Re: [ccp4bb] Difficult data
Stephen, Stephen, Although your peptide is smaller than the one I once worked on, here are some thoughts that might be applicable. 1. Check and play with the radius of integration in the molecular replacement. The default is probably not appropriate for your case but the value should be much smaller than the default. 2. Take a model - any model - of your peptide and make an arbitrary artificial dataset by rotating the model around an arbitrary angle, and put it in an arbitrary unit cell (say P1 for simplicity). Then use the original non-rotated model and see what parameters will give you the best solution and use those as starting parameters for your search. 3. Consider that your model may be very asymmetric, i.e. much longer in one direction than the other. In theory, you want the radius of integration to be such that it covers one copy of the model but not more than one. If the peptide is much longer than it is wide (which is somewhat likely), you might run into the situation where the correct radius for the length would incorporate multiple copies in the other direction(s). If this is the case, I am not sure you can fix it. In my humble opinion, which might be very out-of-date, this might be one of the reasons why MR does not work well on small molecules. 4. I think that it is possible that your crystal could be built from multiple copies of randomly oriented copies of the peptide, which are similar in their nature, but not exactly the same. This sounds odd but I convinced myself a long time ago that such a crystal could be made. A long time ago I worked on a sea anemone toxin that had similar properties. At the time I could not make step 2 above work, that is, I convinced myself that I was unable to find parameters that did the job. That was enough for my mentor to tell me that I should not pursue the project... Of course such molecules are easily(*) resolved with NMR. Mark (*) Relative statement, and not by me. -Original Message- From: Stephen Campbell j...@ualberta.ca To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Tue, Apr 16, 2013 7:39 pm Subject: [ccp4bb] Difficult data Hello, I am having a few issues with a data set I have been working on recently, and was hoping to get some ideas on how to deal with it, if anyone is in the mood. I have been working with a very small bacterocin (about 3 kDa) and set up some crystal trays in hope of getting some high diffracting crystals. I failed, but did manage to get a data set of reasonable quality to about 4A from crystals that reproduce very poorly. Now, this sounds horrendous, but the MR model I have available is of a similar bacteriocin, whose structure is predicted to be essentially identical (different ORF, but almost identical sequence). I was thinking it would be done in a day. The bravais lattice is P4, and 118 x 118 x 165 (which seems HUGE for such a small protein...indeed calling it a protein is generous...peptide). The data seems reasonably nice (nice spots, no visible overlap within 4A, but is very mosaic, about 1.5 degrees. The data integrates and scales nicely, with very good chi2, R-factors and % rejected reflections. It is hard to predict the correct space group, since all the tetragonal options have the same stats. The systematic absences seem to predict p41212 (as does pointless), but it wouldn't be the first time I screwed that up. I can't get a solution, no matter what I try. Is this the nature of such a small peptide in such a large unit cell (placing the first model is difficult for MR since there may be many copies in the AU), or are there some tricks? Is it likely that the unit cell is wrong? Self Rotation functions give 8 peaks, but this is considering a peak fairly generously (approx. 15-20% of origin). Are there some blatantly obvious red flags that I may be missing? Any advice would be great - even if that advice is that it may be time to move on. I should note that I have considered the idea that the peptide may be forming some sort of oligomeric structure such as a coiled coil, but it fails coiled coil predicting software, and there is no evidence that this should be the case. The homologous structure is very rigid, and I would say fairly confidently that mine is likely the same - I just want to confirm it. Thanks so much, Stephen Campbell Post Doctoral Fellow Department of Biochemistry McGill University
Re: [ccp4bb] delete subject
No. :-) When you are a reviewer for structural papers in journals (I do this work sometimes), and when you see an article that has (in this example) Tom's structure in it, but he and/or his mentor is not an author, then you call the editor and tell them you may have a problem. I realize that the case may not be closed with that statement because the manuscript could indeed be totally legitimate and genuine, but it would be a signal in my mind to watch for. A friend could not just run with the data and publish. A competing group could take advantage and get ahead in their project inexpensively (provided that the posted data are what you think they are). But that is sort of the point of publishing result (I must remember to leave my idealism at home tomorrow). Our old approach is to keep a lid on all your data until the paper is published. Although it is hard to imagine, there could be a mechanism by which you make all your data public, immediately when you get it and this public record shows who owns it. The advantage (in my mind) of such a system would be that you would also make public the data that does not make sense to you (it does not fit your scientific model) and this could (and has) lead to great discoveries. The disadvantage to the method is that you will sometimes post experiments that are just completely wrong (you did not measure what you said you measured) and this might make you look dumb (not really, this happens all the time; a favorite saying is 'we all make mistakes, we just make sure they don't leave the room'). And furthermore, you would finally have a journal of unpublishable data, where all the experiments that we should not have done for one reason or another reside and can act as a warning what not to do in the future. It is possible that I am socialist. In the US that is not a good thing, but I don't worry about it. Furthermore, teaching/learning is a concern. More and more places no longer have the resources or the patience to teach or learn crystallography. I once heard a friend say something along these lines: people who did not learn crystallography are now teaching the next generation. As proof for that, he explained that experiments are done at synchrotrons that clearly show that not the beamline is broken, but the operator does not understand the concepts and therefore the data collected are not useful. In my world I see crystallography as a tool, and no longer as a goal all by itself (it was a goal when I was a graduate student). I am frequently concerned that protein crystallography will go the way of small molecule crystallography: a few places provide this service and as an experimentalist you don't much worry about how they do it. Of course, until it becomes super-easy to produce high-quality protein and crystals, this won't happen. Mark With apologies to Tom, I don't have a stop-button, Raji is right about that. -Original Message- From: vellieux frederic.velli...@ibs.fr To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Thu, Mar 28, 2013 1:54 am Subject: Re: [ccp4bb] delete subject Hello, I stayed away from this thread until now - the major reason being that I was fitting snugly under my quilt. However I feel compelled to react now: placing your data in a public repository (thereby proving that you did the work) also means that a colleague, friend or whatever can and will publish your work for you. Once your work has been published you cannot publish it again, you did the work and the colleague, friend or whatever has in fact appropriated your work. In the world of dreams I was living in until a few moments ago (it was night time), this is perhaps the way we should act. In the real world we live in, even your colleague upstairs will publish your work if he / she has a chance to do it because by doing so he / she will improve his / her career while ensuring that yours doesn't take off. Fred. On 28/03/13 01:34, mjvdwo...@netscape.net wrote: Earlier today, I thought this and didnot write it. It is a slightly different theme on your suggestion: I hear there are now (but have not seen examples of) journals (web sites) where you do exactlywhat Tom did: you put your data there, which proves that you did the work (first) and you do not worry about the fact that you are making it public before formal publication, because makingdata public is the reason why you got the datain the first place. And nobody can claim to havedone the work, because everybody knows thatsomeone else was first - the web site is
Re: [ccp4bb] delete subject
Earlier today, I thought this and did not write it. It is a slightly different theme on your suggestion: I hear there are now (but have not seen examples of) journals (web sites) where you do exactly what Tom did: you put your data there, which proves that you did the work (first) and you do not worry about the fact that you are making it public before formal publication, because making data public is the reason why you got the data in the first place. And nobody can claim to have done the work, because everybody knows that someone else was first - the web site is proof. The results are not peer-reviewed of course (even though, in the case of CCP4, things are inherently peer-reviewed to some extent, that is what he asked us to do). And I hear that there are now journals that will accept references to such web sites. Freely sharing unpublished data on a public forum might well be the future, even if in our corner of science this is not yet commonplace. The pivotal point to Tom is that he can learn from the suggestions that have been made. I hope he will. I actually hope that he will follow up on the suggestions (privately maybe). Unlike some, I do not feel that it was bad to find a big file in my inbox, this is what move to is for. I think my reaction was ouch, he did not want to do what he just did and it cannot be undone. But maybe this is not true. There is definitely value in sharing preliminary data, especially for junior people. To have such a function as part of CCP4 might be a very good suggestion, but I agree with you that perhaps it should not land in its full glory in everyone's mailbox. Mark -Original Message- From: William G. Scott wgsc...@ucsc.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Wed, Mar 27, 2013 6:09 pm Subject: Re: [ccp4bb] delete subject Dear Tom et al: Although arriving too late to participate in the snark-fest, it occurred to me that maybe this is almost exactly how we should solve structures and educate graduate students (or others). Instead of attachments, the relevant files could be shared via dropbox. Those of generous spirit could help solve, refine, correct, critique or otherwise improve structures before formal peer review. (If everyone knows the source of the data, it is far less likely to be ripped off, not more.) It might cut down on the number of mistakes (or worse) that appear in the PDB and journals, new mentorships and collaborations might be established, in exceptional cases co-authorship, or more generally, an acknowledgement could be offered. For students like mine who are comparatively isolated in a small institution somewhat off the beaten path, it would be a real asset and advantage to them not to have to rely only upon my limited abilities and increasingly obsolete knowledge. We should all be able to learn from one anther without fear of reproach. All the best, Bill William G. Scott Professor Department of Chemistry and Biochemistry and The Center for the Molecular Biology of RNA 228 Sinsheimer Laboratories University of California at Santa Cruz Santa Cruz, California 95064 USA On Mar 27, 2013, at 3:36 PM, Tom Van den Bergh tom.vandenbe...@student.kuleuven.be wrote: Is it possible to delete my post: refinement protein structure from ccp4 bb, i get too many bad reactions. I think its bettter to just delete the whole topic. Greetings, Tom
Re: [ccp4bb] statistical or systematic? bias or noise?
I think that in statistics you can build a model that describes (and predicts) the uncertainty. So if you have done similar (!) replicate experiments, from which you can build the model, you can apply it to a single observation and provide a reasonably good guess for the value that you were measuring and its variance. Of course that guess would not be as good as the average value and variance from true replicates. With protein crystals (or solutions for that matter), the sample is often too precious to redo the experiment and it is worth thinking about doing replicate experiments with a cheap one, build the model, and then apply it to single expensive observations. That would be statistically justified (provided that the model is valid for all sets of experiments). I have not built such models, but we know that pipetting isn't really as good as we believe. If you randomly dial to a particular value on your pipetteman (say 5 uL), you will get a certain pattern of errors (which is really not a good word for it), while if you consistently dial either from a low (1uL) or a high (10uL) value towards the value you want, you will get another pattern. Those two patterns are not representative of each other, I don't think, and you would need to understand how to do experiments consistently to stay within your error-model (bad word). Among many other things, statisticians try to come up with models that explain the uncertainty so that you know what to think, even if your set of observation is too small to say for sure, with n=1 being the ultimate too small. (Maybe not ultimate, n=0 is really too small.) Mark -Original Message- From: Alexander Aleshin aales...@sanfordburnham.org To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Wed, Mar 13, 2013 3:05 pm Subject: Re: [ccp4bb] statistical or systematic? bias or noise? On Mar 13, 2013, at 1:36 PM, Ed Pozharski wrote: But what if I only have one measurement worth of sample? Is it proper to use statistical analysis for a single measurement? I thought statistics, by definition, means multiple measurements. Alex
Re: [ccp4bb] Protein concentration vs Molecular wt...
I don't think there is such a rule, but in the old days, when we only had Hampton Screen I and II, the rule was: - Set up screen 1, look at the drops and you should expect some kind of precipitation in 50% of the drops. If much less than that, increase your protein concentration. If much more than that, decrease protein concentration. - Set up screen 2, look and expect 30% precipitation. I used to cut corners and do the statistics at 1/2 of a screen (one 24-well plate). You can probably use this method to get within a factor of 2 of the optimal concentration. There are probably good statistics in the papers for the screens that you may use. One of the advantages of structural genomics efforts is that these things are known (and hopefully published). Even older trick is to take a drop of protein and look under a microscope, record how much AmSO4 it takes to cause precipitation. Do the same with PEG. Keep adding a little at a time and look immediately. This will give you an idea if you are near a reasonable concentration. I think that this latter method does not tell you much more than physics-information - which is how many zeroes there are: whether 1 mg/ml or 10mg/ml or 100mg/ml is reasonable. Mark -Original Message- From: james09 pruza james09x...@gmail.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Thu, Jul 19, 2012 1:59 pm Subject: [ccp4bb] Protein concentration vs Molecular wt... Dear Crystallographers, Is there any rule of thumb for Protein concentration and molecular weight for crystallization trials of a soluble protein? Looking for high molecular wt. protein ~50kDa. James.
Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)
BTW, a l-o-n-g time ago, I worked on a project with crystals that only grew in the cold room. BUT... we found out that the crystals could in fact be transferred to a regular lab under condition that you warmed them up very slowly. So I would harvest the crystals into capillaries (this was before cryo) and put them in a petri dish, then put the dish in a cooler with several glass bottles of buffer and put the cooler in the lab. Then you wait. This worked. If you did not warm them up slowly, the crystals would be ruined. Also, we never tried to take the trays in which the crystals were grown out of the cold room, i.e. when the crystals are still swimming. Just some old data that might apply to other projects. Mark -Original Message- From: Radisky, Evette S., Ph.D., Ph.D. radisky.eve...@mayo.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Sat, Jul 14, 2012 7:20 am Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal) As I recall, I used to wear latex gloves, add extra padding to the handle end of the wand (and other tools) with bits of tubing, and take my tools out of the cold room every 20 min or so to de-ice and dry them. I didn’t really have a choice about the cold room because it was the only place my crystals would grow, and my crystallization solution was 20% isopropanol which was too volatile to work with outside the cold room anyway. Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bosch, Juergen Sent: Friday, July 13, 2012 8:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal) This is a very interesting topic I have to say. But what I missed in this discussion is the pain you go through when freezing in the cold room. As the name implies it's supposed to be cold (most of the times). But that's not too much of an issue as you can dress up accordingly. The problem I always had was freezing up of the advertisement Hampton Magnetic Wand /advertisement and icing up towards your fingertips after some time when moisture from the cold room condenses and freezes. I hate wearing gloves when handling crystals so there was not much of a skin protection. How do you guys solve this problem ? Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)
Hi Evette: Technically: The expansion ratio of liquid to gaseous nitrogen is approximately 1:700, that is, 1 liter of liquid becomes 700 liters of gas (at room temperature). When you are in a room that is 3 (~10ft) meters tall, 6 (~18ft) meters wide and 10 (~30ft) meters long and you assume that it is poorly ventilated (i.e. no gas replacement at all), then you will have 3x6x10 = 180m3 volume of gas, which is 180,000 liters. Air consists of 21% oxygen and is considered deficient if it goes down to 19.5%. OSHA recommends having monitors present in the case you might, in worst case scenario, reach 19.5%. Note: I don't know, but it seems unlikely that you are critically injured at 19.5%. In this hypothetical case, you will have about 37800 liters of oxygen. If you displace some of it with 700 liters of nitrogen (you spilled one liter of liquid nitrogen), you will be down to 37100 liters, or approximately 20.5%. So, no worry. If you have cryogenic storage for crystals (typically hundreds of liters) or one of those large tanks to back-fill your cryo-system, the story changes a lot. Large dewars or large tanks for filling do not normally fail, but when they do, you will be at risk. Humans cannot sense the lack of oxygen, you just feel sleepy and keel over. So in small rooms with large amounts of liquid nitrogen, it makes sense to have a monitor (and it does not make sense to be scared of the issue when you have a monitor). Educationally: For each safety risk in your environment you are supposed to do a calculation like the one above and consider how likely (or not) it is that this may happen to you and how bad it will be. Likelihood and severity multiply: if it is very unlikely (that a large nitrogen tank will rupture) but the consequence is severe (you die), then you need to think about how you can make sure that it never happens (install sensor). Conclusion: if you only work with a small open dewar, then even in a small room it is highly unlikely to run out of oxygen. It is an excellent idea to ask questions like you did. It should be expected that your institution has experts who can answer such questions, but some (like ours) do not and you have to figure it out yourself. It is a good idea to document your concern, calculation and recommendation. Hope this helps. Mark PS: nirtogen vendors have excellent reference materials about these things. -Original Message- From: Radisky, Evette S., Ph.D., Ph.D. radisky.eve...@mayo.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Fri, Jul 13, 2012 3:19 pm Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal) Several have mentioned harvesting in the cold room to reduce evaporation. I used to do this also as a postdoc, but I worried whether I risked nitrogen gas poisoning from liquid N2 boil-off, since the cold room did not seem very well-ventilated. I’ve also hesitated to recommend it to trainees in my current lab for the same reason. Does anyone have solid information on this? I would like to be convinced that such fears are unfounded … Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett Sent: Thursday, July 12, 2012 2:11 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] cryo for high salt crystal We frequently crystallize one of our proteins and variants of it in 1.6-1.8 M ammonium sulfate solutions. Cryoprotection with 25-30% glycerol or 25-30% glucose does not cause precipitation of salts. Both KCl (4.6 M) and ammonium sulfate (5.6 M) have enormous solubilities in water, so I would not expect cryoprotectant concentrations of glycerol or glucose to cause precipitation (We can save cryoprotectant solutions of at least 2 M ammonium sulfate indefinitely). How are you introducing cryprotectant? We use one of two methods: Fish the crystal out of the mother liquor and place into artificial mother liquor with the same composition as the well solution + cryoprotectant. For glycerol or other liquids, you have to make this from scratch. For glucose, we just weigh out 300 mg of glucose in a microcentrifuge tube and make to the 1.0 mL mark with well solution. (Mix well of course before use. Gentle heating in a block or sonication will help dissolve the glucose. Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to the drop the crystals are in. You can do this all at once, or in stages, keeping the drop hydrated by placing the hanging drop back in the well between additions. If your drops are drying out during crystal harvesting (very possible in dry conditions), you might try harvesting in the cold room, where evaporation is slower. We often have problems with crystal cracking and drop-drying in the winter months when the humidity is very low indoors.
Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)
FYI, I live at 5500 ft elevation and the oxygen content of air is 21.5% here. The TOTAL amount of oxygen is less where I am than where you are because there is less air (lower density). Therefore my body has to do more work to get the same amount of oxygen to my cells. OSHA has nothing to do with how much air we have and the sensors you can buy will tell you that the percentage is 21.5, even at 5500 feet. No, nitrogen is not toxic. The question is if you can displace enough oxygen so you cannot absorb enough of it anymore. Unlike the experience you may have had when you hold year breath, you can breathe fine, there is lots of gas around you. Just not the right kind. We are not on the cusp of suffocating. On the other hand, paradoxically, oxygen is toxic. When you get too much of it, you will damage your CNS (not the program) and your eyes. When premature babies are given oxygen so they can survive, their eyes may get damaged. Too far removed from CCP4. This cannot happen in the cold room while harvesting crystals. Mark -Original Message- From: Jacob Keller j-kell...@fsm.northwestern.edu To: mjvdwoerd mjvdwo...@netscape.net Cc: CCP4BB CCP4BB@jiscmail.ac.uk Sent: Fri, Jul 13, 2012 5:10 pm Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal) The expansion ratio of liquid to gaseous nitrogen is approximately 1:700, that is, 1 liter of liquid becomes 700 liters of gas (at room temperature). When you are in a room that is 3 (~10ft) meters tall, 6 (~18ft) meters wide and 10 (~30ft) meters long and you assume that it is poorly ventilated (i.e. no gas replacement at all), then you will have 3x6x10 = 180m3 volume of gas, which is 180,000 liters. Air consists of 21% oxygen and is considered deficient if it goes down to 19.5%. OSHA recommends having monitors present in the case you might, in worst case scenario, reach 19.5%. Note: I don't know, but it seems unlikely that you are critically injured at 19.5% How can this OSHA number be right? At fairly high altitude, say 2500 m, the partial pressure of O2 will be about 75% of that at sea level, and most are okay with it--so how can a drop from 21% to 19.5% have any importance? Is N2 competing with O2, perhaps? Never heard of that. Can N2 really be a poison, such that we are constantly poised at the cusp of suffocation? JPK In this hypothetical case, you will have about 37800 liters of oxygen. If you displace some of it with 700 liters of nitrogen (you spilled one liter of liquid nitrogen), you will be down to 37100 liters, or approximately 20.5%. So, no worry. If you have cryogenic storage for crystals (typically hundreds of liters) or one of those large tanks to back-fill your cryo-system, the story changes a lot. Large dewars or large tanks for filling do not normally fail, but when they do, you will be at risk. Humans cannot sense the lack of oxygen, you just feel sleepy and keel over. So in small rooms with large amounts of liquid nitrogen, it makes sense to have a monitor (and it does not make sense to be scared of the issue when you have a monitor). Educationally: For each safety risk in your environment you are supposed to do a calculation like the one above and consider how likely (or not) it is that this may happen to you and how bad it will be. Likelihood and severity multiply: if it is very unlikely (that a large nitrogen tank will rupture) but the consequence is severe (you die), then you need to think about how you can make sure that it never happens (install sensor). Conclusion: if you only work with a small open dewar, then even in a small room it is highly unlikely to run out of oxygen. It is an excellent idea to ask questions like you did. It should be expected that your institution has experts who can answer such questions, but some (like ours) do not and you have to figure it out yourself. It is a good idea to document your concern, calculation and recommendation. Hope this helps. Mark PS: nirtogen vendors have excellent reference materials about these things. -Original Message- From: Radisky, Evette S., Ph.D., Ph.D. radisky.eve...@mayo.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Fri, Jul 13, 2012 3:19 pm Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal) Several have mentioned harvesting in the cold room to reduce evaporation. I used to do this also as a postdoc, but I worried whether I risked nitrogen gas poisoning from liquid N2 boil-off, since the cold room did not seem very well-ventilated. I’ve also hesitated to recommend it to trainees in my current lab for the same reason. Does anyone have solid information on this? I would like to be convinced that such fears are unfounded … Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 From
Re: [ccp4bb] getting larger protein crystals
Rex,
\begin{shameless self-advertizing}
Once upon a time we did a systematic study how to optimize crystal size. The
study was done with knowledge of the protein solubility (which is not normally
available, of course) and we showed that you can still do it when you do not
have this information.
Perhaps worth reading. The paper does not end with a shocking conclusion, but
more or less with if you do a systematic study, you can improve crystal
volume.
http://www.springerlink.com/content/16133650m43x013g/?MUD=MP
\end{shamelss self-advertizing}
Mark
-Original Message-
From: REX PALMER rex.pal...@btinternet.com
To: CCP4BB CCP4BB@JISCMAIL.AC.UK
Sent: Wed, Jul 4, 2012 3:55 am
Subject: [ccp4bb] getting larger protein crystals
Does anyone have any new tips/methods for improving crystal size once the
initial conditions have been established?
Rex Palmer
http://www.bbk.ac.uk/biology/our-staff/emeritus-staff
http://rexpalmer2010.homestead.com
Re: [ccp4bb] Off topic about application of detergent on non-membrane protein crystallization
Hi Donghui, Yes. Crystals for restriction enzyme Bsob I were ugly and did not diffract well without detergent and were beautiful with good diffraction in presence of OBG. You can find it in the publication. We did not find any detergent molecules in the structure. Now, when to try such things... I am not sure I have a good answer. This protein was perfectly soluble in regular buffer without detergent. Detergent is just one of the things to try to improve crystals and it makes sense to try the cheapest detergent first. Hope this helps a little. Mark -Original Message- From: wu donghui wdh0...@gmail.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Fri, Jun 15, 2012 9:03 am Subject: [ccp4bb] Off topic about application of detergent on non-membrane protein crystallization Dear all, I wonder if anyone has successful experience on using detergent to crystallize non-membrane protein. Based on what criteria to choose detergent to help solubilize and crystallize your non-membrane protein. Thanks for any input or comments. Best regards, Donghui
Re: [ccp4bb] Are these xtal conditions worth optimizing?
Hi Christine, I would try to optimize both conditions, provided that you do not have contradicting information (like a diffraction pattern that shows a small lattice - salt). Have you tried an additive screen? Have you tried adding a detergent? My experience is mixed with things of that nature - sometimes you can get better looking crystals, sometimes not. And you should always keep in mind that looks and content (and subsequently diffraction quality) are two different matters. Once I was taught a rule that in the case of protein-DNA complexes, the quality of diffraction is inversely proportional to the prettiness of the crystals and then I proved that rule wrong with nice-looking crystals that did diffract well. But it is true that crystals do not need to be pretty to be useful. So by all means, please do test diffraction. Good luck. Mark -Original Message- From: Harman, Christine christine.har...@fda.hhs.gov To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Wed, Jun 6, 2012 3:53 pm Subject: [ccp4bb] Are these xtal conditions worth optimizing? Hi All, I have these very weird drops that I found from screening (please find pictures attached). I am not sure if they are worth optimizing. I am very interesting to know your opinions of what you think of thesedrops could be (are these what you call spherulites?). If you think these conditions are worth optimizing, I welcome any ideas on how to optimize. I have done some optimization and still get the same result which is many of these weird things growing throughoutthe drop and with no sharp edges, and sometimes a skin forms after 2weeks (with the sodium malonate conditions only). I have also opened the drop and poked around to see if it is phase separation and this things are definitely solid and slightly-very mushy. I haven't had a chance to check for diffraction, but will be very soon. Both of these drops contain the same protein preparation of a Fab/peptide complex @ ~5mg/mL in buffer containing 0.1M Sodium Acetate pH 5, 150mM NaCl. I appreciate any advice, thoughtsor comments that you could provide. Peace, Christine
Re: [ccp4bb] color for metal ions
There is a really nice web site that shows how colors are perceived by various color-blind readers. One of the journals I recently published in recommends it for consideration. If you are interested, have a look. The web site is made especially for people who publish scientific articles with color illustrations. It is not hard to be considerate. http://jfly.iam.u-tokyo.ac.jp/html/color_blind/ Mark -Original Message- From: Artem Evdokimov artem.evdoki...@gmail.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Sun, May 20, 2012 2:54 pm Subject: Re: [ccp4bb] color for metal ions As long as you are considerate of the needs of colorblind people, I would vote that anything goes. Artem On May 20, 2012 3:17 PM, sujata halder halder.suj...@gmail.com wrote: Hi all, I was wondering if there is a rule for coloring metal ions a specific color. I am using pymol and was not sure if I have to use a specific color for calcium or magnesium ions for publication figures. Thanks, Sujata
Re: [ccp4bb] Suggestions for solving a structure with 8-10 copies per asymmetric unit
Provided that you guess the number of copies and your guess is reasonably close, my experience is that Phaser will do the job. But you have to tell it how many copies you expect, or it will never make sense of the data. When I did my structure with 6(?) copies some years ago, I guessed a number that was close enough and then when I inspected the electron density I could see that there were more copies than I had told the software and all was fine after that. It was surprising to see that good solutions were obvious from a packing consideration, while inadequate solutions were obviously wrong. Mark -Original Message- From: Ke, Jiyuan jiyuan...@vai.org To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Mon, Apr 30, 2012 2:28 pm Subject: [ccp4bb] Suggestions for solving a structure with 8-10 copies per asymmetric unit Dear All, I have a question regarding solving a crystal structure by molecular replacement. It is a single protein with a molecular weight of 25.5 kDa. The cell dimension is rather big from the diffraction data ( 90.9 Å, 143.9 Å, 216.3Å, 90°, 90°, 90°). The possible space group is P212121. With such a big unit cell, we predicted that there are 8-10 molecules per asymmetric unit. We have a decent model with sequence similarity of 49%. I tried several times with Phaser search with the current model and had difficulty to find any clear solution. Has anyone seen such cases and any suggestions to solve the structure? Thanks! Jiyuan Ke, Ph.D. Research Scientist Van Andel Research Institute 333 Bostwick Ave NE Grand Rapids, MI 49503
Re: [ccp4bb] Crystal behave funny
Hi Prem, In addition to other remarks made: - You could dissolve one or more crystals in water and have mass spec done to verify that your crystals are a complex. It takes many crystals (20-30) to make sure on an SDS-PAGE. You will probably need to silver stain the gel to enhance the sensitivity. And optimize for visualizing DNA (of course protein and DNA would each be control lanes). - Apart from optimizing your DNA length and overhang as suggested, you could also try to see what a detergent does for you. My experience is that they can dramatically improve the crystal quality for protein-DNA complexes. But you first need to know if the crystal consists of both protein and DNA. I am optimistic about the probability. Mark (who apparently is also a dinosaur because he practices room temperature crystallography) -Original Message- From: Prem kumar pk_ai...@yahoo.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Fri, Apr 13, 2012 5:10 am Subject: [ccp4bb] Crystal behave funny Hi all, I got some Protein + DNA complex crystals (image attached) recently. They are needle shape some times splitted chromosome type crystals. When we pick long needles they bend so much than normal crystal but they dont break. The small needle dissolve very fast as try to open the drop's film. we try to diffract the long needle crystals and they diffract up to 20 A resolution. Any suggestion how to improve those crystal packing. Thanks in advance! -Prem
Re: [ccp4bb] MAD
With the starting remark that Wayne is larger than life in my mind, we could call SAD the Teeter Method? I think it has a very nice ring to it and perhaps Wayne would approve. I learned something new today. Until now I thought that of course it is called dispersion. That is because in the late 1980s I started studying MAD and used it as topic for my PhD qualifier (which was not allowed to be the same topic as one's dissertation). So I read every paper I could get my hands on (this was before internet and electronic access to journals, yes it once was that way, hard to believe these days). I worried a lot at the time about how it works exactly, not what it is (was) called. It is probably the case that crystallography itself isn't intuitive for someone who has never done it and to add MAD (or SAD) to it... I shall try to practice diffraction from now on. It seems scientifically preferable. Mark -Original Message- From: Lawrence Shapiro l...@columbia.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Thu, Jan 19, 2012 2:48 pm Subject: Re: [ccp4bb] MAD I never weigh in, so I don't know if I'll get in trouble here... How would we distinguish MAD (to now be called The Hendrickson Method) from SAD (The Hendrickson Method - remeber crambin? Nature, 1981)? On Thu, Jan 19, 2012 at 3:59 PM, Anastassis Perrakis a.perra...@nki.nl wrote: A, yes, inventor's names. Anyone reading who is less than 40 and knows what MTZ stands for? ;-) My favorite technique remains SADDAM - a side product of Gerard's War On Error, that never did catch-up with the masses - experimentally or as an acronym. A. On 19 Jan 2012, at 21:51, Petr Leiman wrote: It would be so much more convenient to call these techniques (MAD, SAD, etc.) by their inventor's name. This would simplify things immensely simultaneously eliminating CCP4BB MADisagreements. Although in our days of copyrights wars, the journals and perhaps conferences where these methods were presented for the first time would insist on using their names as part of the method's name... Petr On Jan 19, 2012, at 7:42 PM, Ethan Merritt wrote: On Thursday, 19 January 2012, Ian Tickle wrote: So what does this have to do with the MAD acronym? I think it stemmed from a visit by Wayne Hendrickson to Birkbeck in London some time around 1990: he was invited by Tom Blundell to give a lecture on his MAD experiments. At that time Wayne called it multi-wavelength anomalous dispersion. Tom pointed out that this was really a misnomer for the reasons I've elucidated above. Wayne liked the MAD acronym and wanted to keep it so he needed a replacement term starting with D and diffraction was the obvious choice, and if you look at the literature from then on Wayne at least consistently called it multi-wavelength anomalous diffraction. Ian: The change-over from dispersion to diffraction in MAD protein crystallography happened a couple of years earlier, at least with regard to work being done at SSRL. I think the last paper using the term dispersion was the 1988 Lamprey hemoglobin paper. The next two papers, one a collaboration with Wayne's group and the other a collaboration with Hans Freeman's group, used the term diffraction. WA Hendrickson, JL Smith, RP Phizackerley, EA Merritt. Crystallographic structure-analysis of lamprey hemoglobin from anomalous dispersion of synchrotron radiation. PROTEINS-STRUCTURE FUNCTION AND GENETICS, 4(2):77–88, 1988. JM Guss, EA Merritt, RP Phizackerley, B Hedman, M Murata, KO Hodgson, HC Freeman. Phase determination by multiple-wavelength X-ray-diffraction - crystal-structure of a basic blue copper protein from cucumbers. SCIENCE, 241(4867):806–811, AUG 12 1988. WA Hendrickson, A Pahler, JL Smith, Y Satow, EA Merritt, RP Phizackerley. Crystal structure of core streptavidin determined from multiwavelength anomalous diffraction of synchrotron radiation. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 86(7):2190–2194, APR 1989. On the other hand, David and Lilo Templeton continued to use the term anomalous dispersion for at least another decade, describing their diffraction experiments exploring polarization effects and other characteristics of near-edge X-ray scattering by elements all over the periodic table. Ethan Cheers -- Ian On 18 January 2012 18:23, Phil Jeffrey pjeff...@princeton.edu wrote: Can I be dogmatic about this ? Multiwavelength anomalous diffraction from Hendrickson (1991) Science Vol. 254 no. 5028 pp. 51-58 Multiwavelength anomalous diffraction (MAD) from the CCP4 proceedings http://www.ccp4.ac.uk/courses/proceedings/1997/j_smith/main.html Multi-wavelength anomalous-diffraction (MAD) from Terwilliger Acta Cryst. (1994). D50, 11-16 etc. I don't see where the problem lies: a SAD experiment is a single wavelength experiment where you are using the anomalous/dispersive signals for phasing
Re: [ccp4bb] linux upgrade preferences for CCP4
Paul, Wait a while, and then CENTOS 6 (or not wait a while). In my opinion neither of your choices are as stable as CENTOS. The big drawback is that CENTOS does not have the latest gadgets - but gadgets and stability are mutually exclusive, by definition. I have lately been annoyed because I obtained a program that needs C-libraries that are not available in CENTOS5, so I can appreciate your thinking, but I am not planning on moving away from CENTOS. It is completely maintenance-free nothing ever crashes and all standard programs work on it. Uh-oh, I think I just heard a breaking-sound in the computer room. :-) Mark -Original Message- From: Paul Kraft haresea...@yahoo.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Wed, Dec 21, 2011 1:29 pm Subject: [ccp4bb] linux upgrade preferences for CCP4 hello, I'm considering upgrading my linux software from CENTOS5 to perhaps Fedora or UBUNTO. Does anyone have an opinion about the best linux version to upgrade to for not only CCP4 but also for general robustness and for the best standard apps..Thanks Paul Dr. Paul Kraft Structural Biologist cell 586-596-2770 email: haresea...@yahoo.com email: kraft_proteome_resea...@yahoo.com This communication and any attachments contain information which is confidential and may also be privileged. It is for the exclusive use of the intended recipient(s). If you are not the intended recipient(s) please note that any form of disclosure, distribution, copying or use of this communication or the information in it or in any attachments is strictly prohibited and may be unlawful. If you have received this communication in error, please notify the sender and delete the email and destroy any copies of it. E-mail communications cannot be guaranteed to be secure or error free, as information could be intercepted, corrupted, amended, lost, destroyed, arrive late or incomplete, or contain viruses. We do not accept liability for any such matters or their consequences. Anyone who communicates with us by e-mail is taken to accept the risks in doing so.
