[ccp4bb] crystal size
Dear all, I was wondering if anyone had some practical advice in regards to increasing the size of a crystal. Currently my enzyme forms these rather nice cubic and very sharp 0.1mm (in 25% peg 5000, 0.1M ammonium sulphate, 0.1M Tris ph 7.0) crystals following optimisation of the pH and precipitant concentrations of the initial screening condition and by playing with various increasing levels of protein concentration 20-60mg/ml. Has anyone had success with additive screens in increasing crystal size and what were they? Any advice would be useful thanks Vince
Re: [ccp4bb] crystal size
Anything that slows growth potentially could help. In one case, I found by re-dissolving drops with showers of small crystals I could get one or two huge ones. I just added 2 microliters of water to the handing drop and allowed it to re-equilibrate. Changing temperature or any physical variable that effects solubility can help. Adding small amounts of glycerol can help, etc. As with all aspects of crystallization, it is highly empirical. On Sep 9, 2008, at 11:23 PM, Vincenzo Carbone wrote: Dear all, I was wondering if anyone had some practical advice in regards to increasing the size of a crystal. Currently my enzyme forms these rather nice cubic and very sharp 0.1mm (in 25% peg 5000, 0.1M ammonium sulphate, 0.1M Tris ph 7.0) crystals following optimisation of the pH and precipitant concentrations of the initial screening condition and by playing with various increasing levels of protein concentration 20-60mg/ml. Has anyone had success with additive screens in increasing crystal size and what were they? Any advice would be useful thanks Vince
Re: [ccp4bb] crystal size
We've had lots of success with the additive screens (3 boxes of 24 additives) sold by Hampton Research Vincenzo Carbone wrote: Dear all, I was wondering if anyone had some practical advice in regards to increasing the size of a crystal. Currently my enzyme forms these rather nice cubic and very sharp 0.1mm (in 25% peg 5000, 0.1M ammonium sulphate, 0.1M Tris ph 7.0) crystals following optimisation of the pH and precipitant concentrations of the initial screening condition and by playing with various increasing levels of protein concentration 20-60mg/ml. Has anyone had success with additive screens in increasing crystal size and what were they? Any advice would be useful thanks Vince begin:vcard fn:Fred. Vellieux (Ph.D) n:Vellieux (Ph.D);Fred. email;internet:[EMAIL PROTECTED] tel;work:+33 438789605 version:2.1 end:vcard
Re: [ccp4bb] crystal size
I feel compelled to add: rather nice cubic 0.1mm presumably means they're 90x90x90um? At any state-of-the-art synchrotron beamline with a half-way decently-sized beam, that's an absolute stonker, we rarely optimize more if they're that large. phx. Vincenzo Carbone wrote: Dear all, I was wondering if anyone had some practical advice in regards to increasing the size of a crystal. Currently my enzyme forms these rather nice cubic and very sharp 0.1mm (in 25% peg 5000, 0.1M ammonium sulphate, 0.1M Tris ph 7.0) crystals following optimisation of the pH and precipitant concentrations of the initial screening condition and by playing with various increasing levels of protein concentration 20-60mg/ml. Has anyone had success with additive screens in increasing crystal size and what were they? Any advice would be useful thanks Vince
Re: [ccp4bb] crystal size
Oh, another thing you can try is simply increasing the drop volume, especially large sitting drops. This will slow down equilibration, and there will be proportionately more material On Sep 9, 2008, at 11:23 PM, Vincenzo Carbone wrote: Dear all, I was wondering if anyone had some practical advice in regards to increasing the size of a crystal. Currently my enzyme forms these rather nice cubic and very sharp 0.1mm (in 25% peg 5000, 0.1M ammonium sulphate, 0.1M Tris ph 7.0) crystals following optimisation of the pH and precipitant concentrations of the initial screening condition and by playing with various increasing levels of protein concentration 20-60mg/ml. Has anyone had success with additive screens in increasing crystal size and what were they? Any advice would be useful thanks Vince
Re: [ccp4bb] truncate and anisotropy
-Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Eleanor Dodson Sent: 09 September 2008 17:11 To: William G. Scott Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] truncate and anisotropy I dont think you need to worry much about the strong stacking reflections; TRUNCATE really only modifies the weakest data - and anything 3Sigma is barely altered . There is a slightly wrong estimate of sigma for that resolution shell, but in practice it seems to have no observable effect.. Anisotropy is another question and drastically increases the number of weak reflections in the higher resolution shells which must be bad.. Ian - have you analysed any such data sets? I must admit I've not looked specifically at the effect of anisotropy, pseudo-translation etc on the Wilson distribution. But it should (in principle at least) be possible to do something about this, i.e. one could obtain the experimental PDF from the measured intensities and use that in the Bayes integrals to get the corrected intensities, rather than naively using the Wilson PDFs as we are doing now. One can get the CDFs (cumulative distribution function) for the centric acentric data by plotting Z (normalised intensity) = I/(eS) (where e = epsilon factor, S = mean epsilon-corrected intensity for shell) along the x axis, against (i-.5)/n (where i is the serial no of the reflection after sorting in increasing Z value and n is the no of centric or acentric reflections) along y. The PDF is then just the 1st derivative of the CDF, which can be obtained by finite differences, maybe with some smoothing to smooth out any 'noise' due to sampling. I don't know how well this scheme would work in practice, it would need some testing. The question remains whether to do this for all centric data in one go similarly for all acentric data, or to split each type of data into resolution shells. In the first case one is assuming that the PDF is the same at all resolutions, which may not be appropriate say in the case of a pseudo-translation, because usually such a translation applies uniformly to all atoms only at low-medium resolution at high resolution it often breaks down, so one would expect a reversion to the Wilson PDF at high res. How this plays out in the case of anisotropy I've no idea (one would certainly need to derive an overall anisotropic B factor correction), you would have to suck it see. If you decide to do it in resolution shells the centric data might prove to be a problem: there may not be enough of them in each shell to give a reliable estimate of the PDF, in which case you might say have to revert to using all centric data in one go, while still keeping the acentric data in shells. -- Ian Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing [EMAIL PROTECTED] and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
Re: [ccp4bb] crystal size
Hi Vince, There is a very nice paper by Monika Budayova-Spano in Acta Cryst 63D 2007 339-347. A methodology and an instrument for the temperature- controlled optimization of crystal growth. Best, Wally On Sep 10, 2008, at 2:23 AM, Vincenzo Carbone wrote: Dear all, I was wondering if anyone had some practical advice in regards to increasing the size of a crystal. Currently my enzyme forms these rather nice cubic and very sharp 0.1mm (in 25% peg 5000, 0.1M ammonium sulphate, 0.1M Tris ph 7.0) crystals following optimisation of the pH and precipitant concentrations of the initial screening condition and by playing with various increasing levels of protein concentration 20-60mg/ml. Has anyone had success with additive screens in increasing crystal size and what were they? Any advice would be useful thanks Vince Walter R.P. Novak, Ph.D. Postdoctoral Fellow Rosenstiel Basic Medical Research Center Brandeis University 415 South St. MS 029 Waltham, MA 02454-9110 Phone: (781) 736-4944 Fax: (781) 736-2405
Re: [ccp4bb] crystal size
My usual first approach to increasing crystal size is to decrease precipitant and increase protein concentration, keeping the mixture in the proper nucleation zone, but providing more protein for crystal growth. Minimizing dust or particulates by filtration and centrifugation of reagents and protein may help, by reducing nucleation sites in the solution. Crystallization at a lower temperature may also result in fewer, but larger crystals. Or you could try seeding. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED] Walter Novak wrote: Hi Vince, There is a very nice paper by Monika Budayova-Spano in Acta Cryst 63D 2007 339-347. A methodology and an instrument for the temperature-controlled optimization of crystal growth. Best, Wally On Sep 10, 2008, at 2:23 AM, Vincenzo Carbone wrote: Dear all, I was wondering if anyone had some practical advice in regards to increasing the size of a crystal. Currently my enzyme forms these rather nice cubic and very sharp 0.1mm (in 25% peg 5000, 0.1M ammonium sulphate, 0.1M Tris ph 7.0) crystals following optimisation of the pH and precipitant concentrations of the initial screening condition and by playing with various increasing levels of protein concentration 20-60mg/ml. Has anyone had success with additive screens in increasing crystal size and what were they? Any advice would be useful thanks Vince Walter R.P. Novak, Ph.D. Postdoctoral Fellow Rosenstiel Basic Medical Research Center Brandeis University 415 South St. MS 029 Waltham, MA 02454-9110 Phone: (781) 736-4944 Fax: (781) 736-2405 -
