Re: [ccp4bb] Copy NCS chain in coot
Dear Zhang yu, I also found that same problem recently. I think is a small bug from Coot. It will only copy NCS if the molecule's name you want to copy is molecule A. So maybe will work if you rename your built chain as A. Good luck! Javier On 31/03/11 00:16, zhang yu wrote: Dear all, I have two fold NCS in ASU. I could find phase by molecular replacement, but I have to build other chains by myself, since the model is not complete. During model building in coot, I built one chain in one NCS, and I merged this chain into the previous molecule. Now it is the chain M'. When I tried to directly copy this chain to its NCS mate, It didn't work. I followed the instruction posted beforehttp://www.mail-archive.com/coot@jiscmail.ac.uk/msg01984.html;. What I did is 1. Change the master chain to chain M Draw-NCS ghost control-change to chain M (The terminal said 'there are no ghosts when I change the master chain to chain M) 2. go to Extension-NCS-copy chain-, coot ask to fill the master chain ID, and I typed M Nothing happened. Could someone tell me what is the correct procedure to do it ? Bye the way, for those chains present in both NCS maps, I modified one chain and applied the change to its NCS mate by using the same above procedure, and it worked. Thanks -- Yu Zhang HHMI associate Waksman Institute, Rutgers University 190 Frelinghuysen Rd. Piscataway, NJ, 08904
Re: [ccp4bb] [phenixbb] what to do with disordered side chains
If a part of sequence has no density, this part will be cut from coordinates. If the side chain of a residue is lack of density, the opinion is not converged. What about the ligand, if no density is observed? Huanwang Ed Pozharski wrote: The results of the online survey on what to do with disordered side chains (from total of 240 responses): Delete the atoms 43% Let refinement take care of it by inflating B-factors41% Set occupancy to zero12% Other 4% Other suggestions were: - Place atoms in most likely spot based on rotomer and contacts and indicate high positional sigmas on ATMSIG records - To invent refinement that will spread this residues over many rotamers as this is what actually happened - Delet the atoms but retain the original amino acid name - choose the most common rotamer (B-factors don't inflate, they just rise slightly) - Depends. if the disordered region is unteresting, delete atoms. Otherwise, try to model it in one or more disordered model (and then state it clearly in the pdb file) - In case that no density is in the map, model several conformations of the missing segment and insert it into the PDB file with zero occupancies. It is equivalent what the NMR people do. - Model it in and compare the MD simulations with SAXS - I would assumne Dale Tronrod suggestion the best. Sigatm labels. - Let the refinement inflate B-factors, then set occupancy to zero in the last round. Thanks to all for participation, Ed.
Re: [ccp4bb] titering baculovirus ?
I checked with someone in our protein production group and got the following response: We also stopped doing the virus titration with the plaque assay and instead are performing expression test with different concentration of virus from the 3rd amplification. But for some viruses we still have doubts concerning the amplification success, so we are now evaluating a new technology using qPCR with the following kit (http://oetltd.com/products/category/baculoquant/). So you might have a look and see if it could be useful for your group. We would also be curious to hear if anyone else has experience with this approach. I hope this helps. Jay -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nathaniel Clark Sent: Wednesday, March 30, 2011 11:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] titering baculovirus ? We don't have a problem getting them to stick to the plates in serum-free media, or in 5% FBS media. The more challenging part is getting the plating density just right, too low and the plaques are too big, to high and they are too small. Or if the cells dry out, or if your agarose overlay is too hot, etc... However, we have actually stopped titering all together. We find early stocks (from co-transfection, or plaque purification) are 'low', but after ~2 rounds of amplification in adherent culture of the 'low' titer stock(using a large volume of low-titer virus in a t25 flask), we can add ~0.5 ml to a 30-100 ml shake flask of Sf9's (serum free media) and get a high titer stock( ie. 10^8 pfu/ml). From there we amplify with ~ 10 mls of virus in a 1 L shaker culture, and we have our large volume high-titer stock. Sometimes we will incubate the cells in pure virus stock in a t25 flask for 1 hour, take the virus off, and add fresh media, as a way to rescue low-titer stocks. If you are just trying to titer (not plaque-purify), you can just take 10 fold dilutions of your virus, and do several small scale infections in 6 well plates, 10 ml shaker cultures, whatever you prefer. At the lowest virus concentration where you see a synchronous infection (judged by protein expression levels, or cell-diameter if you have a cell counter, or by viewing with a trained eye), you call that an MOI=1. From there you know the number of cells in the plate, and the volume of virus you added, so you can calculate an effective titer. Plaque assays are really difficult and slow, and if you are just trying to make protein, an effective titer is fine, the absolute number isn't that helpful, Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Wed, Mar 30, 2011 at 5:14 PM, Gloria Borgstahl gborgst...@gmail.com wrote: Hi Guys, we are learning to work with Sf9 cells and Carol in my lab wanted me to ask you the following question. Many thanks for any help, G I need to titer a baculovirus stock in my suspension-adapted Sf9 cells. I know that these can be encouraged to attach better to tissue culture plastic if they have added FBS (about 10%), but am not sure that they will not be migrating and hiding plaques. Does anyone have suggestions about how to keep them more firmly anchored during the baculovirus titration, or about another cell line that we could use instead? This message has been scanned for malware by Websense. www.websense.com
Re: [ccp4bb] [phenixbb] what to do with disordered side chains
Judging from some examples in the literature, if there's no density for the ligand, you publish in Nature! On 31 Mar 2011, at 14:32, Huanwang Yang wrote: If a part of sequence has no density, this part will be cut from coordinates. If the side chain of a residue is lack of density, the opinion is not converged. What about the ligand, if no density is observed? Huanwang Ed Pozharski wrote: The results of the online survey on what to do with disordered side chains (from total of 240 responses): Delete the atoms 43% Let refinement take care of it by inflating B-factors41% Set occupancy to zero12% Other 4% Other suggestions were: - Place atoms in most likely spot based on rotomer and contacts and indicate high positional sigmas on ATMSIG records - To invent refinement that will spread this residues over many rotamers as this is what actually happened - Delet the atoms but retain the original amino acid name - choose the most common rotamer (B-factors don't inflate, they just rise slightly) - Depends. if the disordered region is unteresting, delete atoms. Otherwise, try to model it in one or more disordered model (and then state it clearly in the pdb file) - In case that no density is in the map, model several conformations of the missing segment and insert it into the PDB file with zero occupancies. It is equivalent what the NMR people do. - Model it in and compare the MD simulations with SAXS - I would assumne Dale Tronrod suggestion the best. Sigatm labels. - Let the refinement inflate B-factors, then set occupancy to zero in the last round. Thanks to all for participation, Ed. -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
[ccp4bb] Question about TEV cleavage
Hello all: I am curious about what level of recovery is reasonable when performing a TEV cleavage to remove 6HIS tags (N-terminal) from a protein. Currently we are experiencing 50% loss of soluble protein after TEV cleavage, and feel that this is too much. Are there better systems for his tag cleavage that have better soluble protein recoveries than TEV? Many thanks (in advance) Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
[ccp4bb] off topic: protease identification
Hi all: I was wondering if anyone had any tips on identifying proteases. I have a protein for which I know the proteolytic cleavage site. What are the best ways to identify the protease that does the cutting either: 1) bioinformatically (i.e., a good database to search using the cleavage site or a consensus) 2) experimentally (some engineered substrate to trap/identify the substrate or any other method?) Thanks in advance -Tom Tom J. Brett, PhD Assistant Professor of Medicine Division of Pulmonary and Critical Care Washington University School of Medicine Campus Box 8052, 660 S. Euclid Saint Louis, MO 63110 http://brettlab.dom.wustl.edu/
Re: [ccp4bb] what to do with disordered side chains
Dear Quyen, I am afraid you won't get any better answers than you got so far. There is no holy bible telling you what to do with disordered side chains. I fully agree with James that you should try to get the best possible model, which best explains your data and that will be your decision. Here are my 2 cents: -If you see alternative positions, you have to build them. -If you do not see alternative positions, I would not replace one fantasy (some call it most likely) orientation with 2 or 3 fantasy orientations. -I personally belong to the let the B-factors take care of it camp, but that is my personal opinion. Leaving side chains out could lead to misinterpretations by slightly less savy users of our data, especially when charge distributions are being studied. Besides, we know (almost) for sure that the side chain is there, it is only disordered and as we just learned, even slightly less savy users know what flaming red side chains mean. Even if they may not be mathematically entirely correct, huge B-factors clearly indicate that there is disorder involved. -I would not let occupancies take up the slack since even very savy users have never heard of them and again, the side chain is fully occupied, only disordered. Of course if you build alternate positions, you have to divede the occupancies amongst them. Best, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Quyen Hoang Sent: Thursday, March 31, 2011 3:55 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] what to do with disordered side chains We are getting off topic a little bit. Original topic: is it better to not build disordered sidechains or build them and let B-factors take care of it? Ed's poll got almost a 50:50 split. Question still unanswered. Second topic introduced by Pavel: Your B-factors are valid within a harmonic (small) approximation of atomic vibrations. Larger scale motions you are talking about go beyond the harmonic approximation, and using the B-factor to model them is abusing the corresponding mathematical model. And that these large scale motions (disorders) are better represented by alternative conformations and associated with them occupancies. My question is, how many people here do this? If you're currently doing what Pavel suggested here, how do you decide where to keep the upper limit of B-factors and what the occupancies are for each atom (data with resolution of 2.0A or worse)? I mean, do you cap the B-factor at a reasonable number to represent natural atomic vibrations (which is very small as Pavel pointed out) and then let the occupancies pick up the slack? More importantly, what is your reason for doing this? Cheers and thanks for your contribution, Quyen On Mar 30, 2011, at 5:20 PM, Pavel Afonine wrote: Mark, alternative conformations and associated with them occupancies are to describe the larger scale disorder (the one that goes beyond the B-factor's capability to cope with). Multi-model PDB files is another option. Best, Pavel. On Wed, Mar 30, 2011 at 2:15 PM, VAN RAAIJ , MARK JOHAN mjvanra...@cnb.csic.es wrote: yet, apart from (and additionally to) modelling two conformations of the side-chain, the B-factor is the only tool we have (now). Quoting Pavel Afonine: Hi Quyen, (...) And if B-factor is an estimate of thermo-motion (or static disorder), then would it not be reasonable to accept that building the side-chain and let B-factor sky rocket might reflect reality more so than not building it? NO. Your B-factors are valid within a harmonic (small) approximation of atomic vibrations. Larger scale motions you are talking about go beyond the harmonic approximation, and using the B-factor to model them is abusing the corresponding mathematical model. http://www.phenix-online.org/newsletter/CCN_2010_07.pdf Pavel. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco
Re: [ccp4bb] Question about TEV cleavage
Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng
Re: [ccp4bb] titering baculovirus ?
Though I'm not meaning to turn this into a plaque assay burn session, for many reasons we have also abandoned it and instead titer our baculovirus stocks (P3 and P4) using flow cytometry. We use an antibody against viral gp64 that has been labeled with PE and stain infected cells in 96 well plates with the PE-antibody. We then measure PE fluorescence on a flow cytometer (you can also do cell counts, viability determinations, etc.). We equate 1 fluorescent event as 1 infected cell. Since we know how many cells have been plated in each well, we can determine the percentage infected in each well. We calculate a non-normalized titer from this data alone or we compare this data to a standardized virus and determine a normalized titer using a standard curve. From infection to having a titer in hand takes about 24 hours. Of course, the potential bottleneck is access to a flow cytometer! I can give more experimental details off-board. I should say that for getting an idea of relative titers and to test protein expression on a small scale, we also do the effective titer tests as suggested by Nat, with cell morphology and immunoblots as our read-out of virus potency and recombinant protein expression, respectively. No doubt, this will get you a long way but at some point, I argue, you need to determine an actual, absolute titer for duplication of results, troubleshooting, monitoring virus health over time, publications, etc. HTH- Brad On Thu, Mar 31, 2011 at 9:23 AM, Bertrand, Jay Aaron [Nervianoms] jay.bertr...@nervianoms.com wrote: I checked with someone in our protein production group and got the following response: We also stopped doing the virus titration with the plaque assay and instead are performing expression test with different concentration of virus from the 3rd amplification. But for some viruses we still have doubts concerning the amplification success, so we are now evaluating a new technology using qPCR with the following kit ( http://oetltd.com/products/category/baculoquant/). So you might have a look and see if it could be useful for your group. We would also be curious to hear if anyone else has experience with this approach. I hope this helps. Jay -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nathaniel Clark Sent: Wednesday, March 30, 2011 11:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] titering baculovirus ? We don't have a problem getting them to stick to the plates in serum-free media, or in 5% FBS media. The more challenging part is getting the plating density just right, too low and the plaques are too big, to high and they are too small. Or if the cells dry out, or if your agarose overlay is too hot, etc... However, we have actually stopped titering all together. We find early stocks (from co-transfection, or plaque purification) are 'low', but after ~2 rounds of amplification in adherent culture of the 'low' titer stock(using a large volume of low-titer virus in a t25 flask), we can add ~0.5 ml to a 30-100 ml shake flask of Sf9's (serum free media) and get a high titer stock( ie. 10^8 pfu/ml). From there we amplify with ~ 10 mls of virus in a 1 L shaker culture, and we have our large volume high-titer stock. Sometimes we will incubate the cells in pure virus stock in a t25 flask for 1 hour, take the virus off, and add fresh media, as a way to rescue low-titer stocks. If you are just trying to titer (not plaque-purify), you can just take 10 fold dilutions of your virus, and do several small scale infections in 6 well plates, 10 ml shaker cultures, whatever you prefer. At the lowest virus concentration where you see a synchronous infection (judged by protein expression levels, or cell-diameter if you have a cell counter, or by viewing with a trained eye), you call that an MOI=1. From there you know the number of cells in the plate, and the volume of virus you added, so you can calculate an effective titer. Plaque assays are really difficult and slow, and if you are just trying to make protein, an effective titer is fine, the absolute number isn't that helpful, Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Wed, Mar 30, 2011 at 5:14 PM, Gloria Borgstahl gborgst...@gmail.com wrote: Hi Guys, we are learning to work with Sf9 cells and Carol in my lab wanted me to ask you the following question. Many thanks for any help, G I need to titer a baculovirus stock in my suspension-adapted Sf9 cells. I know that these can be encouraged to attach better to tissue culture plastic if they have added FBS (about 10%), but am not sure that they will not be migrating and hiding plaques. Does anyone have suggestions about how to keep them more firmly anchored during the baculovirus titration, or about another cell line that we could use instead? This message has been scanned for malware by Websense.