Re: [ccp4bb] crystallization of synthetic peptides
Hans, Most natural toxins from snakes, scorpions etc are 50+/-some peptides. And quite a few of those have been studied and crystallized (see pdb for a list). Having worked on one of these structures as a graduate student, I can share my experience: - Purification is harder than you would think. You are talking about 10kD, usually around 5kD. Many methods (size exclusion, even concentration over a simple membrane) don't work as easily as you would like. - I did not have much of a problem crystallizing (i.e. no worse than other proteins, maybe even a little easier) - Crystals tend to diffract well (maybe better than average) - Structures can be hard to solve; MIR is very difficult because ions tend to not go into such crystals easily (because the molecules are small and tightly packed?); MR is hard because (again) it does not work very well on very small systems - Crystallization is not necessarily purification - if you have a mixture of peptides to start with, it may be harder to crystallize, or not: you might get a crystal that is a (random-ish) mixture. - If you have more than two cysteines in your sequence (natural toxins typically do), the additional problem is to get the correct folding and disulphide bridges; alternatively it is very hard to discriminate between correctly and incorrectly linked disulphides Finally: These sequence should be small enough for NMR. That may or may not answer your questions, but it avoids your original question. Mark -Original Message- From: H. Raaijmakers hraaijmak...@xs4all.nl To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Thu, Nov 10, 2011 8:16 am Subject: [ccp4bb] crystallization of synthetic peptides Dear crystallographers, Because of the low cost and speed of synthesizing 40- to 60-mer peptides, I wonder whether anyone has (good or bad) experiences crystalizing such peptides. In literature, I've found up to 34-mer synthetic coiled coils, but no other protein class. I can imagine that a protein sample with a few percent random deletion mutants mixed into it won't crystallize easily, but has anyone actually tried? cheers, Hans
Re: [ccp4bb] image compression
Hmmm, so you would, when collecting large data images, say 4 images, 100MB in size, per second, in the middle of the night, from home, reject seeing compressed images on your data collection software, while the real thing is lingering behind somewhere, to be downloaded and stored later? As opposed to not seeing the images (because your home internet access cannot keep up) and only inspecting 1 in a 100 images to see progress? I think there are instances where compressed (lossy or not) images will be invaluable. I know the above situation was not the context, but (y'all may gasp about this) I still have some friends (in the US) who live so far out in the wilderness that only dial-up internet is available. That while synchrotrons and the detectors used get better all the time, which means more MB/s produced. James has already said (and I agree) that the original images (with all information) should not necessarily be thrown away. Perhaps a better question would be which would you use for what purpose, since I am convinced that compressed images are useful. I would want to process the real thing, unless I have been shown by scientific evidence that the compressed thing works equally well. It seems reasonable to assume that such evidence can be acquired and/or that we can be shown by evidence what we gain and lose by lossy-compressed images. Key might be to be able to choose the best thing for your particular application/case/location etc. So yes, James, of course this is useful and not a waste of time. Mark -Original Message- From: Miguel Ortiz Lombardia miguel.ortiz-lombar...@afmb.univ-mrs.fr To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Tue, Nov 8, 2011 12:29 pm Subject: Re: [ccp4bb] image compression Le 08/11/2011 19:19, James Holton a écrit : At the risk of putting this thread back on-topic, my original question was not should I just lossfully compress my images and throw away the originals. My question was: would you download the compressed images first? So far, noone has really answered it. I think it is obvious that of course we would RATHER have the original data, but if access to the original data is slow (by a factor of 30 at best) then can the mp3 version of diffraction data play a useful role in YOUR work? Taking Graeme's request from a different thread as an example, he would like to see stuff in P21 with a 90 degree beta angle. There are currently ~609 examples of this in the PDB. So, I ask again: which one would you download first?. 1aip? (It is first alphabetically). Then again, if you just email the corresponding authors of all 609 papers, the response rate alone might whittle the number of datasets to deal with down to less than 10. Perhaps even less than 1. -James Holton MAD Scientist Hmm, I thought I had been clear. I will try to be more direct: Given the option, I would *only* download the original, non-lossy-compressed data. At the expense of time, yes. I don't think Graeme's example is very representative of our work, sorry. As long as the option between the two is warranted, I don't care. I just don't see the point for the very same reasons Kay has very clearly exposed. Best regards, -- Miguel Architecture et Fonction des Macromolécules Biologiques (UMR6098) CNRS, Universités d'Aix-Marseille I II Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France Tel: +33(0) 491 82 55 93 Fax: +33(0) 491 26 67 20 mailto:miguel.ortiz-lombar...@afmb.univ-mrs.fr http://www.afmb.univ-mrs.fr/Miguel-Ortiz-Lombardia
Re: [ccp4bb] Archiving Images for PDB Depositions
Reluctantly I am going to add my 2 cents to the discussion, with various aspects in one e-mail. - It is easy to overlook that our business is to answer biological/biochemical questions. This is what you (generally) get grants for to do (showing that these questions are of critical importance in your ability to do science). Crystallography is one tool that we use to acquire evidence to answer questions. The time that you could get a Nobel prize for doing a structure or a PhD for doing a structure is gone. Even writing a publication with just a structure is now not as common anymore as it used to be. So the biochemistry drives crystallography. It is not reasonable to say that once you have collected data and you don't publish the data for 5 years, you are no longer interested. What that generally means is that the rest of science is not cooperating. In short: I would be against a strict rule for mandatory deposition of raw data, even after a long time. An example: I have data sets here with low resolution data (~10A) presumably of protein structures that have known structures for prokaryotes, but not for eukaryotes and it would be exciting if we could prove (or disprove) that they look the same. The problem, apart from resolution, is that the spots are so few and fuzzy that I cannot index the images. The main reason why I save the images is that if/when someone comes to me to say that they think they have made better crystals, we have something to compare. (Thanks to Gerard B. for encouragement to write this item :-) - For those that think that we have come to the end of development in crystallography, James Holton (thank you) has described nicely why we should not think this. We are all happy if our model generates an R-factor of 20%. Even small molecule crystallographers would wave that away in an instant as inadequate. However, everybody has come to accept that this is fine for protein crystallography. It would be better if our models were more consistent with the experimental data. How could we make such models without access to lots of data? As a student I was always taught (when asking why 20% is actually good) that we don't (for example) model solvent. Why not? It is not easy. If we did, would the 20% go down to 3%? I am guessing not, there are other errors that come into play. - Gerard K. has eloquently spoken about cost and effort. Since I maintain a small (local) archive of images, I can affirm his words: a large-capacity disk is inexpensive ($100). A box that the disk sits in is inexpensive ($1000). A second box that sits in a different building, away for security reasons) that holds the backup, is inexpensive ($1400, with 4 disks). The infrastructure to run these boxes (power, fiber optics, boxes in between) is slightly more expensive. What is *really* expensive is people maintaining everything. It was a huge surprise to me (and my boss) how much time and effort it takes to annotate all data sets, rename them appropriately and file them away in a logical place so that anyone (who understands the scheme) can find them back. Therefore (!) the reason why this should be centralized is that the cost per data set stored goes down - it is more efficient. One person can process several (many, if largely automated) data sets per day. It is also of interest that we locally (2-5 people for a project) may not agree on what exactly should be stored. Therefore there is no hope that we can find consensus in the world, but we CAN get a reasonably compromise. But it is tough: I have heard the argument that data for published structures should be kept in case someone wants to see and/or go back, while I have also heard the argument that once published it is signed, sealed and delivered and it can go, while UNpublished data should be preserved because eventually it hopefully will get to publication. Each argument is reasonably sensible, but the conclusions are opposite. (I maintain both classes of data sets.) - Granting agencies in the US generally require that you archive scientific data. What is not yet clear is whether they would be willing to pay for a centralized facility that would do that. After all, it is more exciting to NIH to give money for the study of a disease than it is to store data. But if the argument were made that each grant(ee) would be more efficient and could apply more money towards the actual problem, this might convince them. For that we would need a reasonable consensus what we want and why. More power to John. H and The Committee. Thanks to complete silence on the BB today I am finally caught up reading! Mark van der Woerd -Original Message- From: James Holton jmhol...@lbl.gov To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Tue, Nov 1, 2011 11:07 am Subject: Re: [ccp4bb] Archiving Images for PDB Depositions On general scientific principles the reasons for archiving raw data all boil down to one thing: there
Re: [ccp4bb] IUCr committees, depositing images
Phoebe, Just automate the archiving and come up with a reasonable scheme how to. Ours is that data sets are called: userid_yearmonth_projectid_# Userid is derived from the login into CrystalClear (oops, free advertizing), projectid is set by the PI (so she can remember 10 years from now what in the world these data are all about) and the users are asked (threatened) to call their data sets projectid_# (and not the ubiquitous test). We have a script that automatically archives everything away from our data collection computer into an archive - activated by an icon on the desktop - and it adds the userid and date to the filename. This has the nice added advantage that the data collection disk stays clean. This only breaks when we collect synchrotron data (which is all the time) because our synchrotron remote scientist who collects the data cannot (should not) be threatened. :-) I then rename all data sets for archiving so the naming is consistent and you can actually make (say in pdf) an index of all the data you have, organized by user, date, or project. Our policy is that the PI decides if data should be maintained or if it really can go (no diffraction, really a test crystal to see that the crystal is in the beam etc). In practice this doesn't happen so someone else makes the decision. We tend to err on the side of caution. We tend to think that all results should be saved, unless it is blatantly obvious that there is no point. Storage is cheap (and cheaper every time you think of it). After you automate in the previously agreed upon scheme, it is somewhat easier to find things back because if you can remember who collected it, or approximately when it was done, or what the project was, you can find it. The pain was up front: to come up with a scheme, to enable a rigorous naming convention and to implement it (data collection computer and archive are not physically on the same computer etc). Maybe the Committee is also thinking about that issue - how are you going to keep all the data manageable and searchable. Presumably by something like a PDB id (this seems to make sense for published/deposited structures) but for things that did not make it to PDB one would have to come up with another plan. Mark -Original Message- From: Phoebe Rice pr...@uchicago.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Tue, Oct 18, 2011 12:01 pm Subject: Re: [ccp4bb] IUCr committees, depositing images One more consideration: Since organization is not one of my greatest talents, I would be absolutely delighted if a databank took over the burden of archiving my raw data for me. Phoebe = Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Original message Date: Tue, 18 Oct 2011 18:17:14 +0100 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Gerard Bricogne g...@globalphasing.com) Subject: Re: [ccp4bb] IUCr committees, depositing images To: CCP4BB@JISCMAIL.AC.UK Dear Enrico, Frank and colleagues, I am glad to have suggested that everyone's views on this issue should be aired out on this BB rather than sent off-list to an IUCr committee member: this is much more interactive and thought-provoking. There would seem to be clear biases in some of the positions - for instance, the statement that we overvalue individual structures and that there is value only in their ensemble has to be seen to be coming from someone in a structural genomics centre ;-) . However, as Wladek pointed out, when an investigator's project is crucially dependent on a result embodied in a deposited structure, it would be of the greatest value to that investigator to be able to double-check how reliable some features of that structure (especially its ligands) actually are. On the other hand Enrico, as a specialist of crystallisation and modelling, sees value only in improving those contributors to the task of structure determination. This is forgetting (1) an essential capability of crystallography: that, through experimental phasing, it can show you what a protein looks like even if you have never seen nor modelled one before, through the wondrous process of producing model-free electron-density maps; and (2) an essential aspect of the task of structure determination: that it doesn't aim at producing a model with perfect geometry, but one that best explains the measured data and neither under- nor over-interprets them (I realise, though, that Enrico's statement Data just introduces experimental errors into what would otherwise be a perfect structure is likely to be tongue-in-cheek ...). When it comes to making explicit the advantages of archiving at least the raw images that yielded the data against which a
Re: [ccp4bb] Neutron data collection
Rex, There are people more qualified to answer your question 1 than I am, so I am going to politely defer that answer. The answer depends on the unit cell dimensions, detector distance etc, and yes, there are more observations rejected due to overlap than would be the case in monochromatic data collection. As for 2, you should not freeze your crystals but mount them the old-fashioned way in capillaries. In practice neutron diffraction does not cause radiation damage to your crystals so you should not freeze and collect data as much as your time allotment allows for. Hope this helps. Mark van der Woerd -Original Message- From: REX PALMER rex.pal...@btinternet.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Wed, Sep 21, 2011 3:52 am Subject: [ccp4bb] Neutron data collection Re Neutron Data Collection: 1. What are the limits to data set completeness imposed by a Laue experiment versus those of monochromatic data collection? 2. What problems are caused by flash freezing the larger protein crystals used for neutron data collection which do not occur for X-ray data collection ie because smaller crystals can be used. Any help will be greatly appreciated. Rex Palmer http://www.bbk.ac.uk/biology/our-staff/emeritus-staff http://rexpalmer2010.homestead.com
Re: [ccp4bb] Aging PEGs
PEG is a polymer and it can be made by anionic or cationic polymerization. Whichever you use, you go the other way to terminate the reaction at an appropriate time (so you have the molecular weight you want). So when you start with an acid, you terminate with a base and vice versa. If you terminate with a base, your final pH is going to be high (presumably 7) and if you terminate with an acid, the pH is going to low. It is therefore important to keep track of your lot number (because as long as the lot number is the same, the treatment was the same). For crystallization recipes that do not involve buffers (there are some!) this is essential, because PEG and PEG are not the same thing (and you should always pH the solution before you use it, so you have a reference point - remember we do not know what we have in our crystallization drops, but we do know what we put into them to make them). Even if the PEG was made by the same process, the manufacturer is concerned with stopping the polymerization at the right time, but not how hard they stop it. In other words, the solution might be pH 8 or pH 11 when it is done. So when you say that you measured different PEGs and found the pH to be different, that might be accounted for by the way the PEG was made and they may always have been different, irrespective of age. It is probably not known if acid PEG vs basic PEG ages at a different speed and with a different mechanism. As you know, the first thing to observe is whether PEG solutions are clear (water) or slightly colored (dilute lemonade :-) because this is a sign of aging. Remember that PEGMME is much more sensitive to aging than PEG itself (you should store MME solutions in the dark). And if the PEG is stored in dry form, it is not very easy to age it by chemical reaction, because nothing is swimming, but not easy is not the same as impossible. My 2 cents worth. Mark -Original Message- From: Jacob Keller j-kell...@fsm.northwestern.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Wed, Aug 24, 2011 1:18 pm Subject: [ccp4bb] Aging PEGs A while ago I measured the pH's of old and new PEGs and found them very different, and internally attributed all old vs new PEG issues to pH. Upon reflection, this seems too simplistic. Are there other known mechanisms of crystallization capacities of PEGs of various ages? Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] small lysozyme crystals?
