[ccp4bb] KD of dimerization, off topic
Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina
Re: [ccp4bb] KD of dimerization, off topic
Hi Careina, I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged. Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation. Hth, Dave On 14 Feb 2014 08:03, Careina Edgooms careinaedgo...@yahoo.com wrote: Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina
Re: [ccp4bb] Sister CCPs
Seems it's worth thinking about this as an experiment that has actually been done: BB and wiki have been available in parallel for many years now; so where has all the activity happened, where do people go for information - and more to the point, where are other people happy to volunteer information? According to what you say, the experiment has a clear outcome. Even crystallographers are social beings, and thrive on interaction. Wikis don't interact. I should add I'm not at all clear what problem is being addressed here: if I get an email I don't want to read, I make a tiny hand-movement (= hit delete) and it vanishes forever. Are people suggesting we abandon an empirically proven mechanism merely to save me the need for this tiny hand-movement? phx On 14/02/2014 07:19, Kay Diederichs wrote: Nat, that's why I set up the CCP4 wiki at http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Main_Page ! The idea is that everybody benefits: experienced crystallographers/biologists can concentrate on the new and difficult questions coming up on the bulletin board, and novices find answers to those ever-recurring questions. Everybody can contribute answers, or improve existing ones! But the wiki can only be useful in the long run if there are contributors. Why are there (almost) no contributors? It cannot be due to technical difficulty; it's very easy to contribute to a wiki. One guess is that a posting on a BB is more socially rewarding, because the interaction via emails is more immediate. Re-vitalize the wiki! Kay On Thu, 13 Feb 2014 07:21:14 -0800, Nat Echols nathaniel.ech...@gmail.com wrote: One comment (not a complaint) on all this: it seems like the same questions get asked over and over again. If there is a good place for a general crystallography FAQ list it is well past time for one to be put together - or maybe it just needs to be better advertised? At a minimum, for instance: - what cryoprotectant should I use? - how do I get big single crystals? - how do I improve diffraction? - how can I tell if I've solved my structure? - why is my R-free stuck? - is pick random statistic suitable for publication? Some of the other common queries (name my blob!) still need to be handled on a case-by-case basis, but it would be much more efficient for everyone if the standard answers were collected somewhere permanent. -Nat On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov eugene.val...@gmail.comwrote: I absolutely agree with Juergen. Leaving aside methods developers, who are a completely different breed, there is no such thing as a crystallographer sitting in a dark room solving structures all day. If there are, these are anachronisms destined for evolutionary demise. More and more cell biologists, immunologists and all other kinds of biologists are having a go at doing structural work with their molecules of interest themselves without involving the professionals. Typically, they learn on the job and they need advice with all kinds of things ranging from cloning and protein preps through to issues with tetartohedrally-twinned data and interpreting their structures. So, a modern structural biologist is one who is equipped for the wet lab and has some idea of how to go about solving structures. CCP4BB is a wonderful resource that is great for both the quality of the advice offered to those that seek it and for the variety of topics that are addressed in the scope of structural biology. I have learnt greatly from reading posts from very skilled and knowledgeable scientists at this forum and then implemented these insights into my own research. I am very grateful for this. In short, please do not discourage your colleagues, particularly very junior ones, from posting to the CCP4BB. Some of the questions may appear quaint or irrelevant but it is easy to simply ignore topics that are of no interest! Eugene On 13 February 2014 14:41, Bosch, Juergen jubo...@jhsph.edu wrote: Let me pick up Eleanor's comment: is there something like a crystallographer today ? I mean in the true sense ? I think as a crystallographer you won't be able to survive the next decade, you need to diversify your toolset of techniques as pointed out in this article http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a And I'm not quite sure how software developers see themselves, as I would argue they are typically maybe not doing so much wet lab stuff related to crystallography (I may be wrong here) but rather code these days. What type of crystallographer is a software developer ? I think like our beloved crystals we come in different flavors. And we need to train the next generation of students with that perspective in mind. Just my two cents on a snowy day (30cm over night) J�rgen .. J�rgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615
Re: [ccp4bb] Determining concentration of membrane protein
Hi Raji, i the kit i used for this purpose was from Pierce it's call Protein BCA RAC assay. BCA : for the colorimetric parts, en ad the RAC is for Reducing agent compatibility. This RAC is also efficient with detergent. (according my remember) But you if you use at the same time detergent and Reducing Agent, it's inefficient. http://www.piercenet.com/product/bca-protein-assay-reducing-agent-compatible If you use this one, it could be costless to buy the microplate oriented kit. (you can use also without microplate and you have more experiment in the same box) Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Ho Leung Ng [h...@hawaii.edu] Envoyé : vendredi 14 février 2014 01:58 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] Determining concentration of membrane protein Hi Raji, There are also some proprietary stains such as the 660 nm (can't they think of a better product name?) stain from Pierce that are detergent compatible. I used this briefly with success when comparing against Abs 280 nm. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edumailto:h...@hawaii.edu Date:Thu, 13 Feb 2014 10:06:12 -0500 From:Raji Edayathumangalam r...@brandeis.edumailto:r...@brandeis.edu Subject: Determining concentration of membrane protein Dear CC4BBers, I am trying to figure out what is the best way to determine the protein concentration of my membrane protein. My purified membrane protein is in 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%). After reading the friendly manuals and searching online, I've learned that detergents interferes with assays like Bradford but can't find good descriptions of what works best. For now, I am trying to estimate concentration from absorbance at 280nm and using molar extinction coefficients based on aromatic amino acids, but again suspect detergent interference. I would like to know what other folks working on membrane proteins are doing. Thanks very much. Raji
Re: [ccp4bb] Determining concentration of membrane protein
The problem is that if you put detergent or reducing agent in Bradford or BCA, the reaction is complet also without protein. You can't determine the color gradient because every tubes are blue or purple. De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Niks [nik...@gmail.com] Envoyé : vendredi 14 février 2014 07:45 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] Determining concentration of membrane protein Dear All, May be a stupid question. But if we take buffer with detergent as control (Blank), would not the difference in ODs using any of the methods used e.g. Bradford assay, gives protein concentration? Regards Nishant On Thu, Feb 13, 2014 at 9:09 PM, Roger Rowlett rrowl...@colgate.edumailto:rrowl...@colgate.edu wrote: Your basic choices for protein assays are: 1. Alkaline copper methods (e.g., Biuret and micro-biuret) 2. alkaline copper + molybdate methods (e.g., Lowry, BCA assays) 3. Hydrophobic dye methods (e.g. Bradford) 4. UV methods (e.g., A280, A230, A210, etc.) Method 1 is least sensitive to amino acid composition, but is also has highest detection limits. Thiols interfere. Method 2 is very idiosyncratic with amino acid composition, and also subject to interference by thiols. Method 3 is not usable in detergent solutions. Method 4 has many inteferences as most everything absorbs in the far UV region. If you have some special protein cofactors, metals, chromophores, etc. these can be exploited for better measurements. For ecample metalloproteins are easy to quantify by ICP-OES or TXRF if they are reasonably pure. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edumailto:rrowl...@colgate.edu On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote: Dear CC4BBers, I am trying to figure out what is the best way to determine the protein concentration of my membrane protein. My purified membrane protein is in 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%). After reading the friendly manuals and searching online, I've learned that detergents interferes with assays like Bradford but can't find good descriptions of what works best. For now, I am trying to estimate concentration from absorbance at 280nm and using molar extinction coefficients based on aromatic amino acids, but again suspect detergent interference. I would like to know what other folks working on membrane proteins are doing. Thanks very much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- The most difficult phase of life is not when No one understands you;It is when you don't understand yourself
Re: [ccp4bb] KD of dimerization, off topic
I agree with Dave, and i suggest one more method to estimate Kd, The intrinsic fluorescence of proteins thanks to the aromatic chain side. Maybe it's also possible to have an estimation with native gels if you use prot A concentration as fixed and B protein concentration as variable. I am not sure. De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de David Briggs [drdavidcbri...@gmail.com] Envoyé : vendredi 14 février 2014 09:13 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] KD of dimerization, off topic Hi Careina, I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged. Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation. Hth, Dave On 14 Feb 2014 08:03, Careina Edgooms careinaedgo...@yahoo.commailto:careinaedgo...@yahoo.com wrote: Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina
[ccp4bb] AW: [ccp4bb] KD of dimerization, off topic
Dear Carina, An additional problem is that due to the dilution, but also due to the separation of monomers and dimers you get a reequilibration which is dependent on Kon/Koff of the interaction. Unless these are very slow, you cannot use size exclusion to determine the monomer/dimer ratio. Although not perfect, I would try dynamic light scattering. For calculating the Kd, I would just use the standard textbook formula, with A being identical to B. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Careina Edgooms Gesendet: Freitag, 14. Februar 2014 09:04 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] KD of dimerization, off topic Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina
Re: [ccp4bb] Acetyl link problems.