[ccp4bb] problem of conventions
Dear all, I would like to share my experiencde with a rather unexpected problem of indexing conventions. Perhaps I can save people some time I have a crystal in the more unusual P21212 space-group (No 18). Its unit cell lengths are bac (please note). I systematically use XDS for data integration, since so far it was able to handle even the most horrible-looking spots. Now XDS indexed my data in space-group 18, but with the axes order abc! It had, in fact, invented a space-group P22121, which does not exist. I did not realise this until I had spent a couple of weeks with beautiful peaks in rotation functions, but hopeless results in translation functions. It wasn't until I looked more closely into the definition of the screw axes that I realised the problem. POINTLESS does not allow a reindexing of reflexions within the same space-group, but fortunately REINDEX did the trick at the level of intensities, because I like to use SCALA for careful scaling of my data. So, basically, beyond just warning people who might encounter similar problems, I was wo,dering if XDS could perhaps reindex reflexions according to Int. Table conventions once the screw axes of a crystal system have been identified? With best wishes, Anita Anita Lewit-Bentley Unité d'Immunologie Structurale CNRS URA 2185 Département de Biologie Structurale Chimie Institut Pasteur 25 rue du Dr. Roux 75724 Paris cedex 15 FRANCE Tel: 33- (0)1 45 68 88 95 FAX: 33-(0)1 40 61 30 74 email: ale...@pasteur.fr
Re: [ccp4bb] problem of conventions
If you are using CCP4, it can accomodate P22121. However, just reindex in CCP4 to the correct setting with P21212. Bernie Santarsiero On Thu, March 31, 2011 9:28 am, Anita Lewit-Bentley wrote: Dear all, I would like to share my experiencde with a rather unexpected problem of indexing conventions. Perhaps I can save people some time I have a crystal in the more unusual P21212 space-group (No 18). Its unit cell lengths are bac (please note). I systematically use XDS for data integration, since so far it was able to handle even the most horrible-looking spots. Now XDS indexed my data in space-group 18, but with the axes order abc! It had, in fact, invented a space-group P22121, which does not exist. I did not realise this until I had spent a couple of weeks with beautiful peaks in rotation functions, but hopeless results in translation functions. It wasn't until I looked more closely into the definition of the screw axes that I realised the problem. POINTLESS does not allow a reindexing of reflexions within the same space-group, but fortunately REINDEX did the trick at the level of intensities, because I like to use SCALA for careful scaling of my data. So, basically, beyond just warning people who might encounter similar problems, I was wo,dering if XDS could perhaps reindex reflexions according to Int. Table conventions once the screw axes of a crystal system have been identified? With best wishes, Anita Anita Lewit-Bentley Unité d'Immunologie Structurale CNRS URA 2185 Département de Biologie Structurale Chimie Institut Pasteur 25 rue du Dr. Roux 75724 Paris cedex 15 FRANCE Tel: 33- (0)1 45 68 88 95 FAX: 33-(0)1 40 61 30 74 email: ale...@pasteur.fr
Re: [ccp4bb] Copy NCS chain in coot
Dear Javier, Thanks for your reply. I am using coot 0.6.1, and I could copy molecules with names besides A only if chains are already present in both NCS. For chains which are only present in one NCS , I did find out a way to copy to the other NCS by using 'Find NCS ligand' , although it is not a ligand. For example, there are already chain A - chain D in the first NCS, and chain E-chain H in the second NCS. I build chain 'M' from the scratch in the first NCS, and I want to copy it to its NCS mate. 1. Change the master chain to chain A Draw-NCS ghost control-change to chain A 2. go to Extension-NCS-NCS ligands-, In upper dialog box, Protein with NCS, Fill the master chain ID to A. In the lower dialogue box Molecules contain ligands, choose the right molecule and Fill 'M' and 'residue ranges'. Coot will give a few positions to fit the chain, just choose the right one. Yu 2011/3/31 Javier Garcia gar...@ysbl.york.ac.uk Dear Zhang yu, I also found that same problem recently. I think is a small bug from Coot. It will only copy NCS if the molecule's name you want to copy is molecule A. So maybe will work if you rename your built chain as A. Good luck! Javier On 31/03/11 00:16, zhang yu wrote: Dear all, I have two fold NCS in ASU. I could find phase by molecular replacement, but I have to build other chains by myself, since the model is not complete. During model building in coot, I built one chain in one NCS, and I merged this chain into the previous molecule. Now it is the chain M'. When I tried to directly copy this chain to its NCS mate, It didn't work. I followed the instruction posted before http://www.mail-archive.com/coot@jiscmail.ac.uk/msg01984.html;. What I did is 1. Change the master chain to chain M Draw-NCS ghost control-change to chain M (The terminal said 'there are no ghosts when I change the master chain to chain M) 2. go to Extension-NCS-copy chain-, coot ask to fill the master chain ID, and I typed M Nothing happened. Could someone tell me what is the correct procedure to do it ? Bye the way, for those chains present in both NCS maps, I modified one chain and applied the change to its NCS mate by using the same above procedure, and it worked. Thanks -- Yu Zhang HHMI associate Waksman Institute, Rutgers University 190 Frelinghuysen Rd. Piscataway, NJ, 08904 -- Yu Zhang HHMI associate Waksman Institute, Rutgers University 190 Frelinghuysen Rd. Piscataway, NJ, 08904
Re: [ccp4bb] Question about TEV cleavage
I echo this. Some time ago I was working on a target which seemed to precipitate when cleaving overnight with TEV. I wasted a fair bit of time trying to optimise cleavage conditions with a myriad of buffers. In the end, just by not agitating my solution, there was no precipitation and recovery was extremely good. I never agitate my solutions when cleaving now and have not had any problems with cleavage (to do with this issue, anyway) or recovery. cheers charlie Dr. Charles Allerston Genome Integrity Group Structural Genomics Consortium Nuffield Department of Medicine Old Road Campus University of Oxford OX3 7DQ http://www.sgc.ox.ac.uk/ -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiaopeng Hu Sent: 31 March 2011 15:23 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Question about TEV cleavage Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng
Re: [ccp4bb] what to do with disordered side chains
Why not have the b-factors take care of it until some magic cutoff number? When they reach the cutoff, two things happen: 1. Occupancies are set to zero for those side chains, to represent our lack of ability to model the region, 2. B-factors are set to exactly 500, as a flag allowing casual b-factor-savvy users to identify suspicious regions, since they will probably not see occupancies, but *will* see b-factors. Therefore, all 0-occupancy atoms will automatically have b-factors = 500. I believe it is true that if the occupancies are zero, the b-factors are totally irrelevant for all calculations? Doesn't this satisfy both parties? Jacob On Thu, Mar 31, 2011 at 9:22 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Quyen, I am afraid you won't get any better answers than you got so far. There is no holy bible telling you what to do with disordered side chains. I fully agree with James that you should try to get the best possible model, which best explains your data and that will be your decision. Here are my 2 cents: -If you see alternative positions, you have to build them. -If you do not see alternative positions, I would not replace one fantasy (some call it most likely) orientation with 2 or 3 fantasy orientations. -I personally belong to the let the B-factors take care of it camp, but that is my personal opinion. Leaving side chains out could lead to misinterpretations by slightly less savy users of our data, especially when charge distributions are being studied. Besides, we know (almost) for sure that the side chain is there, it is only disordered and as we just learned, even slightly less savy users know what flaming red side chains mean. Even if they may not be mathematically entirely correct, huge B-factors clearly indicate that there is disorder involved. -I would not let occupancies take up the slack since even very savy users have never heard of them and again, the side chain is fully occupied, only disordered. Of course if you build alternate positions, you have to divede the occupancies amongst them. Best, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Quyen Hoang Sent: Thursday, March 31, 2011 3:55 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] what to do with disordered side chains We are getting off topic a little bit. Original topic: is it better to not build disordered sidechains or build them and let B-factors take care of it? Ed's poll got almost a 50:50 split. Question still unanswered. Second topic introduced by Pavel: Your B-factors are valid within a harmonic (small) approximation of atomic vibrations. Larger scale motions you are talking about go beyond the harmonic approximation, and using the B-factor to model them is abusing the corresponding mathematical model. And that these large scale motions (disorders) are better represented by alternative conformations and associated with them occupancies. My question is, how many people here do this? If you're currently doing what Pavel suggested here, how do you decide where to keep the upper limit of B-factors and what the occupancies are for each atom (data with resolution of 2.0A or worse)? I mean, do you cap the B-factor at a reasonable number to represent natural atomic vibrations (which is very small as Pavel pointed out) and then let the occupancies pick up the slack? More importantly, what is your reason for doing this? Cheers and thanks for your contribution, Quyen On Mar 30, 2011, at 5:20 PM, Pavel Afonine wrote: Mark, alternative conformations and associated with them occupancies are to describe the larger scale disorder (the one that goes beyond the B-factor's capability to cope with). Multi-model PDB files is another option. Best, Pavel. On Wed, Mar 30, 2011 at 2:15 PM, VAN RAAIJ , MARK JOHAN mjvanra...@cnb.csic.es wrote: yet, apart from (and additionally to) modelling two conformations of the side-chain, the B-factor is the only tool we have (now). Quoting Pavel Afonine: Hi Quyen, (...) And if B-factor is an estimate of thermo-motion (or static disorder), then would it not be reasonable to accept that building the side-chain and let B-factor sky rocket might reflect reality more so than not building it? NO. Your B-factors are valid within a harmonic (small) approximation of atomic vibrations. Larger scale motions you are talking about go beyond the harmonic approximation, and using the B-factor to model them is abusing the corresponding mathematical model. http://www.phenix-online.org/newsletter/CCN_2010_07.pdf Pavel. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es -- *** Jacob Pearson Keller Northwestern University Medical
Re: [ccp4bb] problem of conventions
The IUCr standard is to make abc, see http://nvl.nist.gov/pub/nistpubs/jres/107/4/j74mig.pdf http://nvl.nist.gov/pub/nistpubs/jres/106/6/j66mig.pdf and P 2 21 21 is a perfectly valid space group Pointless will reindex within the same point group, or you can choose the abc convention (SETTING CELL_BASED) or the reference setting P 21 21 2 (SETTING SYMMETRY_BASED), so you have a choice Most programs are perfectly happy with space group P 2 21 21 (I'm not sure about [auto]Sharp) Phil On 31 Mar 2011, at 15:28, Anita Lewit-Bentley wrote: Dear all, I would like to share my experiencde with a rather unexpected problem of indexing conventions. Perhaps I can save people some time I have a crystal in the more unusual P21212 space-group (No 18). Its unit cell lengths are bac (please note). I systematically use XDS for data integration, since so far it was able to handle even the most horrible-looking spots. Now XDS indexed my data in space-group 18, but with the axes order abc! It had, in fact, invented a space-group P22121, which does not exist. I did not realise this until I had spent a couple of weeks with beautiful peaks in rotation functions, but hopeless results in translation functions. It wasn't until I looked more closely into the definition of the screw axes that I realised the problem. POINTLESS does not allow a reindexing of reflexions within the same space-group, but fortunately REINDEX did the trick at the level of intensities, because I like to use SCALA for careful scaling of my data. So, basically, beyond just warning people who might encounter similar problems, I was wo,dering if XDS could perhaps reindex reflexions according to Int. Table conventions once the screw axes of a crystal system have been identified? With best wishes, Anita Anita Lewit-Bentley Unité d'Immunologie Structurale CNRS URA 2185 Département de Biologie Structurale Chimie Institut Pasteur 25 rue du Dr. Roux 75724 Paris cedex 15 FRANCE Tel: 33- (0)1 45 68 88 95 FAX: 33-(0)1 40 61 30 74 email: ale...@pasteur.fr
Re: [ccp4bb] what to do with disordered side chains
On Thu, Mar 31, 2011 at 7:42 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Why not have the b-factors take care of it until some magic cutoff number? When they reach the cutoff, two things happen: 1. Occupancies are set to zero for those side chains, to represent our lack of ability to model the region, 2. B-factors are set to exactly 500, as a flag allowing casual b-factor-savvy users to identify suspicious regions, since they will probably not see occupancies, but *will* see b-factors. Therefore, all 0-occupancy atoms will automatically have b-factors = 500. I believe it is true that if the occupancies are zero, the b-factors are totally irrelevant for all calculations? Doesn't this satisfy both parties? No, because now you're not only presenting the user with made-up coordinates, you're giving them a made-up B-factor as well, so there is effectively no property of those atoms that is based on experimental data rather than subjective criteria. Regardless of any problems inherent in letting the B-factors take care of all forms of disorder, they are nonetheless a refined parameter. -Nat
Re: [ccp4bb] what to do with disordered side chains
- they all know what B is and how to look for regions of high B (with, say, pymol) and they know not to make firm conclusions about H-bonds to flaming red side chains. But this knowledge may be quite wrong. If the flaming red really indicates large vibrational motion then yes, one whould not bet on stable H-bonds. But if the flaming red indicates that a well-ordered sidechain was incorrectly modeled at full occupancy when in fact it is only present at half-occupancy then no, the H-bond could be strong but only present in that half-occupancy conformation. One presumes that the other half-occupancy location (perhaps missing from the model) would have its own H-bonding network. I beg to differ. If a side chain has 2 or more positions, one should be a bit careful about making firm conclusions based on only one of those, even if it isn't clear exactly why one should use caution. Also, isn't the isotropic B we fit at medium resolution more of a spherical cow approximation to physical reality anyway? Phoebe
Re: [ccp4bb] what to do with disordered side chains
Well, I guess I was thinking to make the b-factor such a preposterous value that no one would possibly believe it. Setting occupancies to zero effectively places a stumbling block, because people see the residues and think they are actually supported by data. So to counter-balance this, I thought putting up a high-b-factor flag would prevent people from tripping over the stumbling block. Look, you could even set the b-factor to 1 if you want--just something so people totally discount those coordinates. Jacob On Thu, Mar 31, 2011 at 9:55 AM, Nat Echols nathaniel.ech...@gmail.com wrote: On Thu, Mar 31, 2011 at 7:42 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Why not have the b-factors take care of it until some magic cutoff number? When they reach the cutoff, two things happen: 1. Occupancies are set to zero for those side chains, to represent our lack of ability to model the region, 2. B-factors are set to exactly 500, as a flag allowing casual b-factor-savvy users to identify suspicious regions, since they will probably not see occupancies, but *will* see b-factors. Therefore, all 0-occupancy atoms will automatically have b-factors = 500. I believe it is true that if the occupancies are zero, the b-factors are totally irrelevant for all calculations? Doesn't this satisfy both parties? No, because now you're not only presenting the user with made-up coordinates, you're giving them a made-up B-factor as well, so there is effectively no property of those atoms that is based on experimental data rather than subjective criteria. Regardless of any problems inherent in letting the B-factors take care of all forms of disorder, they are nonetheless a refined parameter. -Nat -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] what to do with disordered side chains
Dear Jacob, What do we gain? As Dale pointed out, we are already abusing either occupancy, B-factor or delete the side chain to compensate for our inability to tell the user that the side chain is disordered. With your proposal, we would fudge both occupancy and B-factor, which in my eyes is even worse as fudging just one of the two. Also, who should decide on the magic number: the all-knowing gurus at the protein data bank? Maybe we should really start using cif files, which allow to specify coordinate uncertainties. Best regards, Herman -Original Message- From: Jacob Keller [mailto:j-kell...@fsm.northwestern.edu] Sent: Thursday, March 31, 2011 4:43 PM To: Schreuder, Herman RD/DE Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] what to do with disordered side chains Why not have the b-factors take care of it until some magic cutoff number? When they reach the cutoff, two things happen: 1. Occupancies are set to zero for those side chains, to represent our lack of ability to model the region, 2. B-factors are set to exactly 500, as a flag allowing casual b-factor-savvy users to identify suspicious regions, since they will probably not see occupancies, but *will* see b-factors. Therefore, all 0-occupancy atoms will automatically have b-factors = 500. I believe it is true that if the occupancies are zero, the b-factors are totally irrelevant for all calculations? Doesn't this satisfy both parties? Jacob On Thu, Mar 31, 2011 at 9:22 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Quyen, I am afraid you won't get any better answers than you got so far. There is no holy bible telling you what to do with disordered side chains. I fully agree with James that you should try to get the best possible model, which best explains your data and that will be your decision. Here are my 2 cents: -If you see alternative positions, you have to build them. -If you do not see alternative positions, I would not replace one fantasy (some call it most likely) orientation with 2 or 3 fantasy orientations. -I personally belong to the let the B-factors take care of it camp, but that is my personal opinion. Leaving side chains out could lead to misinterpretations by slightly less savy users of our data, especially when charge distributions are being studied. Besides, we know (almost) for sure that the side chain is there, it is only disordered and as we just learned, even slightly less savy users know what flaming red side chains mean. Even if they may not be mathematically entirely correct, huge B-factors clearly indicate that there is disorder involved. -I would not let occupancies take up the slack since even very savy users have never heard of them and again, the side chain is fully occupied, only disordered. Of course if you build alternate positions, you have to divede the occupancies amongst them. Best, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Quyen Hoang Sent: Thursday, March 31, 2011 3:55 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] what to do with disordered side chains We are getting off topic a little bit. Original topic: is it better to not build disordered sidechains or build them and let B-factors take care of it? Ed's poll got almost a 50:50 split. Question still unanswered. Second topic introduced by Pavel: Your B-factors are valid within a harmonic (small) approximation of atomic vibrations. Larger scale motions you are talking about go beyond the harmonic approximation, and using the B-factor to model them is abusing the corresponding mathematical model. And that these large scale motions (disorders) are better represented by alternative conformations and associated with them occupancies. My question is, how many people here do this? If you're currently doing what Pavel suggested here, how do you decide where to keep the upper limit of B-factors and what the occupancies are for each atom (data with resolution of 2.0A or worse)? I mean, do you cap the B-factor at a reasonable number to represent natural atomic vibrations (which is very small as Pavel pointed out) and then let the occupancies pick up the slack? More importantly, what is your reason for doing this? Cheers and thanks for your contribution, Quyen On Mar 30, 2011, at 5:20 PM, Pavel Afonine wrote: Mark, alternative conformations and associated with them occupancies are to describe the larger scale disorder (the one that goes beyond the B-factor's capability to cope with). Multi-model PDB files is another option. Best, Pavel. On Wed, Mar 30, 2011 at 2:15 PM, VAN RAAIJ , MARK JOHAN mjvanra...@cnb.csic.es wrote: yet, apart from (and additionally to) modelling two conformations of the side-chain, the B-factor is the only tool we have (now). Quoting Pavel Afonine: Hi Quyen, (...) And if B-factor is an estimate of thermo-motion
Re: [ccp4bb] Question about TEV cleavage
We have the same experience with the DO NOT AGITATE. We purify our own His-tagged TEV protease, and flash-freeze it IMMEDIATELY after IMAC prep at about 1 mg/mL. Laurie Betts UNC On Thu, Mar 31, 2011 at 10:37 AM, Charles Allerston charles.allers...@sgc.ox.ac.uk wrote: I echo this. Some time ago I was working on a target which seemed to precipitate when cleaving overnight with TEV. I wasted a fair bit of time trying to optimise cleavage conditions with a myriad of buffers. In the end, just by not agitating my solution, there was no precipitation and recovery was extremely good. I never agitate my solutions when cleaving now and have not had any problems with cleavage (to do with this issue, anyway) or recovery. cheers charlie Dr. Charles Allerston Genome Integrity Group Structural Genomics Consortium Nuffield Department of Medicine Old Road Campus University of Oxford OX3 7DQ http://www.sgc.ox.ac.uk/ -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiaopeng Hu Sent: 31 March 2011 15:23 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Question about TEV cleavage Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng
Re: [ccp4bb] off topic: protease identification
Hi Tom, You can try the MEROPS database. There is a search feature called What peptidases can cleave this bond. http://merops.sanger.ac.uk/cgi-bin/specsearch.pl Cheers, Jack On Thu, Mar 31, 2011 at 6:59 AM, Brett, Thomas tbr...@dom.wustl.edu wrote: Hi all: I was wondering if anyone had any tips on identifying proteases. I have a protein for which I know the proteolytic cleavage site. What are the best ways to identify the protease that does the cutting either: 1) bioinformatically (i.e., a good database to search using the cleavage site or a consensus) 2) experimentally (some engineered substrate to trap/identify the substrate or any other method?) Thanks in advance -Tom Tom J. Brett, PhD Assistant Professor of Medicine Division of Pulmonary and Critical Care Washington University School of Medicine Campus Box 8052, 660 S. Euclid Saint Louis, MO 63110 http://brettlab.dom.wustl.edu/
Re: [ccp4bb] Question about TEV cleavage
And not at full moon! Klaus Am 31.03.2011 um 16:23 schrieb Xiaopeng Hu: Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: klaus.pion...@ocbc.uni-freiburg.de Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
Re: [ccp4bb] what to do with disordered side chains