James, I would have a look at the paper by Judge et al: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1300446/pdf/10465769.pdf Specifically, in this paper you will find that the crystallization behavior of lysozyme changes drastically with pH. At the time the paper wasn't really written to manipulate for small crystal size, but looking back at the paper (specifically Fig 5), it appears that you can read the conditions that will give you crystals around the size you want. Not re-reading the paper, quoting from memory (which we all think is better than it really is), it is important to use good quality lysozyme to get reproducible results. Good quality probably means freshly purified from fresh (farm-acquired) eggs. I am not kidding you, it makes a big difference. Also, I am going out on a limb to say (I know you know this) that the buffer preparation method matters a lot. Taking sodium acetate solution and pH-ing it with HCl will give very different results from taking acetic acid and pH-ing it with NaOH (because the ionic strength of the buffer is not the same). Lysozyme crystallizes so easily that we tend to forget tedious details. Hope this helps. This paper will probably give you some ideas in the right direction. Mark van der Woerd -Original Message- From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Tue, Jul 26, 2011 11:55 am Subject: [ccp4bb] small lysozyme crystals? Does anyone out there have a protocol of growing HEWL crystals that are all 50-100 microns wide? I gave this project to a summer student recently, thinking it would be easy, but it is turning out to be more difficult than I thought. Keep getting sphereulites instead of small crystals. Yes, I know you can smash a large lysozyme crystal with a hammer, but that is not exactly what I was going for. What I was hoping for was a well-defined protocol for growing reference crystals that stay evenly illuminated in our x-ray beams as they rotate. The beam is 100 um wide. I'm sure someone has done this before? -James Holton MAD Scientist
[ccp4bb] Question about movie making
All, Pardon the slightly off-topic question. We would like to use Pymol and generate movies with it on a WINDOWS computer. We are very familiar with Pymol and how to make the correct views etc. We write the individual frames out into PNG files. So what is left to do, is to stitch together the PNG images to an MPEG file. On Linux you could do this with mencoder. But we would like to do this on Windows and installing mencoder on windows is possible but not easy. We have found videomach, which costs a very small amount of money to obtain. Similarly, Adobe Premiere is affordable for an educational institution. We don't mind paying, but before we go there, does anyone have experience with making MPEG movies from PNG files on windows? What is your experience with quality of product and especially with user friendliness? If you have any insight, we would appreciate your comments. Thanks! Mark van der Woerd Colorado State University
Re: [ccp4bb] linux flavors
Dave, We have used CentOS for years and I am very happy with it. We also use NVIDIA hardware. CentOS does not work out of the box with NVIDIA, but NVIDIA has an installation package for their drivers on their web site that does work out of the box in combination with CentOS. That is, you run it once and the proper drivers get incorporated in the kernel and it works great. You only have to apply this special step when you upgrade your kernel. As far as stereo goes, we use the old-fashioned emitter-based hardware stereo and this works as long as you do as you are supposed to do (properly configure the X-window system). The thing I have most enjoyed is that CentOS is very, very reliable and stable. Hope this helps. Mark van der Woerd -Original Message- From: David Roberts drobe...@depauw.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tue, Feb 22, 2011 8:16 am Subject: [ccp4bb] linux flavors Hello all, Quick question on linux varieties. For years (and years) I have used fedora (after Ultrix of course). In fact, most of my computers are running FC7 (that long ago), it's very stable and works fine. However, since it is no longer supported, I'm toying with upgrading. I upgraded one machine to FC13. However, this nouveau driver thing is killing me, and getting my nvidia drivers installed is hopeless (I have followed every thread on this and I simply give up - it's not worth it). With a Zalman monitor it doesn't matter - nouveau works fine and my stereo is good - so I don't really care (or do I). The question is this - what flavors of linux out there are simplest to install - work instantly with various hardwares, and run stereo seamlessly (either Zalman stereo or hardware stereo with an emitter). For zalman anything works - which is why I'm going that way - but I still need hardware stereo on a few machines. So, for hardware, I need my nvidia drivers to install easily. I'm downloading ubuntu - is that a good choice? Can I run different flavors of linux with nfs and share drives in a local network (so one has fc7, one has fc13, and another has ubuntu)? Thanks Dave
Re: [ccp4bb] Micro-g Crystal Growth and the literature
Methods for vapor diffusion in microgravity have long existed (and the 'trick' is that you cannot have 'free liquid', as we do in standard vapor diffusion plates, because it does not 'stay'). Having worked at NASA, I have said the same thing: vacuum and cooling should be easy - but alas, they are not. It is not permissible to have 'connections' with the vacuum outside (unless they are very stringently controlled and triply guaranteed) and cooling in general is a huge problem (vacuum is an excellent insulator!). Personal crystal mounting could be fun, but you do have to take into account the number of years(!) you would need for appropriate training (and I don't mean in crystal mounting itself), the fact that you could not afford to be near your scientific experiments (training does not take place in Amsterdam), and that you would have to choose to be away from your family for extended amounts of time or alternatively would have to take your family to a training site (taking kids away from school and their friends). There are many reasons why I was never really interested in all that, but astronauts evidently set EVERYTHING aside for this purpose because it is their dream... Yes, robots have been prototyped. And yes, it is incredibly expensive. Putting ANYTHING in orbit is really expensive (and we do it all the time) so all you have to decide is which experiment(s) (if any) are important enough or rather more important than other experiments. Mark -Original Message- From: Anastassis Perrakis a.perra...@nki.nl To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, May 10, 2010 11:25 am Subject: Re: [ccp4bb] Micro-g Crystal Growth and the literature I am assuming that if the diffractometer is on the space station, the X-ray source will be an no stronger than a standard in-house source. Cryoprotection might not even be necessary assuming that I guess there is room for imagination on how to utilize micro-gravity for setting up vapor or liquid diffusion crystallization experiments ... but given the cooling capacity offered by the room next door, combined with a pretty good and cheap vacuum, it could be fun to build a big rotating anode out there ... and you could save money from the cryo-cooling too, also for free. I would love to have a go mounting a crystal or two out there ... but I guess even that can be done by one of the existing robots modified. would still be a waste of money in terms of practical returns I bet ... one could learn things about crystallization maybe ... A. On May 10, 2010, at 17:58, SIPPEL,KATHERINE H wrote: I am assuming that if the diffractometer is on the space station, the X-ray source will be an no stronger than a standard in-house source. Cryoprotection might not even be necessary assuming that =
Re: [ccp4bb] where I have been going wrong in crystallization?
I was going to comment that I have learned the following: respect does not mean the same thing in all places in the world. Some time back I had a protein here that I thought needed extra respect and I had learned from a Rigaku employee how to do this - I bowed very very deeply in front of the protein before handling it. But it still did not crystallize. So when I complained to the Rigaku employee about the recipe, he asked with appropriate hesitation in his voice: are you saying that you only bowed ONCE? In defense of the original poster, I think the recipe on the Rigaku web site is entirely correct, but it does not specify how to pay proper respect. This was self-evident to the person who wrote down the recipe, but as we all know, what it obvious to one person, is not obvious to the next - especially in such difficult things like respect. Good recipes are indispensable and should be explicit about such important ingredients. Mark -Original Message- From: Mark J. van Raaij mark.vanra...@usc.es To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, Dec 21, 2009 12:18 pm Subject: Re: [ccp4bb] where I have been going wrong in crystallization? - I think the original poster was only calling attention to the fact that some proteins want to be treated respectfully in order to crystallise (and the fact that Rigaku Japan realises this). I find that indeed the case. Other proteins, however, prefer the attitude I don't know why I am setting up these drops, this protein is too crappy to crystallise, i.e. a challenge. Lysozyme, on the other hand, even crystallises under conditions of complete indifference. At least I find that every student in a practical course can get nice crystals of lysozyme, and a majority of these drops have been set up under conditions of complete indifference...maybe lysozyme is not a protein after all, but a salt: lysozyme-chloride / LyCl7 ? Mark PS the detailed protocols and experiences are useful though. Quoting Jeffrey Wilson wil...@ucmail.uc.edu: I was recently testing out the 30% PEGMME 5k, 0.1M NaOAc pH 4.5, 1M NaCl method mentioned in a Hampton Research catalog and attributed to Enrico Stura. I see that he has also just commented on this thread. I found that at 80mg/ml, batch with 1.5ul:1.5ul protein to precipitant ratio, lysozyme crystallized in about 1 hour. Jumping that up to 150mg/ml allowed for crystallization in minutes. Hanging drop behaved similarly. I was using lysozyme from Sigma. Jeff Jeffrey Wilson, Ph.D. University of Cincinnati College of Medicine Molecular Genetics Department 231 Albert Sabin Way MSB 3109A Cincinnati, OH 45267-0524 (513) 558-1360 On Dec 21, 2009, at 11:12 AM, MARTYN SYMMONS wrote: Dear All checking out the Lysozyme crystallization methods on the web I liked the Rigaku Instructions that I found: (http://www.rigaku.com/protein/crystallization.html) ...create a drop of 3ul lysozyme solution, and 3 ul of well solution, respectfully, for a total drop size of 6ul... So perhaps sometimes I am just not respectful enough to deserve crystals ? good wishes to all regards, Martyn --- Martyn Symmons MRC-MBU Cambridge UK 'Chan fhiosrach mur feòraich.' Gaelic proverb - Nothing asked, nothing learned.
Re: [ccp4bb] Methylation of macromolecular complexes
There is a reasonable experiment you can do: Take the unit cell of your (presumed) crystal complex and put the search model into the until cell in a way that seems reasonable (use coot, or your favorite program). Make structure factors (with CCP4 or your favorite program) and throw away the phases. Then rotate your MR search model randomly and put the search model and structure factors into the MR program. See if it finds a solution. You can repeat it with structure factor data to which noise has been added. You can use this method to optimize your MR search parameters. Don't be surprised if you cannot find a MR solution at all (even if you know for sure it should exist in your artificial problem). Once upon a time I tried to solve the structure of a small poly-peptide and with the method above I proved to myself that it was not possible to find a solution (with the programs that were available at that time, when Dinosaurs still roamed the Earth). I found that MR is very finicky when applied to small peptides. At least you will be able to determine what the optimal parameters are (resolution, search radius) and whether it can succeed at all. To pursue Se-Met is smart. Small peptide crystals resist heavy atom soaking. If your resolution is high enough, you can also try direct methods. On such a small peptide that should be easy (provided your crystals are well-behaved). Frequently the diffraction resolution of small peptide crystals is high enough that direct methods work very well. Mark -Original Message- From: Sean Seaver s...@p212121.com To: CCP4BB@JISCMAIL.AC.UK Sent: Tue, Nov 24, 2009 5:13 pm Subject: Re: [ccp4bb] Methylation of macromolecular complexes I have a small complex, one component is 13 kDa with structure available and the other is 7 kDa, which could not be able to grew crystals after lots of efforts. It grew crystals after methylation and diffracted to 2.3 A, however, I could not be able to solve the structure using the structure of the big component as a template for molecular replacement, and heavy atoms soaking was not successful. I plan to do selenomethionine expression next. Does the methylation change protein structures a lot? otherwise, why does molecular replacement not work? I would much appreciate any idea and suggestions how to solve the structure using the data and the template avaliable. --- A couple of questions that I would consider in regards to the molecular replacement not working: Is the binding between the proteins 1:1? Does the Matthews coefficient reflect this? MR maybe difficult if you have a number of 7 kDa proteins binding to a 13 kDa protein. Hope that Helps, Sean
Re: [ccp4bb] Using SAXS data for phasing at mediocre resolution.
Hi Francis, There is an older paper that mentions this idea: Tsao et al, Acta Cryst B48 (1992), 75-88. However, when you look at the paper, small-angle scattering data is not the only thing that was used. In particular, if my memory serves me right, the 60-fold averaging applied to the problem really made all the difference in getting the phases right. The big difference that works to your advantage is that small-angle scattering was not nearly as well developed at the time as it is now. For example, I don't think they could get molecular envelopes in 1992. There is sufficient information available these days that the test could be done (for appropriate systems). Mark -Original Message- From: Francis E Reyes francis.re...@colorado.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, Sep 14, 2009 9:47 am Subject: [ccp4bb] Using SAXS data for phasing at mediocre resolution. Hi all? ? I'm looking for anyone who has had (practical) experience using SAXS data to phase 4.2 A crystals. Please email me.? ? FR? ? -? Francis Reyes M.Sc.? 215 UCB? University of Colorado at Boulder? ? gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D? ? 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D?
