Re: [ccp4bb] Phasing with Many Monomers/AU
Dear all, I don't know how closely this relates to what James pointed at, but regarding the aspect of NCS information usage in the heavy-atom finding/refinement stage, I would like to mention two programs of my knowledgethat are able to detect NCS in a set of putative heavy atom sites froma substructure solution trial: Professs (CCP4) and SitCom. Probaby I missed others ... These are useful in particular for cases like heavy atom-soaked structures, where site occupancies are partial and hence peak heights a weak indicator ofcorrectness, but the binding sites are not random i.e. still comply to the NCS. Both programs are based on the identification of matching triangles among HA sites. (meaning that the approach is less suited for cases with less than 3 sites per monomer). SitCom extracts HA site matches, no matter whether (closed) rotational NCS or purely translational NCS is present; and it can try to determine an unknown NCS order or apply an expected one. No matter which program used, the main benefit in my opinion is telling correct sites (NCS-conform) from wrong ones, so that ideally the filtering results in an improved substructure and HA phase set. Kind regards, Fabio Am 1/23/14 1:00 AM, schrieb CCP4BB automatic digest system: Date:Tue, 21 Jan 2014 18:24:34 -0800 From:James Holton jmhol...@lbl.gov Subject: Re: Phasing with Many Monomers/AU [...] but your initial problems are going to be phasing. Ideally what you'd want is a way of folding back NCS information into the heavy atom finding and phase refinement process, but I know of no programs that actually do that. In fact, both molecular replacement and heavy-atom finding are hindered by this pseodo-translation rather than helped by it. Personally, I blame the fact that methods developers seldom get their hands on interesting datasets like yours. [...] -- Dr. rer. nat. Fabio Dall'Antonia European Molecular Biology Laboratory c/o DESY Notkestraße 85, Bldg. 25a D-22603 Hamburg phone: +49 (0)40 89902-178 fax:+49 (0)40 89902-149 e-mail: fabio.dallanto...@embl-hamburg.de
[ccp4bb] Postdoctoral Research Fellow (Structural Biology)
Dear Colleagues, Description for a postdoctoral position in the lab of Dr Anne-Marie Krachler, University of Birmingham, UK; Post is available from 1 March 2014 Postdoctoral Research Fellow (Structural Biology) Applications are invited for a highly motivated post-doctoral researcher with expertise in structural biology to join the group of Dr Anne-Marie Krachler (https://sites.google.com/site/krachlerlab/) at the Institute of Microbiology and Infection, University of Birmingham. The position is funded by the BBSRC and is part of an exciting project which aims to study a group of bacterial adhesins which bind host cell membrane lipids. This project will entail structural and biophysical studies of this novel adhesin family and adhesin-ligand complexes, with the aim to utilize them as tools to investigate host cellular lipids and potentially as antimicrobials (adhesion inhibitors). Candidates must have a PhD in biology, biochemistry, biophysics or a related discipline and experience in all aspects of protein crystallography, from protein expression and purification through crystallisation and structure determination. They should also have an interest in working on structural and mechanistic questions related to host-pathogen interactions. Previous experience in studying protein-ligand interactions is highly desirable and a background in studying lipid-binding proteins would be advantageous. Applicants will be expected to work closely with colleagues from different disciplines and to assist junior researchers in their work. Candidates should have excellent written and oral communication skills and be highly organized and able to prioritize their own work with minimal supervision. Applications will be accepted until 2 February 2014. The University of Birmingham is a family-friendly work environment and we are happy to discuss flexible work arrangements. To download the details and submit an electronic application online visit: www.hr.bham.ac.uk/jobshttp://www.hr.bham.ac.uk/jobs (post number 47725).