Hello Ian and ccp4 community, I think you must use modified residue, N-acetylmethionine with code AME, instead of LINKR. May be someone find this mildly useful: there is a file called mon_lib_list.cif, located in $CCP4/lib/data/monomers/list/ If you are not sure about particular residue modification or three-letter code for insertion in COOT-check this file P.S.: I hope you are still able to understand my runglish 14.02.2014 02:57, Ian Tickle пишет: All, I'm having problems refining a structure with an N-terminal acetylated MET residue. I'm trying it with both Refmac Buster. Buster works fine gives perfect planar geometry for the ACE-MET linkage. Refmac gives a pyramidal acetyl group after refinement which to my eyes is wrong (sp2 C atom?). I have this line in my input PDB: LINKR C ACE A 0 N MET A 1 ACE_C-N which as I understand it should solve the problem. However, looking at the CIF entry for the ACE_C-N link I see restraints defined for bonds, angles torsion angles but not for the CC(=O)N plane. So the problem seems to be that the planar restraints for this link group are missing - or are they defined elsewhere? Anyway I added planar restraints to the ACE_C-N link entry it solves the problem, at least for regularisation - I still have the same problem with refinement. Refmac in regularisation mode now gives the correct (planar) geometry for the ACE-MET linkage. I'm just puzzled why no-one has noticed this, after all post-translational acetylation is surely not that uncommon (according to Wikipedia 80% of human proteins are N-term acetylated!). Further, looking at the entry for ACE I see: ACE ACE 'ACETYL GROUP ' non-polymer 7 3 . ACE O O O 0.000 0.000 0.000 0.000 ACE C C C1 0.000 -1.044 -0.606 0.000 ACE H H H 0.000 -1.978 -0.069 0.000 ACE CH3 C CH3 0.000 -1.041 -2.113 0.000 ACE H3 H H 0.000 -0.541 -2.464 0.865 ACE H2 H H 0.000 -2.038 -2.468 0.000 ACE H1 H H 0.000 -0.540 -2.464 -0.864 Where did the extra H atom (3rd atom) come from? Acetyl is CH3C=O: the extra H atom would make it acetaldehyde which of course has nothing whatsoever to do with acetylation! Is this the reason for the lack of planar link restraints (though that wouldn't explain why the other link restraints are present)? Any insights appreciated! Cheers -- Ian -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
Re: [ccp4bb] Sister CCPs
I think that more people read wikis than contribute to them, so this 'experiment' (see below) is a little biased. On the other hand, it is often easier to put a question to CCP4bb than to search for the answer in wikis and other documentation, however well organized they are. Kay: can you see how often your XDS wiki (for example) is accessed? The small number of different contributors to a wiki is however a problem. Replying to an email is something that has to be done almost immediately and reaches instantly a large audience. Making a contribution to a wiki lacks the urgency and can be put off for a few weeks, and does not reward the contributor with immediate feedback or lead to controversial discussions. Perhaps someone should make a list of the questions most frequently asked on CCP4bb and the most helpful replies that they generated. This could even be made into a wiki. George On 02/14/2014 09:15 AM, Frank von Delft wrote: Seems it's worth thinking about this as an experiment that has actually been done: BB and wiki have been available in parallel for many years now; so where has all the activity happened, where do people go for information - and more to the point, where are other people happy to volunteer information? According to what you say, the experiment has a clear outcome. Even crystallographers are social beings, and thrive on interaction. Wikis don't interact. I should add I'm not at all clear what problem is being addressed here: if I get an email I don't want to read, I make a tiny hand-movement (= hit delete) and it vanishes forever. Are people suggesting we abandon an empirically proven mechanism merely to save me the need for this tiny hand-movement? phx -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-33021 or -33068 Fax. +49-551-39-22582
Re: [ccp4bb] Sister CCPs
I couldn't help noticing the use of the passive voice in the last sentence... :) Which is the real problem: wikis are an SEP, a Somebody-Else's-Problem. Emails are not. (And this interesting meta-conversation would never have happened on a wiki.) On 14/02/2014 09:15, George Sheldrick wrote: I think that more people read wikis than contribute to them, so this 'experiment' (see below) is a little biased. On the other hand, it is often easier to put a question to CCP4bb than to search for the answer in wikis and other documentation, however well organized they are. Kay: can you see how often your XDS wiki (for example) is accessed? The small number of different contributors to a wiki is however a problem. Replying to an email is something that has to be done almost immediately and reaches instantly a large audience. Making a contribution to a wiki lacks the urgency and can be put off for a few weeks, and does not reward the contributor with immediate feedback or lead to controversial discussions. Perhaps someone should make a list of the questions most frequently asked on CCP4bb and the most helpful replies that they generated. This could even be made into a wiki. George On 02/14/2014 09:15 AM, Frank von Delft wrote: Seems it's worth thinking about this as an experiment that has actually been done: BB and wiki have been available in parallel for many years now; so where has all the activity happened, where do people go for information - and more to the point, where are other people happy to volunteer information? According to what you say, the experiment has a clear outcome. Even crystallographers are social beings, and thrive on interaction. Wikis don't interact. I should add I'm not at all clear what problem is being addressed here: if I get an email I don't want to read, I make a tiny hand-movement (= hit delete) and it vanishes forever. Are people suggesting we abandon an empirically proven mechanism merely to save me the need for this tiny hand-movement? phx
Re: [ccp4bb] 3letter code
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Faisal, I find the files in the directory $CLIB/data/monomers/list very useful for such questions, e.g. # grep -i ribulose * mon_lib_list.cif:5RP 5RP 'RIBULOSE-5-PHOSPHATE non-polymer mon_lib_list.cif:HMS HMS '5-O-phosphono-L-ribulose mon_lib_list.cif:RUB RUB 'RIBULOSE-1,5-DIPHOSPHATE points you at the RUB, as Mario already mentioned. Best, Tim On 02/13/2014 10:07 PM, Faisal Tarique wrote: Hi everyone Can anyone please tell me the three letter code for D-Ribulose 1,5-bisphosphate.. Thanks in advance - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFS/eHBUxlJ7aRr7hoRAjVtAKDJbmndqLGBpIIr/WJwtl0KcWkzpACgyhlp UJUo2t//J/vrs3ZN7epGsCQ= =9P2R -END PGP SIGNATURE-
Re: [ccp4bb] How to find the unfound part of a big protein
Dear Sun, I had a similar problem. If you have a good TaBr dataset this should give you good phases. You can combine the TaBr phases and Molrepl phases in SHARP. What worked in my hands very well and is easy to do: SAD using Phaser and the partial Mol Repl Model. You find here a input file for such a scenario: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Phenix#phenix.phaser_-_SAD_phasing_with_Phaser Or simply use the CCP4 gui Phaser and 'SAD with molecular replacement partial structure' After DM or parrot you might get some interpretable density even at 6.5 A. If you don't have a good Mol Repl Model for the missing part you might try http://toolkit.tuebingen.mpg.de/hhpred to look for structures that are not that closely related. SeMet can help a lot in this case too. You will get the SeMet positions at low resolution with Phaser as described above. The SeMet positions will guide you to place a model into a very crude density. HTH, Guenter Dear Crystallographers, I'm working on a ~90KDa membrane protein, with big extracellular part, probably function as dimer. Now we have dataset to ~4.2 Angstrom and using extracellular homolog structure we can find a solution for this part(~45% of the whole molecule MW) through molecular replacement, and the molecules are packed as layers, and the other part are presumably between these layers. However, we are having trouble to fit the rest of the protein even though there're some density between the solved part. Rfree is at 40% now. We're trying to do heavy atom soaking, such as TaBr. We collected data for MIR but it's not helping so far. (Can I combine these MIR data with the native dataset because the MIR set is only at ~6.5 Angstrom)? Other information: this protein is expressed in Sf9 cells (so very hard to do Se-Met derivatives). The crystals is nice and big and cubic. Any suggestions or examples? Thanks a lot. Bingfa
Re: [ccp4bb] 3letter code
Thanks everybody for sending me the three letter code.. regards Faisal On 2/14/14, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Faisal, I find the files in the directory $CLIB/data/monomers/list very useful for such questions, e.g. # grep -i ribulose * mon_lib_list.cif:5RP 5RP 'RIBULOSE-5-PHOSPHATE non-polymer mon_lib_list.cif:HMS HMS '5-O-phosphono-L-ribulose mon_lib_list.cif:RUB RUB 'RIBULOSE-1,5-DIPHOSPHATE points you at the RUB, as Mario already mentioned. Best, Tim On 02/13/2014 10:07 PM, Faisal Tarique wrote: Hi everyone Can anyone please tell me the three letter code for D-Ribulose 1,5-bisphosphate.. Thanks in advance - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFS/eHBUxlJ7aRr7hoRAjVtAKDJbmndqLGBpIIr/WJwtl0KcWkzpACgyhlp UJUo2t//J/vrs3ZN7epGsCQ= =9P2R -END PGP SIGNATURE- -- Regards Faisal School of Life Sciences JNU
Re: [ccp4bb] Sister CCPs