Re: [ccp4bb] Linux flavour and hard disks
Hi Claudia, There is an option that has not been mentioned yet and has a very obvious advantage: CentOS. I am mentioning this because it is identical in functionality to Red Hat and therefore it will take the least of your time to move to a new system. We like CentOS because it is derived from Red Hat, no surprises, no unexpected bugs. And unlike Red Hat, it is completely free and you don't need to talk to the Red Hat people (which I found to be completely impossible). As far as configuration goes, this is what we do: - Each workstation has a small (100GB or less) drive for the OS. Nothing else. - We have a network-attached disk farm that holds X-ray images on one share, programs on another, and personal data on a third. The advantage is that you need to maintain everything only in one place and it does not matter where you are working - the data and programs are transparently available to all workstations. - We have two storage devices, one primary, one backup. All data get backed up hourly (X-ray images) or daily (user data) or weekly (Programs). We currently have 2TB of space and probably will go to 4TB soon. You do need to make a careful analysis of what your needs are. We determined at the time that network traffic is never a limiting factor. Disk access can be somewhat of a limit, say when you have multiple people processing raw X-ray images at the same time, but that never happens in our group. So in the end we found that our limiting factor was C PU power and this is where we focused our attention to make an efficient system. There are slight advantages to each flavor of Linux, but my personal argument (as care taker) is that it should take as little time as possible to maintain your setup (because maintaining Linux systems is a necessity but not really what we wish to do). It should also be very reliable - it should always work and you want to make a careful plan to deal with disaster recovery - disks fail sooner or later and yes, I have had numerous users who accidentally did 'rm *' when they did not mean to do that... That, of course, works equally well in all Linux flavors. Mark -Original Message- From: Claudia Scotti claudiasco...@hotmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, Aug 24, 2009 7:30 am Subject: [ccp4bb] Linux flavour and hard disks Dear List, I'm planning to migrate soon from Red Hat Linux 7.0 on an HP xw6000 workstation with dual Xeon processor. Please, any suggestion for the best Linux flavour to get the most out of today's crystallographic software? I've seen that both Ubuntu and Fedora are quite common. Also I'm in doubt about the following: will it be safer to use two mirror hard disks (as I'm doing now) or to use one HD for the software and one for the data? And, finally, please, what HD size is today most reasonable (big, but still fast enough)? Thanks a lot, Claudia=0 A Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673 check out the rest of the Windows Live™. More than mail–Windows Live™ goes way beyond your inbox. More than messages=
Re: [ccp4bb] Computer hardware and OS survey
Todd, Once upon a time I studied at an institution of higher learning. Its specialty is (and was) the education of and participation in medical sciences (I guess that?could be?an oxymoron, sorry). With that comes the securely keeping and sharing (as needed) of patient data. The institutional bureaucrats decided that Novell token ring networks?were the?best suited for that purpose?and that, on the other?hand, TCP/IP was inherently insecure, so they were going to do away with TCP/IP networks. Shock was on the face of the workers. All academic and scientific networks need TCP/IP. The same thing was done as Bill says: we had to go in and argue that we didn't work for the computer and network people, but they worked for us. I can't remember if we did this - this was?long?before the time of ssh and sftp- long ago,?but today I would bring up the argument of how much grant money and overhead money (which pays for the computer and network people) scientists bring in and that without the proper tools, these things cannot be perpetuated. It would seem to me that you cannot run crystallography efficiently (!) on one platform alone (no matter which one you choose). Some tasks, like grant writing, are?easily done?on some platforms (windows or Mac, but not Unix/Linux) etc. So the driving force should be what needs to be done and how to best do it. With that should come the realization that making you as a scientist less efficient will translate into less ability to attract funds (because funds are competitive), which does not affect only you, but the entire institution. Things should not be and are not all about money, but that argument always works - hit them in the pocket book and they will reconsider. There are ways of cutting costs without doing away with capabilities. You can have groups of people who use Windows and have support for that. At the same time you can have other groups of people who use Macs with support for that. And you can make a rule that if you want to be different from everyone in your group, you will belong (for computing needs only) to the other group. That is how our University tries to run things. Mark -Original Message- From: William G. Scott wgsc...@chemistry.ucsc.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Fri, 1 May 2009 9:39 am Subject: Re: [ccp4bb] Computer hardware and OS survey Hi Todd:? ? One option on Windows is to install Ubuntu in a mode that lets it run nested as a guest in a window within the host operating system. This is now one of the options on the (free) Ubuntu install CD. I've actually not tried it, so I can't tell you how good it is, but my guess is that it works in a way that is very similar to VMware of Parallels on OS X.? ? But if you already have made the investment in OS X hardware, I really would recommend standing your ground on this. The main arguments to make, I believe, are the following:? ? 1. Scientists really need to have ready access to unix-based operating systems. OS X and Linux are two such variants, but the main arguments in favor of each are the same. I'm flattered you liked my website, but frankly I don't think its existence is a compelling argument. (In fact, I made the thing originally as a publicly accessible log/whine of my trials and tribulations in a do-it-yourself sys admin environment. You could point out that if an idiot like me can do this, anyone can.) You could probably get by with work-around solutions on Windows, but why should you be forced to hobble yourself.? ? 2. Your institutional bureaucrats should not, as a matter of principle, dictate to you what your computer or other equipment needs are. They are supposed to work for you, not vice-versa. As pointed out, you probably only really need their IT support to give you network access. You should be able to work with whatever operating system your needs, tastes and ethics dictate. (The idea that the institution would force you to use an operating system that has been the subject of US Department of Justice litigation and would simultaneously discourage you from using Linux, a Free Software alternative, is particularly troubling).? ? Happy May Day. Time to raise the black flag and start slitting throats.? ? Bill? ? ? On May 1, 2009, at 7:40 AM, Link,Todd M wrote:? ? My home institution, in effort to cut costs, is making an effort to push those of us on Macs onto PCs. Up till now they have been very generous via a lease program for computer hardware, but that is changing given the current economics. The institution currently does not support Linux so we are limited to Mac and Windows OS.? ? We certainly make use of William Scotts crystallography on OS X (thanks so much!) so our main argument is that we would have far more support out there for crystallography on the Mac than we would have for on Windows. But to be fair (and hopefully bolster our argument) I should find out if that is true. I did not find an
Re: [ccp4bb] images
There have been excellent examples given for cases in which the original data would have been very valuable for discussion and understanding. However, it has always been my understanding that scientists are required to keep the original data on which their conclusions are based. It is also my understanding that (in the US) from this logical requirement there is a legal requirement for scientists to keep their original research data on file. This presumably is imposed by the granting agancies, although I have to admit that I have actually never read this rule in writing anywhere. The question is not 'should it be kept', the question is 'how long should it be kept'. It is self-evident that all data are kept for at least 5 years (about the time it takes to get a student to graduate). Should it be 10 years? Should it be 15-20 years? In practice, I think the answer is that when everyone who can remember doing the project has gone (PI retires), then the data are no longer useful because nobody can remember what?they are?for.?I would reluctantly type rm -f * in that case. In addition to this discussion one would have to consider 'should ALL data be preserved'? We all know that it usually?takes more than one diffraction experiment to get a structure. Is it OK to discard the data sets (images) that were not used? My somewhat arbitrary answer is ALL data should be preserved. It is like your lab notebook - do you preserve data on unsuccessful cloning and expression? Yes, you do because you never know what you can learn from this. And also, your unsuccessful ! experiments together with?the successful ones form the record how you?came to an answer/conclusion.?There are recent questions in literature and on this bb that could be answered if we had the next best data set. ? Mark? -Original Message- From: Garib Murshudov ga...@ysbl.york.ac.uk To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 18 Mar 2009 10:41 am Subject: Re: [ccp4bb] images Dear all? ? Before going into and trying to find a technical solution to the problem it would be good if decide if we need images. As far as I know if we face with a problem to solve and we know that it is necessary to solve then we find technical solution to the problem (either from other fields or we find our own solution with some elements of reinvention of new MX wheels).? ? Do we need images to store? What kind of information we can extract from images that we cannot from amplitudes, intensities (even unmerged)? Does anybody have a convincing argument for favour of images?? ? regards? Garib? ? ? On 18 Mar 2009, at 16:32, Herbert J. Bernstein wrote:? ? Actually the radiologists who manage CT and PET scans of brains do have? a solution, called DICOM, see http://medical.nema.org/. If we work? together as a community we should be able to do as well as the? rocket scientists and the brain surgeons' radiologists, perhaps even? better. -- Herbert? ? =? Herbert J. Bernstein, Professor of Computer Science? Dowling College, Kramer Science Center, KSC 121? Idle Hour Blvd, Oakdale, NY, 11769? ? +1-631-244-3035? y...@dowling.edu? =? ? On Wed, 18 Mar 2009, Jacob Keller wrote:? ? Apparently it DOES take a rocket scientist to solve this problem. Maybe the brain surgeons also have a solution?? ? JPK? ? ***? Jacob Pearson Keller? Northwestern University? Medical Scientist Training Program? Dallos Laboratory? F. Searle 1-240? 2240 Campus Drive? Evanston IL 60208? lab: 847.491.2438? cel: 773.608.9185? email: j-kell...@northwestern.edu? ***? ? - Original Message - From: Klaas Decanniere klaas.decanni...@vub.ac.be ? To: CCP4BB@JISCMAIL.AC.UK? Sent: Wednesday, March 18, 2009 5:36 AM? Subject: Re: [ccp4bb] images? ? ? Herbert J. Bernstein wrote:? Other sciences have struggled with this and seem to have found an answer.? Have e.g. a look at http://heasarc.nasa.gov/docs/heasarc/fits.html? kind regards,? Klaas? ? This is a good time to start a major crystallogrpahic image? archiving effort. Money may well be available now that will not be? avialable six month from now, and we have good, if not perfect,? solutions available for many, if not all, of the technical issues? involved. Is it really wise to let this opportunity pass us by?? The deposition of images would be possible providing some consistent? imagecif format was agreed.? This would of course be of great use to developers for certain? pathological cases, but not I suspect much value to the user? community - I down load structure factors all the time for test? purposes but I probably would not bother to go through the data? processing, and unless there were extensive notes associated with? each set of images I suspect it would be hard to reproduce sensible? results.? ?
Re: [ccp4bb] long term data backup
We have a tiered system: a) Personal files. Small and many, change often. Typical: CCP4, coot, CNS and other files. Backed up daily. b) X-ray images. Not so many, but large. Large in total. Never change once established. Backed up every two hours. c) Archive. Mostly X-ray images but also some personal files from people who have left the lab. Projects that have been or are being published and data that need to be preserved 'indefinitely'. Backed up when I have time or when we run low on storage space (whichever comes first). All files reside on a network-attached storage device with currently 2TB of space, can be expanded to 4x largest HD (currently 4x1TB or better, I lose track). We have two of these devices, one primary and one backup in a different building. We archive (are set up to archive) to external HDs. We make two archive copies, one stays in a file cabinet, one goes home to PI, so there are copies at all times. Presumably entire projects will be archived (with multiple data sets, consisting of hundreds of X-ray images) at once. We designed it this way because we wanted 'instant security' once the files are established and we did not want to overwhelm the campus network with large backups overnight when data are collected. In the end, all our storage is on standard HDs, always in duplicate. Our network-attached storage consists of two Infrant (now NetGear) ReadyNAS NV+ systems (they are X-RAIDed). We have run this system for a couple of years now and it works=2 0like a charm. Our local computers do not have disk storage other than O/S, so no local files. Our O/S systems are backed up once in a long while to a VM server so in theory everything should be disaster-proof. I don't know that I would ask 'outsiders' like PDB to keep copies of files. After all, the researcher is responsible to keep good copies of their research data. It is not hard to do, but it requires quite a bit of thinking, probably by an IT specialist. In particular, I can remember when our 9-track tape system was thrown out in grad school. All media (with data) were subsequently useless. So you have to stay with time and upgrade storage once in a while, even if I have to admit that James' clay tablets are 'almost forever'. Technically I think that our 'forever' storage ends when the PI(s) retire(s). Mark -Original Message- From: David Aragao david.ara...@ul.ie To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 11 Mar 2009 5:09 am Subject: [ccp4bb] long term data backup Dear All, I wonder how people currently do their long term backups. I see DATs/DLTs being slowly dropped off at the beamlines and most people brings their data home in external HDs. Anyone using blue-ray or double layer DVDs for long term backups? If so what kind of hardware? Do you use HDs for long term storage? If so, do you do a second copy and how do you store them? I will try to compile the answers and relay back to the list a resume. Thank s, David -- David Aragão, Ph. D. Postdoctoral Researcher Membrane Structural and Functional Biology Group L2-007, Lonsdale Building University of Limerick, Ireland T: 353 (0)61 202302 F: 353 (0)61 234329
Re: [ccp4bb] X-Stream 2000 problem - ICING
Mark, What bothers me about your message is that you already have talked to Rigaku. Until now we have never been able to create a problem that they could not diagnose and help me solve from remote. In danger of offending ccp4 readers: specialized Rigaku experts are a remarkable source for information and solutions, probably better than we are. Your most likely problem is that your nitrogen is not dry? Specifically, check your air dryer (sorry, nitrogen dryer) that it works appropriately. Very specifically, there reside two compressors inside the air dryer and if one no longer works, the quality of your nitrogen stream degrades. It may not be apparent if both compressors work, one can supply all the pressure and volume you need and is sufficiently noisy that you would not notice the second being silent. Of course this problem becomes obvious when you open up the cabinet. (Yes, of course this happened to us once before and in our case the compressor wiring was fickle, as in, working when the cabinet was open and not (always) working when the cabinet was closed; took FOREVER to find the problem.) Your second most likely reason is that the warm stream (outer stream) is not sufficiently protecting your cold stream from humidity, but this is not affected by your phi-axis position. We have two inverted phi-axes and we do not see icing, so there is no fundamental reason why the phi-axis should not be inverted. Mark ? -Original Message- From: Mark Agacan m.aga...@dundee.ac.uk To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, 12 Jan 2009 3:42 am Subject: [ccp4bb] X-Stream 2000 problem - ICING Apologies for this slightly off topic question: I am having a great deal of trouble with my X-Stream 2000 cryostream system and I wondered if other users have similar problems. I've replaced almost all components (new GAST compressors, helium recharges, filters, etc., etc.) in the last couple of months but there is almost always icing of any cryo within 10 - 20 minutes of mounting a loop, and it is adversely affecting data collections. It appears like there is too much moisture in the cold or wam streams but the tubes have been fully dried out as per Rigaku advice. This X-Stream is attached to a generator with inverted phi axis and and i'm wondering if this could be the source of the problem, as the X-Stream for another generator in the same laboratory with normal phi axis does not ice up. Can some sort of turbulence around the loop caused by backdraft from the cryo hitting the inverted phi axis / camera mount cause excess humidity and lead to icing on the pin, loop and crystal? Has anyone else got this problem? Any suggestions would be very gratefully appreciated. Best Wishes, Mark _ Dr Mark Agacan Scientific Officer, Division of Biological Chemistry and Drug Discovery, Wellcome Trust Biocentre, College of Life Sciences, Dow St., University of Dundee, Dundee, DD1 5EH Tel: +44 1382 388751 Fax: +44 1382 345764 _ The University of Dundee is a registered Scottish charity, No: SC015096
Re: [ccp4bb] structure (factor) amplitude
But Tassos, you and Gerard both should know better (Mark vR already knows, clearly). THAT is not Dutch diplomacy, because it always starts like this: No, no, no, you are completely wrong! Actually, the way I learned about Cicero is better explained with Gaius Julius Ceasar. There is a temporal difference in pronunciation. The early schools (such as Leiden Univ.) teach [ˈsiːzɚ] while later schools (such as Nijmegen Univ.) teach [ˈkaɪsar]. Early and late are of course defined on the Roman time scale and it is theoretically possible, after 30 years, that I have my time scale running in the wrong direction, but that would be too diplomatic to add and I don't think so: classical is defined as roughly 100BC to 100AD and the more modern [ˈkaɪsar] eventually stuck. It is of course obvious that the latter has lead to the German Kaiser and Dutch keizer. So if you can have two kinds of Ciceros and two kinds of Ceasars, and we all understand what they are, you can also have Structure (Factor) Amplitude both ways and still understand? Personally I would always leave Factor in there. Somehow in my simplistic mind F comes from Factor. So to BR: please do keep the Factor. Perhaps you will aid consensus by creating more Factor hits in google in the future. Mark (Who now wonders, Nomen est Omen?) -Original Message- From: Anastassis Perrakis a.perra...@nki.nl To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, 12 Jan 2009 7:15 am Subject: Re: [ccp4bb] structu re (factor) amplitude This chain reminds me of another discussion we had during dinner at Grenoble in the late '90s. The topic of the argument was how to pronounce the name 'Cicero'. Namely, my Italian friend (Gino C) was claiming it should be pronounced like in modern Italian, 'Chichero', while I was claiming that since the contemporary Greeks transcribed it as 'Kikero' (with a k) it should indeed sound as in modern Greek, Kikero. My learned Dutch colleague (Mark vR) after a few minutes of this rather dull argument he exclaimed in the well known Dutch diplomatic manner: 'But, who chares?' Not that I don't care, but I would personally understand the same thing in both cases - and I am enjoying the argument. A. PS Wikipedia says: Marcus Tullius Cicero (Classical Latin pronounced [ˈkikeroː], usually pronounced /ˈsɪsəɹəʊ/ in English; January 3, 106 BC – December 7, 43 BC) was a Roman statesman, lawyer, political theorist, philosopher, and Roman constitutionalist. On Jan 12, 2009, at 14:48, Ian Tickle wrote: Hi Gerard Marc My answer was my interpretation of Bernhard's original question what *is* the currently accepted name of the object whose description is 'structure factor amplitude' ?, and was based both on authoritative precedent, i.e. ITC Vol. B, and on frequency of current usage, i .e. Google hits. Carroll was making the point that in logic the name of an object is minimally only an arbitrary string of characters (preferably pronounceable!), like the name of a variable in a program, which minimally need have no semantic connotations whatsoever: a rose by any other name would smell as sweet. The only requirement is that it must not be ambiguous, i.e. you can't have two different objects within the same context with the same name. For example my name 'Ian' provides no semantic clues as to my description (except perhaps that I'm male), and causes no problems provided no other 'Ian's enter the discussion. However alternate names for the same object are clearly allowed (consider names of objects in different languages). In this case I am not offering an opinion on what I think the name *should be*, I am merely reporting on what the name *is* (however illogical), based on precedent and usage. However I do accept your argument that when making up the compound name of an object, it should as far as possible also be accurately descriptive in the way it relates to the names of related objects, consistent with the conflicting needs for abbreviation and lack of ambiguity. You are going much further than me: you are answering a different question what *should be* the=C 2 accepted name of ... ?. In this case you have clearly made a strong argument, which I accept, for establishing an alternate name for this particular object. However one should not create new names or change the names of objects lightly, if misunderstandings are to be avoided. Fortunately in this case it can be done with minimal misunderstanding on the part of the readers of Bernhard's textbook (though others may disagree on that point), provided it is pointed out that there is precedent for an alternative name for the object in question, and perhaps a reference should be made to the original authoritative definition. Cheers -- Ian -Original Message- From: Gerard Bricogne
Re: [ccp4bb] Crystallographic computing platform recommendations?