[ccp4bb] Neutrons in Biology and Biotechnology 2014 - ILL - Final Announcement
Dear all, This is the final announcement for the Neutrons in Biology and Biotechnology meeting which will be held at the Institut Laue Langevin on the 19th - 21st February 2014. The programme has an exciting list of speakers and concludes with an open ILL/PSB colloquium from Tim Hunt (Nobel Laureate 2001). The meeting will highlight recent results in fundamental biology and biotechnology, while also introducing new areas of high biological interest where neutron methods may be of importance. The programme is now available online on http://www.ill.fr/nibb2014. The flyer is available on the website. The deadline for submission of oral contributions has now passed. However there are a few remaining spaces before we hit our limit and poster abstracts can be submitted until the registration deadline which is the 31st January 2014. We hope that you will join us in what is expected to be an exciting meeting. Best wishes, The organizing committee M. Blakeley, O.Byron, T. Forsyth, G. Fragneto, F. Gabel, M. Haertlein, A. Martel, E. Mitchell, E. Mossou, T. Nylander, J. Richardson ps: apologies if you get multiple versions of this mail Estelle Mossou Life sciences group Institut Laue Langevin, Grenoble Tel: +33476209441
Re: [ccp4bb] Two P1 xtals with same xtal contacts give 2 different asymmetric units
You dont say whether the unit cells are the same? In cases like this I sbmit both sets of coordinates to PISA - to get the preferred biological unit and check whether each structure has similar contacts. PISA is distributed with CCP4 but the version at the EBI pdb gives prettier results! Eleanor On 23 January 2014 15:19, Yong Wang wang_yon...@lilly.com wrote: Gabriel, Could this be just different but equivalent way of defining the asu? Ignoring one of the two tetramers and just focusing on the one tetramer that looks different in your case, the following picture assumes objects ABCD form a tetramer and repeat themselves in P1. You can have one trimer (ABC) plus a D from a symmetry related object as enclosed in the blue box. Then the other equivalent assembly is two dimers (BD and AC) as enclosed in the red box. This assumes that the “void” you referred to actually contains electron density for one monomer, not real void as having empty density. The equivalent assembly of asu can happen to any crystal form but if you try to keep the equivalent molecules together, it may appear more easily in P1 due to the arbitrary origin shift in P1. Cheers, *Yong Wang, Ph.D. Research Advisor, Discovery Chemistry Research* Eli Lilly Company Phone: 317-655-9145 Lilly Corporate Center DC 0403 Fax: 317-651-6333 Indianapolis, IN 46285 wang_y...@lilly.com CONFIDENTIALITY NOTICE: This e-mail message from Eli Lilly and Company (including all attachments) is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Gabriel Moreno *Sent:* Wednesday, January 22, 2014 3:50 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Two P1 xtals with same xtal contacts give 2 different asymmetric units Dear CCP4 Contributors, I have a bit of a mystery: Two co-crystals that I picked up from the same grid tray (the two conditions vary slightly in %PEG and [salt], both indexed as P1 (apo structure normally crystallizes in P3221). One dataset was indexed, integrated and scaled with HKL2000. The other was processed with MOSFILM (could not process in HKL2000). Downstream processing for both sets was done exactly the same in PHENIX. Though both asymmetric units contain two complete tetramers, the interesting thing is that the configuration of monomers is different between the solutions. One contains one complete tetramer, one trimer (with a void where the fourth monomer would be), and one monomer on off on its own. The asymmetric unit of the other dataset solution also contains a complete tetramer, but then has two dimers. Close analysis of contacts between symmetrically related molecules reveals that the crystal packing is exactly the same between the two solutions from the two datasets. Also, the Rwork and Rfree for both models are 0.18 and 0.20. Other quality indices are also comparable between the two sets. Here's my question: Does this phenomenon reveal anything important, or is this type of thing just seen sometimes with P1 solutions from crystals of the same protein and condition (more or less). I hope I have been clear. Thanks! Gabriel image001.png
[ccp4bb] Postdoc Opening-Structural Biology/Chemical Biology
We are inviting applications for a postdoctoral position immediately available in Dr. Jiang’s laboratory at Drug Discovery Division of University of Wisconsin-Madison School of Pharmacy. An ideal candidate will undertake a project that applies multidisciplinary approaches (e.g., enzymology, mass spectrometry, X-ray crystallography, high-throughput screening, cell biology) to study the roles of protein glycosylation in epigenetic regulation. Further information can be found at the page: http://pharmacy.wisc.edu/jiang-lab. Highly motivated candidates with PhD degrees in structural biology, chemical biology, biochemistry, molecular biology, cellular biology or related fields are encouraged to apply. Please send CV and the contact information of three references to Dr. Jiang at jji...@pharmacy.wisc.edu.
