[ccp4bb] Vivaspin vs Amicon Ultra
Dear all, sorry, for the slightly off-topic theme, but I wonder if anyone has compared the above mentioned ultracentrifugation devices, thoroughly. Currently, we are using the Amicon Ultra, but as the Vivaspins are considerably cheaper we are considering to change. I used both with extracellular matrix proteins and both worked fine for me; however, some of us are working with membrane proteins, which might behave differently. Has anyone experiences with Vivaspins and membrane proteins? Has anyone compared the two (also with non-membrane proteins) and can share her or his experience? Thanks, Jan -- Dr. Jan Gebauer AG Prof. Baumann Institut für Biochemie / Uni-Köln Otto-Fischer-Str. 12-14 / 50674 Köln Fon: +49 (221) 470 3212 Fax: +49 (221) 470 5066
Re: [ccp4bb] Overlapmap
On Wed, Oct 5, 2011 at 11:03 AM, Adam Ralph adam.ra...@nuim.ie wrote: Hi Brigitte, You are correct, the columns labeled Fobs and Fcal refer to density. The columns should be: averaged density for Obs and Cal for the main chain, then averaged density Obs and Cal for the side chains. I have included a version of overlapmap that calculates the R-factor correctly and have changed the above columns in the output. All the best Adam Documentation should be fixed too :) -- Ian
Re: [ccp4bb] Vivaspin vs Amicon Ultra
Hi Jan I have used Vivaspins for membrane proteins and they generally work fine. The exact pore size might be different though between Vivaspin and Amicon with the same specifications, say e.g. 100,000 MWCO. So you might find that your protein is retained in one of them, while it is in the flowthrough with the other. Best regards Florian Am 05.10.2011 um 11:22 schrieb Jan Gebauer: Dear all, sorry, for the slightly off-topic theme, but I wonder if anyone has compared the above mentioned ultracentrifugation devices, thoroughly. Currently, we are using the Amicon Ultra, but as the Vivaspins are considerably cheaper we are considering to change. I used both with extracellular matrix proteins and both worked fine for me; however, some of us are working with membrane proteins, which might behave differently. Has anyone experiences with Vivaspins and membrane proteins? Has anyone compared the two (also with non-membrane proteins) and can share her or his experience? Thanks, Jan -- Dr. Jan Gebauer AG Prof. Baumann Institut für Biochemie / Uni-Köln Otto-Fischer-Str. 12-14 / 50674 Köln Fon: +49 (221) 470 3212 Fax: +49 (221) 470 5066 -- Dr. Florian Brückner Biomolecular Research Laboratory, OFLG/102 Paul Scherrer Institut CH-5232 Villigen PSI Switzerland Tel.: +41 (0) 56 310 2332 Email: florian.brueck...@psi.ch
Re: [ccp4bb] Vivaspin vs Amicon Ultra
Hi Jan, In our lab we use both the Amicon Ultras and the Vivaspins (soluble and membrane proteins). What I can tell you is that me and a few other colleagues had the experience (and still do) of specific soluble proteins precipitating on the Cellulose membrane of the Amicon. By switching to the Vivaspins, which have a polyethersulfone membrane, the precipitation completely vanished. This seems to be very protein dependend as many seem to be able to use both systems with no or little problems. I don't know if the type of membrane really makes the difference but it could be a possible explanation. I unfortunately have less expierence with these systems on membrane proteins and I only know that both systems seem to work fine for colleagues who work on membrane proteins. Best Regards, Peter -- P.G.M. Gutte PhD student Institute of Biochemistry University of Zurich Winterthurerstrasse 190 CH-8057 Zurich Tel: +41 (0)44 635 55 02
Re: [ccp4bb] Vivaspin vs Amicon Ultra
The vivaspins are the best thing in ultrafiltration since sliced bread. I learned about them while I was at the NIH several years ago and haven't looked back. Fast and low-binding. PES membranes are superior for minimizing protein binding, and are also less subject to pore shrinkage in high salt solutions, unlike cellulose or nylon membranes. I will hardly use anything else for protein concentration. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 10/5/2011 8:59 AM, Peter Gutte wrote: Hi Jan, In our lab we use both the Amicon Ultras and the Vivaspins (soluble and membrane proteins). What I can tell you is that me and a few other colleagues had the experience (and still do) of specific soluble proteins precipitating on the Cellulose membrane of the Amicon. By switching to the Vivaspins, which have a polyethersulfone membrane, the precipitation completely vanished. This seems to be very protein dependend as many seem to be able to use both systems with no or little problems. I don't know if the type of membrane really makes the difference but it could be a possible explanation. I unfortunately have less expierence with these systems on membrane proteins and I only know that both systems seem to work fine for colleagues who work on membrane proteins. Best Regards, Peter
