[ccp4bb] Mac OSX 10.7 Lion
Is there any opinion or experience about whether Lion is ready for crystallographic use? Should I upgrade? Phil
Re: [ccp4bb] Mac OSX 10.7 Lion
Phil, On Fri, Sep 9, 2011 at 4:28 AM, Phil Evans p...@mrc-lmb.cam.ac.uk wrote: Is there any opinion or experience about whether Lion is ready for crystallographic use? Should I upgrade? MacPyMOL works fine on Lion. Cheers, -- Jason -- Jason Vertrees, PhD PyMOL Product Manager Schrodinger, LLC (e) jason.vertr...@schrodinger.com (o) +1 (603) 374-7120
[ccp4bb] drops swelling
Dear Crystallographers, I have set hanging drop trays with 2ul of protein and 2 ul of resorvior solution. I have seen in some cases the drops are swelling. My protein buffer has 15% glycerol in it. This is happening mainly when I have peg 400 or peg MME or MPD or Jeffamine in the buffer condition. Could any one suggest a remedy for this. with regards Anita
Re: [ccp4bb] drops swelling
Yes - add glycerol to the reservoir - 15% glycerol has a huge influence on the vapour diffusion gradients Poul On 09/09/2011, at 13.22, anita p crystals...@gmail.com wrote: Dear Crystallographers, I have set hanging drop trays with 2ul of protein and 2 ul of resorvior solution. I have seen in some cases the drops are swelling. My protein buffer has 15% glycerol in it. This is happening mainly when I have peg 400 or peg MME or MPD or Jeffamine in the buffer condition. Could any one suggest a remedy for this. with regards Anita
Re: [ccp4bb] drops swelling
The following is simply a quote from a recent post by Enrico Stura. Probably has a lot to do with what you are finding: Glycerol is also great to reduce nucleation. If you decide to add glycerol to the protein solution (for solubility, but in your case it might be for stability reasons), you also need to have a higher (double) glycerol concentration in the reservoir else you will risk finding that your drops will get biggger and not smaller. This note of caution applies to vapour diffusion set ups as equilibration can be tricky in such context: Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth Des. 11 :2755–2762. http://pubs.acs.org/doi/abs/10.1021/cg101364m Good luck Dave (but really, this is all Enrico) On 9/9/2011 7:22 AM, anita p wrote: Dear Crystallographers, I have set hanging drop trays with 2ul of protein and 2 ul of resorvior solution. I have seen in some cases the drops are swelling. My protein buffer has 15% glycerol in it. This is happening mainly when I have peg 400 or peg MME or MPD or Jeffamine in the buffer condition. Could any one suggest a remedy for this. with regards Anita
Re: [ccp4bb] drops swelling
Dear Anita You are seeing the effects of a very modest reduction of vapor pressure of water with polymers. You could add salt to your reservoir and you could insure your drops are at the same temperature as your reservoir - not easy in conventional constant temp incubators. George Sent via BlackBerry by ATT -Original Message- From: anita p crystals...@gmail.com Sender: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK Date: Fri, 9 Sep 2011 19:22:53 To: CCP4BB@JISCMAIL.AC.UK Reply-To: anita p crystals...@gmail.com Subject: [ccp4bb] drops swelling Dear Crystallographers, I have set hanging drop trays with 2ul of protein and 2 ul of resorvior solution. I have seen in some cases the drops are swelling. My protein buffer has 15% glycerol in it. This is happening mainly when I have peg 400 or peg MME or MPD or Jeffamine in the buffer condition. Could any one suggest a remedy for this. with regards Anita
Re: [ccp4bb] drops swelling
Anita, see the message I posted yesterday on the CCP4bb: http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg22638.html or google stura glycerol Re: [ccp4bb] How to help crystal grow bigger In your case the sentence you need is: You need to have a higher (double) glycerol concentration in the reservoir else you will risk finding that your drops will get biggger and not smaller. This note of caution applies to vapour diffusion set ups as equilibration can be tricky in such context: Vera,L., Czarny, B., Georgiadis, D., Dive, V., Stura, E.A. (2011) Practical Use of Glycerol in Protein Crystallization. Cryst. Growth Des. 11 :2755–2762. http://pubs.acs.org/doi/abs/10.1021/cg101364m Enrico. On Fri, 09 Sep 2011 13:22:53 +0200, anita p crystals...@gmail.com wrote: Dear Crystallographers, I have set hanging drop trays with 2ul of protein and 2 ul of resorvior solution. I have seen in some cases the drops are swelling. My protein buffer has 15% glycerol in it. This is happening mainly when I have peg 400 or peg MME or MPD or Jeffamine in the buffer condition. Could any one suggest a remedy for this. with regards Anita -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