Dear George, Ever since I read your message that gave rise to this thread I have had a few remarks in mind that other preoccupations have kept me from sharing, but perhaps there is still room for them among the numerous contributions to this topic that have been made since. What attracted me to the field all these years ago was the example of Max Perutz. In the very early stages of his work he could have said We have this dreaded phase problem, and the only way to measure phases (or rather, phase differences) is by interference - therefore we can't advance until we have sources of coherent hard X-rays. He could then have reclined in his chair, feeling absolutely brilliant and extremely important, twiddling his thumbs while leaving it to physicists and funding agencies to scurry around to meet his high-minded demands and come up with facilities to produce this wonderful radiation. However, he reached instead for a modest bottle of a mercury reagent and, with it, managed to do X-ray interferometry by creating a reference beam within the crystal by chemical derivatisation, instead of waiting for that interference phenomenon to be made possible externally by the use of a coherent X-ray beam. I have never tired of thinking back on this glorious messiness of macromolecular crystallography, stretching across the five main disciplines and presenting a permanent tribute to the unity of science - even more so since we became beneficiaries of Special Relativity through our use of synchrotron radiation. As a computational methods developer in X-ray crystallography one is acutely aware from the beginning that these methods' main mandate is to fill in some (phase) information that experiment wasn't able to capture. Even in a broader perspective than the strict phase problem, it is clear that any improvements upstream, at the level of wet stuff or data collection, can achieve more than all the computational massaging we could ever come up with - experimental phasing remaining one of the strongest paradigms here, but with many others such as construct optimisation, or the use of additives or humidity control to stabilise cell parameters, to name only a few. Seeing a different pattern of interplay between wet lab stuff, diffraction physics and computation in each structure determination has been to me and countless others an inexhaustible source of scientific excitement and joy. I must therefore say that I relish the heterogeneity of the questions that come up on this BB, as well as seeing that there is a core of younger scientists who can answer just about the entire spectrum of questions! Trying to make a histogram of the recurring topics and to organise answers in an FAQ on a Wiki is certainly an excellent idea; but I do vote for the preservation of the mixed-bag nature of this BB, where a strictly crystallographic or computational question can almost seem off topic in the middle of the diversity of the others. All Things that (eventually) Serve the Diffraction Pattern - and the Electron Density, to paraphrase David Schuller's motto, should have a place in the CCP4BB and contribute to retaining its spontaneously holistic vision of our field. With best wishes, Gerard. -- On Fri, Feb 14, 2014 at 10:15:54AM +0100, George Sheldrick wrote: I think that more people read wikis than contribute to them, so this 'experiment' (see below) is a little biased. On the other hand, it is often easier to put a question to CCP4bb than to search for the answer in wikis and other documentation, however well organized they are. Kay: can you see how often your XDS wiki (for example) is accessed? The small number of different contributors to a wiki is however a problem. Replying to an email is something that has to be done almost immediately and reaches instantly a large audience. Making a contribution to a wiki lacks the urgency and can be put off for a few weeks, and does not reward the contributor with immediate feedback or lead to controversial discussions. Perhaps someone should make a list of the questions most frequently asked on CCP4bb and the most helpful replies that they generated. This could even be made into a wiki. George On 02/14/2014 09:15 AM, Frank von Delft wrote: Seems it's worth thinking about this as an experiment that has actually been done: BB and wiki have been available in parallel for many years now; so where has all the activity happened, where do people go for information - and more to the point, where are other people happy to volunteer information? According to what you say, the experiment has a clear outcome. Even crystallographers are social beings, and thrive on interaction. Wikis don't interact. I should add I'm not at all clear what problem is being addressed here: if I get an email I don't want to read, I make a tiny hand-movement (= hit delete) and it vanishes forever. Are people
Re: [ccp4bb] How to find the unfound part of a big protein
The very best advice is: get better data if at all possible. Things are much easier and more informative at 3A than at 4.2A and even better at 2.5A. There are various tricks which might help improve diffraction.. But if you are stuck then: The partial MR phases are very useful for checking your derivatives - look at difference Fouriers and anomalous Fouriers to see if anything has bound.. Then if you do have substitution Phaser SAD + MR will help to improve phases. If there is NCS then you can use Parrot or DM to improve the phases substantially. Buccaneer is quite good at building missing parts - we are experimenting with different scenarios to optimise this.. Eleanor On 14 February 2014 09:41, Guenter Fritz guenter.fr...@uni-konstanz.de wrote: Dear Sun, I had a similar problem. If you have a good TaBr dataset this should give you good phases. You can combine the TaBr phases and Molrepl phases in SHARP. What worked in my hands very well and is easy to do: SAD using Phaser and the partial Mol Repl Model. You find here a input file for such a scenario: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Phenix#phenix.phaser_-_SAD_phasing_with_Phaser Or simply use the CCP4 gui Phaser and 'SAD with molecular replacement partial structure' After DM or parrot you might get some interpretable density even at 6.5 A. If you don't have a good Mol Repl Model for the missing part you might try http://toolkit.tuebingen.mpg.de/hhpred to look for structures that are not that closely related. SeMet can help a lot in this case too. You will get the SeMet positions at low resolution with Phaser as described above. The SeMet positions will guide you to place a model into a very crude density. HTH, Guenter Dear Crystallographers, I'm working on a ~90KDa membrane protein, with big extracellular part, probably function as dimer. Now we have dataset to ~4.2 Angstrom and using extracellular homolog structure we can find a solution for this part(~45% of the whole molecule MW) through molecular replacement, and the molecules are packed as layers, and the other part are presumably between these layers. However, we are having trouble to fit the rest of the protein even though there're some density between the solved part. Rfree is at 40% now. We're trying to do heavy atom soaking, such as TaBr. We collected data for MIR but it's not helping so far. (Can I combine these MIR data with the native dataset because the MIR set is only at ~6.5 Angstrom)? Other information: this protein is expressed in Sf9 cells (so very hard to do Se-Met derivatives). The crystals is nice and big and cubic. Any suggestions or examples? Thanks a lot. Bingfa
Re: [ccp4bb] Sister CCPs
Frank, the way you phrase implies that the outcome of the 'experiment', as you call it, is obvious. But that would be throwing out the baby with the bathtub, it seems to me. My point is: the wiki and the bulletin board have complementary roles, and it makes sense to use both. After all, there are on average 500 visits viewing 3.500 pages _every_ _day_ (not counting robots/spiders of search engines) to CCP4wiki + XDSwiki combined: Day Number of visitsPages HitsBandwidth 01 Feb 2014 373 2,363 4,760 58.26 MB 02 Feb 2014 361 2,142 4,806 122.95 MB 03 Feb 2014 483 3,139 6,635 102.01 MB 04 Feb 2014 489 3,224 7,999 118.76 MB 05 Feb 2014 450 3,187 7,722 125.14 MB 06 Feb 2014 451 2,627 5,940 87.03 MB 07 Feb 2014 490 2,999 7,087 185.78 MB 08 Feb 2014 55 368 916 9.20 MB 09 Feb 2014 323 1,928 4,327 58.37 MB 10 Feb 2014 597 3,160 7,986 163.56 MB 11 Feb 2014 535 3,485 8,685 114.64 MB 12 Feb 2014 540 3,231 7,883 123.59 MB 13 Feb 2014 544 3,498 8,367 147.47 MB (awstats statistics; sorry for the bad formatting; column 2 is visits, 3 is pages, 4 is hits, 5 is bandwidth.) So people _do_ turn to the wiki for information - maybe potential contributors should know this! best, Kay On Fri, 14 Feb 2014 08:15:51 +, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: Seems it's worth thinking about this as an experiment that has actually been done: BB and wiki have been available in parallel for many years now; so where has all the activity happened, where do people go for information - and more to the point, where are other people happy to volunteer information? According to what you say, the experiment has a clear outcome. Even crystallographers are social beings, and thrive on interaction. Wikis don't interact. I should add I'm not at all clear what problem is being addressed here: if I get an email I don't want to read, I make a tiny hand-movement (= hit delete) and it vanishes forever. Are people suggesting we abandon an empirically proven mechanism merely to save me the need for this tiny hand-movement? phx On 14/02/2014 07:19, Kay Diederichs wrote: Nat, that's why I set up the CCP4 wiki at http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Main_Page ! The idea is that everybody benefits: experienced crystallographers/biologists can concentrate on the new and difficult questions coming up on the bulletin board, and novices find answers to those ever-recurring questions. Everybody can contribute answers, or improve existing ones! But the wiki can only be useful in the long run if there are contributors. Why are there (almost) no contributors? It cannot be due to technical difficulty; it's very easy to contribute to a wiki. One guess is that a posting on a BB is more socially rewarding, because the interaction via emails is more immediate. Re-vitalize the wiki! Kay On Thu, 13 Feb 2014 07:21:14 -0800, Nat Echols nathaniel.ech...@gmail.com wrote: One comment (not a complaint) on all this: it seems like the same questions get asked over and over again. If there is a good place for a general crystallography FAQ list it is well past time for one to be put together - or maybe it just needs to be better advertised? At a minimum, for instance: - what cryoprotectant should I use? - how do I get big single crystals? - how do I improve diffraction? - how can I tell if I've solved my structure? - why is my R-free stuck? - is pick random statistic suitable for publication? Some of the other common queries (name my blob!) still need to be handled on a case-by-case basis, but it would be much more efficient for everyone if the standard answers were collected somewhere permanent. -Nat On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov eugene.val...@gmail.comwrote: I absolutely agree with Juergen. Leaving aside methods developers, who are a completely different breed, there is no such thing as a crystallographer sitting in a dark room solving structures all day. If there are, these are anachronisms destined for evolutionary demise. More and more cell biologists, immunologists and all other kinds of biologists are having a go at doing structural work with their molecules of interest themselves without involving the professionals. Typically, they learn on the job and they need advice with all kinds of things ranging from cloning and protein preps through to issues with tetartohedrally-twinned data and interpreting their structures. So, a modern structural biologist is one who is equipped for the wet lab and has some idea of how to go about solving structures. CCP4BB is a wonderful resource that is great for both the quality of the advice offered to those that seek it and for the variety of topics that are addressed in the scope of structural
Re: [ccp4bb] Acetyl link problems.