I followed Kay's advice (after deciding that I knew better, of course, but we won't elaborate on that :-) and I am very pleased AND have had no trouble (knock on wood) to get everything working just fine. We make sure we have both 64 bit and 32 bit libraries and so far everything has worked out of the box, no hassle. And that includes coot. Mark -Original Message- From: Kevin Cowtan [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Tue, 18 Nov 2008 3:21 am Subject: Re: [ccp4bb] Crystallographic computing platform recommendations? And I would give exactly the opposite advice, unless you are or have a guru who can devote time to fixing all the little things which still don't work under 64 bit OSs.? ? (Does anyone else have any clues on why 64-bit compiled coot can't calculate a map? I need to look into it, but have a huge backlog of work at the moment.)? ? Kay Diederichs wrote:? Dear Anna,? you didn't ask about that, but I would definitely recommend a 64bit operating system.? My specific recommendations are mostly in the articles Computer_hardware and CentOS, to be found under the more general topic Xtal_computing of the CCP4 wiki (http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Xtal_computing) HTH,? Kay? Anna S Gardberg schrieb:? Dear list,? I haven't seen the crystallographic computing platform thread come up for a while, and I've got a chance to upgrade my desktop to a workstation, so I thought I'd ask the CCP4BB for advice on:? ? 1. Mac vs. Linux (which flavor?) vs. Windows? 2. Graphics cards? 3. Displays? 4. Processors - multiple processors, multiple cores? Speed?? ? About half of what I do involves ~1.0 A X-ray structures - data processing, rebuilding in Coot, refinement, and so forth - so my current desktop (Optiplex GX745, Radeon X1300) machine drags on graphics sometimes. I don't seem to need stereo these days, for what it's worth.? ? Anybody have suggestions or specs they'd like to share? Thanks in anticipation of your advice.? ? Regards,? Anna Gardberg?
Re: [ccp4bb] Crystallographic computing platform recommendations?
I completely agree with Marius. Our (my) constraints are not $500 in price difference, but the fact that I maintain a system for scientific computing AND an X-ray system AND I am expected to be a scientist who publishes and writes grants. Thus, our approach has been to automate and minimize my time and effort spent. We buy out-of-the-box Dell systems WITH support. This has already paid off once when (for hard to explain reasons) a motherboard went out. I diagnosed it, called India, and Dell sent someone out to replace the motherboard and with minimal effort we were back in business. Our Dell boxes came with standard NVIDIA quadro graphics cards. Hardware-wise our only real problem is the non-existance of CRTs that can do stereo and the exhorbitant prices for the flat screens. We run CentOS because (I think) RH is impossible to deal with, but I do like the stability. We have shared, network attached file storage? (which backs up automagically and is RAIDed) and we authenticate off a Windows server via LDAP, so I do not need to maintain user records, someone else already does that and all I need to do is e-mail a note that person X (or Y , gender neutral) can have access to the Linux systems and voila work is done. The system creates everything needed. It took a LOT of effort to get where we are. We thought long and hard about what exactly we needed and why. The most difficult operation is currently to get data from synchrotrons to our system. All the firewalls (existing for good reasons, of course) make it so hard these days to do anything useful in data transfer. We use Rsync and then I manually archive raw image data away. We sometimes have dreams about distributed computing (not so much for crystallography but for small angle scattering). Not enough experience to tell you how it should be done well. Mark -Original Message- From: Mischa Machius [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Tue, 18 Nov 2008 8:01 am Subject: Re: [ccp4bb] Crystallographic computing platform recommendations? After having dealt, over the years, with several dozens of 'crystallographic computing platforms' and having setup and maintained quite a few myself, I would recommend to not be cheap. I would recommend to go with well supported hardware and OS. For linux, I would recommend a commercial solution, and the hardware could come from a vendor such as Dell. In our lab, we use Macs practically exclusively, except for a few legacy Linux boxes.? ? I don't think it is worth saving a few hundred dollars when you end up spending/wasting so much time down the road assembling and fixing the machine as well as trying to keep up with the latest OS patches and drivers. I'd rather spend my time doing something else than being a computer support person. I realize I am not using the latest, greatest, pimped-out number-crunching monster, but a quad-core Mac is plenty sufficient. I like the fact that a refinement takes a few minutes longer, because that gives me time to fetch a cup of coffee or chat with a colleague.? ? Just a thought.? ? Best - MM? ? ? Dear list,? I haven't seen the crystallographic computing platform thread come? up for a while, and I've got a chance to upgrade my desktop to a? workstation, so I thought I'd ask the CCP4BB for advice on:? ? 1. Mac vs. Linux (which flavor?) vs. Windows? 2. Graphics cards? 3. Displays? 4. Processors - multiple processors, multiple cores? Speed?? ? About half of what I do involves ~1.0 A X-ray structures - data? processing, rebuilding in Coot, refinement, and so forth - so my? current desktop (Optiplex GX745, Radeon X1300) machine drags on? graphics sometimes. I don't seem to need stereo these days, for what? it's worth.? ? Anybody have suggestions or specs they'd like to share? Thanks in? anticipation of your advice.? ? Regards,? Anna Gardberg? ? ? ? Mischa Machius, PhD? Associate Professor? Department of Biochemistry? UT Southwestern Medical Center at Dallas? 5323 Harry Hines Blvd.; ND10.214A? Dallas, TX 75390-8816; U.S.A.? Tel: +1 214 645 6381? Fax: +1 214 645 6353?
Re: [ccp4bb] Crystallogrphy today
I agree with Bill. After a few minutes thinking, in between jobs working in the yard: It depends if you need to understand everything (I guess that's impossible these days) --- are you comfortable with publishing and defending research results that you do not understand? I am not. In quite a few labs there are crystallographers available who understand and can make sure that they can defend the results/science. If you have such people on staff, I guess (but don't like the sound of it) that you can treat macromolecular crystallography as a service and you can focus on other things. But you cannot (should not) publish or defend things you do not understand. A little more involved is the answer what if the problem is too difficult for standard approaches? We tend to see a lot of those. Problems where you have to sit and think because all standard approaches do not work. Of course these problems cannot be solved without a thorough understanding of standard problems and procedures. My $0.02 (soon to be $-0.02) Mark -Original Message- From: William G. Scott [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Sat, 20 Sep 2008 3:44 pm Subject: Re: [ccp4bb] Crystallogrphy today On Sep 20, 2008, at 2:18 PM, Jayashankar wrote:? ? Dear friends and crystallographers,? ? Are they mutually exclusive?? ? ? ? During One of my lab meeting ,? ? I told twinning in crystals are ok, because ccp4's recent releases just need? the keyword TWIN to solve them,? ? I believe the closer you get to complete twinning, the more intractable the problem gets. I don't know if the pain scales linearly with twinning fraction.? ? ? ? As a new generation research student, I am now confused,? ? This is both normal and proper, but has nothing to do with generation.? ? is that I need to? learn and understand all programs(so many...but research does not mean? relaying on them)? to solve my crystallographic problems(is that all)? if you see all the queries in ccp4BB is just about undocumented or? misunderstood program oriented questions.? ? Actually there are many lively discussions about fundamental problems. These will often arise in the context of a specific program, but you still have to understand the problem the program is designed to solve.? ? ? is that all i have to learn in crystallography in future.? ? That's up to you, but I would say no. Learn the fundamentals. Programs will come and go.? ? ? Still upto what limitations we are now in crystallography.? this is my very naive and prime question.? ? 1.Phase problem? ? This is still the problem. Some inroads have been made toward ab initio solutions, but the traditional heavy-atom methods, variations like MAD phasing, and molecular replacement remain in practice the standard approaches for what you usually find in the PDB.? ? ? 2.twin problem? ? see above.? ? ? 3.solving intrinsically disordered proteins? ? Crystals give a spatial average, so there is nothing magical you can do to overcome intrinsic disorder.? ? 4.hetro multimeric proteins? ? ribosomes are I think the current upper bound? ? ? 5.high order oligomers? ? Chromatin fibers maybe?? ? ? 6.cryo crystallography? ? This is routine.? ? ? 7.automation in high through put crystallography? ? The main problem is finding strong enough amphetamines to keep one awake while reading the papers.? ? ? 8.radiation damage? ? see cryocrystallography, and take lots of vitamin C? ? 9.kinetic crystallography? ? Laue? There is now a fair body of work, but development for irreversible enzyme systems is probably a worthwhile future goal.? ? ? 10. crystal growth research (antigravity, pressure )? ? Anti-gravity?? ? ? 11.stereo graphics? ? In the land of the blind, the one-eyed Macintosh user is king (as long as the program is not X-windows-based).? ? ? if i am right all the above has been studied (what we are not clear? still about them),? ? I need an answer to motivate me in doing my research in Crystallography.? ? S.Jayashankar? (A confused new generation research student)? Research Student? Institute for Biophysical Chemistry? Hannover Medical School? Germany.?
Re: [ccp4bb] Progresss with Stereo 3D under Mac OS X Leopard
I would agree with this statement, my preference is VERY STRONG. We mostly
work on Nucleosome and Nucleosome-nuclear protein complexes. By definition
these are low-resolution structures. They are very difficult to interpret, even
with stereo and I could not imagine even trying to work on things like this
without stereo. And yes, I am old - I learned O on SGIs with dials (which we
have kept as a momento, but nobody uses them anymore) and yes, most of us have
changed to Coot.
This reminds me: it would REALLY be nice to have a program that is better at
fitting ('non-standard') DNA in electron density. Any suggestions?
Mark
-Original Message-
From: Patrick Loll [EMAIL PROTECTED]
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wed, 17 Sep 2008 6:01 am
Subject: Re: [ccp4bb] Progresss with Stereo 3D under Mac OS X Leopard
People's feelings about using stereo seem to be highly idiosyncratic, but for
many of us who are pro-stereo, the preference is VERY STRONG.
Pat
On Sep 16, 2008, at 8:26 PM, Warren DeLano wrote:
CCP4bb,
Some graphics news:? Full operating-system support for stereo 3D has at
last been restored on Mac OS X Leopard, but NOT in Leopard's X11 just
yet (the Xquartz open-source community will hopefully soon remedy
this...).
While today's fix is great for native Mac OpenGL applications like
MacPyMOL, my fear is that other critical applications will still be
unable to support stereo 3D under Leopard because of a reliance upon
X11/GLX for OpenGL. ?
While Qt-based programs may now work fine in stereo 3D, X11-based apps
such as open-source PyMOL are definitely still mono 3D-only under
Leopard 10.5.5.
Perhaps some of the other software developers can chime in soon
regarding their support for stereo 3D under Mac OS X Leopard 10.5.5?? At
the very least, I am thinking of: ?
ccp4mg, O, coot, and VIDA/AFITT, as well as perhaps Chimera, VMD, and
MOE.
Apologies if I left out something important!? Along those lines, what
other go to molecular graphics apps are crystallographers using on
Macs these days?? In other words, who else should we/Apple lean on?
Also, do crystallographers still consider stereo 3D to be a
high-priority or must-have feature in a graphics workstation? ?
Cheers,
Warren
[EMAIL PROTECTED]
-
Patrick J. Loll, Ph. D.?? ? ? ? ? ? ? ? ?