Thank you for your post, Herman. Since there is no holy bible to provide guidance, perhaps we should hold off the idea of electing a powerful dictator to enforce this? - at least until we all can come to a consensus on how the dictator should dictate... Cheers, Quyen On Mar 31, 2011, at 10:22 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Quyen, I am afraid you won't get any better answers than you got so far. There is no holy bible telling you what to do with disordered side chains. I fully agree with James that you should try to get the best possible model, which best explains your data and that will be your decision. Here are my 2 cents: -If you see alternative positions, you have to build them. -If you do not see alternative positions, I would not replace one fantasy (some call it most likely) orientation with 2 or 3 fantasy orientations. -I personally belong to the let the B-factors take care of it camp, but that is my personal opinion. Leaving side chains out could lead to misinterpretations by slightly less savy users of our data, especially when charge distributions are being studied. Besides, we know (almost) for sure that the side chain is there, it is only disordered and as we just learned, even slightly less savy users know what flaming red side chains mean. Even if they may not be mathematically entirely correct, huge B-factors clearly indicate that there is disorder involved. -I would not let occupancies take up the slack since even very savy users have never heard of them and again, the side chain is fully occupied, only disordered. Of course if you build alternate positions, you have to divede the occupancies amongst them. Best, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Quyen Hoang Sent: Thursday, March 31, 2011 3:55 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] what to do with disordered side chains We are getting off topic a little bit. Original topic: is it better to not build disordered sidechains or build them and let B-factors take care of it? Ed's poll got almost a 50:50 split. Question still unanswered. Second topic introduced by Pavel: Your B-factors are valid within a harmonic (small) approximation of atomic vibrations. Larger scale motions you are talking about go beyond the harmonic approximation, and using the B-factor to model them is abusing the corresponding mathematical model. And that these large scale motions (disorders) are better represented by alternative conformations and associated with them occupancies. My question is, how many people here do this? If you're currently doing what Pavel suggested here, how do you decide where to keep the upper limit of B-factors and what the occupancies are for each atom (data with resolution of 2.0A or worse)? I mean, do you cap the B-factor at a reasonable number to represent natural atomic vibrations (which is very small as Pavel pointed out) and then let the occupancies pick up the slack? More importantly, what is your reason for doing this? Cheers and thanks for your contribution, Quyen On Mar 30, 2011, at 5:20 PM, Pavel Afonine wrote: Mark, alternative conformations and associated with them occupancies are to describe the larger scale disorder (the one that goes beyond the B-factor's capability to cope with). Multi-model PDB files is another option. Best, Pavel. On Wed, Mar 30, 2011 at 2:15 PM, VAN RAAIJ , MARK JOHAN mjvanra...@cnb.csic.es wrote: yet, apart from (and additionally to) modelling two conformations of the side-chain, the B-factor is the only tool we have (now). Quoting Pavel Afonine: Hi Quyen, (...) And if B-factor is an estimate of thermo-motion (or static disorder), then would it not be reasonable to accept that building the side- chain and let B-factor sky rocket might reflect reality more so than not building it? NO. Your B-factors are valid within a harmonic (small) approximation of atomic vibrations. Larger scale motions you are talking about go beyond the harmonic approximation, and using the B-factor to model them is abusing the corresponding mathematical model. http://www.phenix-online.org/newsletter/CCN_2010_07.pdf Pavel. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] what to do with disordered side chains
On Thu, 2011-03-31 at 17:04 +0200, herman.schreu...@sanofi-aventis.com wrote: Maybe we should really start using cif files, which allow to specify coordinate uncertainties. PDB has SIGATM record for that purpose -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] what to do with disordered side chains
On Thu, 2011-03-31 at 11:27 -0400, Quyen Hoang wrote: Thank you for your post, Herman. Since there is no holy bible to provide guidance, perhaps we should hold off the idea of electing a powerful dictator to enforce this? - at least until we all can come to a consensus on how the dictator should dictate... Well, that is partly what we have the IUCr for, isn't it? A couple of people have referred to the wwPDB in this context, but IMHO the IUCr is a much better forum to try to reach some kind of decision about these issues. If the IUCr has a clear policy, the wwPDB can enforce it (like they did with the deposition of structure factors). If the wwPDB takes a lead a lot of people will get annoyed at them, when the real problem is that crystallographic practitioners haven't come to any agreement amongst themselves. And yes, I know that balancing questions of scientific correctness with the needs of more or less naive consumers of the data isn't straightforward :-) Regards, Peter. -- Peter Keller Tel.: +44 (0)1223 353033 Global Phasing Ltd., Fax.: +44 (0)1223 366889 Sheraton House, Castle Park, Cambridge CB3 0AX United Kingdom
[ccp4bb] The meaning of B-factor, was Re: [ccp4bb] what to do with disordered side chains
The B-factor in crystallography represents the convolution (sum) of two types of uncertainties about the atom (electron cloud) position: 1) dispersion of atom positions in crystal lattice 2) uncertainty of the experimenter's knowledge about the atom position. In general, uncertainty needs not to be described by Gaussian function. However, communicating uncertainty using the second moment of its distribution is a widely accepted practice, with frequently implied meaning that it corresponds to a Gaussian probability function. B-factor is simply a scaled (by 8 times pi squared) second moment of uncertainty distribution. In the previous, long thread, confusion was generated by the additional assumption that B-factor also corresponds to a Gaussian probability distribution and not just to a second moment of any probability distribution. Crystallographic literature often implies the Gaussian shape, so there is some justification for such an interpretation, where the more complex probability distribution is represented by the sum of displaced Gaussians, where the area under each Gaussian component corresponds to the occupancy of an alternative conformation. For data with a typical resolution for macromolecular crystallography, such multi-Gaussian description of the atom position's uncertainty is not practical, as it would lead to instability in the refinement and/or overfitting. Due to this, a simplified description of the atom's position uncertainty by just the second moment of probability distribution is the right approach. For this reason, the PDB format is highly suitable for the description of positional uncertainties, the only difference with other fields being the unusual form of squaring and then scaling up the standard uncertainty. As this calculation can be easily inverted, there is no loss of information. However, in teaching one should probably stress more this unusual form of presenting the standard deviation. A separate issue is the use of restraints on B-factor values, a subject that probably needs a longer discussion. With respect to the previous thread, representing poorly-ordered (so called 'disordered') side chains by the most likely conformer with appropriately high B-factors is fully justifiable, and currently is probably the best solution to a difficult problem. Zbyszek Otwinowski - they all know what B is and how to look for regions of high B (with, say, pymol) and they know not to make firm conclusions about H-bonds to flaming red side chains. But this knowledge may be quite wrong. If the flaming red really indicates large vibrational motion then yes, one whould not bet on stable H-bonds. But if the flaming red indicates that a well-ordered sidechain was incorrectly modeled at full occupancy when in fact it is only present at half-occupancy then no, the H-bond could be strong but only present in that half-occupancy conformation. One presumes that the other half-occupancy location (perhaps missing from the model) would have its own H-bonding network. I beg to differ. If a side chain has 2 or more positions, one should be a bit careful about making firm conclusions based on only one of those, even if it isn't clear exactly why one should use caution. Also, isn't the isotropic B we fit at medium resolution more of a spherical cow approximation to physical reality anyway? Phoebe Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
Re: [ccp4bb] what to do with disordered side chains
Regarding suggestions that the pdb or the IUCR to tell us what to do: IMO - Neither of the usual solutions - (a) deleting side chains when there is no density or (b) letting B factors go where they will - is without problems (this is clear from the ongoing discussion). I would be really unhappy if some authority unilaterally imposed either of these solutions on the protein crystallographic community. Sue Dr. Sue A. Roberts Dept. of Chemistry and Biochemistry University of Arizona 1041 E. Lowell St., Tucson, AZ 85721 Phone: 520 621 8171 s...@email.arizona.edu http://www.biochem.arizona.edu/xray
Re: [ccp4bb] what to do with disordered side chains
Dear Quyen, On Thu, Mar 31, 2011 at 11:27:58AM -0400, Quyen Hoang wrote: Thank you for your post, Herman. Since there is no holy bible to provide guidance, perhaps we should hold off the idea of electing a powerful dictator to enforce this? - at least until we all can come to a consensus on how the dictator should dictate... ... but that might well be even harder than to decide what to do with disordered side chains ... . With best wishes, Gerard. -- === * * * Gerard Bricogne g...@globalphasing.com * * * * Global Phasing Ltd. * * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * * * === On Mar 31, 2011, at 10:22 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Quyen, I am afraid you won't get any better answers than you got so far. There is no holy bible telling you what to do with disordered side chains. I fully agree with James that you should try to get the best possible model, which best explains your data and that will be your decision. Here are my 2 cents: -If you see alternative positions, you have to build them. -If you do not see alternative positions, I would not replace one fantasy (some call it most likely) orientation with 2 or 3 fantasy orientations. -I personally belong to the let the B-factors take care of it camp, but that is my personal opinion. Leaving side chains out could lead to misinterpretations by slightly less savy users of our data, especially when charge distributions are being studied. Besides, we know (almost) for sure that the side chain is there, it is only disordered and as we just learned, even slightly less savy users know what flaming red side chains mean. Even if they may not be mathematically entirely correct, huge B-factors clearly indicate that there is disorder involved. -I would not let occupancies take up the slack since even very savy users have never heard of them and again, the side chain is fully occupied, only disordered. Of course if you build alternate positions, you have to divede the occupancies amongst them. Best, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Quyen Hoang Sent: Thursday, March 31, 2011 3:55 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] what to do with disordered side chains We are getting off topic a little bit. Original topic: is it better to not build disordered sidechains or build them and let B-factors take care of it? Ed's poll got almost a 50:50 split. Question still unanswered. Second topic introduced by Pavel: Your B-factors are valid within a harmonic (small) approximation of atomic vibrations. Larger scale motions you are talking about go beyond the harmonic approximation, and using the B-factor to model them is abusing the corresponding mathematical model. And that these large scale motions (disorders) are better represented by alternative conformations and associated with them occupancies. My question is, how many people here do this? If you're currently doing what Pavel suggested here, how do you decide where to keep the upper limit of B-factors and what the occupancies are for each atom (data with resolution of 2.0A or worse)? I mean, do you cap the B-factor at a reasonable number to represent natural atomic vibrations (which is very small as Pavel pointed out) and then let the occupancies pick up the slack? More importantly, what is your reason for doing this? Cheers and thanks for your contribution, Quyen On Mar 30, 2011, at 5:20 PM, Pavel Afonine wrote: Mark, alternative conformations and associated with them occupancies are to describe the larger scale disorder (the one that goes beyond the B-factor's capability to cope with). Multi-model PDB files is another option. Best, Pavel. On Wed, Mar 30, 2011 at 2:15 PM, VAN RAAIJ , MARK JOHAN mjvanra...@cnb.csic.es wrote: yet, apart from (and additionally to) modelling two conformations of the side-chain, the B-factor is the only tool we have (now). Quoting Pavel Afonine: Hi Quyen, (...) And if B-factor is an estimate of thermo-motion (or static disorder), then would it not be reasonable to accept that building the side-chain and let B-factor sky rocket might reflect reality more so than not building it? NO. Your B-factors are valid within a harmonic (small) approximation of atomic vibrations. Larger scale motions you are talking about go beyond the harmonic approximation, and using the
Re: [ccp4bb] titering baculovirus ?
A related question: how do most people amplify their baculovirus stocks? Adherent cultures vs suspension? Fold dilution at each stage (P1 to P2, P2 to P3)? Duration of each amplification stage? We have some viral stocks that go off rather quickly (1-2 months) despite being stored with FBS in a cool, dark place. Katya On Thu, Mar 31, 2011 at 10:23 AM, Brad Bennett bradbennet...@gmail.com wrote: Though I'm not meaning to turn this into a plaque assay burn session, for many reasons we have also abandoned it and instead titer our baculovirus stocks (P3 and P4) using flow cytometry. We use an antibody against viral gp64 that has been labeled with PE and stain infected cells in 96 well plates with the PE-antibody. We then measure PE fluorescence on a flow cytometer (you can also do cell counts, viability determinations, etc.). We equate 1 fluorescent event as 1 infected cell. Since we know how many cells have been plated in each well, we can determine the percentage infected in each well. We calculate a non-normalized titer from this data alone or we compare this data to a standardized virus and determine a normalized titer using a standard curve. From infection to having a titer in hand takes about 24 hours. Of course, the potential bottleneck is access to a flow cytometer! I can give more experimental details off-board. I should say that for getting an idea of relative titers and to test protein expression on a small scale, we also do the effective titer tests as suggested by Nat, with cell morphology and immunoblots as our read-out of virus potency and recombinant protein expression, respectively. No doubt, this will get you a long way but at some point, I argue, you need to determine an actual, absolute titer for duplication of results, troubleshooting, monitoring virus health over time, publications, etc. HTH- Brad On Thu, Mar 31, 2011 at 9:23 AM, Bertrand, Jay Aaron [Nervianoms] jay.bertr...@nervianoms.com wrote: I checked with someone in our protein production group and got the following response: We also stopped doing the virus titration with the plaque assay and instead are performing expression test with different concentration of virus from the 3rd amplification. But for some viruses we still have doubts concerning the amplification success, so we are now evaluating a new technology using qPCR with the following kit (http://oetltd.com/products/category/baculoquant/). So you might have a look and see if it could be useful for your group. We would also be curious to hear if anyone else has experience with this approach. I hope this helps. Jay -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nathaniel Clark Sent: Wednesday, March 30, 2011 11:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] titering baculovirus ? We don't have a problem getting them to stick to the plates in serum-free media, or in 5% FBS media. The more challenging part is getting the plating density just right, too low and the plaques are too big, to high and they are too small. Or if the cells dry out, or if your agarose overlay is too hot, etc... However, we have actually stopped titering all together. We find early stocks (from co-transfection, or plaque purification) are 'low', but after ~2 rounds of amplification in adherent culture of the 'low' titer stock(using a large volume of low-titer virus in a t25 flask), we can add ~0.5 ml to a 30-100 ml shake flask of Sf9's (serum free media) and get a high titer stock( ie. 10^8 pfu/ml). From there we amplify with ~ 10 mls of virus in a 1 L shaker culture, and we have our large volume high-titer stock. Sometimes we will incubate the cells in pure virus stock in a t25 flask for 1 hour, take the virus off, and add fresh media, as a way to rescue low-titer stocks. If you are just trying to titer (not plaque-purify), you can just take 10 fold dilutions of your virus, and do several small scale infections in 6 well plates, 10 ml shaker cultures, whatever you prefer. At the lowest virus concentration where you see a synchronous infection (judged by protein expression levels, or cell-diameter if you have a cell counter, or by viewing with a trained eye), you call that an MOI=1. From there you know the number of cells in the plate, and the volume of virus you added, so you can calculate an effective titer. Plaque assays are really difficult and slow, and if you are just trying to make protein, an effective titer is fine, the absolute number isn't that helpful, Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Wed, Mar 30, 2011 at 5:14 PM, Gloria Borgstahl gborgst...@gmail.com wrote: Hi Guys, we are learning to work with Sf9 cells and Carol in my lab wanted me to ask you the following question. Many thanks for any help, G I need to titer a baculovirus stock in my suspension-adapted Sf9
Re: [ccp4bb] The meaning of B-factor, was Re: [ccp4bb] what to do with disordered side chains
This is a lovely summary, and we should make our students read it. - But I'm afraid I do not see how it supports the closing statement in the last paragraph... phx. On 31/03/2011 17:06, Zbyszek Otwinowski wrote: The B-factor in crystallography represents the convolution (sum) of two types of uncertainties about the atom (electron cloud) position: 1) dispersion of atom positions in crystal lattice 2) uncertainty of the experimenter's knowledge about the atom position. In general, uncertainty needs not to be described by Gaussian function. However, communicating uncertainty using the second moment of its distribution is a widely accepted practice, with frequently implied meaning that it corresponds to a Gaussian probability function. B-factor is simply a scaled (by 8 times pi squared) second moment of uncertainty distribution. In the previous, long thread, confusion was generated by the additional assumption that B-factor also corresponds to a Gaussian probability distribution and not just to a second moment of any probability distribution. Crystallographic literature often implies the Gaussian shape, so there is some justification for such an interpretation, where the more complex probability distribution is represented by the sum of displaced Gaussians, where the area under each Gaussian component corresponds to the occupancy of an alternative conformation. For data with a typical resolution for macromolecular crystallography, such multi-Gaussian description of the atom position's uncertainty is not practical, as it would lead to instability in the refinement and/or overfitting. Due to this, a simplified description of the atom's position uncertainty by just the second moment of probability distribution is the right approach. For this reason, the PDB format is highly suitable for the description of positional uncertainties, the only difference with other fields being the unusual form of squaring and then scaling up the standard uncertainty. As this calculation can be easily inverted, there is no loss of information. However, in teaching one should probably stress more this unusual form of presenting the standard deviation. A separate issue is the use of restraints on B-factor values, a subject that probably needs a longer discussion. With respect to the previous thread, representing poorly-ordered (so called 'disordered') side chains by the most likely conformer with appropriately high B-factors is fully justifiable, and currently is probably the best solution to a difficult problem. Zbyszek Otwinowski - they all know what B is and how to look for regions of high B (with, say, pymol) and they know not to make firm conclusions about H-bonds to flaming red side chains. But this knowledge may be quite wrong. If the flaming red really indicates large vibrational motion then yes, one whould not bet on stable H-bonds. But if the flaming red indicates that a well-ordered sidechain was incorrectly modeled at full occupancy when in fact it is only present at half-occupancy then no, the H-bond could be strong but only present in that half-occupancy conformation. One presumes that the other half-occupancy location (perhaps missing from the model) would have its own H-bonding network. I beg to differ. If a side chain has 2 or more positions, one should be a bit careful about making firm conclusions based on only one of those, even if it isn't clear exactly why one should use caution. Also, isn't the isotropic B we fit at medium resolution more of a spherical cow approximation to physical reality anyway? Phoebe Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
Re: [ccp4bb] The meaning of B-factor, was Re: [ccp4bb] what to do with disordered side chains
Dear Zbyszek: Thanks a lot for your good summary. It is very interesting but, do you think there are some references for more detailed description, especially from mathematics point of view about correlating B-factor to the Gaussian probability distribution (the B-factor unit of A^2 is my first doubt as for the probability distribution description)? Thanks again for your efforts! Best Regards, Hailiang The B-factor in crystallography represents the convolution (sum) of two types of uncertainties about the atom (electron cloud) position: 1) dispersion of atom positions in crystal lattice 2) uncertainty of the experimenter's knowledge about the atom position. In general, uncertainty needs not to be described by Gaussian function. However, communicating uncertainty using the second moment of its distribution is a widely accepted practice, with frequently implied meaning that it corresponds to a Gaussian probability function. B-factor is simply a scaled (by 8 times pi squared) second moment of uncertainty distribution. In the previous, long thread, confusion was generated by the additional assumption that B-factor also corresponds to a Gaussian probability distribution and not just to a second moment of any probability distribution. Crystallographic literature often implies the Gaussian shape, so there is some justification for such an interpretation, where the more complex probability distribution is represented by the sum of displaced Gaussians, where the area under each Gaussian component corresponds to the occupancy of an alternative conformation. For data with a typical resolution for macromolecular crystallography, such multi-Gaussian description of the atom position's uncertainty is not practical, as it would lead to instability in the refinement and/or overfitting. Due to this, a simplified description of the atom's position uncertainty by just the second moment of probability distribution is the right approach. For this reason, the PDB format is highly suitable for the description of positional uncertainties, the only difference with other fields being the unusual form of squaring and then scaling up the standard uncertainty. As this calculation can be easily inverted, there is no loss of information. However, in teaching one should probably stress more this unusual form of presenting the standard deviation. A separate issue is the use of restraints on B-factor values, a subject that probably needs a longer discussion. With respect to the previous thread, representing poorly-ordered (so called 'disordered') side chains by the most likely conformer with appropriately high B-factors is fully justifiable, and currently is probably the best solution to a difficult problem. Zbyszek Otwinowski - they all know what B is and how to look for regions of high B (with, say, pymol) and they know not to make firm conclusions about H-bonds to flaming red side chains. But this knowledge may be quite wrong. If the flaming red really indicates large vibrational motion then yes, one whould not bet on stable H-bonds. But if the flaming red indicates that a well-ordered sidechain was incorrectly modeled at full occupancy when in fact it is only present at half-occupancy then no, the H-bond could be strong but only present in that half-occupancy conformation. One presumes that the other half-occupancy location (perhaps missing from the model) would have its own H-bonding network. I beg to differ. If a side chain has 2 or more positions, one should be a bit careful about making firm conclusions based on only one of those, even if it isn't clear exactly why one should use caution. Also, isn't the isotropic B we fit at medium resolution more of a spherical cow approximation to physical reality anyway? Phoebe Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
Re: [ccp4bb] titering baculovirus ?