Hi Evgeny Thanks a lot for responding. It's a nice idea but sadly it doesn't work. For sure it makes the acetyl group planar at what was the A0-A1 link (now a single AME residue), but the amide group at the A1-A2 link is now pyramidal! Presumably now planar link restraints for this link are missing! Also Buster now doesn't recognise the A1-A2 (AME-ASN) link. It would be nice to be able to use the same input PDB file for both programs. Ho hum! Cheers -- Ian On 14 February 2014 08:35, Evgeny Osipov e.m.osi...@gmail.com wrote: Hello Ian and ccp4 community, I think you must use modified residue, N-acetylmethionine with code AME, instead of LINKR. May be someone find this mildly useful: there is a file called mon_lib_list.cif, located in $CCP4/lib/data/monomers/list/ If you are not sure about particular residue modification or three-letter code for insertion in COOT-check this file P.S.: I hope you are still able to understand my runglish 14.02.2014 02:57, Ian Tickle пишет: All, I'm having problems refining a structure with an N-terminal acetylated MET residue. I'm trying it with both Refmac Buster. Buster works fine gives perfect planar geometry for the ACE-MET linkage. Refmac gives a pyramidal acetyl group after refinement which to my eyes is wrong (sp2 C atom?). I have this line in my input PDB: LINKR C ACE A 0 N MET A 1 ACE_C-N which as I understand it should solve the problem. However, looking at the CIF entry for the ACE_C-N link I see restraints defined for bonds, angles torsion angles but not for the CC(=O)N plane. So the problem seems to be that the planar restraints for this link group are missing - or are they defined elsewhere? Anyway I added planar restraints to the ACE_C-N link entry it solves the problem, at least for regularisation - I still have the same problem with refinement. Refmac in regularisation mode now gives the correct (planar) geometry for the ACE-MET linkage. I'm just puzzled why no-one has noticed this, after all post-translational acetylation is surely not that uncommon (according to Wikipedia 80% of human proteins are N-term acetylated!). Further, looking at the entry for ACE I see: ACE ACE 'ACETYL GROUP ' non-polymer 7 3 . ACE O O O 0.000 0.000 0.000 0.000 ACE C C C1 0.000 -1.044 -0.606 0.000 ACE H H H 0.000 -1.978 -0.069 0.000 ACE CH3 C CH3 0.000 -1.041 -2.113 0.000 ACE H3 H H 0.000 -0.541 -2.464 0.865 ACE H2 H H 0.000 -2.038 -2.468 0.000 ACE H1 H H 0.000 -0.540 -2.464 -0.864 Where did the extra H atom (3rd atom) come from? Acetyl is CH3C=O: the extra H atom would make it acetaldehyde which of course has nothing whatsoever to do with acetylation! Is this the reason for the lack of planar link restraints (though that wouldn't explain why the other link restraints are present)? Any insights appreciated! Cheers -- Ian -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
[ccp4bb] Check waters for possibly being sodium ions and similar
In the past I remember using a simple program or webserver to check whether modelled water molecules might be metal ions, especially sodium, magnesium etc. as they do not differ much in density to waters. Based on coordination. However, I can't remember the name and can't find it in google or the ccp4bb archives. What_if seems to have something similar implemented, but I remember it as a stand-alone program with a rather simple name. Anyone have better memory or searching skills than me? Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij
Re: [ccp4bb] Acetyl link problems.
Hi Ian dod you try without link and standard ACE atom naming. Refmac should be able to deal N-terminal activation and few other things. At least it was the intention when it was written. Bugs may have been (self)introduced to prevent this from happening. If it is so then I would like to know. Regards Garib On 13 Feb 2014, at 22:57, Ian Tickle ianj...@gmail.com wrote: All, I'm having problems refining a structure with an N-terminal acetylated MET residue. I'm trying it with both Refmac Buster. Buster works fine gives perfect planar geometry for the ACE-MET linkage. Refmac gives a pyramidal acetyl group after refinement which to my eyes is wrong (sp2 C atom?). I have this line in my input PDB: LINKRC ACE A 0 N MET A 1 ACE_C-N which as I understand it should solve the problem. However, looking at the CIF entry for the ACE_C-N link I see restraints defined for bonds, angles torsion angles but not for the CC(=O)N plane. So the problem seems to be that the planar restraints for this link group are missing - or are they defined elsewhere? Anyway I added planar restraints to the ACE_C-N link entry it solves the problem, at least for regularisation - I still have the same problem with refinement. Refmac in regularisation mode now gives the correct (planar) geometry for the ACE-MET linkage. I'm just puzzled why no-one has noticed this, after all post-translational acetylation is surely not that uncommon (according to Wikipedia 80% of human proteins are N-term acetylated!). Further, looking at the entry for ACE I see: ACE ACE 'ACETYL GROUP' non-polymer 7 3 . ACE O OO 0.000 0.0000.0000.000 ACE C CC10.000 -1.044 -0.6060.000 ACE H HH 0.000 -1.978 -0.0690.000 ACE CH3CCH3 0.000 -1.041 -2.1130.000 ACE H3 HH 0.000 -0.541 -2.4640.865 ACE H2 HH 0.000 -2.038 -2.4680.000 ACE H1 HH 0.000 -0.540 -2.464 -0.864 Where did the extra H atom (3rd atom) come from? Acetyl is CH3C=O: the extra H atom would make it acetaldehyde which of course has nothing whatsoever to do with acetylation! Is this the reason for the lack of planar link restraints (though that wouldn't explain why the other link restraints are present)? Any insights appreciated! Cheers -- Ian Dr Garib N Murshudov MRC-LMB Francis Crick Avenue Cambridge CB2 0QH UK Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
Re: [ccp4bb] Check waters for possibly being sodium ions and similar
turns out some people indeed have better memory than me: WASP inside STAN server http://xray.bmc.uu.se/cgi-bin/gerard/rama_server.pl thanks! On 14 Feb 2014, at 12:49, Mark van Raaij wrote: In the past I remember using a simple program or webserver to check whether modelled water molecules might be metal ions, especially sodium, magnesium etc. as they do not differ much in density to waters. Based on coordination. However, I can't remember the name and can't find it in google or the ccp4bb archives. What_if seems to have something similar implemented, but I remember it as a stand-alone program with a rather simple name. Anyone have better memory or searching skills than me? Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij
Re: [ccp4bb] Sister CCPs
Gerard's beautifully worded message underscores in my mind what a great resource for all kinds of information this bulletin board is. I have many times been impressed by the time people take to answer queries, even if they sometimes seem perfectly googlable to me. As a community we can be proud of CCP4BB! Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Gerard Bricogne [g...@globalphasing.com] Sent: Friday, February 14, 2014 10:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Sister CCPs Dear George, Ever since I read your message that gave rise to this thread I have had a few remarks in mind that other preoccupations have kept me from sharing, but perhaps there is still room for them among the numerous contributions to this topic that have been made since. What attracted me to the field all these years ago was the example of Max Perutz. In the very early stages of his work he could have said We have this dreaded phase problem, and the only way to measure phases (or rather, phase differences) is by interference - therefore we can't advance until we have sources of coherent hard X-rays. He could then have reclined in his chair, feeling absolutely brilliant and extremely important, twiddling his thumbs while leaving it to physicists and funding agencies to scurry around to meet his high-minded demands and come up with facilities to produce this wonderful radiation. However, he reached instead for a modest bottle of a mercury reagent and, with it, managed to do X-ray interferometry by creating a reference beam within the crystal by chemical derivatisation, instead of waiting for that interference phenomenon to be made possible externally by the use of a coherent X-ray beam. I have never tired of thinking back on this glorious messiness of macromolecular crystallography, stretching across the five main disciplines and presenting a permanent tribute to the unity of science - even more so since we became beneficiaries of Special Relativity through our use of synchrotron radiation. As a computational methods developer in X-ray crystallography one is acutely aware from the beginning that these methods' main mandate is to fill in some (phase) information that experiment wasn't able to capture. Even in a broader perspective than the strict phase problem, it is clear that any improvements upstream, at the level of wet stuff or data collection, can achieve more than all the computational massaging we could ever come up with - experimental phasing remaining one of the strongest paradigms here, but with many others such as construct optimisation, or the use of additives or humidity control to stabilise cell parameters, to name only a few. Seeing a different pattern of