[ccp4bb] PhD studentship opportunities in Leeds
Dear BB, If you are, or know, a keen, talented, motivated student looking for a PhD, then there are several opportunities within the Faculty of Biological Sciences at the University of Leeds. One project involves the use of stapled peptides to investigate inhibition of protein-protein interactions in cancer development and progression, another will look at Structure based drug design of anti vitals against a Hemorrhagic Fever Virus. See: http://www.fbs.leeds.ac.uk/staff/tae/EPSRC_Advert_JB_TE_RF.pdf http://www.fbs.leeds.ac.uk/staff/tae/EPSRC_Advert_TAE_AJW.pdf For more info. The deadline is short (20th Feb), appointments will be competitive,and the student must be in place at Leeds by April… Email me with informal enquiries. Ed T.A.Edwards Ph.D. Deputy Director Astbury Centre for Structural Molecular Biology Ass. Professor, School of Molecular and Cellular Biology Garstang 8.53d University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.fbs.leeds.ac.uk/staff/tae/ http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE http://www.fbs.leeds.ac.uk/staff/profile.php?un=bmbtae --You can't possibly be a scientist if you mind people thinking that you're a fool. - Wonko the Sane
[ccp4bb] Crystallography Comics
Hi all, I thought the bulletin board might be interested to know that one of the contributors on Popular Science is featuring a webcomic series on X-ray crystallography. The first post went up yesterday and can be found here ( http://www.popsci.com/blog-network/boxplot/x-tallographers ). If nothing else it may provide amusing material for the undergrad lectures. Cheers, Katherine -- Nil illegitimo carborundum* - *Didactylos
Re: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules
Chen, Dioxane is not easy to work with, exactly for the reasons you describe. There is one thing you did not mention, which I know to be an additional issue: the quality of the dioxane. I do not know if you need good quality (whatever that is) but it is a fact that crystallization works with dioxane from some manufacturer/lots and not with others. I have never figured out why this is so. For a paper on kinetics and reservoir volume discussion, I would read the work by Forsythe et al (http://journals.iucr.org/d/issues/2002/10/01/ic0013/vidsup.html, doi:10.1107/S0907444902014208) Basically you will find that this paper says that well volume does not matter much. Content of your drop matters a lot. In your case, having a very volatile component, equilibration would be really fast. One way to get away from all that is to do batch experiments, where there is no active transport from a drop into a well. But your question remains very good: you need an oil (?) that will not permit water or a volatile organic substance to escape. I am not sure what would do the trick. Dioxane is a decent cryo-protectant by itself. I would think about adding gooey things like low molecular weight PEG to try to get the drops better behaved. Evaporation of the dioxane will still be an issue, but with a higher viscosity you may not have to chase your crystals through the drop. Hope this helps a little. Mark -Original Message- From: Chen Zhao c.z...@yale.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Wed, Jan 22, 2014 11:00 pm Subject: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules Dear all, I am now optimizing a hit which contains about 30% 1,4-dioxane using hanging-drop vapor diffusion at 25 degree. I am having a hard time to reproduce the results: most of the times the drops are either dry in one day or full of precipitate, and only occasionally could I get small crystals. Is there a way to control the vapor diffusion process, like using oil to seal the reservoir? (I know paraffin is permeable to dioxane) Also if someone could refer me to studies on the effects of reservoir volume and surface area to the crystallization kinetics, that would be very helpful. I am also seeking for recommendations for freezing crystals in this condition. What kind of cryoprotectant has a higher chance? Another problem is that when I tried to freeze the crystals, the drop dries super rapidly, and the crystals will dissolve if I add reservoir buffer. But I would assume good cryoprotectant could do the job. On the other hand, this points back to my previous question on dioxane-impermeable oil. If this magic oil exists, I could use it to seal the drop when freezing. Thank you for help! Sincerely, Chen
Re: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules
Dear Mark, Thank you so much for your reply. Interestingly, after several days I posted this question, I found both my previous crystals and precipitant completely dissolved, but instead much larger crystals start to appear. However, most of them are intergrown together, only few are single. And I think I have to get the right timing and freeze them soon, otherwise it may dissolve again. For cryoprotectant, I am also thinking of starting from low molecular weight PEG. Hopefully at least I could manage to get one or two crystals out of a drop. Finger crossed... Best, Chen On Fri, Jan 24, 2014 at 6:31 PM, Mark van der Woerd mjvdwo...@netscape.netwrote: Chen, Dioxane is not easy to work with, exactly for the reasons you describe. There is one thing you did not mention, which I know to be an additional issue: the quality of the dioxane. I do not know if you need good quality (whatever that is) but it is a fact that crystallization works with dioxane from some manufacturer/lots and not with others. I have never figured out why this is so. For a paper on kinetics and reservoir volume discussion, I would read the work by Forsythe et al ( http://journals.iucr.org/d/issues/2002/10/01/ic0013/vidsup.html, doi:10.1107/S0907444902014208)http://dx.doi.org/10.1107/S0907444902014208 Basically you will find that this paper says that well volume does not matter much. Content of your drop matters a lot. In your case, having a very volatile component, equilibration would be really fast. One way to get away from all that is to do batch experiments, where there is no active transport from a drop into a well. But your question remains very good: you need an oil (?) that will not permit water or a volatile organic substance to escape. I am not sure what would do the trick. Dioxane is a decent cryo-protectant by itself. I would think about adding gooey things like low molecular weight PEG to try to get the drops better behaved. Evaporation of the dioxane will still be an issue, but with a higher viscosity you may not have to chase your crystals through the drop. Hope this helps a little. Mark -Original Message- From: Chen Zhao c.z...