[ccp4bb] TCA or acetone precipitation of proteins
Hi Everyone, Sorry for the naive and non-CCP4 question. Is it possible to precipitate proteins (TCA, acetone) from a sample that has already been stored in protein loading dye? The protein is too dilute in my current sample and I basically want to load all of the sample (100uL) in a single well in the gel. Unfortunately, I already added protein dye with SDS and all. Cheers and thanks. Raji -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] TCA or acetone precipitation of proteins
Are you sure that 100 uL won't fit in your loading well? Commercial 10x10 cm minigels have quite large loading wells. I think it's possible to squeeze as much as 50 uL into a standard 10-well, 1 mm gel. If you are using typical discontinuous SDS-PAGE with a stacking gel the sample will be concentrated to a tight band shortly after applying running current. While you may be able to precipitate your protein with TCA, incomplete precipitation and losses on resolubilization may not help you as much as you would like. Small centrifugal filters may get you down to 50 uL dead-stop volume, but that might be enough to load into a single well on a standard gel. A possible option is to add some dry BioGel with a low exclusion volume (e.g., P-6) but this will be tricky to get just right with such a small volume of sample. I've done this kind of volume reduction successfully with larger samples (1-2 mL), before there were centrifugal ultrafilters. 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 9/9/2011 8:57 AM, Raji Edayathumangalam wrote: Hi Everyone, Sorry for the naive and non-CCP4 question. Is it possible to precipitate proteins (TCA, acetone) from a sample that has already been stored in protein loading dye? The protein is too dilute in my current sample and I basically want to load all of the sample (100uL) in a single well in the gel. Unfortunately, I already added protein dye with SDS and all. Cheers and thanks. Raji
[ccp4bb] Postdoctoral post available
Structural studies on ribosomal RNA modification enzymes: A postdoctoral position in X-ray crystallography is available immediately in the group of Dr. Graeme L. Conn, in the Department of Biochemistry at Emory University School of Medicine in Atlanta GA, USA. This opening is part of a NIH-funded project to study aminoglycoside-resistance enzyme structures and interactions with the 30S ribosomal subunit. The successful candidate will coordinate joint efforts and work directly with Dr. Christine Dunham’s group (also at Emory) who are experts in ribosome crystallography and collaborators on this project. Both laboratories are located in recently renovated space in the Department of Biochemistry where we are two of four groups with diverse research interests that form a critical mass in the area of structural biology. We have shared state-of-the-art facilities for crystallization, in-house X-ray data collection and other biophysical approaches. We also have regular synchrotron beamtime at the Advanced Photon Source (APS) through membership of SER-CAT and at the NE-CAT beamline (awarded to the Dunham lab). Interested applicants must have a Ph.D. in biochemistry, molecular biology or structural biology and experience in X-ray crystallographic. Experience working with RNA would be an advantage but not essential. The ideal candidate will be highly motivated, possess excellent communication skills and the ability to work in a collaborative and team-oriented environment. To apply, please e-mail a CV including a list of publications, a brief statement of experience and scientific interests, and contact information for three references to: gc...@emory.edu. _ Graeme L. Conn, Ph.D. Department of Biochemistry Emory University School of Medicine 1510 Clifton Road NE - Room 4135 Atlanta GA 30322 USA http://www.biochem.emory.edu/conn
Re: [ccp4bb] TCA or acetone precipitation of proteins
Is it possible to precipitate proteins (TCA, acetone) from a sample that has already been stored in protein loading dye? The protein is too dilute in my current sample and I basically want to load all of the sample (100uL) in a single well in the gel. Unfortunately, I already added protein dye with SDS and all. Methanol/chloroform precipitation is both more effiecient than TCA or acetone and it can do exactly what you want it to do. Wessel, D., and Fugge, U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Analytical Biochemistry, 1984, 138:141-143 Or google it - there are many online protocols. - Dima
Re: [ccp4bb] TCA or acetone precipitation of proteins