Re: [ccp4bb] software for surface curvature
On Tue, 2011-10-04 at 15:25 -0700, Jun Liao wrote: I want to calculate the surface curvature for my proteins in a quantitative way and show the results in a graphics software such as Pymol Surface Racer http://apps.phar.umich.edu/tsodikovlab/index_files/Page756.htm will output curvature per atom. The easiest way to get this info rendered in pymol is to move the curvature values into the b-factor column. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Overlapmap
Adam, sorry I don't use overlapmap, for the reasons you mention (and many others!). In fact I decided it was in such a mess that it was irrecoverable so I wrote my own program EDSTATS to do all all these electron density stats (and more). I talked about it at the last CSW ( http://www.cse.scitech.ac.uk/events/CCP4_2011/talks/tickle.pdf), and submitted it to CCP4 in January but it doesn't seem to have made the latest release yet (or even the pre-release), so at the moment I'm just distributing it to anyone who's interested. Cheers -- Ian On Wed, Oct 5, 2011 at 12:31 PM, Adam Ralph adam.ra...@maths.nuim.iewrote: Hi Ian, Yes I agree. The whole program is a bit of a mess and could do with some updating. I am not an official CCP4 maintainer any more but I might send them something. Do you know how to use SFALL ATMMAP? I tired to test overlapmap's residue correlation but did not work properly. Adam - Original Message - From: Ian Tickle ianj...@gmail.com To: Adam Ralph adam.ra...@nuim.ie Cc: CCP4BB@jiscmail.ac.uk Sent: Wednesday, 5 October, 2011 11:36:30 AM Subject: Re: [ccp4bb] Overlapmap On Wed, Oct 5, 2011 at 11:03 AM, Adam Ralph adam.ra...@nuim.ie wrote: Hi Brigitte, You are correct, the columns labeled Fobs and Fcal refer to density. The columns should be: averaged density for Obs and Cal for the main chain, then averaged density Obs and Cal for the side chains. I have included a version of overlapmap that calculates the R-factor correctly and have changed the above columns in the output. All the best Adam Documentation should be fixed too :) -- Ian
Re: [ccp4bb] Database access failure during program execution
On Wed, 2011-10-05 at 10:36 +0800, Jobichen Chacko wrote: I looked into our previous posts and tried to locate the ccp4.LOCK file, but I cannot find one in my system. Curiously, neither can I with the ccp4i currently running. Maybe this info is outdated, and you should try deleting database.LOCK instead in the CCP4_DATABASE folder of the project that you have listed under CURRENT_PROJECT in ~/.CCP4/unix/status.def ? Although my expectation is that if a project is locked ccp4i will offer you an override option. As a random thought, check if you don't have any disk access problems (stale NFS handles and such). A drive that drops into read-only mode for some reason could cause all kinds of otherwise mysterious problems. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
[ccp4bb] Parrot NCS averaging
Dear CCP4'ers, I am working on refining the structure of a protein complex consisting of two different chains. Data were collected to 2.3A in space group C2 and phased by molecular replacement without any problem, but the ASU contains 6 complexes (so 12 chains in total). In the ASU, the 6 complexes form 3 dimers. Dimer 1 is related to dimer 2 by a rotation operation and to dimer 3 by translation - I hope that makes sense! When I run Parrot to do density improvement and NCS averaging, Parrot works beautifully (final FOM is 0.87) but NCS averaging causes the average NCS correlation coefficient to drop (from 0.94 to 0.64) and the average mask volume to increase from 0.31 to 0.7 (minimum volume increases from 0.05 to 0.07 and maximum volume goes from 1.2 to 2), which seems a bit high. Unless I have misunderstood something, I thought the NCS correlation coefficient should increase during averaging and the average mask volume should decrease. If this is the case, has anyone any idea what I'm doing wrong? Also, at the risk of asking a dumb question, is there a particular way my Parrot modified file should be input into Refmac to get the best results? The FOM from my refmac runs never seems to be as good as that output by Parrot. Thanks in advance, all advice gratefully received, etc etc... Peter Canning SGC