Re: [ccp4bb] crystal size
Hi! I would suggest to try another temperature or seeding. It worked for me before. Good luck! Nurit. Anyone who has never made a mistake has never tried anything new. Albert Einstein Nurit Mirkin Bril Date: Wed, 10 Sep 2008 08:02:53 -0400 From: [EMAIL PROTECTED] Subject: Re: [ccp4bb] crystal size To: CCP4BB@JISCMAIL.AC.UK My usual first approach to increasing crystal size is to decrease precipitant and increase protein concentration, keeping the mixture in the proper nucleation zone, but providing more protein for crystal growth. Minimizing dust or particulates by filtration and centrifugation of reagents and protein may help, by reducing nucleation sites in the solution. Crystallization at a lower temperature may also result in fewer, but larger crystals. Or you could try seeding. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED] Walter Novak wrote: Hi Vince, There is a very nice paper by Monika Budayova-Spano in Acta Cryst 63D 2007 339-347. A methodology and an instrument for the temperature-controlled optimization of crystal growth.Best, Wally On Sep 10, 2008, at 2:23 AM, Vincenzo Carbone wrote: Dear all, I was wondering if anyone had some practical advice in regards to increasing the size of a crystal. Currently my enzyme forms these rather nice cubic and very sharp 0.1mm (in 25% peg 5000, 0.1M ammonium sulphate, 0.1M Tris ph 7.0) crystals following optimisation of the pH and precipitant concentrations of the initial screening condition and by playing with various increasing levels of protein concentration 20-60mg/ml. Has anyone had success with additive screens in increasing crystal size and what were they? Any advice would be useful thanks VinceWalter R.P. Novak, Ph.D. Postdoctoral Fellow Rosenstiel Basic Medical Research Center Brandeis University 415 South St. MS 029 Waltham, MA 02454-9110 Phone: (781) 736-4944 Fax: (781) 736-2405 - _ Stay up to date on your PC, the Web, and your mobile phone with Windows Live. http://clk.atdmt.com/MRT/go/msnnkwxp1020093185mrt/direct/01/
[ccp4bb] program to rotate atoms with anisotropic B factors
Dear all, I am looking for a program that can rotate atoms with anisotropic B factors - not only change coordinates of the atom, but to modify the Anisou coefficients too. If you are confident that there is none let me know too ;-) Pavel
Re: [ccp4bb] program to rotate atoms with anisotropic B factors
Pavel There is a program from the computer stone-age called XP (nothing to do with Microsoft who though of the name many years later) that does exactly what you need (with the SGEN instruction). It is part of the Bruker SHELXTL software, maybe you can find a nearby small-molecule crystallographer who happens to have it. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Wed, 10 Sep 2008, Pavel Plevka wrote: Dear all, I am looking for a program that can rotate atoms with anisotropic B factors - not only change coordinates of the atom, but to modify the Anisou coefficients too. If you are confident that there is none let me know too ;-) Pavel
[ccp4bb] Lab-techician position
Laboratory Technician Position at the CIC bioGUNE (Bilbao-Spain) Applications are invited for a Laboratory Technician in the group of Dr. Nicola G. A. Abrescia at the Structural Biology Unit at the CIC bioGUNE (http://www.cicbiogune.es/), a multidisciplinary research institute located near Bilbao, Spain. Candidates should hold an academic qualification in biosciences, good communication skills, be computer literate, enthusiastic and committed. Job requirements: Experience in tissue culture methods (cell maintenance and passaging, transfection techniques, etc) and in protein expression (E.coli, mammalian or insect cells) are required. Experience in recombinant DNA techniques (PCR, cloning, mutagenesis, plasmid preparations etc.) and in protein purification methods will be of additional benefit. The selected candidate will provide assistance to the researchers ensuring the smooth operational activities of the wet-lab. The postholder’s duties will range from basic laboratory management (reagent ordering, liaison with Spanish and international suppliers) to independent research in line with the activities of the group. Environment: The CIC bioGUNE carries basic and biomedical research at international level. Since it was first opened in January 2005 the Institute has been able to build an exciting and vibrant multidisciplinary environment with more