We do adherent if we have a small volume of low titer virus, but as soon as we have a decent titer we will start with a 30 ml suspension flask. Normally I add something like 0.5 ml, which is probably more then I need. We only use serum free media right now. As I mentioned, if you need to boost the titer of a low titer stock, we plate a t25 flask with Sf9s, take off the media, and incubate it in 1 ml of pure virus (or two-fold diluted) for an hour or so, then remove (and save) that virus stock and give the cells fresh media. For fold dilution, just do a rough calculation of titer. Once you are at a high titer assume you have 10^8-10^9 pfu/ml, so you can estimate the ballpark volume to infect a given number of cells at an MOI of 1. From early stocks you might have a titer of 10^6-10^7 pfu/ml. If you do an amplification that doesn't work, decide if you were to high (MOI=10 not great for amplifying) or too low (MOI=0.1) and try again. Better yet, just set up 3 amplifications at the same time, whichever worked the best, use for another round of amplification, or for infections. For duration, we do 3 days amplification (post-infection), but sometimes we let them go a few days longer, which allows a secondary infection of the cells that weren't infected by the viral innoculum. There doesn't seem to be any harm to letting infections go longer; at some point all the cells die, but since the virus is pretty stable, it's no problem. We have a CEDEX cell counter which tells you average cell diameter, which is a great read out for infection. Also looking and density and viability versus an uninfected control will tell you if the infection worked. Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Thu, Mar 31, 2011 at 12:36 PM, Katya Heldwein katya.heldw...@gmail.com wrote: A related question: how do most people amplify their baculovirus stocks? Adherent cultures vs suspension? Fold dilution at each stage (P1 to P2, P2 to P3)? Duration of each amplification stage? We have some viral stocks that go off rather quickly (1-2 months) despite being stored with FBS in a cool, dark place. Katya On Thu, Mar 31, 2011 at 10:23 AM, Brad Bennett bradbennet...@gmail.com wrote: Though I'm not meaning to turn this into a plaque assay burn session, for many reasons we have also abandoned it and instead titer our baculovirus stocks (P3 and P4) using flow cytometry. We use an antibody against viral gp64 that has been labeled with PE and stain infected cells in 96 well plates with the PE-antibody. We then measure PE fluorescence on a flow cytometer (you can also do cell counts, viability determinations, etc.). We equate 1 fluorescent event as 1 infected cell. Since we know how many cells have been plated in each well, we can determine the percentage infected in each well. We calculate a non-normalized titer from this data alone or we compare this data to a standardized virus and determine a normalized titer using a standard curve. From infection to having a titer in hand takes about 24 hours. Of course, the potential bottleneck is access to a flow cytometer! I can give more experimental details off-board. I should say that for getting an idea of relative titers and to test protein expression on a small scale, we also do the effective titer tests as suggested by Nat, with cell morphology and immunoblots as our read-out of virus potency and recombinant protein expression, respectively. No doubt, this will get you a long way but at some point, I argue, you need to determine an actual, absolute titer for duplication of results, troubleshooting, monitoring virus health over time, publications, etc. HTH- Brad On Thu, Mar 31, 2011 at 9:23 AM, Bertrand, Jay Aaron [Nervianoms] jay.bertr...@nervianoms.com wrote: I checked with someone in our protein production group and got the following response: We also stopped doing the virus titration with the plaque assay and instead are performing expression test with different concentration of virus from the 3rd amplification. But for some viruses we still have doubts concerning the amplification success, so we are now evaluating a new technology using qPCR with the following kit (http://oetltd.com/products/category/baculoquant/). So you might have a look and see if it could be useful for your group. We would also be curious to hear if anyone else has experience with this approach. I hope this helps. Jay -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nathaniel Clark Sent: Wednesday, March 30, 2011 11:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] titering baculovirus ? We don't have a problem getting them to stick to the plates in serum-free media, or in 5% FBS media. The more challenging part is getting the plating density just right, too low and the plaques are too big, to high and they are too small. Or if the cells dry
Re: [ccp4bb] step refine speed of wincoot - a little drawback of the new interruptible_fit_protein() function in wincoot
Thank you for the comment. You are correct that there are being backups made in the newer interruptable fit function (they were't previously in the uninterruptable fitting). I wasnt thinking about that (*). This may (especially on windows) slow down things a great deal as we write and gzip these files as well. To note here, gzip may be slow on windows as is writing files. In the pre-release versions I disabled the gzip on windows by default (and in general made the compression of backup files optional). This may speed up things (in certain circumstances). I hope this clarifies things a bit... B (*) It's debatable if we want backups here or not. If you can and want to interrupt the fitting you may want to go back one step, which requires a backup. On the other side this will write a lot of backup files which is slowing thing down by writing and compressing files, as well as producing a lot of backup files. Discuss... (or Paul and I just decide). Hi Xiaopeng, and those who are using the new interruptible fit_protein... or stepped_refine... in wincoot 0.6.1: I just took a look at wincoot 0.6.1's fitting.py file (WinCoot\share\coot\python\fitting.py). It seems that in the old fit_protein() and stepped_refine_protein() functions, the backup was programmed to be turned off during the cycles when it steps through the protein. However when using the Stepped refine... from the Extensions menu, the program starts to spit out tons of backup files. I then checked the extension.py. It turns out that these menu items are calling the new interruptible_fit_protein() function instead of the old uninterruptible ones. Some further experiment showed that this behavior was dependent on the backup state variable of the model being refined. When the backup state is 1 (the default value?), these new interruptible fitting functions will save a compressed backup file on each step. So, in order to save time when using the new interruptible protein fitting functions, one can type the following command in the python script window to tweak the backup state to 0: make_backup(imol) #to save a backup turn_off_backup(imol) where imol is the number associated with the pdb file being fitted. Do not forget to turn it back on after the fitting: turn_on_backup(imol) To check the current model's backup state: backup_state(imol) Zhijie -- From: Xiaopeng Hu huxp...@mail.sysu.edu.cn Sent: Tuesday, March 29, 2011 9:02 AM To: Zhijie Li zhijie...@utoronto.ca Subject: Re: [ccp4bb] step refine speed of wincoot Dear Zhijie, Thanks for there suggestions. I tried to turn off backup (with script command) and it did help although not much. Is there any way to add this perference to setting of coot/wincoot? best xiaopeng - 原始邮件 - 发件人: Zhijie Li zhijie...@utoronto.ca 收件人: Xiaopeng Hu huxp...@mail.sysu.edu.cn 发送时间: 星期二, 2011年 3 月 29日 下午 7:46:05 主题: Re: [ccp4bb] step refine speed of wincoot Hi, If you feel the refinement to be too slow, you may turn off the smooth centering (in preferences) or change the centering steps to a smaller number to save unnecessary graphical calculations. To go extreme, you may even remove the real time display commands in the scripts - also a way to test if the difference observed is due to different graphical efficiency. Reducing the size of the displayed map also helps. The other thing you may need to consider is that coot/wincoot will save a backup file each time it updates the model, which means on each step of the refinement you have a new file generated (take a look at the coot-backup directory when doing stepped refinement). If your windows disk is terribly fragmented then sure you will spend a lot of time on writing these files. The other thing is, windows has a different file caching mechanism than linux, this can also cause a huge difference when a few hundred small temporary files are queued for writing. My impression is that both the ext2/3 file system and the way linux handles caching are more efficient for this kind of situations. You may try deleting the files in wincoot\coot-backup periodically and defragmenting that partition. Making a virtual disk in the RAM to put your backup directory there could be something to experiment on too. Zhijie -- From: Xiaopeng Hu huxp...@mail.sysu.edu.cn Sent: Sunday, March 20, 2011 9:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] step refine speed of wincoot Dear all, I found the step refine speed of wincoot is much slower than that of linux coot (with the same pc). Is it normal or I need to configure something with the wincoot? best, Xiaopeng Hu
Re: [ccp4bb] what to do with disordered side chains
What do we gain? As Dale pointed out, we are already abusing either occupancy, B-factor or delete the side chain to compensate for our inability to tell the user that the side chain is disordered. With your proposal, we would fudge both occupancy and B-factor, which in my eyes is even worse as fudging just one of the two. We gain clarity to the non-crystallographer user: a b-factor of 278.9 sounds like possibly something real. A b-factor of exactly 1000 does not. Both probably have the same believability, viz., ~zero. Also, setting occupancy = zero is not fudging but rather respectfully declining to comment based on lack of data. I think it is exactly the same as omitting residues one cannot see in the density. Also, who should decide on the magic number: the all-knowing gurus at the protein data bank? Maybe we should really start using cif files, which allow to specify coordinate uncertainties. I think a reasonable number could be derived and agreed upon, and would not be surprised if there is such a derivation or analysis in the literature answering the question: At what b-factor does modelling an atom become insignificant with respect to explaining/predicting/fitting the data? That point would be the b-factor/occupancy cutoff. JPK
Re: [ccp4bb] problem of conventions
Dear Anita, I happen to have a very similar problem today. Does XDS use the desired setting if you provide it with the correct cell and space group during the IDXREF step? You can otherwise re-index in CORRECT. To comment on Phil: I fed the mtz-file from pointless into ctruncate (or maybe it was scala) which left the space group string (P2 21 21) but turned the space group number 18 into 3018 - this does screw up autosharp and maybe also other programs which use the space group number/ symbol and not the symmetry operators. Tim On Thu, Mar 31, 2011 at 04:28:18PM +0200, Anita Lewit-Bentley wrote: Dear all, I would like to share my experiencde with a rather unexpected problem of indexing conventions. Perhaps I can save people some time I have a crystal in the more unusual P21212 space-group (No 18). Its unit cell lengths are bac (please note). I systematically use XDS for data integration, since so far it was able to handle even the most horrible-looking spots. Now XDS indexed my data in space-group 18, but with the axes order abc! It had, in fact, invented a space-group P22121, which does not exist. I did not realise this until I had spent a couple of weeks with beautiful peaks in rotation functions, but hopeless results in translation functions. It wasn't until I looked more closely into the definition of the screw axes that I realised the problem. POINTLESS does not allow a reindexing of reflexions within the same space-group, but fortunately REINDEX did the trick at the level of intensities, because I like to use SCALA for careful scaling of my data. So, basically, beyond just warning people who might encounter similar problems, I was wo,dering if XDS could perhaps reindex reflexions according to Int. Table conventions once the screw axes of a crystal system have been identified? With best wishes, Anita Anita Lewit-Bentley Unité d'Immunologie Structurale CNRS URA 2185 Département de Biologie Structurale Chimie Institut Pasteur 25 rue du Dr. Roux 75724 Paris cedex 15 FRANCE Tel: 33- (0)1 45 68 88 95 FAX: 33-(0)1 40 61 30 74 email: ale...@pasteur.fr -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] titering baculovirus ?
We don't do anything special to store the virus, just a dark fridge. You do need to mix well as the virus can settle. You might want to try this 'TIPS' approach. Basically you freeze baculovirus infected cells, and use them as your virus innoculum. I think I tried it once and it didn't really work, but I didn't really try too hard... There is a research paper on the subject that I can't find, but here is the first thing I found on google: http://www.abrf.org/ResearchGroups/ProteinExpressionResearchGroup/Activities/Wasilko-PERG2007.pdf Nat On Thu, Mar 31, 2011 at 1:12 PM, chitra shintre chitra.shin...@gmail.com wrote: Hi, We have observed this virus going off within 2 months as well. ...any idea why this happens? Chitra On 31 March 2011 18:07, Nathaniel Clark nathanielcl...@gmail.com wrote: We do adherent if we have a small volume of low titer virus, but as soon as we have a decent titer we will start with a 30 ml suspension flask. Normally I add something like 0.5 ml, which is probably more then I need. We only use serum free media right now. As I mentioned, if you need to boost the titer of a low titer stock, we plate a t25 flask with Sf9s, take off the media, and incubate it in 1 ml of pure virus (or two-fold diluted) for an hour or so, then remove (and save) that virus stock and give the cells fresh media. For fold dilution, just do a rough calculation of titer. Once you are at a high titer assume you have 10^8-10^9 pfu/ml, so you can estimate the ballpark volume to infect a given number of cells at an MOI of 1. From early stocks you might have a titer of 10^6-10^7 pfu/ml. If you do an amplification that doesn't work, decide if you were to high (MOI=10 not great for amplifying) or too low (MOI=0.1) and try again. Better yet, just set up 3 amplifications at the same time, whichever worked the best, use for another round of amplification, or for infections. For duration, we do 3 days amplification (post-infection), but sometimes we let them go a few days longer, which allows a secondary infection of the cells that weren't infected by the viral innoculum. There doesn't seem to be any harm to letting infections go longer; at some point all the cells die, but since the virus is pretty stable, it's no problem. We have a CEDEX cell counter which tells you average cell diameter, which is a great read out for infection. Also looking and density and viability versus an uninfected control will tell you if the infection worked. Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Thu, Mar 31, 2011 at 12:36 PM, Katya Heldwein katya.heldw...@gmail.com wrote: A related question: how do most people amplify their baculovirus stocks? Adherent cultures vs suspension? Fold dilution at each stage (P1 to P2, P2 to P3)? Duration of each amplification stage? We have some viral stocks that go off rather quickly (1-2 months) despite being stored with FBS in a cool, dark place. Katya On Thu, Mar 31, 2011 at 10:23 AM, Brad Bennett bradbennet...@gmail.com wrote: Though I'm not meaning to turn this into a plaque assay burn session, for many reasons we have also abandoned it and instead titer our baculovirus stocks (P3 and P4) using flow cytometry. We use an antibody against viral gp64 that has been labeled with PE and stain infected cells in 96 well plates with the PE-antibody. We then measure PE fluorescence on a flow cytometer (you can also do cell counts, viability determinations, etc.). We equate 1 fluorescent event as 1 infected cell. Since we know how many cells have been plated in each well, we can determine the percentage infected in each well. We calculate a non-normalized titer from this data alone or we compare this data to a standardized virus and determine a normalized titer using a standard curve. From infection to having a titer in hand takes about 24 hours. Of course, the potential bottleneck is access to a flow cytometer! I can give more experimental details off-board. I should say that for getting an idea of relative titers and to test protein expression on a small scale, we also do the effective titer tests as suggested by Nat, with cell morphology and immunoblots as our read-out of virus potency and recombinant protein expression, respectively. No doubt, this will get you a long way but at some point, I argue, you need to determine an actual, absolute titer for duplication of results, troubleshooting, monitoring virus health over time, publications, etc. HTH- Brad On Thu, Mar 31, 2011 at 9:23 AM, Bertrand, Jay Aaron [Nervianoms] jay.bertr...@nervianoms.com wrote: I checked with someone in our protein production group and got the following response: We also stopped doing the virus titration with the plaque assay and instead are performing expression test with different concentration of virus
Re: [ccp4bb] titering baculovirus ?