Is it ok for you to heat your sample? If so, you could near-boil your sample in a heating block, say at 95 degrees C, in order to concentrate it. I've done this in regular 1.5 mL eppendorf tubes with either the cap open or where I've punctured the top of the cap with a narrow bore needle (in order to relieve vapor pressure from water evaporation). At 95C, reducing 100 uL down to ~20 uL probably will take 15-30 minutes. If you're working on a membrane protein, this is probably not a route you can take. Alternatively, maybe a speed vac will work? HTH- Brad On Fri, Sep 9, 2011 at 8:57 AM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Everyone, Sorry for the naive and non-CCP4 question. Is it possible to precipitate proteins (TCA, acetone) from a sample that has already been stored in protein loading dye? The protein is too dilute in my current sample and I basically want to load all of the sample (100uL) in a single well in the gel. Unfortunately, I already added protein dye with SDS and all. Cheers and thanks. Raji -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] How to help crystal grow bigger
Hi SnowDeer- What's your precipitant? Have you tried lowering that as much as possible? This will likely delay nucleation and crystal growth but the crystals will also likely be larger (and perhaps more well ordered). Of course, you may reach a lower limit where you prohibit nucleation. You'll have to determine this empirically. How about smaller reservoir volumes while keeping the drop volume the same? Have you tried setting up and storing trays at lower temperatures, 10 C or less? Sitting drop vs. hanging drop? Different oils in your vapor diffusion experiments, like Al's oil? Also, I had success with growing large crystals by setting up microbatch drops under oil. If you'd like details, I can provide them off-board. Cheers- Brad On Thu, Sep 8, 2011 at 3:52 AM, SnowDeer huanxu...@gmail.com wrote: To Boaz: I seperated the large crystals and checked its diffraction already. While the diffraction is quite poor, only several dots could be seen. T_T I washed the seeds twice with my buffer and seed the drop immediately after setting it. Thanks for your advices and I will try the additives. To Charles, Enrico, Bernie Tiantian: Thanks for your kindly advices, I set different conditions for the buffers with glycerol and different protein/reservoir volume ratios already following your instructions. :) To David: Hmm...my crystals are smaller than the smallest loop T_T. It's quite hard for me to pick them up (due to my clumsy fingers...lol). Thanks for your advices and the review. I have another question: I usually stored my protein samples aliquots at -80 degree and thaw the small aliquots when I need to use. While my senior said it will harm the protein so she suggested to keep them at 4 degree. So it is possible that I got the small crystals coz the freezing and thawing alter the proteins? Thanks very much. SnowDeer On Tue, Sep 6, 2011 at 9:39 PM, David Waterman dgwater...@gmail.comwrote: Dear SnowDeer, Just how small is too small? If you have access to a microfocus beamline you might find you can collect decent diffraction data from crystals with dimensions in the single digit microns. Fishing tiny crystals is difficult, but something like the MicroMesh tennis racquet mounts can help. Having multiple crystals on the same pin is in fact a rather helpful way of screening lots of samples. Please don't discount your crystals _only_ because they are small! Shameless plug: there is a review on microcrystallography here that you might find interesting. http://www.tandfonline.com/doi/abs/10.1080/0889311X.2010.527964 Best wishes -- David On 5 September 2011 09:06, SnowDeer huanxu...@gmail.com wrote: Dear All: Recently I am working on a protein which can already grow nice pyramid-like crystals after the condition was optimized, while the crystals are too small to be picked up. The crystals grew quite fast and densely, so I tried to put 100ul paraffin oil inside the 600ul reservoir solution or put the plate under 16 degree to slow down the evaporation, while the crystals were still the same. I also tried macro or micro seeding with or without the paraffin oil. Macroseeding would give a larger crystal (not very nice) with many small crystals in the drop even I washed the seeds carefully. For microseeding, the same small crystals grew. I don't have many experiences in crystallography, so I have no idea how to make it grow bigger... Any suggestion is most welcome. Thanks. SnowDeer
[ccp4bb] ALMN
Dear CCP4'ers In the input instructions for ALMN it says:FIND peak maxpek [RMS] [OUTPUT filename] Read peak threshold and maximum number of peaks. MAXPEK is the maximum number of peaks to find (default = 20). Up to MAXPEK peaks above PEAK will be found, and all symmetry related peaks generated. If the keyword RMS is present, then the peak threshold is PEAK * RMS density, otherwise PEAK is the absolute threshold in the scaled map. Question: RMS density of what and what is the formula used? Rex Palmer http://www.bbk.ac.uk/biology/our-staff/emeritus-staff http://rexpalmer2010.homestead.com
