Re: [ccp4bb] Superpositions: Deviation by Residue
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Jacob, Coot prints this information to the terminal, so you can start coot and 'tee' its output into a file. Tim On 11/28/2011 11:53 PM, Jacob Keller wrote: Let me refine my question (sorry for my lack of clarity): is there a program that will output the distances between the corresponding ca's of a superposition on a residue-by-residue basis, and not just a global RMSD value (doubtless these numbers are part of the superposition algorithm itself)? I want to plot these values as a function of residue number to show which parts of the structures deviate more or less from each other. Jacob - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO1KmAUxlJ7aRr7hoRAjy0AJ0WepnWXxMHVFfAE1oSX0rQ5BV4zQCggo/N OQ67cZNE7jMPZyXL4v1zOjE= =JXFJ -END PGP SIGNATURE-
Re: [ccp4bb] Disappearing crystals
Dear Christine, I had guessed that you had more salt in the protein solution than in the reservoir with a relatively low protein concentration. The protein is in: 100mM Sodium Acetate pH 5.5 with 150mM NaCl at a protein concentration of ~4.3mg/mL. and the reservoir is: (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and 30% PEG MME 550). We can suspect that no matter what temperature the equilibrium will move towards dilution in the drop. Basic suggestion: Set up a range from 20-30% PEG MME 550 with at least 150mM NaCl in the reservoir. Fine tuning: Add high MW PEG (10K or 20K 1-5%) it should have a stabilizing effect Try a range of salts apart from NaCl: The clasics are: Li2SO4, KSCN, MgCl2. Try additive amounts od MPD 0.5-3%. Try a small tight range of various pH. Enrico. On Mon, 28 Nov 2011 21:43:51 +0100, Harman, Christine christine.har...@fda.hhs.gov wrote: Hi, Thank you so much for your replies. A lot of you have mentioned fluctuations in temp as the major contributor. And a few of you have asked for more details of my protein/buffer and set up. To my knowledge, the tray has been kept at constant 20 C (in an incubator) with exception of course to when I remove the tray to view the drops. It could be possible that my inspection of the tray might have contributed to an increase in temp, but only temporarily. I am very careful about the time the drop sees intense light, but it is possible the temp could have changed enough to cause this problem. Just to give a few more details. My protein (a Fab fragment/peptide complex (hopefully) is in buffer containing 100mM Sodium Acetate pH 5.5 with 150mM NaCl at a protein concentration of ~4.3mg/mL. After setting up my drop with reservoir solution I add NaCl to well to give ~75mM NaCl to match ionic strength of protein in drop which is diluted 1:1 with well solution. I do hope this problem is temperature. Although I am a little sad to not be able to freeze those crystals I did see, I still consider myself lucky to get such good result from a condition right from the screen so there will be some definite optimization set ups with this condition. Could I safely say though that the crystals I observed are not salt..:) I guess that is one good thing to take from this. Any more suggestions on optimization would be very welcomed. Thanks again to all you, Christine -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Enrico Stura Sent: Monday, November 28, 2011 1:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Disappearing crystals When advice on crystallization is needed, it is important to give details of the protein concentration, the buffer the protein is in as well as the method used to grow the crystals. Problem: The crystallization conditions are essentially low salt: 100mM buffer and only 50mM CaCl2. So the buffer that the protein is in is very important !! Fluctuation in the reservoir/drop environment will lead to crystals dissolving. Solution: Balance the salt in the reservoir and in the protein:precipitant drop and make sure the temperature is kept constant. Since I do not have all the necessary information, the diagnosis and the solution proposed are likely to be wrong! Enrico. On Mon, 28 Nov 2011 19:19:49 +0100, YoungJin yj...@brandeis.edu wrote: On 11/28/11 12:04 PM, Harman, Christine wrote: Hi All, I have just noticed a very strange thing and need some help in understanding it. I recently found two crystals in a condition from a screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and 30% PEG MME 550). The small crystals appeared after a month and started to grow over the next 5 days after I first saw them (see pictures attached). I just check the same drop today and now the crystals are gone. So I was wondering what happened and if anyone experienced this before. Any insight or advice on what to do would be greatly appreciated. Thanks Christine Small 5 days later Hi Christine, I had similar experience. In my case, another crystal showed again with different size a few days later. Sometimes, it seems like it is a common event to others as well as I heard although my case only takes about a week to be crystallized. I'd rather wait or just set up again or in a slightly different way. Wish you well. Young-Jin -- 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