Hi, We have observed this virus going off within 2 months as well. ...any idea why this happens? Chitra On 31 March 2011 18:07, Nathaniel Clark nathanielcl...@gmail.com wrote: We do adherent if we have a small volume of low titer virus, but as soon as we have a decent titer we will start with a 30 ml suspension flask. Normally I add something like 0.5 ml, which is probably more then I need. We only use serum free media right now. As I mentioned, if you need to boost the titer of a low titer stock, we plate a t25 flask with Sf9s, take off the media, and incubate it in 1 ml of pure virus (or two-fold diluted) for an hour or so, then remove (and save) that virus stock and give the cells fresh media. For fold dilution, just do a rough calculation of titer. Once you are at a high titer assume you have 10^8-10^9 pfu/ml, so you can estimate the ballpark volume to infect a given number of cells at an MOI of 1. From early stocks you might have a titer of 10^6-10^7 pfu/ml. If you do an amplification that doesn't work, decide if you were to high (MOI=10 not great for amplifying) or too low (MOI=0.1) and try again. Better yet, just set up 3 amplifications at the same time, whichever worked the best, use for another round of amplification, or for infections. For duration, we do 3 days amplification (post-infection), but sometimes we let them go a few days longer, which allows a secondary infection of the cells that weren't infected by the viral innoculum. There doesn't seem to be any harm to letting infections go longer; at some point all the cells die, but since the virus is pretty stable, it's no problem. We have a CEDEX cell counter which tells you average cell diameter, which is a great read out for infection. Also looking and density and viability versus an uninfected control will tell you if the infection worked. Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Thu, Mar 31, 2011 at 12:36 PM, Katya Heldwein katya.heldw...@gmail.com wrote: A related question: how do most people amplify their baculovirus stocks? Adherent cultures vs suspension? Fold dilution at each stage (P1 to P2, P2 to P3)? Duration of each amplification stage? We have some viral stocks that go off rather quickly (1-2 months) despite being stored with FBS in a cool, dark place. Katya On Thu, Mar 31, 2011 at 10:23 AM, Brad Bennett bradbennet...@gmail.com wrote: Though I'm not meaning to turn this into a plaque assay burn session, for many reasons we have also abandoned it and instead titer our baculovirus stocks (P3 and P4) using flow cytometry. We use an antibody against viral gp64 that has been labeled with PE and stain infected cells in 96 well plates with the PE-antibody. We then measure PE fluorescence on a flow cytometer (you can also do cell counts, viability determinations, etc.). We equate 1 fluorescent event as 1 infected cell. Since we know how many cells have been plated in each well, we can determine the percentage infected in each well. We calculate a non-normalized titer from this data alone or we compare this data to a standardized virus and determine a normalized titer using a standard curve. From infection to having a titer in hand takes about 24 hours. Of course, the potential bottleneck is access to a flow cytometer! I can give more experimental details off-board. I should say that for getting an idea of relative titers and to test protein expression on a small scale, we also do the effective titer tests as suggested by Nat, with cell morphology and immunoblots as our read-out of virus potency and recombinant protein expression, respectively. No doubt, this will get you a long way but at some point, I argue, you need to determine an actual, absolute titer for duplication of results, troubleshooting, monitoring virus health over time, publications, etc. HTH- Brad On Thu, Mar 31, 2011 at 9:23 AM, Bertrand, Jay Aaron [Nervianoms] jay.bertr...@nervianoms.com wrote: I checked with someone in our protein production group and got the following response: We also stopped doing the virus titration with the plaque assay and instead are performing expression test with different concentration of virus from the 3rd amplification. But for some viruses we still have doubts concerning the amplification success, so we are now evaluating a new technology using qPCR with the following kit (http://oetltd.com/products/category/baculoquant/). So you might have a look and see if it could be useful for your group. We would also be curious to hear if anyone else has experience with this approach. I hope this helps. Jay -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nathaniel Clark Sent: Wednesday, March 30, 2011 11:38 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb]
Re: [ccp4bb] what to do with disordered side chains
On Thu, Mar 31, 2011 at 10:14 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Also, setting occupancy = zero is not fudging but rather respectfully declining to comment based on lack of data. I think it is exactly the same as omitting residues one cannot see in the density. No, it's not the same. If you have placed any atoms, even with zero occupancy, you have said something about where you expect the atoms to be, or at least where the refinement program thinks they should be. Declining to comment would be deleting them, not guessing. I think a reasonable number could be derived and agreed upon, and would not be surprised if there is such a derivation or analysis in the literature answering the question: At what b-factor does modelling an atom become insignificant with respect to explaining/predicting/fitting the data? That point would be the b-factor/occupancy cutoff. Although atoms with very high B-factors may have almost no impact on F(calc), if the occupancy is non-zero they will still be driven by gradients with respect to X-ray data, and their positions (or changes thereof) will in turn affect other atoms, through geometry restraints if not F(calc). So there is no point at which these atoms cease to be relevant to the task of fitting. -Nat
Re: [ccp4bb] The meaning of B-factor, was Re: [ccp4bb] what to do with disordered side chains
On Thursday, March 31, 2011 10:05:22 am Hailiang Zhang wrote: Dear Zbyszek: Thanks a lot for your good summary. It is very interesting but, do you think there are some references for more detailed description, especially from mathematics point of view about correlating B-factor to the Gaussian probability distribution (the B-factor unit of A^2 is my first doubt as for the probability distribution description)? Thanks again for your efforts! Best Regards, Hailiang I already cited the IUCr standard once, but here it is again: Trueblood, et al, 1996; Acta Cryst. A52, 770-781 http://dx.doi.org/10.1107/S0108767396005697 -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] problem of conventions
To comment on Phil: I fed the mtz-file from pointless into ctruncate (or maybe it was scala) which left the space group string (P2 21 21) but turned the space group number 18 into 3018 - this does screw up autosharp and maybe also other programs which use the space group number/ symbol and not the symmetry operators. If that's the case autosharp should be fixed so it recognises the correct space group! (in this P22121 or #3018 in CCP4-ese). It has been fixed in autoBuster so maybe you're using an old version of autosharp? -- Ian
Re: [ccp4bb] The meaning of B-factor, was Re: [ccp4bb] what to do with disordered side chains
While what you say here is quite true and is useful for us to remember, your list is quite short. I can add another 3) The systematic error introduced by assuming full occupancy for all sites. There are, of course, many other factors that we don't account for that our refinement programs tend to dump into the B factors. The definition of that number in the PDB file, as listed in the mmCIF dictionary, only includes your first factor -- http://mmcif.rcsb.org/dictionaries/mmcif_std.dic/Items/_atom_site.B_iso_or_equiv.html and these numbers are routinely interpreted as though that definition is the law. Certainly the whole basis of TLS refinement is that the B factors are really Atomic Displacement Parameters. In addition the stereochemical restraints on B factors are derived from the assumption that these parameters are ADPs. Convoluting all these other factors with the ADPs causes serious problems for those who analyze B factors as measures of motion. The fact that current refinement programs mix all these factors with the ADP for an atom to produce a vaguely defined B factor should be considered a flaw to be corrected and not an opportunity to pile even more factors into this field in the PDB file. Dale Tronrud On 3/31/2011 9:06 AM, Zbyszek Otwinowski wrote: The B-factor in crystallography represents the convolution (sum) of two types of uncertainties about the atom (electron cloud) position: 1) dispersion of atom positions in crystal lattice 2) uncertainty of the experimenter's knowledge about the atom position. In general, uncertainty needs not to be described by Gaussian function. However, communicating uncertainty using the second moment of its distribution is a widely accepted practice, with frequently implied meaning that it corresponds to a Gaussian probability function. B-factor is simply a scaled (by 8 times pi squared) second moment of uncertainty distribution. In the previous, long thread, confusion was generated by the additional assumption that B-factor also corresponds to a Gaussian probability distribution and not just to a second moment of any probability distribution. Crystallographic literature often implies the Gaussian shape, so there is some justification for such an interpretation, where the more complex probability distribution is represented by the sum of displaced Gaussians, where the area under each Gaussian component corresponds to the occupancy of an alternative conformation. For data with a typical resolution for macromolecular crystallography, such multi-Gaussian description of the atom position's uncertainty is not practical, as it would lead to instability in the refinement and/or overfitting. Due to this, a simplified description of the atom's position uncertainty by just the second moment of probability distribution is the right approach. For this reason, the PDB format is highly suitable for the description of positional uncertainties, the only difference with other fields being the unusual form of squaring and then scaling up the standard uncertainty. As this calculation can be easily inverted, there is no loss of information. However, in teaching one should probably stress more this unusual form of presenting the standard deviation. A separate issue is the use of restraints on B-factor values, a subject that probably needs a longer discussion. With respect to the previous thread, representing poorly-ordered (so called 'disordered') side chains by the most likely conformer with appropriately high B-factors is fully justifiable, and currently is probably the best solution to a difficult problem. Zbyszek Otwinowski - they all know what B is and how to look for regions of high B (with, say, pymol) and they know not to make firm conclusions about H-bonds to flaming red side chains. But this knowledge may be quite wrong. If the flaming red really indicates large vibrational motion then yes, one whould not bet on stable H-bonds. But if the flaming red indicates that a well-ordered sidechain was incorrectly modeled at full occupancy when in fact it is only present at half-occupancy then no, the H-bond could be strong but only present in that half-occupancy conformation. One presumes that the other half-occupancy location (perhaps missing from the model) would have its own H-bonding network. I beg to differ. If a side chain has 2 or more positions, one should be a bit careful about making firm conclusions based on only one of those, even if it isn't clear exactly why one should use caution. Also, isn't the isotropic B we fit at medium resolution more of a spherical cow approximation to physical reality anyway? Phoebe Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
Re: [ccp4bb] problem of conventions
I would like to share my experiencde with a rather unexpected problem of indexing conventions. Perhaps I can save people some time I have a crystal in the more unusual P21212 space-group (No 18). Its unit cell lengths are bac (please note). I systematically use XDS for data integration, since so far it was able to handle even the most horrible-looking spots. Now XDS indexed my data in space-group 18, but with the axes order abc! It had, in fact, invented a space-group P22121, which does not exist. I did not realise this until I had spent a couple of weeks with beautiful peaks in rotation functions, but hopeless results in translation functions. It wasn't until I looked more closely into the definition of the screw axes that I realised the problem. POINTLESS does not allow a reindexing of reflexions within the same space-group, but fortunately REINDEX did the trick at the level of intensities, because I like to use SCALA for careful scaling of my data. I was wo,dering if XDS could perhaps reindex reflexions according to Int. Table conventions once the screw axes of a crystal system have been identified? The International Tables / IUCr / NIST convention _is_ a=b=c for orthorhombic so no re-indexing is necessary or desirable. See IT vol. A 5th ed. (2002), table 9.3.4.1 (p. 758 in my edition) for all the conventional cells. The problem may be that some programs are not sticking to the agreed convention - but then the obvious solution is to fix the program (or use a different one). Is the problem that XDS is indexing it correctly as P22121 but calling it SG #18 (i.e. instead of the correct #3018). That would certainly confuse all CCP4 programs which generally tend to use the space-group number first if it's available. I'm not clear what you mean when you say P22121 doesn't exist? It's clearly shown in my edition of IT (p. 202). Maybe your lab needs to invest in the most recent edition of IT? Cheers -- Ian
Re: [ccp4bb] what to do with disordered side chains
On 3/31/2011 10:14 AM, Jacob Keller wrote: What do we gain? As Dale pointed out, we are already abusing either occupancy, B-factor or delete the side chain to compensate for our inability to tell the user that the side chain is disordered. With your proposal, we would fudge both occupancy and B-factor, which in my eyes is even worse as fudging just one of the two. We gain clarity to the non-crystallographer user: a b-factor of 278.9 sounds like possibly something real. A b-factor of exactly 1000 does not. Both probably have the same believability, viz., ~zero. Also, setting occupancy = zero is not fudging but rather respectfully declining to comment based on lack of data. I think it is exactly the same as omitting residues one cannot see in the density. These things are never clear unless there is a solid definition of the terms you are using. I don't think you can come up with an out of band value for the B factor that doesn't have a legitimate meaning as an atomic displacement parameter for someone. How large a B factor you can meaningfully define depends on your lower resolution limit. People working with electron microscopy or small angle X-ray scattering could easily build models with ADPs far larger than anything we normally encounter. In addition, you can't define 1000 as a magic value since the PDB format will only allow values up to 999.99, and I presume maintaining the PDB format is one of your goals. Of course, you could choose -99.99 as the magic value but that would break all of our existing software and I presume you don't want that either. Actually defining any value for the B factor as the magic value would break all of our software. The only advantage of a large, positive, number is that it would create bugs that are more subtle. The fundamental problem with your solution is that you are trying to cram two pieces of information into a single number. Such density always causes problems. Each concept needs its own value. You could implement your solution easily in mmCIF. Just create a new tag, say _atom_site.imaginary_site, which is either true or false for every atom. Then everyone would be able to either filter out the fake atoms or leave them in, without ambiguity or confusion. If you object that the naive user of structural models wouldn't know to check this tag - they aren't going to know about your magic B factor either. You can't out-think someone who's not paying attention. At some point you have to assume that people being paid to perform research will learn the basics of the data they are using, even if you know that assumption is not 100% true. Dale Tronrud
Re: [ccp4bb] problem of conventions
Interesting. My IT, both volume I and volume A (1983) only have P21212 for space group #18. Do I have to purchase a new volume A every year to keep up with the new conventions? Cheers, Bernie On Thu, March 31, 2011 12:57 pm, Ian Tickle wrote: I would like to share my experiencde with a rather unexpected problem of indexing conventions. Perhaps I can save people some time I have a crystal in the more unusual P21212 space-group (No 18). Its unit cell lengths are bac (please note). I systematically use XDS for data integration, since so far it was able to handle even the most horrible-looking spots. Now XDS indexed my data in space-group 18, but with the axes order abc! It had, in fact, invented a space-group P22121, which does not exist. I did not realise this until I had spent a couple of weeks with beautiful peaks in rotation functions, but hopeless results in translation functions. It wasn't until I looked more closely into the definition of the screw axes that I realised the problem. POINTLESS does not allow a reindexing of reflexions within the same space-group, but fortunately REINDEX did the trick at the level of intensities, because I like to use SCALA for careful scaling of my data. I was wo,dering if XDS could perhaps reindex reflexions according to Int. Table conventions once the screw axes of a crystal system have been identified? The International Tables / IUCr / NIST convention _is_ a=b=c for orthorhombic so no re-indexing is necessary or desirable. See IT vol. A 5th ed. (2002), table 9.3.4.1 (p. 758 in my edition) for all the conventional cells. The problem may be that some programs are not sticking to the agreed convention - but then the obvious solution is to fix the program (or use a different one). Is the problem that XDS is indexing it correctly as P22121 but calling it SG #18 (i.e. instead of the correct #3018). That would certainly confuse all CCP4 programs which generally tend to use the space-group number first if it's available. I'm not clear what you mean when you say P22121 doesn't exist? It's clearly shown in my edition of IT (p. 202). Maybe your lab needs to invest in the most recent edition of IT? Cheers -- Ian
[ccp4bb] Question about GST cleavage
Just an offshoot of the same Question.. I would like to ask whether the same applies for GST-tag digestion using thrombin.. No agitation gives better results in the above case too... Any personal experiences On Thu, Mar 31, 2011 at 11:29 AM, Klaus Piontek klaus.pion...@ocbc.uni-freiburg.de wrote: And not at full moon! Klaus Am 31.03.2011 um 16:23 schrieb Xiaopeng Hu: Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: klaus.pion...@ocbc.uni-freiburg.de Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
Re: [ccp4bb] titering baculovirus ?
