your input in advance and I greatly appreciate it! With all my best wishes, Bei 2011-04-08 joybeiyang
Re: [ccp4bb] Assigning secondary structure
Just use dssp and cite it. For cartoon figures, if you provide a disclaimer, it doesn't hurt to tweak it a bit to make it look better. The idea is that readers are supposed to know that a protein doesn't really look like a cartoon--and those who don't understand the distinction probably won't have enough knowledge to have an opinion about your assignments anyway. James On Apr 8, 2011, at 9:19 AM, Cale Dakwar wrote: Hello all, Given a PDB file of a newly solved protein structure, what is the standard procedure for assigning regions of secondary structure? And by this I mean to ask, how does one decide which residues form beta strands, which alpha helices, and so on? Is DSSP sufficient for this? Are we supposed to manually walk through the entire molecule and assign secondary structure as we deem appropriate based on hydrogen bonding behaviour? Some other procedure? And what of structures solved to ~2.7 A (or worse) where we can't be sure of H-bonding. Cheers, Cale
Re: [ccp4bb] program to calculate electron density at x,y,z (SUMMARY)
Hi Ed, thanks for nice summary! Just a quick update while on this subject: (using nightly build dev-724 an up) you will be able to get density value at a given point using just one command: phenix.map_value_at_point map_coeffs.mtz label=2FOFC point=1 2 3 point=4 5 6 where you can specify as many points as you wish. You can request to use either sigma or volume scaled map, as well as you can specify the grid_step for map finess. You can use a parameter file if the number of points is too large to be specified in the command line. Pavel. On Fri, Apr 8, 2011 at 9:07 AM, Ed Pozharski epozh...@umaryland.edu wrote: Thanks to everyone for their suggestions. The closest solution was 1. Expand dataset in P1 using SFTOOLS (keyword EXPAND) 2. Write it out in text file (WRITE data.hkl format(3i5,2f16.3) col col1 col2) 3. Use program HYDENS (Bart Hazes) It should be noted that the current version of HYDENS only works in orthorhombic spacegroups (although there may be a workaround of pre-calculating fractional coordinates with coordconv and then setting the unit cell to 1x1x1). This was the only solution that actually calculates electron density at the atomic positions. The rest of options were all to calculate the map and then interpolate it to the select (x,y,z). Several options exist for that (in the order they were received): 1. MAPMAN (PEEK keyword) (Gerard Kleywegt, James Holton, Edward Berry) 2. CCTBX (Pavel Afonin) 3. COOT (density_at_point, probably uses clipper libs) (Paul Emsley) 4. CHIMERA (Values at Atom Positions) (Eric Pettersen) On Fri, 2011-04-01 at 11:16 -0400, Ed Pozharski wrote: I need to calculate the electron density values for a list of spatial locations (e.g. atom positions in a model) using an mtz-file that already contains map coefficients. To write my own code may be easier than I think (if one can manipulate mtz columns, isn't the only problem left how to incorporate symmetry-related reflections?), but I would need an alternative at least for troubleshooting purposes. So, Does anyone know of a software tool that can calculate point electron density for every atom in a structure? If I would have to bring a dependency into this, the best choice for me would be clipper libs. Thanks in advance, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] Tev Cleavage issue !!
Hi Anita, so you tested your crystals inhouse, any idea how they do at the synchrotron ? Still no diffraction ? Since it's a hexamer I would expect the His-tag to be not so important and would rather rescreen with seeding first to see if any other conditions might result in diffracting crystals. Annealing did not help ? Can you slow down the growth of the crystals ? Another option you could try out is limited proteolysis and see if you get a stable fragment, then purify it via SEC and try it with the initial conditions but also rescreening. How big is your monomer ? Do you have a structural homolog / prediction ? Can you make better guesses what you should trim of by design ? Jürgen On Apr 8, 2011, at 11:43 PM, anita p wrote: Hi Jürgen I tried it by RT capillary as well as under liquid nitrogen, both dont diffract. I ran those on gel and then I confirmed that it is protein. On the SEC it runs as a hexamer.and the DLS shows that it is polydispersed. with regards Anita On Fri, Apr 8, 2011 at 10:03 PM, Jürgen Bosch jubo...@jhsph.edumailto:jubo...@jhsph.edu wrote: I'd say since you obtained crystals with your tag it is not a disturbing factor and either disordered or making contacts. So removing the tag you might end up not getting crystals in the worst case. Now to the question why they don't diffract. Did you test the old fashioned way at RT in capillary ? Maybe your freezing is the problem. The inability to purify TEVed protein suggests that you have at least a dimer perhaps or higher multimer. Since you observe three bands on the gel, uncleaved, cleaved and TEV itself. Any idea about DLS or migration behavior on a SEC column for your uncleaved protein ? Since you have crystals what I would do is crush them up and rescreen whatever commercial screens you have available using some of the crushed crystals as seed. A second option would be to take your current condition and modify it with say the most frequent 12 cryo solutions added in maybe 5-10% effective concentration and see if you still obtain crystals. Before freezing them I would then freeze directly from the drop and a second crystal would be soaked into whatever concentration of the cryo is required to properly freeze. I bet you will find something that works. Of course you don't stop there. Once your crystals are on the gonio and diffract or don't diffract you will flash anneal them once or multiple times and report back in a nice table which condition resulted in the 2.3A dataset. So you have roughly 146 experiments to do alone from cryo- optimization. Good luck ! .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-3655tel:%2B1-410-955-3655 http://web.mac.com/bosch_lab/ On Apr 7, 2011, at 22:37, anita p crystals...@gmail.commailto:crystals...@gmail.com wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. Is it probable that they dont diffract because of the extra his tag and the tev site? I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. Is there a possible way to approach this problem? Suggestions awaited Anita .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