interplay between wet lab stuff, diffraction physics and computation in each structure determination has been to me and countless others an inexhaustible source of scientific excitement and joy. I must therefore say that I relish the heterogeneity of the questions that come up on this BB, as well as seeing that there is a core of younger scientists who can answer just about the entire spectrum of questions! Trying to make a histogram of the recurring topics and to organise answers in an FAQ on a Wiki is certainly an excellent idea; but I do vote for the preservation of the mixed-bag nature of this BB, where a strictly crystallographic or computational question can almost seem off topic in the middle of the diversity of the others. All Things that (eventually) Serve the Diffraction Pattern - and the Electron Density, to paraphrase David Schuller's motto, should have a place in the CCP4BB and contribute to retaining its spontaneously holistic vision of our field. With best wishes, Gerard. -- On Fri, Feb 14, 2014 at 10:15:54AM +0100, George Sheldrick wrote: I think that more people read wikis than contribute to them, so this 'experiment' (see below) is a little biased. On the other hand, it is often easier to put a question to CCP4bb than to search for the answer in wikis and other documentation, however well organized they are. Kay: can you see how often your XDS wiki (for example) is accessed? The small number of different contributors to a wiki is however a problem. Replying to an email is something that has to be done almost immediately and reaches instantly a large audience. Making a contribution to a wiki lacks the urgency and can be put off for a few weeks, and does not reward the contributor with immediate feedback or lead to controversial discussions. Perhaps someone should make a list of the questions most frequently asked on CCP4bb and the most helpful replies that they generated. This could even be made into a wiki. George On 02/14/2014 09:15 AM, Frank von Delft wrote: Seems it's worth thinking about this as an experiment that has actually been done: BB and wiki have been available in
Re: [ccp4bb] KD of dimerization, off topic
Sedimentation equilibrium or sedimentation velocity experiments by analytical centrifugation is the best method for this. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of FOOS Nicolas [nicolas.f...@synchrotron-soleil.fr] Sent: Friday, February 14, 2014 12:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] KD of dimerization, off topic I agree with Dave, and i suggest one more method to estimate Kd, The intrinsic fluorescence of proteins thanks to the aromatic chain side. Maybe it's also possible to have an estimation with native gels if you use prot A concentration as fixed and B protein concentration as variable. I am not sure. De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de David Briggs [drdavidcbri...@gmail.com] Envoyé : vendredi 14 février 2014 09:13 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] KD of dimerization, off topic Hi Careina, I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged. Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation. Hth, Dave On 14 Feb 2014 08:03, Careina Edgooms careinaedgo...@yahoo.commailto:careinaedgo...@yahoo.com wrote: Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina - *SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. (fpc5p) -
Re: [ccp4bb] Sister CCPs
On 14/02/14 09:15, George Sheldrick wrote: Perhaps someone should make a list of the questions most frequently asked on CCP4bb and the most helpful replies that they generated. This could even be made into a wiki. RIP minor-groove :-(
[ccp4bb] What really happens in XDSCONV?
Hi, I am a long time user of XDS (20 years this year) but all the same I find that I have constant angst about losing observations because I don't understand what goes in in the conversion steps to get to CCP4 format. I used to believe that XSCALE was always necessary, and I always use it in my workflow even if there is only one dataset (after all, there's nothing to lose), but my Ph.D. students pointed out to me that XDSCONV could take output directly from CORRECT, and they often do it this way. The XDS wiki and XDSCONV docs seems to confirm this: using MERGE=TRUE in XDSCONV should output the geometrical mean of the observations. However I am worried by what the XDSCONV output says. For today's example CORRECT gives me this: NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES 190093 NUMBER OF REJECTED MISFITS3842 NUMBER OF SYSTEMATIC ABSENT REFLECTIONS 34 NUMBER OF ACCEPTED OBSERVATIONS 186217 NUMBER OF UNIQUE ACCEPTED REFLECTIONS44059 XDSCONV says the following: FRIEDEL'S_LAW=TRUE MERGE=TRUE NUMBER OF REFLECTION RECORDS ON INPUT FILE 190093 NUMBER OF IGNORED REFLECTIONS (I -3.0*SIGMA) 19 NUMBER OF REFLECTIONS ACCEPTED FROM INPUT FILE 44047 To my literal mind this says it is throwing away most of the observations. Now if I merge the reflections in XSCALE first it says this: FRIEDEL'S_LAW=TRUE MERGE=TRUE NUMBER OF REFLECTION RECORDS ON INPUT FILE 44046 NUMBER OF IGNORED REFLECTIONS (I -3.0*SIGMA)0 NUMBER OF REFLECTIONS ACCEPTED FROM INPUT FILE 44046 which makes more sense. If I compare the first few reflections of the output file with structure factor amplitudes from XDSCONV for each scenario they are different, but I believe that is because XSCALE has put the intensities on an absolute scale and CORRECT has not. Basically all I want to know is that the output from XDSCONV is misleadingly worded, i.e. that even if it appears to say that only the asymmetric unit has been accepted, actually all observations have gone in and the geometric mean is indeed output. That would put my mind to rest! /Derek Derek Logan tel: +46 46 222 1443 Associate Professor mob: +46 76 8585 707 Dept. of Biochemistry and Structural Biology www.cmps.lu.sehttp://www.cmps.lu.se Centre for Molecular Protein Science www.maxlab.lu.se/node/307http://www.maxlab.lu.se/node/307 Lund University, Box 124, 221 00 Lund, Sweden
Re: [ccp4bb] Determining concentration of membrane protein
No, because Bradford is based on the increase in absorbance when the dye moves from a hydrophilic environment to a hydrophobic one (like the protein interior, or like the interior of a micelle). When detergents are present in excess of their CMC, the change in absorbance from partitioning into the micelles is generally large compared to any signal due to protein binding; plus preparing a perfectly matched blank solution is challenging when dealing with protein-detergent solutions. I second Michael's recommendation--BCA works well. On 14 Feb 2014, at 1:45 AM, Niks wrote: Dear All, May be a stupid question. But if we take buffer with detergent as control (Blank), would not the difference in ODs using any of the methods used e.g. Bradford assay, gives protein concentration? Regards Nishant On Thu, Feb 13, 2014 at 9:09 PM, Roger Rowlett rrowl...@colgate.edu wrote: Your basic choices for protein assays are: Alkaline copper methods (e.g., Biuret and micro-biuret) alkaline copper + molybdate methods (e.g., Lowry, BCA assays) Hydrophobic dye methods (e.g. Bradford) UV methods (e.g., A280, A230, A210, etc.) Method 1 is least sensitive to amino acid composition, but is also has highest detection limits. Thiols interfere. Method 2 is very idiosyncratic with amino acid composition, and also subject to interference by thiols. Method 3 is not usable in detergent solutions. Method 4 has many inteferences as most everything absorbs in the far UV region. If you have some special protein cofactors, metals, chromophores, etc. these can be exploited for better measurements. For ecample metalloproteins are easy to quantify by ICP-OES or TXRF if they are reasonably pure. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote: Dear CC4BBers, I am trying to figure out what is the best way to determine the protein concentration of my membrane protein. My purified membrane protein is in 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%). After reading the friendly manuals and searching online, I've learned that detergents interferes with assays like Bradford but can't find good descriptions of what works best. For now, I am trying to estimate concentration from absorbance at 280nm and using molar extinction coefficients based on aromatic amino acids, but again suspect detergent interference. I would like to know what other folks working on membrane proteins are doing. Thanks very much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- The most difficult phase of life is not when No one understands you;It is when you don't understand yourself
[ccp4bb] CCP4-6.4.0 Update 008
Dear CCP4 Users, An update for the CCP4-6.4.0 series has just been released, consisting of the following changes: * phaser * update to 2.5.6 * ccp4i * phaser_EP: corrected HySS output parsing Note that auto-updates work only with CCP4 6.4.0 series, therefore please upgrade if necessary. The Update Manager is now included in the package so you do not need to install it separately. In addition, all available updates will be installed automatically if you are using Setup Manager for CCP4 installation. Please report any bugs to c...@stfc.ac.ukmailto:c...@stfc.ac.uk. Many thanks for using CCP4. Charles -- Scanned by iCritical.