than 20 Group Leaders and with state-of-the-art infrastructure from protein production and structural analysis to functional studies. Salary ranges between 14.588 to 20.072 euros (gross salary/year) depending on experience and with an annual salary increase similar to that of the cost of living in Spain (presently an annual ~4%). The contract is permanent but the position will be reviewed based on performance every three years. Applications (in English) including the CV and 2 references should be sent before October, the 8th 2008 to [EMAIL PROTECTED] with Ref.3302 in the subject. Selected candidates will be invited for an on-site interview at the end of October. Nicola G. A. Abrescia, PhD Division Structural Biology Wellcome Trust Centre for Human Genetics University of Oxford Roosevelt Drive Headington OX3 7BN = New address from 1/10/2008: Nicola G. A. Abrescia, PhD Structural Biology Unit CIC Biogune Bizkaia Technology Park, Bld 800 48160 Derio - Spain email: [EMAIL PROTECTED]
[ccp4bb] Art Robbins Phoenix User Group
To the CCP4bb members who are/have been users of Art Robbins' Phoenix robot: Would anyone be interested in joining a Phoenix robot user group/bulletin board? Someplace where users would be able to discuss their experiences, as well as to learn from the experiences of others. I will be tallying posted responses and be in contact with Art Robbins Instruments. Cheers, David Critton Department of Molecular Biology, Cell Biology Biochemistry Brown University, Providence, RI
Re: [ccp4bb] xds and Saturn troubles
Hi all, thanks a lot for all the replies! Using the very current version of XDS fixed all the problems, really nice data, structure solved, yet another one for SSGCID. re input file: attached the XDS.INP file that worked for the Saturn 944+ detector. Thanks again! Jan 2008/9/4 Jan Abendroth [EMAIL PROTECTED] Hi all, a bit off-topic, but maybe someone knows an answer. I am trying to run xds on data collected on our new sparkling Saturn detector. Using the basic xds script that worked beautifully for older Saturn94 detectors, xds now dies with the following error message during the INIT phase: !!! ERROR !!! ILLEGAL RAXIS_COMPRESSION_RATIO Any ideas what this might be? Thanks a lot Jan -- Jan Abendroth deCODE biostructures Seattle / Bainbridge Island WA, USA work: JAbendroth_at_decode.is home: Jan.Abendroth_at_gmail.com -- -- Jan Abendroth deCODE biostructures Seattle / Bainbridge Island, WA, USA work: JAbendroth_at_decode.com home: Jan.Abendroth_at_gmail.com Saturn2_XDS.INP Description: Binary data
Re: [ccp4bb] truncate ignorance
Well, I was pointing to the Sivia David (1994) paper because I thought it might be helpful in the discussion about how to convert intensities to amplitudes. The paper is probably not so well known in the PX community, so I decided that I would advertise it on this BB. However, since I am not one of the authors, I feel that it is inappropriate for me to go into a detailed defense of every sentence and equation which is written in it. The paper is clear and speaks for itself. I can only recommend a careful reading of it. I will nevertheless make some general comments in response to the criticism that was raised: Quoting Ian Tickle [EMAIL PROTECTED]: But there's a fundamental difference in approach, the authors here assume the apparently simpler prior distribution P(I) = 0 for I 0 P(I) = const for I = 0. As users of Bayesian priors well know this is an improper prior since it integrates to infinity instead of unity. Despite of their disparaging name, improper priors can be used in Bayesian analysis without major difficulties (at least for estimation problems), provided that the posterior integrates to a finite value. If you object to the use of an improper prior in the Sivia David paper, I suggest to use a prior where P(I) = 0 for I 0 as well as for I 10^30 and P(I) = constant in between these two boundaries. Technically speaking this would then be a proper prior, but for all intents and purposes it would not make any difference at all. This means that, unlike the case I described for the French Wilson formula based on the Wilson distribution which gives unbiased estimates of the true I's and their average, the effect on the corrected intensities of using this prior really will be to increase all intensities (since the mean I for this prior PDF is also infinite!), hence the intensities and their average must be biased ( I'm sure the same goes for the corresponding F's). Two different bias concepts in this statement : ... unbiased estimates of the true I's and their average... (1) Regarding unbiased estimates of the true I's: The use of a Wilson prior does by no means guarantee that the posterior