Re: [ccp4bb] refmac and DNA
On Thu, 2011-09-08 at 16:58 +0200, Miguel Ortiz Lombardía wrote: Perhaps the Nd/DN issue was solved between revisions 3470 and 3628 ? Indeed. I just built the rev 3633 and it works fine. Autobuild scripts used to have some problems on Lucid, but this time it worked perfectly. This evolved into a coot question, but I am still perplexed as to why refmac would complain only about T and not A/G/C. Cheers, Ed -- After much deep and profound brain things inside my head, I have decided to thank you for bringing peace to our home. Julian, King of Lemurs
Re: [ccp4bb] ALMN
Hi Rex I would assume it's the RMS deviation of the rotation function value from the mean. If so then it's defined in the same way as the population standard deviation as given here: http://en.wikipedia.org/wiki/Standard_deviation Cheers -- Ian On Fri, Sep 9, 2011 at 4:20 PM, REX PALMER rex.pal...@btinternet.comwrote: Dear CCP4'ers In the input instructions for ALMN it says: FIND peak maxpek [RMS] [OUTPUT filename] Read peak threshold and maximum number of peaks. MAXPEK is the maximum number of peaks to find (default = 20). Up to MAXPEK peaks above PEAK will be found, and all symmetry related peaks generated. If the keyword RMS is present, then the peak threshold is PEAK * RMS density, otherwise PEAK is the absolute threshold in the scaled map. Question: RMS density of what and what is the formula used? Rex Palmer http://www.bbk.ac.uk/biology/our-staff/emeritus-staff http://rexpalmer2010.homestead.com
Re: [ccp4bb] ALMN
Rex, sorry, addendum to my last post: the grid points in the Eulerian space of the cross-RF (which is what I assume you're talking about) do not occupy equal volumes in angular space as they would do in a Euclidean space (e.g. a normal electron density map). For example the volume of any grid point on the beta=0 180 sections is zero (these sections actually each consist of a line of points, and a line of course occupies no volume). Therefore it's necessary to weight the values in both the mean and the RMSD by the volume of each grid point (= sin(beta)). Whether ALMN actually does this or not would require an examination of the source code, though it's probably quicker to ask Eleanor! Cheers -- Ian On Fri, Sep 9, 2011 at 5:26 PM, Ian Tickle ianj...@gmail.com wrote: Hi Rex I would assume it's the RMS deviation of the rotation function value from the mean. If so then it's defined in the same way as the population standard deviation as given here: http://en.wikipedia.org/wiki/Standard_deviation Cheers -- Ian On Fri, Sep 9, 2011 at 4:20 PM, REX PALMER rex.pal...@btinternet.comwrote: Dear CCP4'ers In the input instructions for ALMN it says: FIND peak maxpek [RMS] [OUTPUT filename] Read peak threshold and maximum number of peaks. MAXPEK is the maximum number of peaks to find (default = 20). Up to MAXPEK peaks above PEAK will be found, and all symmetry related peaks generated. If the keyword RMS is present, then the peak threshold is PEAK * RMS density, otherwise PEAK is the absolute threshold in the scaled map. Question: RMS density of what and what is the formula used? Rex Palmer http://www.bbk.ac.uk/biology/our-staff/emeritus-staff http://rexpalmer2010.homestead.com
Re: [ccp4bb] Mac OSX 10.7 Lion
Hi Phil: I've found few, if any advantages. I fear for the future. I've had problems getting coot to run stereo due to the X11 implementation in 10.7. Apart from that, no major problems with crystallographic software. Lion greedily uses memory, and any computer I have with less than 4 gig of memory has become extremely sluggish as a consequence of the upgrade. Ideally, you need 8 gig. Even with that, on my 2010 mini that I use for music playback, I regressed to 10.6.8, because of the audio interface. (It seems less robust, more prone to dropouts and now lacks integer mode output). Sara has been screaming at me for the last two weeks (nothing us usual in of itself) because Apple decided to get rid of Save As. Xcode and the compiler set is free again on 10.7. I've put some suggestions here for how to get rid of the most annoying new features: http://sage.ucsc.edu/~wgscott/xtal/wiki/index.php/Lion_upgrade_notes All the best, Bill On Sep 9, 2011, at 1:28 AM, Phil Evans wrote: Is there any opinion or experience about whether Lion is ready for crystallographic use? Should I upgrade? Phil William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/
Re: [ccp4bb] Mac OSX 10.7 Lion