As many have mentioned, there is no need to titer baculoviruses for protein expression purpose. Plaque assay or other titering methods can give 10-fold variation between two operators. Cells can be different from time to time as well. Sf9 cells in different labs are quite different. MOI reported in literature is pretty much meaningless. I think most authors and especially their labmates agree on this point. The important thing is how insect cells are infected. In this aspect, industry is at least 10 year in advance than academia. We have developed methods to quantify the infection and ensure high quality virus production and protein expression. Nat has described some steps how to do it. When properly made and stored, recombinant baculoviruses are stable for at least a year without the need of adding FBS or other stabilizer. We have used several 2 year old viruses and got the exact same results as when they were freshly made. TIPS is one great way for long term virus storage, but reqires expensive equipment and know-how. Bacmids can be stored cheaply (or plasmids if not using Bac-to-Bac system). It takes only a week to generate enough high tiger viruses sufficient for 5-10L expression. For most academic research, there may be no need to do any viral amplification. Cheers, Chun Accelagen -Original Message- From: Nathaniel Clark nathanielcl...@gmail.com Sender: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK Date: Thu, 31 Mar 2011 13:20:14 To: CCP4BB@JISCMAIL.AC.UK Reply-To: Nathaniel Clark nathanielcl...@gmail.com Subject: Re: [ccp4bb] titering baculovirus ? We don't do anything special to store the virus, just a dark fridge. You do need to mix well as the virus can settle. You might want to try this 'TIPS' approach. Basically you freeze baculovirus infected cells, and use them as your virus innoculum. I think I tried it once and it didn't really work, but I didn't really try too hard... There is a research paper on the subject that I can't find, but here is the first thing I found on google: http://www.abrf.org/ResearchGroups/ProteinExpressionResearchGroup/Activities/Wasilko-PERG2007.pdf Nat On Thu, Mar 31, 2011 at 1:12 PM, chitra shintre chitra.shin...@gmail.com wrote: Hi, We have observed this virus going off within 2 months as well. ...any idea why this happens? Chitra On 31 March 2011 18:07, Nathaniel Clark nathanielcl...@gmail.com wrote: We do adherent if we have a small volume of low titer virus, but as soon as we have a decent titer we will start with a 30 ml suspension flask. Normally I add something like 0.5 ml, which is probably more then I need. We only use serum free media right now. As I mentioned, if you need to boost the titer of a low titer stock, we plate a t25 flask with Sf9s, take off the media, and incubate it in 1 ml of pure virus (or two-fold diluted) for an hour or so, then remove (and save) that virus stock and give the cells fresh media. For fold dilution, just do a rough calculation of titer. Once you are at a high titer assume you have 10^8-10^9 pfu/ml, so you can estimate the ballpark volume to infect a given number of cells at an MOI of 1. From early stocks you might have a titer of 10^6-10^7 pfu/ml. If you do an amplification that doesn't work, decide if you were to high (MOI=10 not great for amplifying) or too low (MOI=0.1) and try again. Better yet, just set up 3 amplifications at the same time, whichever worked the best, use for another round of amplification, or for infections. For duration, we do 3 days amplification (post-infection), but sometimes we let them go a few days longer, which allows a secondary infection of the cells that weren't infected by the viral innoculum. There doesn't seem to be any harm to letting infections go longer; at some point all the cells die, but since the virus is pretty stable, it's no problem. We have a CEDEX cell counter which tells you average cell diameter, which is a great read out for infection. Also looking and density and viability versus an uninfected control will tell you if the infection worked. Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Thu, Mar 31, 2011 at 12:36 PM, Katya Heldwein katya.heldw...@gmail.com wrote: A related question: how do most people amplify their baculovirus stocks? Adherent cultures vs suspension? Fold dilution at each stage (P1 to P2, P2 to P3)? Duration of each amplification stage? We have some viral stocks that go off rather quickly (1-2 months) despite being stored with FBS in a cool, dark place. Katya On Thu, Mar 31, 2011 at 10:23 AM, Brad Bennett bradbennet...@gmail.com wrote: Though I'm not meaning to turn this into a plaque assay burn session, for many reasons we have also abandoned it and instead titer our baculovirus stocks (P3 and P4) using flow cytometry. We use an antibody against viral gp64 that
Re: [ccp4bb] Question about TEV cleavage
Properly made TEV protease should work with or without rocking. We have done QC of our TurboTEV, a dual GST- and His-tagged TEV, under many conditions and used on hundreds of proteins. The most common cause of incomplete digestion is the insolubility of target protein or bad construct design. Shaking or agitation may oxidize the protein. TEV is a Cys protease that can be oxidized as well. Adding reducing agents or EDTA may well prevent some of the effects of agitation. Unlike plasmid DNA, protein solutions should be mixed GENTLY. Regarding the original question, 50% loss may not be a bad thing if it is about total protein. In our experience, it is not uncommn to have only 50% recovery after tag removal of a Ni pool. Some proteins aggregate after tag removal even it's His-tag. Again the loss can be a good thing. Chun Accelagen -Original Message- From: Laurie Betts laurie.betts0...@gmail.com Sender: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK Date: Thu, 31 Mar 2011 11:11:33 To: CCP4BB@JISCMAIL.AC.UK Reply-To: Laurie Betts laurie.betts0...@gmail.com Subject: Re: [ccp4bb] Question about TEV cleavage We have the same experience with the DO NOT AGITATE. We purify our own His-tagged TEV protease, and flash-freeze it IMMEDIATELY after IMAC prep at about 1 mg/mL. Laurie Betts UNC On Thu, Mar 31, 2011 at 10:37 AM, Charles Allerston charles.allers...@sgc.ox.ac.uk wrote: I echo this. Some time ago I was working on a target which seemed to precipitate when cleaving overnight with TEV. I wasted a fair bit of time trying to optimise cleavage conditions with a myriad of buffers. In the end, just by not agitating my solution, there was no precipitation and recovery was extremely good. I never agitate my solutions when cleaving now and have not had any problems with cleavage (to do with this issue, anyway) or recovery. cheers charlie Dr. Charles Allerston Genome Integrity Group Structural Genomics Consortium Nuffield Department of Medicine Old Road Campus University of Oxford OX3 7DQ http://www.sgc.ox.ac.uk/ -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiaopeng Hu Sent: 31 March 2011 15:23 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Question about TEV cleavage Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng
Re: [ccp4bb] titering baculovirus ?
On another note, we have moved from baculovirus to stable poly or monoclonal Tn5 cell lines using the pIB/V5-HIS vector. It removes all concerns about titering and viral propagation, and for most of our proteins, the expression level is the same or better. We secrete all our proteins, so the stable cell line is great, you just grow several liters of the cells to a high density, harvest the supernatant, and purify. It's much simpler. Nat On Thu, Mar 31, 2011 at 3:52 PM, Chun Luo c...@accelagen.com wrote: As many have mentioned, there is no need to titer baculoviruses for protein expression purpose. Plaque assay or other titering methods can give 10-fold variation between two operators. Cells can be different from time to time as well. Sf9 cells in different labs are quite different. MOI reported in literature is pretty much meaningless. I think most authors and especially their labmates agree on this point. The important thing is how insect cells are infected. In this aspect, industry is at least 10 year in advance than academia. We have developed methods to quantify the infection and ensure high quality virus production and protein expression. Nat has described some steps how to do it. When properly made and stored, recombinant baculoviruses are stable for at least a year without the need of adding FBS or other stabilizer. We have used several 2 year old viruses and got the exact same results as when they were freshly made. TIPS is one great way for long term virus storage, but reqires expensive equipment and know-how. Bacmids can be stored cheaply (or plasmids if not using Bac-to-Bac system). It takes only a week to generate enough high tiger viruses sufficient for 5-10L expression. For most academic research, there may be no need to do any viral amplification. Cheers, Chun Accelagen -Original Message- From: Nathaniel Clark nathanielcl...@gmail.com Sender: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK Date: Thu, 31 Mar 2011 13:20:14 To: CCP4BB@JISCMAIL.AC.UK Reply-To: Nathaniel Clark nathanielcl...@gmail.com Subject: Re: [ccp4bb] titering baculovirus ? We don't do anything special to store the virus, just a dark fridge. You do need to mix well as the virus can settle. You might want to try this 'TIPS' approach. Basically you freeze baculovirus infected cells, and use them as your virus innoculum. I think I tried it once and it didn't really work, but I didn't really try too hard... There is a research paper on the subject that I can't find, but here is the first thing I found on google: http://www.abrf.org/ResearchGroups/ProteinExpressionResearchGroup/Activities/Wasilko-PERG2007.pdf Nat On Thu, Mar 31, 2011 at 1:12 PM, chitra shintre chitra.shin...@gmail.com wrote: Hi, We have observed this virus going off within 2 months as well. ...any idea why this happens? Chitra On 31 March 2011 18:07, Nathaniel Clark nathanielcl...@gmail.com wrote: We do adherent if we have a small volume of low titer virus, but as soon as we have a decent titer we will start with a 30 ml suspension flask. Normally I add something like 0.5 ml, which is probably more then I need. We only use serum free media right now. As I mentioned, if you need to boost the titer of a low titer stock, we plate a t25 flask with Sf9s, take off the media, and incubate it in 1 ml of pure virus (or two-fold diluted) for an hour or so, then remove (and save) that virus stock and give the cells fresh media. For fold dilution, just do a rough calculation of titer. Once you are at a high titer assume you have 10^8-10^9 pfu/ml, so you can estimate the ballpark volume to infect a given number of cells at an MOI of 1. From early stocks you might have a titer of 10^6-10^7 pfu/ml. If you do an amplification that doesn't work, decide if you were to high (MOI=10 not great for amplifying) or too low (MOI=0.1) and try again. Better yet, just set up 3 amplifications at the same time, whichever worked the best, use for another round of amplification, or for infections. For duration, we do 3 days amplification (post-infection), but sometimes we let them go a few days longer, which allows a secondary infection of the cells that weren't infected by the viral innoculum. There doesn't seem to be any harm to letting infections go longer; at some point all the cells die, but since the virus is pretty stable, it's no problem. We have a CEDEX cell counter which tells you average cell diameter, which is a great read out for infection. Also looking and density and viability versus an uninfected control will tell you if the infection worked. Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst On Thu, Mar 31, 2011 at 12:36 PM, Katya Heldwein katya.heldw...@gmail.com wrote: A related question: how do most people amplify their baculovirus stocks? Adherent cultures vs suspension? Fold
Re: [ccp4bb] problem of conventions
There are no 'new' conventions to keep up with: recent editions of the old volume 1 or new A do not disagree on the question of the unit cell conventions (except for minor details which don't affect the majority of the common space groups), where by recent I mean going back ~ 70 years. So it's certainly not the case that the conventions are changing every year (that would be silly!) - they have been defined exactly once in the last 100 years! I believe the unit cell conventions currently in use were actually first defined by the 1952 edition of International Tables, so both the 1969 edition (volume '1') and the 1983 edition (1st of volume 'A') will certainly describe them. I have only the 2002 edition (the 5th) so I can't tell you exactly where to find the relevant info in the older editions. The very first edition of IT (1935 I believe) did not define the unit cell conventions, only the space groups, so I wouldn't recommend that! The older editions did not include information on alternate space-group settings simply in order to save paper: the 1952 edition was published in the years following WW2 when there was a paper shortage, so this was an important consideration! Only one setting (the 'standard setting') of each space group, chosen arbitrarily, was described and the crystallographer was expected to permute it to get the desired setting. If you need to see all the alternate settings laid out explicitly then you need to get hold of a recent (e.g. the 5th printed or 1st online) edition; failing that you have to work them out yourself! I thought the alternate settings were first described (though possibly without the diagrams) in the 1st (1983) edition of volume A, but I'm relying on memory and could well be wrong. The setting was often chosen to be consistent with a pre-existing isomorphous structure (i.e. generally isomorphism overrides convention); if there was none either the setting was defined by the unit cell convention, or often it was simply easiest to use the standard setting. Of course not everyone followed the conventions: it was common to write programs that could handle only the standard settings (and it still is!). Wiser programmers allowed space-groups to be defined arbitrarily by the equivalent positions instead of the number or symbol, so then it was straightforward to select any desired alternate setting. Note that the convention describes the unit cells, from which the space-group symbols are then derived, not the other way around. The ratiionale behind this is simple: there was a time not so long ago (which I remember!) when data collection and structure solution for even routine structures was actually non-trivial (I'm not implying that it's always trivial even nowadays!). However it was possible relatively straightforwardly to obtain the unit cell from precession photos (i.e. the indexing). It used to be common practice to publish an initial communication giving the unit cell and possibly a tentative space group; this would be followed up (often several years later!) by structures determined to successively higher resolution as more data was collected. Of course it was not possible to be 100% certain of the space-group assignment from the precession photos (and for several space-groups there is of course no unique space-group determinable from the systematic absences alone); final space-group assignment often had to wait several years for the structure determination. Hence it made sense to define the setting from the unit cell, not the space group. I recommend the 2 papers from the US National Institute of Standards Technology (see Phil's posting) for more on this: the NIST conventions are the same as the IUCr ones, i.e. based on the unit cell (in fact Alan Mighell when he was at NIST wrote much of unit-cell convention material in IT). -- Ian On Thu, Mar 31, 2011 at 7:36 PM, Santarsiero, Bernard D. b...@uic.edu wrote: Interesting. My IT, both volume I and volume A (1983) only have P21212 for space group #18. Do I have to purchase a new volume A every year to keep up with the new conventions? Cheers, Bernie On Thu, March 31, 2011 12:57 pm, Ian Tickle wrote: I would like to share my experiencde with a rather unexpected problem of indexing conventions. Perhaps I can save people some time I have a crystal in the more unusual P21212 space-group (No 18). Its unit cell lengths are bac (please note). I systematically use XDS for data integration, since so far it was able to handle even the most horrible-looking spots. Now XDS indexed my data in space-group 18, but with the axes order abc! It had, in fact, invented a space-group P22121, which does not exist. I did not realise this until I had spent a couple of weeks with beautiful peaks in rotation functions, but hopeless results in translation functions. It wasn't until I looked more closely into the definition of the screw axes that I realised the problem. POINTLESS does not allow a
Re: [ccp4bb] Question about TEV cleavage
Totally agree with Chun. We are using a His tagged S219V construct that's very similary to the one described in the Waugh paper. To my experience, agitation, 37C incubation, low salt buffer, (etc.?), should all be avoided when using TEV. When using relatively large amount of enzyme(0.1~0.2mg/mL final, in total about 1/50 -1/10 of the total substrate by mol), we can often cleave our ProteinA fusions to nearly 90% complete on IgG beads. We do it at 4C overnight, elute, check the cleavage by SDS gel, if not complete, we add enzyme, cut O/N again, elute, check on SDS again. Then that's the 90% cleavage I am refering to. I also did O/N cleavage on column at 22C, and the cleave was quite good. In solution, my experience was positive too. In some cases even added at less than 1:200 molar ratio, the enzyme can still achieve a nearly complete cleavage at 4C in one or two days. One thing that could greatly affect the cleavage is the accessibility of the cleavage site. Looking from the crystal structure of TEV protease, the Q in the ENLYFQ-G/S has better be at least 2-3 aa away from the folded protein domain coming up. I had one construct made by LIC, which had only one G outside the folded domain (revealed by later crystal structure), then it could not be cleaved at all. When two more amino acids were added, it cleaved fine. Zhijie From: Chun Luo Sent: Thursday, March 31, 2011 4:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Question about TEV cleavage Properly made TEV protease should work with or without rocking. We have done QC of our TurboTEV, a dual GST- and His-tagged TEV, under many conditions and used on hundreds of proteins. The most common cause of incomplete digestion is the insolubility of target protein or bad construct design. Shaking or agitation may oxidize the protein. TEV is a Cys protease that can be oxidized as well. Adding reducing agents or EDTA may well prevent some of the effects of agitation. Unlike plasmid DNA, protein solutions should be mixed GENTLY. Regarding the original question, 50% loss may not be a bad thing if it is about total protein. In our experience, it is not uncommn to have only 50% recovery after tag removal of a Ni pool. Some proteins aggregate after tag removal even it's His-tag. Again the loss can be a good thing. Chun Accelagen From: Laurie Betts laurie.betts0...@gmail.com Sender: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK Date: Thu, 31 Mar 2011 11:11:33 -0400 To: CCP4BB@JISCMAIL.AC.UK ReplyTo: Laurie Betts laurie.betts0...@gmail.com Subject: Re: [ccp4bb] Question about TEV cleavage We have the same experience with the DO NOT AGITATE. We purify our own His-tagged TEV protease, and flash-freeze it IMMEDIATELY after IMAC prep at about 1 mg/mL. Laurie Betts UNC On Thu, Mar 31, 2011 at 10:37 AM, Charles Allerston charles.allers...@sgc.ox.ac.uk wrote: I echo this. Some time ago I was working on a target which seemed to precipitate when cleaving overnight with TEV. I wasted a fair bit of time trying to optimise cleavage conditions with a myriad of buffers. In the end, just by not agitating my solution, there was no precipitation and recovery was extremely good. I never agitate my solutions when cleaving now and have not had any problems with cleavage (to do with this issue, anyway) or recovery. cheers charlie Dr. Charles Allerston Genome Integrity Group Structural Genomics Consortium Nuffield Department of Medicine Old Road Campus University of Oxford OX3 7DQ http://www.sgc.ox.ac.uk/ -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiaopeng Hu Sent: 31 March 2011 15:23 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Question about TEV cleavage Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng
Re: [ccp4bb] problem of conventions