expectation values will be unbiased estimates of the true I's. Whether one uses the Wilson prior or the naive prior of Sivia David, the posterior probability distribution on I will be a truncated normal distribution (see French Wilson, appendix A). There is nothing which allows us to claim that the expectation value (which is what we use as estimate of the true intensity) over such a posterior will be unbiased (whichever prior was used !). Simple example: take a reflection which has true F=0. The posterior probability distribution p_J(J|I) (here I am using the French Wilson notation) will be a truncated normal (see French Wilson, appendix A) and its expectation value E_J(J|I) will thus always be greater than 0, even if the Wilson prior is used ! Both the the French Wilson and the Sivia David procedures will yield a biased estimate of the true intensity: the estimate will always be greater than 0 (the true value), whatever the measured I is. (2) Regarding intensity averages: Here, your argument about bias seems to be about averages of intensities computed in resolution shells, i.e. you are concerned that the corrected I's, averaged over all reflections in a given resolution bin, should equal the average of the uncorrected intensities in the same resolution bin. I would like to see a proof that the French Wilson procedure actually achieves this goal (none is given in the French Wilson paper - they are actually not addressing this issue). But apart from this, I wonder whether this is of any relevance at all. Why would this be so important ? Why are you so concerned that the intensity averages over many different reflections in a resolution bin is a quantity which should at all price be conserved ? In any event, I think that the discussions about bias on corrected intensities is a somewhat academic side-issue. The real reason why we use the truncate procedure is not so much do get corrected I's, but rather to get estimates of the amplitudes. In that sense, I think that the important message conveyed in the Sivia David paper is the following: the awkward truncated Gaussian pdf's in intensity space (whichever prior was used...) are transformed to well-behaved Gaussian-like pdf's in amplitude-space. This is an argument in favouring F's rather than I's (even corrected I's) for subsequent crystallographic computations. In that regime (i.e. in the regime where we accept that the posterior probability distribution on F's is close to a Gaussian), the estimator given by equation (11) in Sivia David is actually unbiased ! Side argument: to use the French Wilson procedure, it is necessary to know the crystal spacegroup (in order to apply the correct statistical weights for the various classes of reflections). To use the Sivia David procedure, you don't need to know the
[ccp4bb] (Off topic) lN2 generator for sale
Hi all (sorry, don't know where else to post, suggestions welcome) We have for sale a 3 year-old Rigaku LN40 liquid nitrogen generator, which we no longer need thanks to new house lN2 supply. For a picture, see here: http://www.rigaku.com/cryo/nitrogen.html It was working reliably when we last used it (3 months ago), but it requires: A) a cooling water supply B) (recommended) a service contract with Rigaku (it needed two significant fixes while we had it) Price is negotiable, about 1/3rd of its cost new -- but it's a buyer's market :) And you'd have to arrange transport for something the size of a large cupboard. Enquiries to me directly. Cheers
Re: [ccp4bb] Art Robbins Phoenix User Group
Hi, Whatever happened to the Yahoo group pxrbtx (PX robotics), that was started by Ingo Koendorfer in 2006? There haven't been any postings since Feb. 2007. Maybe time to revive it? Derek On Sep 10, 2008, at 15:19, Critton, David wrote: To the CCP4bb members who are/have been users of Art Robbins' Phoenix robot: Would anyone be interested in joining a Phoenix robot user group/ bulletin board? Someplace where users would be able to discuss their experiences, as well as to learn from the experiences of others. I will be tallying posted responses and be in contact with Art Robbins Instruments. Cheers, David Critton Department of Molecular Biology, Cell Biology Biochemistry Brown University, Providence, RI
[ccp4bb] postdoctoral position at the Salk Institute
We would like to invite applications for postdoctoral position in Prof. Senyon Choe's Structural Biology Laboratory at The Salk Institute (http://sbl.salk.edu/~choe) to study structures of membrane proteins. Strong background in membrane protein biochemistry is required. Those interested should send CV to [EMAIL PROTECTED] or directly to [EMAIL PROTECTED]
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