Professor?of Biochemistry Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA??19102-1192??USA
(215) 762-7706
[EMAIL PROTECTED]
=
Re: [ccp4bb] Refinement problem
Hi Sampath, You are asking many questions at once. Since I am right now trying to solve a very difficult Se-Met structure, here are some ideas: - Do you have an energy scan on your crystal, showing that there is absorbance at the correct wavelength for Se? If yes, you have proof that there was indeed Se in your crystal; - You describe that you cannot find the Se atom. In theory it is possible that the atom is in the crystal, but not in an ordered fashion and therefore you would not be able to find it. That is theory, I think that in the majority of cases it works fine. You have not told us how the data were collected (synchrotron? wavelength? Inverse beam protocol to optimize SAD?) and whether or not a statistical analysis (with scaleit) tells you if there is a signal there. If you see a signal, then you know Se is present AND ordered. - If you have a MR solution, you can try to use those phases to find the Se atom in a phased anomalous difference map and then (provided that everything is consistent) use a combination of experimental and MR phases; but... - You also did not tell us how you know that your MR solution is correct. Specifically, can you see any features in the structure that are not part of the search model and are sensible? If yes, your solution is useful. If no, you should try omit maps to convince yourself that the MR solution isn't (too) biased and in fact correct - Your statistics given suggest that the MR solution is very poor, with a cons iderable chance that it is not valid. Remember that ~55% R-factor is equivalent to a random solution. If you do refinement and there is no improvement and the statistics are poor, chances are good that the solution wasn't correct in the first place. I worry that you have concluded that since the model fits the density nicely, based on MR, then things must be good, but if you look at Kevin Cowtan's web page with the duck and cat, you will be reminded of the fact that with phases from MR you (almost?) always get nice density from MR, but that does not mean at all that it is correct density. So yes, you should pursue the SAD phases. Remember that the signal will be weak (we cannot judge how weak, depends on the number of Se and size of your problem and the wavelength used), but (good news) you should not have problems with non-isomorphism (since you are not comparing two data sets). The SAD phases, after appropriate density modification, may show you a partial structure - I just tried this for my problem and it did not work for me, but then again, you must try to see if it can be done. Also, assuming that you have native, S-containing protein (as opposed to Se) there is the option of comparing the S- versus Se-protein and you might be able to get phases from that. Finally, others on this forum are better in this matter: 1.6A is fairly high resolution and I wonder if it is possible to pursue direct methods. Probably too low resolution, but I wouldn't know all that w ell, my maps are 5A resolution, so I don't worry about that option. Mark -Original Message- From: Sampath Natarajan [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Fri, 25 Jul 2008 11:05 am Subject: [ccp4bb] Refinement problem Dear all, Now I'm solving a structure with 1.6A resolution. The data seems good with R-sym (12.4) and all other parameters. Actually the data was collected with SAD phasing. When we checked the data we couldn't find the Se atom in the structure. Since the data resolution is good, we tried to do molecular replacement using Balbes program. It was selected a model with 25% sequence identity and we got the good solution too. I could find all residues in the density and also checked the Ramachandran map which shows almost all residues are in the allowed region. The problem is, I have done refinement many times, the R-factor (45.3) and R-free (51.4) is not reducing during the refinement and also figure of merit is not increasing. Still it remains what I got during the first refinement. The density is also not improving much. Also I could find many cuts in the density. My question is…….. 1. Can we use SAD phasing data for MR solution? 2. Is there any other way to reduce the R/R-free? 3. Why the figure of merit is not increasing even after modeled the residues exactly into the electron density? Thanks, Regards, Sampath
[ccp4bb] Acorn question
All, In the past couple of days I have been trying to use Acorn (in the CCP4 suite of programs). Consistently it starts up fine and after some small amount of time (5-10 minutes) it has taken up all the physical memory and then it starts to slowly gobble up all the swap space until the only option is to switch the computer off, since all processes (including X and such) are being pushed out of memory and they somehow are not swapped back in. Is there a way to restrict the amount of memory that Acorn is allowed to use? Any other suggestions? Parameters to restrict memory I do not see and the Acorn web page does also not mention that they exist, neither does it have any suggestions what to do. We are using CentOS operating system on 64-bit hardware (I think CentOS is vanilla Linux but 64-bit may not be vanilla). We are using CCP4 version 6.0. Any suggestions are appreciated. Thanks, Mark Mark van der Woerd, PhD Research Scientist II Dept. of Biochemistry and Molecular Biology Colorado State University Fort Collins, CO 80523 Phone (970) 491-0469
[ccp4bb] Post-doctoral job opportunity
All, Below you will find the pertinent information for a job opening at the Howard Hughes Medical Research Institute. All information can be found at this site: http://www.hhmi.org/jobs/main?action=jobjob_id=548. If you are interested, please follow the instructions in the advertizement and do NOT e-mail applications to me. Thanks, Mark Mark van der Woerd, PhD Research Scientist II Dept. of Biochemistry and Molecular Biology Colorado State University Fort Collins, CO 80523 Phone (970) 491-0469 ? Job Summary: Looking for a highly motivated individual with a strong interest in integrated approaches to problems in structural biology. The lab has extensive crystallographic and spectroscopic resources, and is part of the W.M. Keck Center for Chromatin Structure and Function. Principal Responsibilities: Investigate the structure, function, and dynamic properties of eukaryotic chromatin, using a wide variety of biochemical, biophysical and in vivo approaches. Investigate how cellular or viral factors interact with histones, nucleosomes, or chromatin and how these interactions may lead to cancer or other diseases. Use multipronged approaches such as x-ray crystallography, small angle x-ray scattering, atomic force microscopy, fluorescence spectroscopy, analytical untracentrifugation, and methods in mechanistic biochemistry/molecular biology as well as genetic approaches to understand the mechanism by which structural transitions in chromatin occur. Preferred Qualifications: Ph.D. in Molecular Biology, Biochemistry, Biophysics or an appropriately related field. Researchers with a strong background in biochemistry preferred. Previous experience with the structure determination of protein/protein and/or nucleic-acid complexes preferred. Extensive biochemical experience with protein purification, functional characterization, and yeast genetics would be highly valued.? Applicants should be strongly motivated, ambitious and function well in a highly collaborative environment. Additional Information: Please send cover letter, CV, and names of three references to Dr. Karolin Luger. Be sure to reference job #099100-01. To Apply To apply for this position, please email or send your resume to: Dr. Karolin Luger, PhD Investigator HHMI at Colorado State University Dept of Biochemistry Molecular Biology 1870 Campus Delivery, 383 MRB Fort Collins, Colorado 80523-1870 E-mail: [EMAIL PROTECTED] Application Deadline: Open Until Filled We are an Equal Opportunity Employer
Re: [ccp4bb] Codon Optimized Expression
I have seen procedures in a reputable lab where they do indeed pick multiple colonies and mix them; the people with the most experience claim(ed) that this MUST be done for the particular prep they were doing or else they get very low yields. The concern would be (in my mind) that if the different clones do not express exactly the same protein, but for example truncations, then you are in big trouble. But apparently that is not the case and the differences are in some other part of the little critter that expresses protein. I'd much rather make different cultures, each based on a single colony and see which works best. It makes somewhat sense that they might not all be exactly equal, but the standard protocol to get one, presumably homogeneous colony as a start makes good sense too. Mark -Original Message- From: Andreas Förster [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Sat, 2 Feb 2008 9:51 am Subject: Re: [ccp4bb] Codon Optimized Expression There has been a somewhat related discussion in the lab the other day. If some colonies might express well and other not so well, why don't I just scoop up loads and start my culture from them? This way I'll be more certain to get the clones that express. The clones that don't express will dilute the yield, but I doubt they'd outcompete the rest, would they? For me, the start-with-plenty procedure has always worked well, but some insist on starting from single colonies. Andreas James Stroud wrote: On Feb 2, 2008, at 2:15 AM, M T wrote: One classical way to optimize expression level is to screen culture conditions. For my proteins, I solved my expression problems by changing the expression vector to a pET or changing a pET 20 to a pET 30 (if the protein is toxic). But keep in mind that a low but folded expression is better than a high expression to inclusion body. Anecdotal evidence also suggests to try picking several colonies from the original cloning procedure and doing test expressions with each. Some clones express better than others even though they should in principle express identically. I have no idea what the mechanism for this differential expression could be. -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com More new features than ever. Check out the new AIM(R) Mail ! - http://webmail.aim.com
Re: [ccp4bb] crystallisation robot
Vaheh, I don't recall precipitation at all, but I do remember that we were prepared to change the crystallization recipes (i.e. adjust the recipes from previous 'large volume' crystallization to make more nuclei). For example, our first tests were with lysozyme (sorry, hardly a representative protein, I know) and it is known (paper by RA Judge et al) that buffer pH is a major determinant in nucleation (for that protein). Our initial concern was: is it possible to grow crystals in exceedingly small volumes? So we changed the pH to increase the nucleation rate. I think I recall that in case of lysozyme the nucleation rate is roughly inversely proportional to acid concentration, i.e. increase pH by 1 unit (10X less acid), gives ~10 times more nuclei. In general (for other proteins) we did make some minor adjustments in crystallization, but generally only in protein/precipitant concentrations. 'Minor' means adjustments of about 10% or so - going from 10% precipitant to 9% or 11%. When you go to very small volumes there is another consideration that you have to think about: mixing. If you assume that mixing takes place by diffusion (no stirring, it is very difficult to stir a very small volume reliably, i.e. to the same extent every time, unless you use microfluidic flow, in which case complete mixing to homogeneous mixtures is trivial - note: the Fluidigm system is 'microstatic', not microfluidic), so - if you assume mixing to take place by diffusion only, then the diffusion length in 100nL drops is very small and you can calculate in the worst case scenario how long it will take to accomplish full mixing by diffusion only. The time scale will be in the order of seconds. If you do the same calculation for a large drop (1 uL), the time scale is MUCH longer. So in case of batch crystallization, the 'end point' is reached very quickly in small volumes, while it takes much longer in large drops. If the end point causes protein precipitation, then this will happen very quickly after starting the experiment. In the larger scale experiment it will take much longer to reach precipitation and your system may go slowly through a process of enabling crystal growth before you reach drastic insolubility that causes precipitation. So as someone else said, the kinetics are completely different, the end point is not, and this can significantly affect the outcome of the experiment. Mark -Original Message- From: Oganesyan, Vaheh [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Thu, 17 Jan 2008 7:40 am Subject: Re: [ccp4bb] crystallisation robot Mark, What was the state of the larger drops when tiny counterparts had crystals? My guess - they all precipitated. I’m trying to understand why some proteins or some conditions require change in protein concentration while others do not when migrating from smaller drops to larger ones. If it is protein dependent then I’m afraid there might be no one answer; if it is not then there should be a trend and explanation of phenomena. Vaheh From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Wednesday, January 16, 2008 8:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] crystallisation robot Once upon a time I worked in a group that was interested in developing crystallization in microfluidics. This was before the time that Fluidigm existed and we had not heard of crystallization with the aid of microfluidics at the time. We had good reason to try to make a system that was as small and light as possible - it had something to do with the cost of shipping proteins and precipitants - less was better. And we also wanted all protein drops to be fully enclosed, out of safety considerations. Like Tassos, we were very worried what would happen if you scaled back drops along the lines of this discussion - several uL downto tens of nanoliters. If the stochastic process had a major influence over this process, we thought that we would never get any crystals. So we set up side-by-side experiments at larger volumes and smaller volumes - basically scanning several orders of magnitude - expecting a decrease of the number of crystals when volumes decrease. To our great surprise the outcome was that smaller volumes almost always gave MORE (I almost want to say 'dramatically more') crystals, more nucleation, and indeed in various cases the crystals grew much faster also. Indeed, it was trivial to observe that the surface-to-volume ratio was the primary driver for the nucleation process. We had control over geometry to some extent and were able to observe surfaces while crystals grow. The crystals would most commonly nucleate on a surface. So although there probably is something to stochastic aspects, it is clear that other aspects can be more important and overrule the stochastic considerations. The somewhat
Re: [ccp4bb] crystallisation robot
Once upon a time I worked in a group that was interested in developing crystallization in microfluidics. This was before the time that Fluidigm existed and we had not heard of crystallization with the aid of microfluidics at the time. We had good reason to try to make a system that was as small and light as possible - it had something to do with the cost of shipping proteins and precipitants - less was better. And we also wanted all protein drops to be fully enclosed, out of safety considerations. Like Tassos, we were very worried what would happen if you scaled back drops along the lines of this discussion - several uL downto tens of nanoliters. If the stochastic process had a major influence over this process, we thought that we would never get any crystals. So we set up side-by-side experiments at larger volumes and smaller volumes - basically scanning several orders of magnitude - expecting a decrease of the number of crystals when volumes decrease. To our great surprise the outcome was that smaller volumes almost always gave MORE (I almost want to say 'dramatically more') crystals, more nucleation, and indeed in various cases the crystals grew much faster also. Indeed, it was trivial to observe that the surface-to-volume ratio was the primary driver for the nucleation process. We had control over geometry to some extent and were able to observe surfaces while crystals grow. The crystals would most commonly nucleate on a surface. So although there probably is something to stochastic aspects, it is clear that other aspects can be more important and overrule the stochastic considerations. The somewhat unpleasant consquence is of course that results acquired in very small volumes (with larger surface-to-volume ratio) cannot necessarily be repeated in larger volumes (smaller surface-to-volume ratio). This is not a flame, even if heat might be a good thing on a night with temperatures predicted far below 0F. ?:-) Mark -Original Message- From: Anastassis Perrakis [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 16 Jan 2008 6:17 am Subject: Re: [ccp4bb] crystallisation robot Oryxnano 50+50 nL? ? Demetres? ? ? Which, indirectly, brings up an interesting (but not relevant to the Oryx) question.? ? Nucleation is a process that does have a stochastic aspect.? ? Thus, one could argue that compromising to 200-300 nl might be better than either extremes of 50nl (too small volume and less chance for nucleation) or 1000 nl (too much sample).? ? any comments ? (let the flames begin).? ? A.? ? PS1? another interesting issue that has has been hardly touched in these emails is the real sample loss: left in wells and not easy to recover, lost because of contamination with system liquid, etc ...? ? PS2? I see lots of people with new robots. please do have a look at the www.BIOXHIT.org page and if you have a few minutes to assemble a table we will be happy to add your specs to our pages. it can be a nice resource and it has already enough things and already one response to my last email ;-) To make life easier to potential contributors we can provide an Excel sheet to fill up with your specs - just ask.? ? On Jan 16, 2008, at 12:46, Demetres D. Leonidas wrote:? ? ? David Briggs wrote:? I'll defend the honour of the phoenix... (again)? ? Bernhard Rupp 100+100 nl? Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl? Others..? ? Only time we have ANY problems is when the nano dispensing tip gets clogged. Often a good wash whilst still on the machine will clear the blockage.? ? Dave? ? ? ? ? -- ? David C. Briggs PhD? Father Crystallographer? http://www.dbriggs.talktalk.net http://www.dbriggs.talktalk.net? AIM ID: dbassophile? ? ? -- Demetres D. Leonidas, Ph.D.? Structural Biology Chemistry Group? Institute of Organic and Pharmaceutical Chemistry? The National Hellenic Research Foundation? 48, Vassileos Constantinou Avenue? Athens 116 35, Greece? ==? Tel. +30 210 7273841 (office)? +30 210 7273895 (lab) Fax. +30 210 7273831? E-mail: [EMAIL PROTECTED] URL: http://athena.eie.gr? ==? More new features than ever. Check out the new AIM(R) Mail ! - http://webmail.aim.com