[ccp4bb] fluorescent pedal
I am trying to find out where I can get the fluorescent material (just a small flat piece) I can glue to the tip of a pin for aligning the X-ray beam of our home source. Does anyone know? Thanks in advance! Ronnie
Re: [ccp4bb] KD of dimerization, off topic
Hi Careina, Since alternative methods are being suggested... ITC can be good for quantitating a monomer-dimer equilibrium by diluting dimers out from a concentrated solution (which obviously favours the dimer) - and presuming a reasonable Kon/Koff. Alan Cooper has kindly figured out the data fitting for the rest of us: http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf I think Alan was using a VP-ITC when he was doing this stuff. Lower volumes - and presumably concentrations if the KD is small enough - are feasible in an ITC200. The protein is recoverable anyway. All the best, Will. On 14 February 2014 12:40, Williams, John Charles jcwilli...@coh.org wrote: Sedimentation equilibrium or sedimentation velocity experiments by analytical centrifugation is the best method for this. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of FOOS Nicolas [nicolas.f...@synchrotron-soleil.fr] Sent: Friday, February 14, 2014 12:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] KD of dimerization, off topic I agree with Dave, and i suggest one more method to estimate Kd, The intrinsic fluorescence of proteins thanks to the aromatic chain side. Maybe it's also possible to have an estimation with native gels if you use prot A concentration as fixed and B protein concentration as variable. I am not sure. De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de David Briggs [drdavidcbri...@gmail.com] Envoyé : vendredi 14 février 2014 09:13 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] KD of dimerization, off topic Hi Careina, I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged. Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation. Hth, Dave On 14 Feb 2014 08:03, Careina Edgooms careinaedgo...@yahoo.commailto:careinaedgo...@yahoo.com wrote: Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina - *SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender. (fpc5p) -
Re: [ccp4bb] fluorescent pedal
Hi Ronnie, We use YAG discs (Yttrium Aluminum Garnet). We buy small 10mm x 100um or 500um thick discs, break them into shards, glue them to various alignment jigs and they provide a very effective X-ray visualization tool. Our latest supplier is Star Tech Instruments (http://www.startechinstruments.com) There are other suppliers, but it does take some calling around because Google doesn't appear to be very helpful… unless you want 5000 lbs from a supplier in China. Good luck, Scott On Feb 14, 2014, at 7:54 AM, Ronnie wrote: I am trying to find out where I can get the fluorescent material (just a small flat piece) I can glue to the tip of a pin for aligning the X-ray beam of our home source. Does anyone know? Thanks in advance! Ronnie smime.p7s Description: S/MIME cryptographic signature
Re: [ccp4bb] What really happens in XDSCONV?
Hi Tim, I would actually recommend using pointless (or xprep) instead of xdsconv. It is much easier to use and maybe even less error prone. I take the point about pointless but XPREP is commercial software sold by Bruker and costs €700 (as I remember), so is not really an option for everyone. All your quotes from the output are perfectly consistent. The first table tells you there are 190093 unmerged reflections in total, but only 44059 unique reflections. Hence if you ask xdsconv to merge the output (MERGE=TRUE), it will do so and only write 44047 (a few less than 44059 because it rejects those 19 unmerged reflections with I-3sigma). I beg to disagree, and this was the point of my question. The output of XDSCONV literally says that 190093 reflections are read, and [of those, my interpretation] 44047 are accepted. I may be a pedant but I can't read that output in any other way. To me it looks like it is reading only the reflections that already fall into the asymmetric unit and is ignoring all the others. So if XDSCONV is really doing what it is supposed to, I would suggest rephrasing that output line. The documentation tells you: MERGE=TRUE means that the weighted mean of symmetry equivalent reflection intensities appearing in the input file will be determined and used in the output file. Yes, I paraphrased this in my original mail. I like to believe that the manual describes what the program is doing, but the output isn't consistent with this in my view. xscale does not scale data in a crystallographic sense since scaling is already done in the CORRECT step of XDS. It put the data on a common scale (in a sophisticated manner), i.e. if you only have one data set there is no need to run xscale except for the thinner shells for the statistics table compared to CORRECT. No need indeed, except that including XSCALE doesn't leave me sleepless about the data having been merged. And maybe correction for radiation damage when you have sufficient multiplicity? But yes, pedantry aside, I will start using pointless in my csh pipelines. Best wishes Derek On 02/14/2014 03:57 PM, Derek Logan wrote: Hi, I am a long time user of XDS (20 years this year) but all the same I find that I have constant angst about losing observations because I don't understand what goes in in the conversion steps to get to CCP4 format. I used to believe that XSCALE was always necessary, and I always use it in my workflow even if there is only one dataset (after all, there's nothing to lose), but my Ph.D. students pointed out to me that XDSCONV could take output directly from CORRECT, and they often do it this way. The XDS wiki and XDSCONV docs seems to confirm this: using MERGE=TRUE in XDSCONV should output the geometrical mean of the observations. However I am worried by what the XDSCONV output says. For today's example CORRECT gives me this: NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES 190093 NUMBER OF REJECTED MISFITS3842 NUMBER OF SYSTEMATIC ABSENT REFLECTIONS 34 NUMBER OF ACCEPTED OBSERVATIONS 186217 NUMBER OF UNIQUE ACCEPTED REFLECTIONS44059 XDSCONV says the following: FRIEDEL'S_LAW=TRUE MERGE=TRUE NUMBER OF REFLECTION RECORDS ON INPUT FILE 190093 NUMBER OF IGNORED REFLECTIONS (I -3.0*SIGMA) 19 NUMBER OF REFLECTIONS ACCEPTED FROM INPUT FILE 44047 To my literal mind this says it is throwing away most of the observations. Now if I merge the reflections in XSCALE first it says this: FRIEDEL'S_LAW=TRUE MERGE=TRUE NUMBER OF REFLECTION RECORDS ON INPUT FILE 44046 NUMBER OF IGNORED REFLECTIONS (I -3.0*SIGMA) 0 NUMBER OF REFLECTIONS ACCEPTED FROM INPUT FILE 44046 which makes more sense. If I compare the first few reflections of the output file with structure factor amplitudes from XDSCONV for each scenario they are different, but I believe that is because XSCALE has put the intensities on an absolute scale and CORRECT has not. Basically all I want to know is that the output from XDSCONV is misleadingly worded, i.e. that even if it appears to say that only the asymmetric unit has been accepted, actually all observations have gone in and the geometric mean is indeed output. That would put my mind to rest! /Derek Derek Logan tel: +46 46 222 1443 Associate Professor mob: +46 76 8585 707 Dept. of Biochemistry and Structural Biology www.cmps.lu.sehttp://www.cmps.lu.se Centre for Molecular Protein Science www.maxlab.lu.se/node/307http://www.maxlab.lu.se/node/307 Lund University, Box 124, 221 00 Lund, Sweden - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12
Re: [ccp4bb] KD of dimerization, off topic
What a nice idea this ITC dilution is--a great example of a wet lab technique learned en passant on the ccp4bb. I wonder what range of Kds could feasibly be measured with existing calorimeter sensitivities? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Will Stanley Sent: Friday, February 14, 2014 12:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] KD of dimerization, off topic Hi Careina, Since alternative methods are being suggested... ITC can be good for quantitating a monomer-dimer equilibrium by diluting dimers out from a concentrated solution (which obviously favours the dimer) - and presuming a reasonable Kon/Koff. Alan Cooper has kindly figured out the data fitting for the rest of us: http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf I think Alan was using a VP-ITC when he was doing this stuff. Lower volumes - and presumably concentrations if the KD is small enough - are feasible in an ITC200. The protein is recoverable anyway. All the best, Will. On 14 February 2014 12:40, Williams, John Charles jcwilli...@coh.org wrote: Sedimentation equilibrium or sedimentation velocity experiments by analytical centrifugation is the best method for this. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of FOOS Nicolas [nicolas.f...@synchrotron-soleil.fr] Sent: Friday, February 14, 2014 12:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] KD of dimerization, off topic I agree with Dave, and i suggest one more method to estimate Kd, The intrinsic fluorescence of proteins thanks to the aromatic chain side. Maybe it's also possible to have an estimation with native gels if you use prot A concentration as fixed and B protein concentration as variable. I am not sure. De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de David Briggs [drdavidcbri...@gmail.com] Envoyé : vendredi 14 février 2014 09:13 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] KD of dimerization, off topic Hi Careina, I'm not sure you can assume that the ratio of monomer and dimer will stay constant through the column - as you say, the protein is diluted during the run, the ratio will change, unless you have a super tight dimer - which clearly you do not. Also, as the mass and the molar extinction coefficient will both double in the dimer, the relationship between absorbance and concentration will be unchanged. Typically, such these sorts of questions are answered (at least me) by equilibrium analytical centrifugation. Hth, Dave On 14 Feb 2014 08:03, Careina Edgooms careinaedgo...@yahoo.commailto:careinaedgo...@yahoo.com wrote: Dear CCP4 board I have a protein that exists in equilibrium between monomer and dimer and I'm trying to calculate KD using size exclusion. The problem is that the column dilutes my sample so that if I put 20 uM on to the column, I only recover 0.5 uM in dimer fraction and 2 uM in monomer fraction. I am getting confused as to how to plot my KDs. Do I not regard my initial concentrations at all and work only with the final concentrations that come off the column? I would plot [monomer] squared vs [dimer] and I will assume that the ratio of monomer to dimer will stay constant as the protein passes through the column. (also I would calculate [dimer] using 2x monomer extinction coefficient) Does this seem a reasonable way to calculate KDs and reasonable argument? Also I am looking for good references for calculating Kds when dealing with dimerization Thanks and sorry for off topic question Careina - *SECURITY/CONFIDENTIALITY WARNING: This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law (e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the
Re: [ccp4bb] Acetyl link problems.