Re: [ccp4bb] Parrot NCS averaging
Dear Herman In some cases density modification starting with MR phases can indeed prove very powerful. We recently used Parrot to bootstrap a difficult case at 4.3 angs and were able to obtain very good maps for entire missing domains. See Supplementary Fig 1 in:Verstraete et al 2011 BLOOD 118:60-68 and more technical info and fig5 in: Verstraete et al 2011 Acta F67:325-331. The Ncs mask radius card in parrot proved to be an important one to experiment with, and is probably more important to do so at medium-low resolution. We provide details of our observations/implementation on page 330 of the actaF paper. We also found that post-MR HL coefficients written out by Phaser were also key. So in subsequent runs, after having built and refined partial models, we would do a phase calculation run in Phaser to obtain HL by 'fixing' the partial model via a template .sol file with 0 values for rotation/translation across the board. Best wishes Savvas On 05 Oct 2011, at 17:13, herman.schreu...@sanofi-aventis.com wrote: Dear CCP4'ers, I do have a similar case with 8 molecules in the asymmetric unit, related by improper symmetry and the structure is solved by molecular replacement. I do have 2 questions: 1) Does it make sense to average to improve phases? The molecular replacement phases perfectly obey the non-crystallographic symmetry? 2) For refinement I do not need average maps since Buster and other refinement programs are very capable to handle ncs. However, I am not a computer and for manual rebuilding and display purposes it would be very good to use averaged maps. Which program could I best use for this: Parrot, DM, Solomon, other? None of them seem to be really made for molecular replacement phases. Also my many thanks for any help, advice etc. Herman Schreuder From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Peter Canning Sent: Wednesday, October 05, 2011 4:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Parrot NCS averaging Dear CCP4’ers, I am working on refining the structure of a protein complex consisting of two different chains. Data were collected to 2.3A in space group C2 and phased by molecular replacement without any problem, but the ASU contains 6 complexes (so 12 chains in total). In the ASU, the 6 complexes form 3 dimers. Dimer 1 is related to dimer 2 by a rotation operation and to dimer 3 by translation – I hope that makes sense! When I run Parrot to do density improvement and NCS averaging, Parrot works beautifully (final FOM is 0.87) but NCS averaging causes the average NCS correlation coefficient to drop (from 0.94 to 0.64) and the average mask volume to increase from 0.31 to 0.7 (minimum volume increases from 0.05 to 0.07 and maximum volume goes from 1.2 to 2), which seems a bit high. Unless I have misunderstood something, I thought the NCS correlation coefficient should increase during averaging and the average mask volume should decrease. If this is the case, has anyone any idea what I’m doing wrong? Also, at the risk of asking a dumb question, is there a particular way my Parrot modified file should be input into Refmac to get the best results? The FOM from my refmac runs never seems to be as good as that output by Parrot. Thanks in advance, all advice gratefully received, etc etc… Peter Canning SGC
Re: [ccp4bb] Parrot NCS averaging