Re: [ccp4bb] crystallisation robot
Once upon a time I worked in a group that was interested in developing crystallization in microfluidics. This was before the time that Fluidigm existed and we had not heard of crystallization with the aid of microfluidics at the time. We had good reason to try to make a system that was as small and light as possible - it had something to do with the cost of shipping proteins and precipitants - less was better. And we also wanted all protein drops to be fully enclosed, out of safety considerations. Like Tassos, we were very worried what would happen if you scaled back drops along the lines of this discussion - several uL downto tens of nanoliters. If the stochastic process had a major influence over this process, we thought that we would never get any crystals. So we set up side-by-side experiments at larger volumes and smaller volumes - basically scanning several orders of magnitude - expecting a decrease of the number of crystals when volumes decrease. To our great surprise the outcome was that smaller volumes almost always gave MORE (I almost want to say 'dramatically more') crystals, more nucleation, and indeed in various cases the crystals grew much faster also. Indeed, it was trivial to observe that the surface-to-volume ratio was the primary driver for the nucleation process. We had control over geometry to some extent and were able to observe surfaces while crystals grow. The crystals would most commonly nucleate on a surface. So although there probably is something to stochastic aspects, it is clear that other aspects can be more important and overrule the stochastic considerations. The somewhat unpleasant consquence is of course that results acquired in very small volumes (with larger surface-to-volume ratio) cannot necessarily be repeated in larger volumes (smaller surface-to-volume ratio). This is not a flame, even if heat might be a good thing on a night with temperatures predicted far below 0F. ?:-) Mark -Original Message- From: Anastassis Perrakis [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 16 Jan 2008 6:17 am Subject: Re: [ccp4bb] crystallisation robot Oryxnano 50+50 nL? ? Demetres? ? ? Which, indirectly, brings up an interesting (but not relevant to the Oryx) question.? ? Nucleation is a process that does have a stochastic aspect.? ? Thus, one could argue that compromising to 200-300 nl might be better than either extremes of 50nl (too small volume and less chance for nucleation) or 1000 nl (too much sample).? ? any comments ? (let the flames begin).? ? A.? ? PS1? another interesting issue that has has been hardly touched in these emails is the real sample loss: left in wells and not easy to recover, lost because of contamination with system liquid, etc ...? ? PS2? I see lots of people with new robots. please do have a look at the www.BIOXHIT.org page and if you have a few minutes to assemble a table we will be happy to add your specs to our pages. it can be a nice resource and it has already enough things and already one response to my last email ;-) To make life easier to potential contributors we can provide an Excel sheet to fill up with your specs - just ask.? ? On Jan 16, 2008, at 12:46, Demetres D. Leonidas wrote:? ? ? David Briggs wrote:? I'll defend the honour of the phoenix... (again)? ? Bernhard Rupp 100+100 nl? Dave Briggs (and all users at Univ of Manchester, UK) 100+100nl? Others..? ? Only time we have ANY problems is when the nano dispensing tip gets clogged. Often a good wash whilst still on the machine will clear the blockage.? ? Dave? ? ? ? ? -- ? David C. Briggs PhD? Father Crystallographer? http://www.dbriggs.talktalk.net http://www.dbriggs.talktalk.net? AIM ID: dbassophile? ? ? -- Demetres D. Leonidas, Ph.D.? Structural Biology Chemistry Group? Institute of Organic and Pharmaceutical Chemistry? The National Hellenic Research Foundation? 48, Vassileos Constantinou Avenue? Athens 116 35, Greece? ==? Tel. +30 210 7273841 (office)? +30 210 7273895 (lab) Fax. +30 210 7273831? E-mail: [EMAIL PROTECTED] URL: http://athena.eie.gr? ==? More new features than ever. Check out the new AIM(R) Mail ! - http://webmail.aim.com
[ccp4bb] HHMI Post-doc Job opening
All, I am posting on behalf of Dr. Karolin Luger. Dr. Luger has a job opening for a post-doctoral researcher. Job Summary: Looking for a highly motivated individual with a strong interest in integrated approaches to problems in structural biology. The lab has extensive crystallographic and spectroscopic resources, and is part of the W.M. Keck Center for Chromatin Structure and Function. Please look at the following web site for additional information and for guidelines how to apply. http://www.hhmi.org/jobs/main?action=jobjob_id=548 Please DO NOT RESPOND to me. Thank you. Mark Mark van der Woerd, PhD Research Scientist II Dept. of Biochemistry and Molecular Biology Colorado State University Fort Collins, CO 80523 Phone (970) 491-0469 More new features than ever. Check out the new AIM(R) Mail ! - http://webmail.aim.com
Re: [ccp4bb] coot in stereo
Paul: We are running Coot under Centos with Nvidia video cards, emitters and glasses. Although our system is relatively new and therefore has not been used as extensively as I would like to see, we have never encountered your problem and hopefully will not. There are programs (VMD) in which you can deliberately change the eyes in stereo and out of curiosity I have done this and it consistently worked well even if I cannot really think of why you would want to have your eyes 'the wrong way around'. In short no instability. We are using Quadro FX3450 cards with the most recent drivers supplied by Nvidia. Note that they regularly (weekly?) issue upgraded drivers. To follow up on Carsten's observation, perhaps a driver upgrade will help? The only thing that I have noticed as 'abnormal' is that coot sometimes freezes when you have another window in front of the coot window and after minimizing this front window, you cannot make the coot window work unless you either minimize coot and open it up again or alternatively you move the window around on your desktop - simply clicking on it does not help. But no stereo problems. Mark -Original Message- From: Paul D. Cook [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Tue, 14 Aug 2007 7:22 am Subject: [ccp4bb] coot in stereo Hello, I'm running stereocoot on linux (centos) machines with nvidia video cards and stereographics emitters and glasses. The setup seems to work fine most of the time, but the stereo will invert every now and then (the right eye is shown the left image and vice versa). This seems to happen especially after a computation such as realspace refine is performed. The stereo will usually spontaneously correct itself, but sometimes I have to minimize the window and restore it. This is occuring on three machines with the above configuration. Swapping emitters and glasses seems to do nothing. Has anyone had such a problem? Thanks, Paul D. Cook Check Out the new free AIM(R) Mail -- Unlimited storage and industry-leading spam and email virus protection.
Re: [ccp4bb] need help for viewing hardware stereo
It could be a hardware problem or it could be a mis-configuration. The card you list DOES support stereo viewing in all crystallographic applications, I am sure, because we have the same hardware (except our computers are 64 bit and we run CentOS operating system, but otherwise identical). As others suggested, you might want to download the latest update for the nVidia drivers - even if you have one, updates are always good. Follow their instructions (i.e. boot to init level 3, install the driver with the supplied install file, answer the questions the script asks you (they are very simple, usually default is fine) and boot back to level 5. Then check your xorg.conf file and make sure that it has the Option Stereo 3 entry. Please feel free to e-mail me directly if you cannot get it to work. I have not heard before that the Dell/nVidia combination is shipped defective, but everything is possible of course. Mark -Original Message- From: venkadesan krishnan [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, 16 Jul 2007 5:56 pm Subject: Re: [ccp4bb] need help for viewing hardware stereo Dear all, ? I think our earlier posting is not clear. So we would like to draw to your attention regarding our stereo viewing problem. As Dr. Paul Emsley suggested it seems to be a harware problem, we are experiencing the same problem not only using coot but other programs too. We will be thankful to you if you could anyone help us to fix the problem for your kind information here is our system graphics card specification 256MB PClex16 nVidia Quadro Fx? 3450, Dual DVI or Dual VGA or DVI + VGA. Thanking you vengadesan On 7/16/07, Paul Emsley [EMAIL PROTECTED] wrote: Hello venkadesan krishnan, On Mon, 2007-07-16 at 15:57 -0500, venkadesan krishnan wrote: Hello everyone, ?? We have bought a new Dell Precision Workstation 690 (32-bit) and installed Fedora core 6. We are having problem while viewing molecules in harware stereo mode using programs coot, pymol, etc. The graphics card came with system is, 256MB PClex16 nVidia Quadro Fx 3450, Dual DVI or Dual VGA or DVI + VGA. In coot it shows the following message in the terminal, WARNING:: Can't enable stereo visual - falling back INFO:: Hardware stereo widget opened successfully INFO:: switch to hardware_stereo_mode succeeded That is a confusing message.??I can't imagine how you got it if you simply installed a binary package :-/ However, that code path does mean that Coot failed to get a hardware stereo graphics context, which leads me to think that it's a hardware issue, not software. (Oh, we are talking about 0.3.3, aren't we?) Paul. Check Out the new free AIM(R) Mail -- Unlimited storage and industry-leading spam and email virus protection.
Re: [ccp4bb] : Crystallographic Programs on AMD computers
We have made the change to 64 bit computers and indeed it is a bit confusing to have to think 'twice' (32/64 bits). I guess that the argument can be made that eventually all computers will be 64 (or more) bit and therefore staying with 32 bits on a new computer is like holding back 'progress'? :-) I agree with Kay that (today) there really is not all that much advantage to running under 64 bit. We purchased the Intel Fortran compiler so we can compile and optimize CNS. I did some tests comparing CNS performance when compiled with g77 (which I could not get to work at all on 64 bit machines), gfortran (works, but not so fast), and Intel (works and very fast). We felt that spending the money to purchase an Intel compiler for CNS was worthwhile. For academics and government they have special rates. All other programs I have used 'out of the box'. We use the standard C-compiler. Intel sells those too, but I could not find as big a difference and saved that money. Mark -Original Message- From: [EMAIL PROTECTED] To: [EMAIL PROTECTED] Sent: Thu, 12 Apr 2007 4:54 PM Subject: Re: [ccp4bb] : Crystallographic Programs on AMD computers Kay Diederichs wrote: the CPU (AMD versus Intel) does not play any role for crystallographic computing, but you'll have to decide whether you want to install the 64bit or the 32bit version of RHEL4. 32bit programs run a bit faster on a 64bit operating system, and with a 64bit OS you can run programs which require arrays of more than 3.5 GB (seldomly needed in xtallography), but you will have to install in parallel many 32- and 64bit libraries. This is no problem by itself, but quite confusing at first ... So I recommend the 32bit version. Any reccommendation for compilers ( c and fortran)? Probably a dumb question, but is there any advantage in using the 64-bit-aware intel compilers on an AMD processor? Ed Check Out the new free AIM(R) Mail -- 2 GB of storage and industry-leading spam and email virus protection.
Re: [ccp4bb] x86-64
Although I have not yet tried to compile coot or CCP4, I have found that the
GNU provided packages (gcc, g77) do not make life convenient (how is that for a
euphamism for 'banging your head against the wall)?
Things worked better with gfortran than g77 and again better with the Intel
compilers (both fortran and C(++)). When I say 'worked better' this means 'less
effort to get it working' and also (particularly in case of Intel) 'faster'. My
experience was not with Fedora but with RHEL (similar problems as described
below, not the same though).
In my humble opinion it is worth to spend the money, get the paid-for
compiler, and get around the problems like the one you describe. Life gets even
better: if you want to try, you can get a free trial license for either fortran
or C(++) or both and convince yourself that it is better. The Intel compilers
are a one-time expense with an indefinite license, but you must pay annually if
you want support. Academic licenses are (appropriately) inexpensive.
My 2 cents worth.
Mark
-Original Message-
From: [EMAIL PROTECTED]
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wed, 14 Feb 2007 1:09 PM
Subject: Re: [ccp4bb] x86-64
Dear Phil,
Good luck . . . I have been fighting an x86_64 system for some time, and
have just figured out what some of the problems are. I am running Fedora
Core 5.
I believe that if you use the -m32 flag for gcc you can compile 32-bit code
for 32-bit libraries. The default is to compile 64-bit and link 64-bit.
The real joker in the deck is the file system layout:
/usrDefault root prefix
/usr/includeUsed for both 32 and 64 bit systems
/usr/libLibraries for 32-bit code
/usr/lib64 Libraries for 64-bit code
/usr/binFor both -- the operating environment is encoded in the file
This breaks the standard prefix scheme for prefix/{include,lib,src,...}
because it is not easy to tell when you need lib and when you need lib64.
I was unable to compile Coot from source until the last day or so because
the linker kept putting the 32-bit libGL.so in the search path. This is a
fatal error.
I finally tracked this to a bug in libtool, which figures out about the
32/64 bit issues *nearly* all of the time. Sigh.
Short answer: get the latest, bleeding-edge Autoconf package from the GNU
web site and install it. It is alpha, but seems to work, and the configure
scripts once generated can be run almost anywhere. (Oh, you may also have
to upgrade M4.)
*Note* I got Autoconf 2.61, but the real key seems to be the version number
on the libtool macros. Version 1.2248 does not work, but Version 1.2381
does work on my system. Unfortunately the latest versions are also more
picky about the macros, so if autoupdate can't fix them, you have to do some
hand editing.
I will be happy to follow up on this off-line, and expect to post a summary
on the Coot bulletin board once I have some loose ends tidied up. I suspect
this may be why I have had problems trying to build ccp4mg from source on
this machine, as well.
Overall the machine runs really well, but you do hit the occasional package
that is not 64-bit clean.
Best regards,
Lynn Ten Eyck
On 2/14/07 10:00 AM, Phil Evans [EMAIL PROTECTED] wrote:
I'm just starting to use a 64-bit Linux machine (running some sort of
RedHat Enterprise system) as a development machine
Our general CCP4 installation is from the binary download (redHat
option) (presumably built on a 32-bit machine), which seems to run OK
on a range of different Linux machines
However if I compile on the 64-bit machine try to link with these
libraries, it doesn't work
r/bin/ld: skipping incompatible /public/xtal/ccp4-6.0/ccp4-6.0.2-
linux/lib/libccp4f.a when searching for -lccp4f
/usr/bin/ld: cannot find -lccp4f
collect2: ld returned 1 exit status
make: *** [scala] Error 1
Is it possible to set compile flags to produce something (.o) which
will link with th distributed libraries, and produce an executable
which will run on other (32-bit) Linux machines?
In the mean time, I'm doing a complete source build on the 64-bit
machine
Phil
--
Lynn F. Ten Eyck[EMAIL PROTECTED]
San Diego Supercomputer Center (858) 534-5141 (Voice)
University of California, San Diego (858) 822-3610 (Fax)
9500 Gilman Drive #0444 Office: 3131 Atkinson Hall
La Jolla, CA 92093-0444
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Re: crystal friendly solvents that are useful for dissolving hydrophobic small molecules?
Toluenes and derivatized benzenes may absorp into your plastic tray? Or into the tape covering your tray? Just few other destinations. It would make sense that if the binding of the 'drug' to the protein is tight, then you do not need much in immediate contact, it will get there. The method below sounds very promising to me. Mark -Original Message- From: [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Mon, 22 Jan 2007 3:57 PM Subject: Re: crystal friendly solvents that are useful for dissolving hydrophobic small molecules? So I've never actually tried this proposed extension to the idea, but: I (and many others) have gotten small hydrophobics (toluene, iodobenzene etc.) into proteins, and these things typically have very small partition coefficients, and they aren't horribly volatile (that's why I am a little partial to iodobenzene). Why not saturate a small solution of your small molecule in iodobenzene and just add a few microliters on top; if the binding is tight enough you can pull it through and not bug your protein with a denaturing co-solvent. I've noticed that the iodobenzene does largely disappear overnight (in hanging drop), I don't know if this is because of evaporation or the iodobenzene just falls into the reservoir. Maybe stick to sitting drop. -Original Message- From: CCP4 bulletin board on behalf of Green, Todd Sent: Mon 1/22/2007 12:40 PM To: CCP4BB@JISCMAIL.AC.UK Subject: crystal friendly solvents that are useful for dissolving hydrophobic small molecules? Hello All, I am trying to soak some crystals with a small molecule that is quite hydrophobic. I am having trouble with solubilty of the small molecule. It will dissolve up to about 1 mM in 100 % DMSO, but precipitates at concentrations of less than 15 micromolar when the DMSO concentration is below 20 percent in my crystal growth solutions(which are peg 4k, low pH, low salt). Can anyone suggest solvents other than DMSO which might help dissolve the inhibitor and might be somewhat friendly to my crystals. Thanks in advance- Todd Green Check Out the new free AIM(R) Mail -- 2 GB of storage and industry-leading spam and email virus protection.