I have the 2002 edition, and indeed it only contains space group numbers up to 230. The page numbers quoted by Ian contain space group numbers 17 and 18. Although I am all for program authors building in support for the screwy orthorhombics (as I call them), I should admit that my fuddy-duddy strategy for dealing with them remains simply to use space groups 17 and 18, and permute the cell edges around with REINDEX to put the unique (screw or non-screw) axis on the c position. I have yet to encounter a program that gets broken when presented with data that doesn't have abc, but there are many non-CCP4 programs out there that still don't seem to understand P22121, P21221, P2122 and P2212. This is not the only space group convention issue out there! The R3x vs H3x business continues to be annoying to this day! -James Holton MAD Scientist On Thu, Mar 31, 2011 at 11:36 AM, Santarsiero, Bernard D. b...@uic.edu wrote: Interesting. My IT, both volume I and volume A (1983) only have P21212 for space group #18. Do I have to purchase a new volume A every year to keep up with the new conventions? Cheers, Bernie On Thu, March 31, 2011 12:57 pm, Ian Tickle wrote: I would like to share my experiencde with a rather unexpected problem of indexing conventions. Perhaps I can save people some time I have a crystal in the more unusual P21212 space-group (No 18). Its unit cell lengths are bac (please note). I systematically use XDS for data integration, since so far it was able to handle even the most horrible-looking spots. Now XDS indexed my data in space-group 18, but with the axes order abc! It had, in fact, invented a space-group P22121, which does not exist. I did not realise this until I had spent a couple of weeks with beautiful peaks in rotation functions, but hopeless results in translation functions. It wasn't until I looked more closely into the definition of the screw axes that I realised the problem. POINTLESS does not allow a reindexing of reflexions within the same space-group, but fortunately REINDEX did the trick at the level of intensities, because I like to use SCALA for careful scaling of my data. I was wo,dering if XDS could perhaps reindex reflexions according to Int. Table conventions once the screw axes of a crystal system have been identified? The International Tables / IUCr / NIST convention _is_ a=b=c for orthorhombic so no re-indexing is necessary or desirable. See IT vol. A 5th ed. (2002), table 9.3.4.1 (p. 758 in my edition) for all the conventional cells. The problem may be that some programs are not sticking to the agreed convention - but then the obvious solution is to fix the program (or use a different one). Is the problem that XDS is indexing it correctly as P22121 but calling it SG #18 (i.e. instead of the correct #3018). That would certainly confuse all CCP4 programs which generally tend to use the space-group number first if it's available. I'm not clear what you mean when you say P22121 doesn't exist? It's clearly shown in my edition of IT (p. 202). Maybe your lab needs to invest in the most recent edition of IT? Cheers -- Ian
Re: [ccp4bb] what to do with disordered side chains
On 3/31/2011 12:52 PM, Jacob Keller wrote: The only advantage of a large, positive, number is that it would create bugs that are more subtle. Although most of the users on this BB probably know more about the software coding, I am surprised that bugs--even subtle ones--would be introduced by residues flagged with 0 occupancy and b-factor = 500. Can you elaborate/enumerate? The principle problems with defining a particular value of the B factor as magical have already been listed in this thread. B factors are usually restrained to the values of the atoms their atom is bonded to and sometimes to other atoms they pack against. You may set the B factor equal to 500.00 but it will not stick. At worst its presence will pull up the B factors of nearby atoms that do have density. In addition, the only refinement program I know of that takes occupancy into account when restraining bond lengths and angles is CNX. The presence of atoms with occ=0 will affect the location of atoms they share geometry restraints with. Of course you could modify the refinement programs, and every other program that reads crystallographic models, to deal with your redefinition of _atom_site.B_iso_or_equiv. In fact you would have to, just as you would have to when you change the definition of any of the parameters in our models. If we have to modify code, why not create a solution that is explicit, clear, and as consistent with previous practices as possible? I think that the worst that could happen is that the unexperienced yet b-factor-savvy user would be astonished by the huge b-factors, even if he did not realize they were flags. At best, being surprised at the precise number 500, he would look into the pdb file and see occupancy = zero, google it, and learn something new about crystallography. How about positive difference map peaks on neighboring atoms? How about values for B factors that don't relate to the mean square motion of the atom, despite that being the direct definition of the B factor? The concept of an unexperienced yet b-factor-savvy user is amusing. I'm not b-factor-savvy. Atomic displacement values are easy, but I'm learning new subtleties about B factors all the time. The fundamental problem with your solution is that you are trying to cram two pieces of information into a single number. Such density always causes problems. Each concept needs its own value. What two pieces of information into what single number? Occupancy = 0 tells you that the atom cannot be modelled, and B=500 is merely a flag for same, and always goes with occ=0. What is so dense? On the contrary, I think the info is redundant if anything... To be honest I had forgotten that you were proposing that the occupancy be set to zero at the same time. Besides putting two pieces of information in the B factor column (The B factor's value and a flag for imaginaryness.) You do the same for occupancy (the occupancy's value and a flag for imaginaryness.) This violates another rule of data structures - that each concept be stored in one, and only one, place. How do you interpret an atom with an occupancy of zero but a B factor of 250? How about an atom with a B factor of 500.00 and an occupancy of 1.00? Now we have the confusing situation that the B factor can only be interpreted in the context of the the value of the occupancy and vice versa. Database-savvy people (and I'm not one of them either) are not going to like this. If you want to calculate the average B factor for a model, certainly those atoms with their B factor = 500 should not be included. However, I gather we do need to include those equal to 500 if their occupancy is not equal to 0.0. This is a mess. In a database application we can't simply SELECT the row with the B factors and average them. We have to SELECT both the B factor and occupancy rows and perform some really weird if statements element by element - just to calculate an average! What should be a simple task becomes very complex. Will a graduate student code the calculation correctly? Probably not. They will likely not recall all the complicated interpretations of special values your convention would require. Now consider this. Refinement is running along and the occupancy for an atom happens to overshoot and, in the middle of refinement, assumes a value of 0.00. There is positive difference density the next cycle. (I did say that it overshot.) Should the refinement program interpret that Occ=0.00 to mean that the atom is imaginary and should not be considered as part of the crystallographic model? Wouldn't it be bad if the atom suddenly disappeared because of a fluctuation? Or should the refinement program use one definition of occupancy during refinement, but write a PDB file occupancy that has a different definition? (It might be relevant to this line of thought to recall that the TNT refinement package writes each intermediate coordinate file to disk
Re: [ccp4bb] Question about GST cleavage
I once cleaved a GST tag on the resin using TEV by rocking overnight at 4 C. I would say it is 100% cutting judging from the gel. One thing to add is that the protein bound so tight to the beads that cutting tag is the only way to elute it except by SDS. I haven't had any trouble with TEV and thrombin with rocking or shaking. The important part is that you have to have good protease to start with. Nian Huang, Ph.D. UT Southwestern Medical Center On Thu, Mar 31, 2011 at 2:41 PM, gauri misra kamga...@gmail.com wrote: Just an offshoot of the same Question.. I would like to ask whether the same applies for GST-tag digestion using thrombin.. No agitation gives better results in the above case too... Any personal experiences On Thu, Mar 31, 2011 at 11:29 AM, Klaus Piontek klaus.pion...@ocbc.uni-freiburg.de wrote: And not at full moon! Klaus Am 31.03.2011 um 16:23 schrieb Xiaopeng Hu: Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: klaus.pion...@ocbc.uni-freiburg.de Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
[ccp4bb] xds question: inverse beam, lots of wedges
We've just collected a number of inverse beam data sets. It turns out the crystals showed little radiation damage, so we have a lot of data: 2 x 360 deg for each crystal, broken up into 30 deg wedges. The collection order went like this: 0-30 deg, 180-210, 30-60, 210-240, etc. Now, assuming no slippage, I could simply integrate the first set of data (non-inverse?) in one run: 0-360 deg. However, since the 12 individual wedges making up this 360 deg sweep were not collected immediately one after the other, I don't expect the scale factors for individual images to vary smoothly (there should be discontinuities at the boundaries between wedges). If I do integrate the data in one fell swoop, am I in danger of introducing errors? For example, I seem to recall that denzo had built-in restraints to ensure that scale factors for adjacent images didn't vary by too much. Is there a similar restraint that in XDS that I might run afoul of? The alternative is to integrate each each wedge separately, but with 24 wedges per xtal, this is starting to look a little tedious. Cheers, Pat
Re: [ccp4bb] The meaning of B-factor, was Re: [ccp4bb] what to do with disordered side chains
Dale Tronrud wrote: While what you say here is quite true and is useful for us to remember, your list is quite short. I can add another 3) The systematic error introduced by assuming full occupancy for all sites. You are right that structural heterogeneity is an additional factor. Se-Met expression is one of the examples where the Se-Met residue is often not fully incorporated, and therefore its side chains have mixed with Met composition. Obviously, solvent molecules may have partial occupancies. Also, in heavily exposed crystals chemical reactions result in loss of the functional groups (e.g. by decarboxylation). However, in most cases even if side chains have multiple conformations their total occupancy is 1.0. There are, of course, many other factors that we don't account for that our refinement programs tend to dump into the B factors. The definition of that number in the PDB file, as listed in the mmCIF dictionary, only includes your first factor -- http://mmcif.rcsb.org/dictionaries/mmcif_std.dic/Items/_atom_site.B_iso_or_equiv.html and these numbers are routinely interpreted as though that definition is the law. Certainly the whole basis of TLS refinement is that the B factors are really Atomic Displacement Parameters. In addition the stereochemical restraints on B factors are derived from the assumption that these parameters are ADPs. Convoluting all these other factors with the ADPs causes serious problems for those who analyze B factors as measures of motion. The fact that current refinement programs mix all these factors with the ADP for an atom to produce a vaguely defined B factor should be considered a flaw to be corrected and not an opportunity to pile even more factors into this field in the PDB file. B-factors describe overall uncertainty of the current model. Refinement programs, which do not introduce or remove parts of the model (e.g. are not able to add additional conformations) intrinsically pile up all uncertainties into B-factors. Solutions, which you would like to see implemented, require a model-building like approach. The test of the success of such approach would be a substantial decrease of R-free values. If anybody can show it, it would be great. Zbyszek Dale Tronrud On 3/31/2011 9:06 AM, Zbyszek Otwinowski wrote: The B-factor in crystallography represents the convolution (sum) of two types of uncertainties about the atom (electron cloud) position: 1) dispersion of atom positions in crystal lattice 2) uncertainty of the experimenter's knowledge about the atom position. In general, uncertainty needs not to be described by Gaussian function. However, communicating uncertainty using the second moment of its distribution is a widely accepted practice, with frequently implied meaning that it corresponds to a Gaussian probability function. B-factor is simply a scaled (by 8 times pi squared) second moment of uncertainty distribution. In the previous, long thread, confusion was generated by the additional assumption that B-factor also corresponds to a Gaussian probability distribution and not just to a second moment of any probability distribution. Crystallographic literature often implies the Gaussian shape, so there is some justification for such an interpretation, where the more complex probability distribution is represented by the sum of displaced Gaussians, where the area under each Gaussian component corresponds to the occupancy of an alternative conformation. For data with a typical resolution for macromolecular crystallography, such multi-Gaussian description of the atom position's uncertainty is not practical, as it would lead to instability in the refinement and/or overfitting. Due to this, a simplified description of the atom's position uncertainty by just the second moment of probability distribution is the right approach. For this reason, the PDB format is highly suitable for the description of positional uncertainties, the only difference with other fields being the unusual form of squaring and then scaling up the standard uncertainty. As this calculation can be easily inverted, there is no loss of information. However, in teaching one should probably stress more this unusual form of presenting the standard deviation. A separate issue is the use of restraints on B-factor values, a subject that probably needs a longer discussion. With respect to the previous thread, representing poorly-ordered (so called 'disordered') side chains by the most likely conformer with appropriately high B-factors is fully justifiable, and currently is probably the best solution to a difficult problem. Zbyszek Otwinowski - they all know what B is and how to look for regions of high B (with, say, pymol) and they know not to make firm conclusions about H-bonds to flaming red side chains. But this knowledge may be quite wrong. If the flaming red really indicates large vibrational motion then yes, one whould not bet
Re: [ccp4bb] xds question: inverse beam, lots of wedges
Pat, at least give it a try with the one sweep approach. We have collected plenty of 360deg data sets on a Rigaku system which requires two omega sweeps at phi 0 and 180 deg. These data sets are for in-house phasing. We haven't seen big issues with running XDS over these images as one continuous sweep. Monitoring scalefactors might be a good indicator. Good luck Jan On Mar 31, 2011, at 3:08 PM, Patrick Loll wrote: We've just collected a number of inverse beam data sets. It turns out the crystals showed little radiation damage, so we have a lot of data: 2 x 360 deg for each crystal, broken up into 30 deg wedges. The collection order went like this: 0-30 deg, 180-210, 30-60, 210-240, etc. Now, assuming no slippage, I could simply integrate the first set of data (non-inverse?) in one run: 0-360 deg. However, since the 12 individual wedges making up this 360 deg sweep were not collected immediately one after the other, I don't expect the scale factors for individual images to vary smoothly (there should be discontinuities at the boundaries between wedges). If I do integrate the data in one fell swoop, am I in danger of introducing errors? For example, I seem to recall that denzo had built-in restraints to ensure that scale factors for adjacent images didn't vary by too much. Is there a similar restraint that in XDS that I might run afoul of? The alternative is to integrate each each wedge separately, but with 24 wedges per xtal, this is starting to look a little tedious. Cheers, Pat -- Jan Abendroth Emerald BioStructures Seattle / Bainbridge Island WA, USA home: Jan.Abendroth_at_gmail.com work: JAbendroth_at_embios.com http://www.emeraldbiostructures.com
Re: [ccp4bb] what to do with disordered side chains
Hi All, Notwithstanding the stimulating discussion about the B-factor, I'd like to chime in with my $0.02 on the original question of to build or not to build and what are the rules and standards... and sorry for the lengthy e-mail - I was trying to respond to several comments at once. I thought there was a very well-defined rule: models based on experimental data should represent the experimental data correctly. If a model has parts that are not substantiated by experimental data and are based only on assumptions, it's no longer an experimental model. Based on this, one should leave out the atoms for which there is no observable electron density. And one need not say that we were unable to build a model of a missing side chains (or any other segments of the structure). There is also no need to guess or fake most probable conformations of the unobserved parts. Instead, it should be reported that the segment in question was so flexible that it could not be described by just one or two (and may be three) conformers. As such, this observation stands on its own feet just like any other observation of visible segments and there is no need to fake a model. If the work was done properly, a model with missing parts is not intrinsically inferior to other, more complete model. The fact that a side chain displays flexibility may be biologically much more relevant than some well-defined Ile in the core of the molecule. Omitting unobserved side chains from the model would also help avoid assumptions as if we know for sure that the side chain is there. Given side chain actually may not be there for some reason or another. Sequence errors and radiation-induced damage come to mind, for example. The latter is also often the reason that the side chain may not be fully occupied in the structure derived from a specific data set (i.e. the sum of occupancies of all its existing conformations may not be 1, contrary to earlier suggestions in the thread). Back in the day I personally spent large amounts of time and effort constructing and refining multi-conformational models of some side chains because I was sure they were there somewhere. Later on, as we learned more, I realized that some of them have been sheered by radiation damage and actually were not there. As knowledge advances, many of our assumptions may crumble and that's why we ought to keep experimentally visible models apart from those with assumed parts. As for the downstream consumers of our models, we may not need to confuse them with strange B factors or occupancies. We just need to give them correct information. Namely, that the given part(s) of the molecule could not be seen experimentally due to its flexibility (or, in some cases, to radiation damage). There was an interesting suggestion of two models - one accurately describing the experimental observations and the other for the downstream users. It would be a good way to separate Sci from Fi but there may be a problem. When theories are derived further downstream, it'll be impossible to keep track of what came from Sci and what came from Fi versions. Best regards, N. Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dale Tronrud Sent: Thursday, March 31, 2011 4:51 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] what to do with disordered side chains On 3/31/2011 12:52 PM, Jacob Keller wrote: The only advantage of a large, positive, number is that it would create bugs that are more subtle. Although most of the users on this BB probably know more about the software coding, I am surprised that bugs--even subtle ones--would be introduced by residues flagged with 0 occupancy and b-factor = 500. Can you elaborate/enumerate? The principle problems with defining a particular value of the B factor as magical have already been listed in this thread. B factors are usually restrained to the values of the atoms their atom is bonded to and sometimes to other atoms they pack against. You may set the B factor equal to 500.00 but it will not stick. At worst its presence will pull up the B factors of nearby atoms that do have density. In addition, the only refinement program I know of that takes occupancy into account when restraining bond lengths and angles is CNX. The presence of atoms with occ=0 will affect the location of atoms they share geometry restraints with. Of course you could modify the refinement programs, and every other program that reads crystallographic models, to deal with your redefinition of _atom_site.B_iso_or_equiv. In fact you would have to, just as you would have to when you change the definition of any of the parameters in our models. If we have to modify code, why not create a solution that is explicit, clear, and as consistent with previous practices as
[ccp4bb] Jrh input Re: [ccp4bb] what to do with disordered side chains
Dear Ed, Thankyou for this and apologies for late reply. If one has chemical evidence for the presence of residues but these residues are disordered I find the delete atoms option disagreeable. Such a static disorder situation should be described by a high atomic displacement parameter, in my view. (nb the use of ADP is better than B factor terminology). Yours sincerely, John Prof John R Helliwell DSc On 29 Mar 2011, at 22:43, Ed Pozharski epozh...@umaryland.edu wrote: The results of the online survey on what to do with disordered side chains (from total of 240 responses): Delete the atoms 43% Let refinement take care of it by inflating B-factors41% Set occupancy to zero12% Other 4% Other suggestions were: - Place atoms in most likely spot based on rotomer and contacts and indicate high positional sigmas on ATMSIG records - To invent refinement that will spread this residues over many rotamers as this is what actually happened - Delet the atoms but retain the original amino acid name - choose the most common rotamer (B-factors don't inflate, they just rise slightly) - Depends. if the disordered region is unteresting, delete atoms. Otherwise, try to model it in one or more disordered model (and then state it clearly in the pdb file) - In case that no density is in the map, model several conformations of the missing segment and insert it into the PDB file with zero occupancies. It is equivalent what the NMR people do. - Model it in and compare the MD simulations with SAXS - I would assumne Dale Tronrod suggestion the best. Sigatm labels. - Let the refinement inflate B-factors, then set occupancy to zero in the last round. Thanks to all for participation, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] The meaning of B-factor, was Re: [ccp4bb] what to do with disordered side chains