On 10/05/2011 03:42 PM, Peter Canning wrote: When I run Parrot to do density improvement and NCS averaging, Parrot works beautifully (final FOM is 0.87) but NCS averaging causes the average NCS correlation coefficient to drop (from 0.94 to 0.64) and the average mask volume to increase from 0.31 to 0.7 (minimum volume increases from 0.05 to 0.07 and maximum volume goes from 1.2 to 2), which seems a bit high. Not impossible if the NCS is along a special direction. I've rewritten the logfile stats in the latest version to give you a more meaningful and less confusing number. Unless I have misunderstood something, I thought the NCS correlation coefficient should increase during averaging and the average mask volume should decrease. If this is the case, has anyone any idea what I’m doing wrong? That's true for experimental phasing. When you start from MR, your initial phases contain the NCS implicitly. The structure factor magnitudes in this case have something to say about the differences between the molecules. Again, I think the stats in the new version may be more informative. Also, at the risk of asking a dumb question, is there a particular way my Parrot modified file should be input into Refmac to get the best results? The FOM from my refmac runs never seems to be as good as that output by Parrot. FOM from density modification is overestimated! From refmac less so! K
[ccp4bb] Industry position job posting
Hi All, The protein engineering department at Zymeworks Inc. (www.zymeworks.com), is actively searching for talented structural biologists who are interested in doing in silco protein engineering. If interested please visit [http://zymeworks.com/careers/postings.html] Cheers, Paula Lario Ph.D. Zymeworks, Inc.
Re: [ccp4bb] Database access failure during program execution
If I'm not mistaken it is caused by /tmp/'username' not existing or being writable... Jon -- Jonathan P. Schuermann, Ph. D. Beamline Scientist NE-CAT, Building 436E Advanced Photon Source (APS) Argonne National Laboratory 9700 South Cass Avenue Argonne, IL 60439 email: schue...@anl.gov Tel: (630) 252-0682 Fax: (630) 252-0687 On 10/05/2011 08:51 AM, Ed Pozharski wrote: On Wed, 2011-10-05 at 10:36 +0800, Jobichen Chacko wrote: I looked into our previous posts and tried to locate the ccp4.LOCK file, but I cannot find one in my system. Curiously, neither can I with the ccp4i currently running. Maybe this info is outdated, and you should try deleting database.LOCK instead in the CCP4_DATABASE folder of the project that you have listed under CURRENT_PROJECT in ~/.CCP4/unix/status.def ? Although my expectation is that if a project is locked ccp4i will offer you an override option. As a random thought, check if you don't have any disk access problems (stale NFS handles and such). A drive that drops into read-only mode for some reason could cause all kinds of otherwise mysterious problems. -- Jonathan P. Schuermann, Ph. D. Beamline Scientist NE-CAT, Building 436E Advanced Photon Source (APS) Argonne National Laboratory 9700 South Cass Avenue Argonne, IL 60439 email: schue...@anl.gov Tel: (630) 252-0682 Fax: (630) 252-0687
Re: [ccp4bb] Vivaspin vs Amicon Ultra
Hello Jan, I've used both with soluble and membrane proteins happily. I haven't seen significant differences with protein behavior or performance in one or the other, but of course, your favorite protein may differ. I've found that buffer condition is a more important variable than membrane type. Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry holeung...@hawaii.edu
[ccp4bb] not so good news
http://www.apple.com/stevejobs/ May innovation continue to lead the future. 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://web.mac.com/bosch_lab/
Re: [ccp4bb] not so good news (Steve Jobs RIP)
On Oct 5, 2011, at 5:52 PM, Bosch, Juergen wrote: http://www.apple.com/stevejobs/ May innovation continue to lead the future. Jürgen I've been quite saddened about this all evening. Even though we knew it was coming, it is still incredibly sad, especially at his age (only 56). Although I never met him, through OS X, he made a fairly significant impact upon my life and career development (such as it is). The advent of OS X has had a significant impact upon both my professional and personal lives. Although I have worked with unix systems since the mid 1980s, it was only when OS X came along, and I made the switch (from SGI's Irix, which many of us remember fondly as a total pig of an OS), did I really come to enjoy using computers. As a crystallographer, a unix operating system really is essential I think, and when OS X appeared, a fundamental change took place in my office as a young assistant professor: I only needed one computer. Before, I had an enormous SGI sitting on a table, for doing science. With its stereographic hardware and dials, it put me back (well, actually, the taxpayer) about $12K for an R10,000, which had a 150MHz RISC processor. The sony trinatron monitor did a good job heating the room, especially in the summer. Then I had this little colored hunk of plastic, the first generation bottom-of-the-line iMac, upon which I wrote my papers and grant proposals, sitting on my desk. This left very little room in my office to mope and feel sorry for myself, which my wife tells me are the only things I am good at. OS X came on an extra CD packaged with an iBook I bought just before the Sept 11th attack. I remember, because I gave my first presentation using OS X 10.0.3 or something like that on that day, in Grenada, Spain, right next to the Alhambra. It was the worst talk I ever gave, but it wasn't the fault of the software. I was excessively nervous, so much so that my arch-competitor I guess took pity and tried to calm me down. Maybe I sensed something bad was about to happen. Anyway, it was an inauspicious start. I eventually made it home and put it on that colored piece of plastic in my office and discovered it really was a real unix operating system, albeit in its infancy. I sort of got lost in it for months at a time, and was determined to get all of the software used in macromolecular crystallography running on OS X. O was about the only thing at the time that seemed ready. What evolved from that was a crystallography on os x website, which started off as a chronicle of my pain, which I seem to feel an almost moral obligation to share and inflict upon everyone around me. (I work at a university that, frankly, isn't very good at infrastructural support, to put it mildly, and I wound up having to learn to do everything the hard way, by myself.) I think it is fair to say that Steve Jobs and OS X were some of the main things that made this, and my survival, possible. I've primarily used OS X I guess from the bottom up, but at the same time I was a bit of an Apple fan-boy in the sense that I got the first iPod when it came out, the first iPod Touch, the first MacBook Air, the first iPad, etc. I've never regretted any of those purchases, nor the purchase of the 20 or so Apple computers that I've acquired for work and family over the last decade or so. My newest OS X adventure has been into computer audio. I really still don't know much of anything about it, but again Apple has made it an enjoyable experience. I bitch and moan about iTunes as much as the next guy, but if you stop to think about it, it transformed the entire music industry, and turned what began as a network of illegal file sharing (Napster), something I regrettably missed out completely, into a legitimate business model. Some might look at this as co-opting, but I think there is a bit more to it than that. In any case, both from the perspective of hardware and software, Apple/Jobs completely changed how I listen to music. During that same decade I was starting out as an academic scientist, I had more or less given up on music almost completely. My oldest son now likes music (Pearl Jam, Joy Division, REM), but at the time he couldn't handle listening to it at all, probably as a consequence of being somewhere on the autistic spectrum. OS X provided an escape from all that for me, but it eventually also provided a relief for him I think, and through it he came to enjoy (some) music and now plays cello and electric guitar. So, I guess Steve Jobs has had a large, albeit indirect, influence on my life, and that of my family and research group. I never met him, but I've learned a bit about him through a former VP. I doubt I would have liked him very much personally. Obsessive secrecy and temper tantrums aren't my favorite personality traits. But as I continue to age, I realize superficial likability isn't really that much of an asset. I'm kind of worried about
Re: [ccp4bb] not so good news (Steve Jobs RIP)