I have noticed, in new versions of OSes, that there generally is rampant violation of the concept of if it ain't broken, don't fix. Shouldn't there be more moments of delight, when you see they have solved a previous poorly-engineered feature with an elegant solution? But a lot of the time, you have to try to figure out how to do what you did in the last version in this version, albeit for no net gain. Back to punch cards! The Friday Curmudgeon On Fri, Sep 9, 2011 at 1:09 PM, William Scott wgsc...@ucsc.edu wrote: Hi Phil: I've found few, if any advantages. I fear for the future. I've had problems getting coot to run stereo due to the X11 implementation in 10.7. Apart from that, no major problems with crystallographic software. Lion greedily uses memory, and any computer I have with less than 4 gig of memory has become extremely sluggish as a consequence of the upgrade. Ideally, you need 8 gig. Even with that, on my 2010 mini that I use for music playback, I regressed to 10.6.8, because of the audio interface. (It seems less robust, more prone to dropouts and now lacks integer mode output). Sara has been screaming at me for the last two weeks (nothing us usual in of itself) because Apple decided to get rid of Save As. Xcode and the compiler set is free again on 10.7. I've put some suggestions here for how to get rid of the most annoying new features: http://sage.ucsc.edu/~wgscott/xtal/wiki/index.php/Lion_upgrade_notes All the best, Bill On Sep 9, 2011, at 1:28 AM, Phil Evans wrote: Is there any opinion or experience about whether Lion is ready for crystallographic use? Should I upgrade? Phil William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/ -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Mac OSX 10.7 Lion
On Sep 9, 2011, at 11:09 AM, William Scott wrote: (nothing us usual in of itself) I forgot to mention how delightful the spelling auto-correction feature can be. (It should have read nothing unusual in and of itself). That, at least, can be turned off.
[ccp4bb] Postdoctoral fellow position in structural biology
Peking University, College of Life Sciences seeks to recruit dedicated postdoctoral fellows to carry out structural and functional investigation of cell surface receptors in nervous systems. The successful candidates will focus his/her research on elucidation of molecular mechanism with which these receptors play the part in neuronal development and the immune function in central nervous system (CNS). These include their functions in axon guidance, synapse formation and neuron-glia interaction that mediates key immunological protection for CNS. The project is the close collaborative efforts between Professor Jia-huai Wang's structural biology lab and Professor Yan Zhang's neuroscience lab. Position requires PhD. degree with a good background in molecular biology, protein chemistry and crystallography. Highly motivated individuals are encouraged to apply. The position will be based at Peking University in Beijing, China, with opportunity of doing some research at Harvard Medical School in Boston. Peking University is one of the leading academic institutions in China. The College of Life Sciences is equipped with the state-of-art facilities in structural biology for X-ray crystallography, NMR, EM and single molecule studies. The well-known beautiful campus is located at Chinas most advanced academic center with more than 100 research institutes and a dozen highly respected universities. Historically Peking University also has a large international community and attracts students and postdoctoral fellows from all over the world. Interested candidates please email a cover letter, CV, 3 reference names, as well as email address and telephone number to Drs. Jia-huai Wang or Yan Zhang at jw...@red.dfci.harvard.edu and yanzh...@pku.edu.cn. For more information on Wangs group, please see the website: http://wang.dfci.harvard.edu The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.
Re: [ccp4bb] Mac OSX 10.7 Lion
Still doesn't beat my all-time favorite, an early Microsoft spell-checker that changed diffract to defrocked. I forgot to mention how delightful the spelling auto-correction feature can be. (It should have read nothing unusual in and of itself). That, at least, can be turned off.
Re: [ccp4bb] Mac OSX 10.7 Lion
Shoshana Wodak once told me that Word kept suggesting Shoeshine Kodak as the correct spelling of her name. I just tried my copy of Word and it seems to have improved. Frances = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** f...@bernstein-plus-sons.com *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Fri, 9 Sep 2011, Patrick Loll wrote: Still doesn't beat my all-time favorite, an early Microsoft spell-checker that changed diffract to defrocked. I forgot to mention how delightful the spelling auto-correction feature can be. (It should have read nothing unusual in and of itself). That, at least, can be turned off.