Regarding the closing statement about the best solution to poorly ordered side chains: I described in the previous e-mail the probabilistic interpretation of B-factors. In the case of very high uncertainty = poorly ordered side chains, I prefer to deposit the conformer representing maximum a posteriori, even if it does not represent all possible conformations. Maximum a posteriori will have significant contribution from the most probable conformation of side chain (prior knowledge) and should not conflict with likelihood (electron density map). Thus, in practice I model the most probable conformation as long as it it in even very weak electron density, does not overlap significantly with negative difference electron density and do not clash with other residues. As a user of PDB files I much prefer the simplest and the most informative representation of the result. Removing parts of side chains that carry charges, as already mentioned, is not particularly helpful for the downstream uses. NMR-like deposits are not among my favorites, either. Having multiple conformations with low occupancies increases potential for a confusion, while benefits are not clear to me. Zbyszek Frank von Delft wrote: This is a lovely summary, and we should make our students read it. - But I'm afraid I do not see how it supports the closing statement in the last paragraph... phx. On 31/03/2011 17:06, Zbyszek Otwinowski wrote: The B-factor in crystallography represents the convolution (sum) of two types of uncertainties about the atom (electron cloud) position: 1) dispersion of atom positions in crystal lattice 2) uncertainty of the experimenter's knowledge about the atom position. In general, uncertainty needs not to be described by Gaussian function. However, communicating uncertainty using the second moment of its distribution is a widely accepted practice, with frequently implied meaning that it corresponds to a Gaussian probability function. B-factor is simply a scaled (by 8 times pi squared) second moment of uncertainty distribution. In the previous, long thread, confusion was generated by the additional assumption that B-factor also corresponds to a Gaussian probability distribution and not just to a second moment of any probability distribution. Crystallographic literature often implies the Gaussian shape, so there is some justification for such an interpretation, where the more complex probability distribution is represented by the sum of displaced Gaussians, where the area under each Gaussian component corresponds to the occupancy of an alternative conformation. For data with a typical resolution for macromolecular crystallography, such multi-Gaussian description of the atom position's uncertainty is not practical, as it would lead to instability in the refinement and/or overfitting. Due to this, a simplified description of the atom's position uncertainty by just the second moment of probability distribution is the right approach. For this reason, the PDB format is highly suitable for the description of positional uncertainties, the only difference with other fields being the unusual form of squaring and then scaling up the standard uncertainty. As this calculation can be easily inverted, there is no loss of information. However, in teaching one should probably stress more this unusual form of presenting the standard deviation. A separate issue is the use of restraints on B-factor values, a subject that probably needs a longer discussion. With respect to the previous thread, representing poorly-ordered (so called 'disordered') side chains by the most likely conformer with appropriately high B-factors is fully justifiable, and currently is probably the best solution to a difficult problem. Zbyszek Otwinowski - they all know what B is and how to look for regions of high B (with, say, pymol) and they know not to make firm conclusions about H-bonds to flaming red side chains. But this knowledge may be quite wrong. If the flaming red really indicates large vibrational motion then yes, one whould not bet on stable H-bonds. But if the flaming red indicates that a well-ordered sidechain was incorrectly modeled at full occupancy when in fact it is only present at half-occupancy then no, the H-bond could be strong but only present in that half-occupancy conformation. One presumes that the other half-occupancy location (perhaps missing from the model) would have its own H-bonding network. I beg to differ. If a side chain has 2 or more positions, one should be a bit careful about making firm conclusions based on only one of those, even if it isn't clear exactly why one should use caution. Also, isn't the isotropic B we fit at medium resolution more of a spherical cow approximation to physical reality anyway? Phoebe Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353 -- Zbyszek
Re: [ccp4bb] problem of conventions
On Thu, Mar 31, 2011 at 10:43 PM, James Holton jmhol...@lbl.gov wrote: I have the 2002 edition, and indeed it only contains space group numbers up to 230. The page numbers quoted by Ian contain space group numbers 17 and 18. You need to distinguish the 'IT space group number' which indeed goes up to 230 (i.e. the number of unique settings), from the 'CCP4 space group number' which, peculiar to CCP4 (which is why I called it 'CCP4-ese'), adds a multiple of 1000 to get a unique number for the alternate settings as used in the API. The page I mentioned show the diagrams for IT SG #18 P22121 (CCP4 #3018), P21221 (CCP4 #2018) and P21212 (CCP4 #18), so they certainly are all there! Although I am all for program authors building in support for the screwy orthorhombics (as I call them), I should admit that my fuddy-duddy strategy for dealing with them remains simply to use space groups 17 and 18, and permute the cell edges around with REINDEX to put the unique (screw or non-screw) axis on the c position. Re-indexing is not an option for us (indeed if there were no alternative, it would be a major undertaking), because the integrity of our LIMS database requires that all protein-ligand structures from the same target crystal form are indexed with the same (or nearly the same) cell and space group (and it makes life so much easier!). With space-groups such as P22121 it can happen (indeed it has happened) that it was not possible to define the space group correctly at the processing stage due to ambiguous absences; indeed it was only after using the SGALternative ALL option in Phaser and refining each TF solution that we identified the space group correctly as P22121. Having learnt the lesson the hard way, we routinely use P222 for all processing of orthorhombics, which of course always gives the conventional abc setting, and only assign the space group well down the pipeline and only when we are 100% confident; by that time it's too late to re-index (indeed why on earth would we want to give ourselves all that trouble?). This is therefore totally analogous to the scenario of yesteryear that I described where it was common to see a 'unit cell' communication followed some years later by the structure paper (though we have compressed the gap somewhat!), and we base the setting on the unit cell convention for exactly the same reason. It's only if you're doing 1 structure at a time that you can afford the luxury of re-indexing - and also the pain: many times I've seen even experienced people getting their files mixed up and trying to refine with differently indexed MTZ PDB files (why is my R factor so high?)! My advice would be - _never_ re-index! -- Ian I have yet to encounter a program that gets broken when presented with data that doesn't have abc, but there are many non-CCP4 programs out there that still don't seem to understand P22121, P21221, P2122 and P2212. I find that surprising! Exactly which 'many' programs are those? You really should report them to CCP4 (or to me if it's one of mine) so they can be fixed! We've been using CCP4 programs as integral components of our processing pipeline (from data processing through to validation) for the last 10 years and I've never come across one that's broken in the way you describe (I've found many broken for other reasons and either fixed it myself or reported it - you should do the same!). Any program which uses csymlib with syminfo.lib can automatically handle all space groups defined in syminfo, which includes all the common alternates you mentioned (and others such as I2). The only program I'm aware of that's limited to the standard settings is sftools (because it has its own internal space group table - it would be nice to see it updated to use syminfo!). This is not the only space group convention issue out there! The R3x vs H3x business continues to be annoying to this day! Yeah to that! H centring was defined in IT long ago (look it up) and it has nothing to do with the R setting! -- Ian
Re: [ccp4bb] unknown electron density~
Try fitting a xylitol molecule in it but watch out for the distortions caused by proximity to symmetry axis. Artem On Thu, Mar 31, 2011 at 1:16 PM, Shu XU xushuh...@gmail.com wrote: Hi, there. I'm refining a 1.76 A structure. The r-work stuck at 21%, rfree is 24% after adding waters. But there is something between the interfaces of the dimers. The space group is I4122, so it looks like something small, five or six atoms all together (without hydrogens). I tried to fit Tris in, however, it didn't look good at all. The crystal came out from Tris-cl buffer, ammonium sulfate, and there is some NaCl in the protein buffer too. The crystals were dropped into 20% xylitol and flash frozen. Fo-Fc maps are attached at 3 sigma level. Let me know what you're thoughts are. Thank you for your help. Shu -- Shu Xu Ph.D candidate in Chemistry Department of Chemistry The University of Toledo Toledo, OH 43606 Tel 419-530-1524
Re: [ccp4bb] step refine speed of wincoot - a little drawback of the new interruptible_fit_protein() function in wincoot
Hi Bernhard, I realized this when comparing the functions yesterday. Exactly like what you said, when a user interrupts the process, what he/she wants at the moment is most likely to be going back a few residues instead of starting all over again. I am quite happy to have both interruptible and uninterruptible functions to choose from. Thanks for the effort. But maybe it would make the impatient ones happier by presenting both versions in the Extension menu? Besides speed, one other advantage I can see for the old function is that by having a simpler structure, it is way easier for the users to make their own modifications. May I beg you not to remove it from future versions? Regards, Zhijie -- From: Bernhard C. Lohkamp bernh...@chem.gla.ac.uk Sent: Thursday, March 31, 2011 1:11 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] step refine speed of wincoot - a little drawback of the new interruptible_fit_protein() function in wincoot Thank you for the comment. You are correct that there are being backups made in the newer interruptable fit function (they were't previously in the uninterruptable fitting). I wasnt thinking about that (*). This may (especially on windows) slow down things a great deal as we write and gzip these files as well. To note here, gzip may be slow on windows as is writing files. In the pre-release versions I disabled the gzip on windows by default (and in general made the compression of backup files optional). This may speed up things (in certain circumstances). I hope this clarifies things a bit... B (*) It's debatable if we want backups here or not. If you can and want to interrupt the fitting you may want to go back one step, which requires a backup. On the other side this will write a lot of backup files which is slowing thing down by writing and compressing files, as well as producing a lot of backup files. Discuss... (or Paul and I just decide). Hi Xiaopeng, and those who are using the new interruptible fit_protein... or stepped_refine... in wincoot 0.6.1: I just took a look at wincoot 0.6.1's fitting.py file (WinCoot\share\coot\python\fitting.py). It seems that in the old fit_protein() and stepped_refine_protein() functions, the backup was programmed to be turned off during the cycles when it steps through the protein. However when using the Stepped refine... from the Extensions menu, the program starts to spit out tons of backup files. I then checked the extension.py. It turns out that these menu items are calling the new interruptible_fit_protein() function instead of the old uninterruptible ones. Some further experiment showed that this behavior was dependent on the backup state variable of the model being refined. When the backup state is 1 (the default value?), these new interruptible fitting functions will save a compressed backup file on each step. So, in order to save time when using the new interruptible protein fitting functions, one can type the following command in the python script window to tweak the backup state to 0: make_backup(imol) #to save a backup turn_off_backup(imol) where imol is the number associated with the pdb file being fitted. Do not forget to turn it back on after the fitting: turn_on_backup(imol) To check the current model's backup state: backup_state(imol) Zhijie -- From: Xiaopeng Hu huxp...@mail.sysu.edu.cn Sent: Tuesday, March 29, 2011 9:02 AM To: Zhijie Li zhijie...@utoronto.ca Subject: Re: [ccp4bb] step refine speed of wincoot Dear Zhijie, Thanks for there suggestions. I tried to turn off backup (with script command) and it did help although not much. Is there any way to add this perference to setting of coot/wincoot? best xiaopeng - 原始邮件 - 发件人: Zhijie Li zhijie...@utoronto.ca 收件人: Xiaopeng Hu huxp...@mail.sysu.edu.cn 发送时间: 星期二, 2011年 3 月 29日 下午 7:46:05 主题: Re: [ccp4bb] step refine speed of wincoot Hi, If you feel the refinement to be too slow, you may turn off the smooth centering (in preferences) or change the centering steps to a smaller number to save unnecessary graphical calculations. To go extreme, you may even remove the real time display commands in the scripts - also a way to test if the difference observed is due to different graphical efficiency. Reducing the size of the displayed map also helps. The other thing you may need to consider is that coot/wincoot will save a backup file each time it updates the model, which means on each step of the refinement you have a new file generated (take a look at the coot-backup directory when doing stepped refinement). If your windows disk is terribly fragmented then sure you will spend a lot of time on writing these files. The other thing is, windows has a different file caching mechanism than linux, this can also cause a huge difference when a few hundred small temporary files are queued for writing. My impression is that both the
Re: [ccp4bb] off topic: protease identification
This can be very hard to do because quite a few proteases are promiscuous and will cut substrates solely based on masking of the polypeptide within the structure of the protein. Typically these proteases will not stop cutting at a single nick - they often proceed until they can't 'dig into' a buried or obstructed section. Often one protease nicks the chain and other proteases (or amino/carboxy peptidases) extend the gap. I would base the search on the observed phenomenon - i.e. is it a single nick, a few residues missing, or a whole swath or domain? Theoretical aspects (i.e. searching for sequences) have been mentioned already - practical ones of course include fractionating the environmental factors (i.e. cell juice) where the cutting takes place and exposing the uncut substrate protein (if you can get it!) to fractions, then sub-fractionating; you can use class-specific inhibitors to further narrow down the selection of enzymes; in some cases a trap (crosslinking, suicide substrate, etc.) can be used to identify the perpetrator. In some cases it may be possible to identify the enzyme by separating all possibilities on e.g. a 2D gel then exposing a fluorogenic peptide to the gel and trying to find a glowing spot (or spots). But again, this is not a very clean solution due to promiscuous nature of many proteases and the sad fact that post-gel renaturation is not guarranteed (not to mention that the protease in question may be multisubunit, or activated by cofactors and gel will destroy these interactions). What do you actually see? Artem On Thu, Mar 31, 2011 at 8:59 AM, Brett, Thomas tbr...@dom.wustl.edu wrote: Hi all: I was wondering if anyone had any tips on identifying proteases. I have a protein for which I know the proteolytic cleavage site. What are the best ways to identify the protease that does the cutting either: 1) bioinformatically (i.e., a good database to search using the cleavage site or a consensus) 2) experimentally (some engineered substrate to trap/identify the substrate or any other method?) Thanks in advance -Tom Tom J. Brett, PhD Assistant Professor of Medicine Division of Pulmonary and Critical Care Washington University School of Medicine Campus Box 8052, 660 S. Euclid Saint Louis, MO 63110 http://brettlab.dom.wustl.edu/
Re: [ccp4bb] problem of conventions
Ian, I think it's amazing that we can program computers to resolve a b c but it would be a major undertaking to store the matrix transformations for 22121 to 21212 and reindex a cell to a standard setting. I was also told that I was lazy to not reindex to the standard setting when I was a grad student. Now it takes less than a minute to enter a transformation and re-index. The orthorhombic rule of a b c makes sense in 222 or 212121, but when there is a standard setting of the 2-fold along the c-axis, then why not adopt that? Often we chose a non-setting when there was a historical precedence, as in the comparison of one structure to another, e.g., P21/c with beta greater than 120deg vs. P21/n, etc. That is no more difficult with modern computing than dragging along three space groups for #18. There was a compactness to 230, and only 230 space groups. (I cheat, since I agree there is both the rhombohedral and hexagonal cell settings for R3bar.) Bernie On Thu, March 31, 2011 5:48 pm, Ian Tickle wrote: On Thu, Mar 31, 2011 at 10:43 PM, James Holton jmhol...@lbl.gov wrote: I have the 2002 edition, and indeed it only contains space group numbers up to 230. The page numbers quoted by Ian contain space group numbers 17 and 18. You need to distinguish the 'IT space group number' which indeed goes up to 230 (i.e. the number of unique settings), from the 'CCP4 space group number' which, peculiar to CCP4 (which is why I called it 'CCP4-ese'), adds a multiple of 1000 to get a unique number for the alternate settings as used in the API. The page I mentioned show the diagrams for IT SG #18 P22121 (CCP4 #3018), P21221 (CCP4 #2018) and P21212 (CCP4 #18), so they certainly are all there! Although I am all for program authors building in support for the screwy orthorhombics (as I call them), I should admit that my fuddy-duddy strategy for dealing with them remains simply to use space groups 17 and 18, and permute the cell edges around with REINDEX to put the unique (screw or non-screw) axis on the c position. Re-indexing is not an option for us (indeed if there were no alternative, it would be a major undertaking), because the integrity of our LIMS database requires that all protein-ligand structures from the same target crystal form are indexed with the same (or nearly the same) cell and space group (and it makes life so much easier!). With space-groups such as P22121 it can happen (indeed it has happened) that it was not possible to define the space group correctly at the processing stage due to ambiguous absences; indeed it was only after using the SGALternative ALL option in Phaser and refining each TF solution that we identified the space group correctly as P22121. Having learnt the lesson the hard way, we routinely use P222 for all processing of orthorhombics, which of course always gives the conventional abc setting, and only assign the space group well down the pipeline and only when we are 100% confident; by that time it's too late to re-index (indeed why on earth would we want to give ourselves all that trouble?). This is therefore totally analogous to the scenario of yesteryear that I described where it was common to see a 'unit cell' communication followed some years later by the structure paper (though we have compressed the gap somewhat!), and we base the setting on the unit cell convention for exactly the same reason. It's only if you're doing 1 structure at a time that you can afford the luxury of re-indexing - and also the pain: many times I've seen even experienced people getting their files mixed up and trying to refine with differently indexed MTZ PDB files (why is my R factor so high?)! My advice would be - _never_ re-index! -- Ian I have yet to encounter a program that gets broken when presented with data that doesn't have abc, but there are many non-CCP4 programs out there that still don't seem to understand P22121, P21221, P2122 and P2212. I find that surprising! Exactly which 'many' programs are those? You really should report them to CCP4 (or to me if it's one of mine) so they can be fixed! We've been using CCP4 programs as integral components of our processing pipeline (from data processing through to validation) for the last 10 years and I've never come across one that's broken in the way you describe (I've found many broken for other reasons and either fixed it myself or reported it - you should do the same!). Any program which uses csymlib with syminfo.lib can automatically handle all space groups defined in syminfo, which includes all the common alternates you mentioned (and others such as I2). The only program I'm aware of that's limited to the standard settings is sftools (because it has its own internal space group table - it would be nice to see it updated to use syminfo!). This is not the only space group convention issue out there! The R3x vs H3x business continues to be annoying to this day!