Bill So eloquently put, much of what you say resonates with me. Very sad day. Cheers Ashley Sent from my iPhone On 06/10/2011, at 3:13 PM, William G. Scott wgsc...@ucsc.edu wrote: On Oct 5, 2011, at 5:52 PM, Bosch, Juergen wrote: http://www.apple.com/stevejobs/ May innovation continue to lead the future. Jürgen I've been quite saddened about this all evening. Even though we knew it was coming, it is still incredibly sad, especially at his age (only 56). Although I never met him, through OS X, he made a fairly significant impact upon my life and career development (such as it is). The advent of OS X has had a significant impact upon both my professional and personal lives. Although I have worked with unix systems since the mid 1980s, it was only when OS X came along, and I made the switch (from SGI's Irix, which many of us remember fondly as a total pig of an OS), did I really come to enjoy using computers. As a crystallographer, a unix operating system really is essential I think, and when OS X appeared, a fundamental change took place in my office as a young assistant professor: I only needed one computer. Before, I had an enormous SGI sitting on a table, for doing science. With its stereographic hardware and dials, it put me back (well, actually, the taxpayer) about $12K for an R10,000, which had a 150MHz RISC processor. The sony trinatron monitor did a good job heating the room, especially in the summer. Then I had this little colored hunk of plastic, the first generation bottom-of-the-line iMac, upon which I wrote my papers and grant proposals, sitting on my desk. This left very little room in my office to mope and feel sorry for myself, which my wife tells me are the only things I am good at. OS X came on an extra CD packaged with an iBook I bought just before the Sept 11th attack. I remember, because I gave my first presentation using OS X 10.0.3 or something like that on that day, in Grenada, Spain, right next to the Alhambra. It was the worst talk I ever gave, but it wasn't the fault of the software. I was excessively nervous, so much so that my arch-competitor I guess took pity and tried to calm me down. Maybe I sensed something bad was about to happen. Anyway, it was an inauspicious start. I eventually made it home and put it on that colored piece of plastic in my office and discovered it really was a real unix operating system, albeit in its infancy. I sort of got lost in it for months at a time, and was determined to get all of the software used in macromolecular crystallography running on OS X. O was about the only thing at the time that seemed ready. What evolved from that was a crystallography on os x website, which started off as a chronicle of my pain, which I seem to feel an almost moral obligation to share and inflict upon everyone around me. (I work at a university that, frankly, isn't very good at infrastructural support, to put it mildly, and I wound up having to learn to do everything the hard way, by myself.) I think it is fair to say that Steve Jobs and OS X were some of the main things that made this, and my survival, possible. I've primarily used OS X I guess from the bottom up, but at the same time I was a bit of an Apple fan-boy in the sense that I got the first iPod when it came out, the first iPod Touch, the first MacBook Air, the first iPad, etc. I've never regretted any of those purchases, nor the purchase of the 20 or so Apple computers that I've acquired for work and family over the last decade or so. My newest OS X adventure has been into computer audio. I really still don't know much of anything about it, but again Apple has made it an enjoyable experience. I bitch and moan about iTunes as much as the next guy, but if you stop to think about it, it transformed the entire music industry, and turned what began as a network of illegal file sharing (Napster), something I regrettably missed out completely, into a legitimate business model. Some might look at this as co-opting, but I think there is a bit more to it than that. In any case, both from the perspective of hardware and software, Apple/Jobs completely changed how I listen to music. During that same decade I was starting out as an academic scientist, I had more or less given up on music almost completely. My oldest son now likes music (Pearl Jam, Joy Division, REM), but at the time he couldn't handle listening to it at all, probably as a consequence of being somewhere on the autistic spectrum. OS X provided an escape from all that for me, but it eventually also provided a relief for him I think, and through it he came to enjoy (some) music and now plays cello and electric guitar. So, I guess Steve Jobs has had a large, albeit indirect, influence on my life, and that of my family and research group. I never met him, but I've learned a bit about him through a