Hi Garib I think that was the first thing we tried but it gave very poor geometry for the ACE and also it's giving a VDW outlier for the ACE C - MET N bond. So it looks like it's not recognising the link. Cheers -- Ian On 14 February 2014 11:51, Garib Murshudov ga...@mrc-lmb.cam.ac.uk wrote: Hi Ian dod you try without link and standard ACE atom naming. Refmac should be able to deal N-terminal activation and few other things. At least it was the intention when it was written. Bugs may have been (self)introduced to prevent this from happening. If it is so then I would like to know. Regards Garib On 13 Feb 2014, at 22:57, Ian Tickle ianj...@gmail.com wrote: All, I'm having problems refining a structure with an N-terminal acetylated MET residue. I'm trying it with both Refmac Buster. Buster works fine gives perfect planar geometry for the ACE-MET linkage. Refmac gives a pyramidal acetyl group after refinement which to my eyes is wrong (sp2 C atom?). I have this line in my input PDB: LINKRC ACE A 0 N MET A 1 ACE_C-N which as I understand it should solve the problem. However, looking at the CIF entry for the ACE_C-N link I see restraints defined for bonds, angles torsion angles but not for the CC(=O)N plane. So the problem seems to be that the planar restraints for this link group are missing - or are they defined elsewhere? Anyway I added planar restraints to the ACE_C-N link entry it solves the problem, at least for regularisation - I still have the same problem with refinement. Refmac in regularisation mode now gives the correct (planar) geometry for the ACE-MET linkage. I'm just puzzled why no-one has noticed this, after all post-translational acetylation is surely not that uncommon (according to Wikipedia 80% of human proteins are N-term acetylated!). Further, looking at the entry for ACE I see: ACE ACE 'ACETYL GROUP' non-polymer 7 3 . ACE O OO 0.000 0.0000.0000.000 ACE C CC10.000 -1.044 -0.6060.000 ACE H HH 0.000 -1.978 -0.0690.000 ACE CH3CCH3 0.000 -1.041 -2.1130.000 ACE H3 HH 0.000 -0.541 -2.4640.865 ACE H2 HH 0.000 -2.038 -2.4680.000 ACE H1 HH 0.000 -0.540 -2.464 -0.864 Where did the extra H atom (3rd atom) come from? Acetyl is CH3C=O: the extra H atom would make it acetaldehyde which of course has nothing whatsoever to do with acetylation! Is this the reason for the lack of planar link restraints (though that wouldn't explain why the other link restraints are present)? Any insights appreciated! Cheers -- Ian Dr Garib N Murshudov MRC-LMB Francis Crick Avenue Cambridge CB2 0QH UK Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
Re: [ccp4bb] Sister CCPs
Limited contributions are a common problem with wikis. This paper describes a way to add some incentive to contributing to a wiki: http://bioinformatics.oxfordjournals.org/content/29/14/1837.full Cheers, Robbie Sent from my Windows Phone Van: Kay Diederichs Verzonden: 14-2-2014 8:21 Aan: CCP4BB@JISCMAIL.AC.UK Onderwerp: Re: [ccp4bb] Sister CCPs Nat, that's why I set up the CCP4 wiki at http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Main_Page ! The idea is that everybody benefits: experienced crystallographers/biologists can concentrate on the new and difficult questions coming up on the bulletin board, and novices find answers to those ever-recurring questions. Everybody can contribute answers, or improve existing ones! But the wiki can only be useful in the long run if there are contributors. Why are there (almost) no contributors? It cannot be due to technical difficulty; it's very easy to contribute to a wiki. One guess is that a posting on a BB is more socially rewarding, because the interaction via emails is more immediate. Re-vitalize the wiki! Kay On Thu, 13 Feb 2014 07:21:14 -0800, Nat Echols nathaniel.ech...@gmail.com wrote: One comment (not a complaint) on all this: it seems like the same questions get asked over and over again. If there is a good place for a general crystallography FAQ list it is well past time for one to be put together - or maybe it just needs to be better advertised? At a minimum, for instance: - what cryoprotectant should I use? - how do I get big single crystals? - how do I improve diffraction? - how can I tell if I've solved my structure? - why is my R-free stuck? - is pick random statistic suitable for publication? Some of the other common queries (name my blob!) still need to be handled on a case-by-case basis, but it would be much more efficient for everyone if the standard answers were collected somewhere permanent. -Nat On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov eugene.val...@gmail.comwrote: I absolutely agree with Juergen. Leaving aside methods developers, who are a completely different breed, there is no such thing as a crystallographer sitting in a dark room solving structures all day. If there are, these are anachronisms destined for evolutionary demise. More and more cell biologists, immunologists and all other kinds of biologists are having a go at doing structural work with their molecules of interest themselves without involving the professionals. Typically, they learn on the job and they need advice with all kinds of things ranging from cloning and protein preps through to issues with tetartohedrally-twinned data and interpreting their structures. So, a modern structural biologist is one who is equipped for the wet lab and has some idea of how to go about solving structures. CCP4BB is a wonderful resource that is great for both the quality of the advice offered to those that seek it and for the variety of topics that are addressed in the scope of structural biology. I have learnt greatly from reading posts from very skilled and knowledgeable scientists at this forum and then implemented these insights into my own research. I am very grateful for this. In short, please do not discourage your colleagues, particularly very junior ones, from posting to the CCP4BB. Some of the questions may appear quaint or irrelevant but it is easy to simply ignore topics that are of no interest! Eugene On 13 February 2014 14:41, Bosch, Juergen jubo...@jhsph.edu wrote: Let me pick up Eleanor's comment: is there something like a crystallographer today ? I mean in the true sense ? I think as a crystallographer you won't be able to survive the next decade, you need to diversify your toolset of techniques as pointed out in this article http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a And I'm not quite sure how software developers see themselves, as I would argue they are typically maybe not doing so much wet lab stuff related to crystallography (I may be wrong here) but rather code these days. What type of crystallographer is a software developer ? I think like our beloved crystals we come in different flavors. And we need to train the next generation of students with that perspective in mind. Just my two cents on a snowy day (30cm over night) J�rgen .. J�rgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Feb 13, 2014, at 6:41 AM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: I agree with Frank - it keeps crystallographers modest to know how challenging wet lab stuff still is.. Eleanor On 12 February 2014 19:23, Robbie Joosten
Re: [ccp4bb] KD of dimerization, off topic
That's an extremely useful link - thanks to Will Stanley for posting that one. For a VP-ITC machine I'd guess that you need to load the injector with about 500ul of protein at a concentration of 80x the Kd or more. Notice that Alan Cooper was injecting 10 microliters of protein at 2mM with a 12 microMolar dissociation constant, per injection. You would probably want to maintain that approximate ratio - ~170 because it's mostly a question of measuring deltaH with a decent signal-to-noise per injection. I recall that it takes up to 500 microLiters to load the injection syringe on a VP-ITC without air gap between plunger tip and injection point - unless someone's got a nice trick to reduce that. The rule of thumb from the VP-ITC manual - and from practical experience on our machine here - for A+B = AB is using at least 10x the Kd in the sample chamber and about 80x the Kd in the injector. That's not exactly the same situation, but 80x vs 170x suggests the the considerations are much the same. Phil Jeffrey Princeton On 2/14/14 12:52 PM, Keller, Jacob wrote: What a nice idea this ITC dilution is--a great example of a wet lab technique learned en passant on the ccp4bb. I wonder what range of Kds could feasibly be measured with existing calorimeter sensitivities? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Will Stanley Sent: Friday, February 14, 2014 12:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] KD of dimerization, off topic Hi Careina, Since alternative methods are being suggested... ITC can be good for quantitating a monomer-dimer equilibrium by diluting dimers out from a concentrated solution (which obviously favours the dimer) - and presuming a reasonable Kon/Koff. Alan Cooper has kindly figured out the data fitting for the rest of us: http://www.chem.gla.ac.uk/staff/alanc/itcdil.pdf I think Alan was using a VP-ITC when he was doing this stuff. Lower volumes - and presumably concentrations if the KD is small enough - are feasible in an ITC200. The protein is recoverable anyway. All the best, Will.