[ccp4bb] Phaser problem child killed: segmentation violation in phaser
Hi CCP4 community. While running the Phaser in the CCP4 GUI, It was terminated and giving the error message. #CCP4I TERMINATION STATUS 0 child killed: segmentation violation #CCP4I VERSION CCP4Interface 2.0.6 CCP4 Version ccp4-6.1.13 Thanks in advance. Sita Ram
[ccp4bb] Leader of Biophysics Facility at the Biozentrum Basel
The Biozentrum of the University of Basel, an internationally renowned Life Sciences research institute, invites applications for the Leader of its new Biophysics Facility. We are looking for an enthusiastic person that combines technical expertise with communication skills. The ideal candidate is a PhD level scientist with practical experience and excellent theoretical knowledge in biophysical methods for the characterization of biomacromolecules and their interactions, including optical spectroscopy (fluorescence, circular dichroism), microcalorimetry, analytical ultracentrifugation and surface plasmon resonance. He/she enjoys providing excellent user training and support for measurements and data evaluation, maintains and develops an assembly of state-of-the art instruments, contributes to practical and theoretical training for students and promotes scientific collaboration in the Biozentrum. The Biozentrum offers an outstanding scientific environment and a competitive salary, while Basel provides a high standard of living and a superb cultural atmosphere. Applications including CV, list of publications and a short summary of past experience, have to be transmitted in electronic form to zs-biozent...@unibas.ch. This position is open until filled; we aim for a starting date in 2012. For informal enquiries please contact Prof. S. Grzesiek (stephan.grzesiek-at-unibas.ch, phone: +41 (0) 61 267 21 00). The University of Basel is an equal opportunity employer and encourages applications from female candidates.
[ccp4bb] Postdoctoral position, Institut de Biologie Stucturale, Grenoble, France
Postdoctoral position, Institut de Biologie Stucturale, Grenoble, France A two year postdoctoral position for a biochemist/structural biologist is available in the Synchrotron Group at the Institute for Structural Biology (IBS) in Grenoble (France). The IBS has state-of-the-art equipments and facilities, including crystallization robots, NMR, EM and easy access to synchrotron beamlines. The subject is focused on the structural analysis of a CDK/Cyclin complex, in a drug design approach. The candidates should hold a Ph.D. in biochemistry or biophysics. A solid experience in protein _expression_, purification and biochemical characterization is required. Knowledge and experience in protein crystallography and in in silico docking will be an advantage. The candidates must be motivated, well-organized and able to work independently as well as a part of the team. Salary is in the range 30-35 k€, depending on experience. The position is immediately available and interested candidates should send their CV to franck.bo...@ibs.fr
[ccp4bb] Job opening for a structural biologist to join the “Biocrystallography and Structural Biology of Therapeutic Targets” group at the Institute for the Biology and Chemistry of Proteins, Lyon in
A 2 year postdoctoral position with support from the French National Research Agency (ANR) is available to study regulatory mechanisms of BMP-1/tolloid like proteinases ((BTPs; also known as procollagen C-proteinases) which are enzymes implicated in a number of key events during tissue morphogenesis and tissue repair. The research project proposed aims at a better understanding of the mechanism by which specific enhancer proteins regulate BTP activity, by solving the 3D structure of the complex of the substrate:enhancer complex. Our studies involve a multidisciplinary approach, which combines molecular- and structural biology along with classical biochemical procedures. A Ph.D. in biochemistry or biophysics, and a solid experience in crystallization and X-ray structure determination are required. Background knowledge in protein expression and purification is highly desirable. Salary in the range 30-35 k€, depending on experience. The position is immediately available (application deadline December 15, 2011) and interested candidates should send their CV, a letter of interest and contact information of 3 references to Dr. Nushin Aghajari; n.aghaj...@ibcp.fr and Dr. David Hulmes; d.hul...@ibcp.fr.