@yale.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Wed, Jan 22, 2014 11:00 pm Subject: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules Dear all, I am now optimizing a hit which contains about 30% 1,4-dioxane using hanging-drop vapor diffusion at 25 degree. I am having a hard time to reproduce the results: most of the times the drops are either dry in one day or full of precipitate, and only occasionally could I get small crystals. Is there a way to control the vapor diffusion process, like using oil to seal the reservoir? (I know paraffin is permeable to dioxane) Also if someone could refer me to studies on the effects of reservoir volume and surface area to the crystallization kinetics, that would be very helpful. I am also seeking for recommendations for freezing crystals in this condition. What kind of cryoprotectant has a higher chance? Another problem is that when I tried to freeze the crystals, the drop dries super rapidly, and the crystals will dissolve if I add reservoir buffer. But I would assume good cryoprotectant could do the job. On the other hand, this points back to my previous question on dioxane-impermeable oil. If this magic oil exists, I could use it to seal the drop when freezing. Thank you for help! Sincerely, Chen
Re: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules
Another thing to try when cryoprotecting any crystals grown at room temp using a volatile precipitant is to first transfer the crystal tray to 4 degrees, then quickly add a cold aliquot of a viscous cryoprotectant to the drop. Evaporation is considerably slower at 4 degrees, and I prefer to use glycerol as cryoprotectant because it significantly slows down the random walk of the crystals through the drop. It makes it so much easier to fish crystals out of the drop if they're not moving at lightning speed... Place the tray at 4 degrees about 20-30 minutes prior to opening the drop. Any longer incubation at lower temp can result in extra nucleation or crystal dissolution. BTW, good luck trying to use a really volatile organic as a cryoprotectant. Every few years I try it and the crystals just get blown out of the cryoloop by the nitrogen gas stream. Diana On Jan 24, 2014, at 5:31 PM, Mark van der Woerd mjvdwo...@netscape.netmailto:mjvdwo...@netscape.net wrote: Chen, Dioxane is not easy to work with, exactly for the reasons you describe. There is one thing you did not mention, which I know to be an additional issue: the quality of the dioxane. I do not know if you need good quality (whatever that is) but it is a fact that crystallization works with dioxane from some manufacturer/lots and not with others. I have never figured out why this is so. For a paper on kinetics and reservoir volume discussion, I would read the work by Forsythe et al (http://journals.iucr.org/d/issues/2002/10/01/ic0013/vidsup.html, doi:10.1107/S0907444902014208)http://dx.doi.org/10.1107/S0907444902014208 Basically you will find that this paper says that well volume does not matter much. Content of your drop matters a lot. In your case, having a very volatile component, equilibration would be really fast. One way to get away from all that is to do batch experiments, where there is no active transport from a drop into a well. But your question remains very good: you need an oil (?) that will not permit water or a volatile organic substance to escape. I am not sure what would do the trick. Dioxane is a decent cryo-protectant by itself. I would think about adding gooey things like low molecular weight PEG to try to get the drops better behaved. Evaporation of the dioxane will still be an issue, but with a higher viscosity you may not have to chase your crystals through the drop. Hope this helps a little. Mark -Original Message- From: Chen Zhao c.z...@yale.edumailto:c.z...@yale.edu To: CCP4BB CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Sent: Wed, Jan 22, 2014 11:00 pm Subject: [ccp4bb] Control the crystallization process in the presence of small volatile organic molecules Dear all, I am now optimizing a hit which contains about 30% 1,4-dioxane using hanging-drop vapor diffusion at 25 degree. I am having a hard time to reproduce the results: most of the times the drops are either dry in one day or full of precipitate, and only occasionally could I get small crystals. Is there a way to control the vapor diffusion process, like using oil to seal the reservoir? (I know paraffin is permeable to dioxane) Also if someone could refer me to studies on the effects of reservoir volume and surface area to the crystallization kinetics, that would be very helpful. I am also seeking for recommendations for freezing crystals in this condition. What kind of cryoprotectant has a higher chance? Another problem is that when I tried to freeze the crystals, the drop dries super rapidly, and the crystals will dissolve if I add reservoir buffer. But I would assume good cryoprotectant could do the job. On the other hand, this points back to my previous question on dioxane-impermeable oil. If this magic oil exists, I could use it to seal the drop when freezing. Thank you for help! Sincerely, Chen UT Southwestern Medical Center The future of medicine, today.