Re: [ccp4bb] Sister CCPs
Hi Robbie, I think lack of feedback is a serious problem for wikis. I once started an XDS tutorial at Kay's XDS-wiki, stating that it was still under development and encouraging readers to send me an email if they want to see faster progress in the completion of the tutorial. I never received a single email, so I did not see the point of spending more time there and the tutorial is still pretty incomplete (although Kay also contributed to it from time to time). It may be hard to believe (why?) for newcomers, but crystallographers are actually very open to criticism and discussions (cf. ccp4bb ;-). Cheers, Tim On 02/14/2014 07:14 PM, Robbie Joosten wrote: Limited contributions are a common problem with wikis. This paper describes a way to add some incentive to contributing to a wiki: http://bioinformatics.oxfordjournals.org/content/29/14/1837.full Cheers, Robbie Sent from my Windows Phone Van: Kay Diederichs Verzonden: 14-2-2014 8:21 Aan: CCP4BB@JISCMAIL.AC.UK Onderwerp: Re: [ccp4bb] Sister CCPs Nat, that's why I set up the CCP4 wiki at http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Main_Page ! The idea is that everybody benefits: experienced crystallographers/biologists can concentrate on the new and difficult questions coming up on the bulletin board, and novices find answers to those ever-recurring questions. Everybody can contribute answers, or improve existing ones! But the wiki can only be useful in the long run if there are contributors. Why are there (almost) no contributors? It cannot be due to technical difficulty; it's very easy to contribute to a wiki. One guess is that a posting on a BB is more socially rewarding, because the interaction via emails is more immediate. Re-vitalize the wiki! Kay On Thu, 13 Feb 2014 07:21:14 -0800, Nat Echols nathaniel.ech...@gmail.com wrote: One comment (not a complaint) on all this: it seems like the same questions get asked over and over again. If there is a good place for a general crystallography FAQ list it is well past time for one to be put together - or maybe it just needs to be better advertised? At a minimum, for instance: - what cryoprotectant should I use? - how do I get big single crystals? - how do I improve diffraction? - how can I tell if I've solved my structure? - why is my R-free stuck? - is pick random statistic suitable for publication? Some of the other common queries (name my blob!) still need to be handled on a case-by-case basis, but it would be much more efficient for everyone if the standard answers were collected somewhere permanent. -Nat On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov eugene.val...@gmail.comwrote: I absolutely agree with Juergen. Leaving aside methods developers, who are a completely different breed, there is no such thing as a crystallographer sitting in a dark room solving structures all day. If there are, these are anachronisms destined for evolutionary demise. More and more cell biologists, immunologists and all other kinds of biologists are having a go at doing structural work with their molecules of interest themselves without involving the professionals. Typically, they learn on the job and they need advice with all kinds of things ranging from cloning and protein preps through to issues with tetartohedrally-twinned data and interpreting their structures. So, a modern structural biologist is one who is equipped for the wet lab and has some idea of how to go about solving structures. CCP4BB is a wonderful resource that is great for both the quality of the advice offered to those that seek it and for the variety of topics that are addressed in the scope of structural biology. I have learnt greatly from reading posts from very skilled and knowledgeable scientists at this forum and then implemented these insights into my own research. I am very grateful for this. In short, please do not discourage your colleagues, particularly very junior ones, from posting to the CCP4BB. Some of the questions may appear quaint or irrelevant but it is easy to simply ignore topics that are of no interest! Eugene On 13 February 2014 14:41, Bosch, Juergen jubo...@jhsph.edu wrote: Let me pick up Eleanor's comment: is there something like a crystallographer today ? I mean in the true sense ? I think as a crystallographer you won't be able to survive the next decade, you need to diversify your toolset of techniques as pointed out in this article http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a And I'm not quite sure how software developers see themselves, as I would argue they are typically maybe not doing so much wet lab stuff related to crystallography (I may be wrong here) but rather code these days. What type of crystallographer is a software developer ? I think like our beloved crystals we come in different flavors. And we
Re: [ccp4bb] Sister CCPs
It is an interesting observation, Tim. The fact that people do not read tutorials or manuals, is somewhat related to the fact that many questions asked in the ccp4bb could be answered by a more experienced colleague, a supervisor, a manual, a wiki, St Google the revealer, not to mention the somewhat extreme suggestion of an actual text book. Tassos PS Many questions can also be answered by coming to the Gordon Conference for Diffraction Methods in Structural Biology this summer!!! A great investment of your time, suitable for both seasoned crystallographers and wannabe stars of the next edition of Cell alike !!! https://www.grc.org/programs.aspx?year=2014program=diffrac But more on that next week!!! On 14 Feb 2014, at 19:25, Tim Gruene wrote: Hi Robbie, I think lack of feedback is a serious problem for wikis. I once started an XDS tutorial at Kay's XDS-wiki, stating that it was still under development and encouraging readers to send me an email if they want to see faster progress in the completion of the tutorial. I never received a single email, so I did not see the point of spending more time there and the tutorial is still pretty incomplete (although Kay also contributed to it from time to time). It may be hard to believe (why?) for newcomers, but crystallographers are actually very open to criticism and discussions (cf. ccp4bb ;-). Cheers, Tim On 02/14/2014 07:14 PM, Robbie Joosten wrote: Limited contributions are a common problem with wikis. This paper describes a way to add some incentive to contributing to a wiki: http://bioinformatics.oxfordjournals.org/content/29/14/1837.full Cheers, Robbie Sent from my Windows Phone Van: Kay Diederichs Verzonden: 14-2-2014 8:21 Aan: CCP4BB@JISCMAIL.AC.UK Onderwerp: Re: [ccp4bb] Sister CCPs Nat, that's why I set up the CCP4 wiki at http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Main_Page ! The idea is that everybody benefits: experienced crystallographers/biologists can concentrate on the new and difficult questions coming up on the bulletin board, and novices find answers to those ever-recurring questions. Everybody can contribute answers, or improve existing ones! But the wiki can only be useful in the long run if there are contributors. Why are there (almost) no contributors? It cannot be due to technical difficulty; it's very easy to contribute to a wiki. One guess is that a posting on a BB is more socially rewarding, because the interaction via emails is more immediate. Re-vitalize the wiki! Kay On Thu, 13 Feb 2014 07:21:14 -0800, Nat Echols nathaniel.ech...@gmail.com wrote: One comment (not a complaint) on all this: it seems like the same questions get asked over and over again. If there is a good place for a general crystallography FAQ list it is well past time for one to be put together - or maybe it just needs to be better advertised? At a minimum, for instance: - what cryoprotectant should I use? - how do I get big single crystals? - how do I improve diffraction? - how can I tell if I've solved my structure? - why is my R-free stuck? - is pick random statistic suitable for publication? Some of the other common queries (name my blob!) still need to be handled on a case-by-case basis, but it would be much more efficient for everyone if the standard answers were collected somewhere permanent. -Nat On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov eugene.val...@gmail.comwrote: I absolutely agree with Juergen. Leaving aside methods developers, who are a completely different breed, there is no such thing as a crystallographer sitting in a dark room solving structures all day. If there are, these are anachronisms destined for evolutionary demise. More and more cell biologists, immunologists and all other kinds of biologists are having a go at doing structural work with their molecules of interest themselves without involving the professionals. Typically, they learn on the job and they need advice with all kinds of things ranging from cloning and protein preps through to issues with tetartohedrally-twinned data and interpreting their structures. So, a modern structural biologist is one who is equipped for the wet lab and has some idea of how to go about solving structures. CCP4BB is a wonderful resource that is great for both the quality of the advice offered to those that seek it and for the variety of topics that are addressed in the scope of structural biology. I have learnt greatly from reading posts from very skilled and knowledgeable scientists at this forum and then implemented these insights into my own research. I am very grateful for this. In short, please do not discourage your colleagues, particularly very junior ones, from posting to the CCP4BB. Some of the questions may appear quaint or irrelevant but it is easy to simply ignore topics that are of no
Re: [ccp4bb] fluorescent pedal
We've used CdWO4 crystals for years. They are highly radiation resistant and colorless and give VERY bright fluorescence. Saint-Gobain has them in various forms. In principle you can mix your own CdWO4 from CdNO3 and NaWO4 ... under proper conditions you let the solution digest to form microcrystals. I tried this once, with no success, but it has been done in the literature. Another option is terbium-doped borosilicate glass. Can be bought in first-draw fiber form from a company called Collimated Holes. This material is bright, colorless, can be melted and formed, but photobleaches in bright x-ray beams over time. Of course there is always cutting thin slivers of the plastic fluorescent sheet material everyone uses at beamlines. Not sure where they get that. Richard Gillilan MacCHESS On Feb 14, 2014, at 12:18 PM, Scott Classen wrote: Hi Ronnie, We use YAG discs (Yttrium Aluminum Garnet). We buy small 10mm x 100um or 500um thick discs, break them into shards, glue them to various alignment jigs and they provide a very effective X-ray visualization tool. Our latest supplier is Star Tech Instruments (http://www.startechinstruments.com) There are other suppliers, but it does take some calling around because Google doesn't appear to be very helpful… unless you want 5000 lbs from a supplier in China. Good luck, Scott On Feb 14, 2014, at 7:54 AM, Ronnie wrote: I am trying to find out where I can get the fluorescent material (just a small flat piece) I can glue to the tip of a pin for aligning the X-ray beam of our home source. Does anyone know? Thanks in advance! Ronnie