Re: [ccp4bb] Disappearing crystals
Christine, I have a couple more comments First, if those crystals come back, I would certainly try microseeding into *random *screens. You should be able to pick up new conditions that have a little more salt in them, and are therefore stable regarding the movement of water. (If you can't get the crystals back, I would try microseeding with the precipitate that you have.) Second, it's helpful to understand that the movement of water in and out of the drop is connected to heat *flows*, not temperature. The heat flow drives the movement of moisture (in addition to the salt etc. of course). Once you understand that you can easily get rid of e.g. condensation on the tape by putting e.g. a warm book on top of the plate in the cold room (or a book from the cold room under the plate at room temp). Similarly, you can prevent dilution of your drops by thinking about heat flows. Some say that it's essential to use Blundell and Johnson. Others say only Bergfors on crystallization, Lord of the Rings or even Harry Potter can provide the necessary inspiration. Good luck Patrick Ref for microseeding: Allan D’Arcy, Frederic Villarda, May Marsh. An automated microseed matrix-screening method for protein crystallization. Acta Crystallographica section D 63 (2007), 550–554. http://www.douglas.co.uk/mms.htm On Mon, Nov 28, 2011 at 8:43 PM, Harman, Christine christine.har...@fda.hhs.gov wrote: Hi, Thank you so much for your replies. A lot of you have mentioned fluctuations in temp as the major contributor. And a few of you have asked for more details of my protein/buffer and set up. To my knowledge, the tray has been kept at constant 20 C (in an incubator) with exception of course to when I remove the tray to view the drops. It could be possible that my inspection of the tray might have contributed to an increase in temp, but only temporarily. I am very careful about the time the drop sees intense light, but it is possible the temp could have changed enough to cause this problem. Just to give a few more details. My protein (a Fab fragment/peptide complex (hopefully) is in buffer containing 100mM Sodium Acetate pH 5.5 with 150mM NaCl at a protein concentration of ~4.3mg/mL. After setting up my drop with reservoir solution I add NaCl to well to give ~75mM NaCl to match ionic strength of protein in drop which is diluted 1:1 with well solution. I do hope this problem is temperature. Although I am a little sad to not be able to freeze those crystals I did see, I still consider myself lucky to get such good result from a condition right from the screen so there will be some definite optimization set ups with this condition. Could I safely say though that the crystals I observed are not salt..:) I guess that is one good thing to take from this. Any more suggestions on optimization would be very welcomed. Thanks again to all you, Christine -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Enrico Stura Sent: Monday, November 28, 2011 1:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Disappearing crystals When advice on crystallization is needed, it is important to give details of the protein concentration, the buffer the protein is in as well as the method used to grow the crystals. Problem: The crystallization conditions are essentially low salt: 100mM buffer and only 50mM CaCl2. So the buffer that the protein is in is very important !! Fluctuation in the reservoir/drop environment will lead to crystals dissolving. Solution: Balance the salt in the reservoir and in the protein:precipitant drop and make sure the temperature is kept constant. Since I do not have all the necessary information, the diagnosis and the solution proposed are likely to be wrong! Enrico. On Mon, 28 Nov 2011 19:19:49 +0100, YoungJin yj...@brandeis.edu wrote: On 11/28/11 12:04 PM, Harman, Christine wrote: Hi All, I have just noticed a very strange thing and need some help in understanding it. I recently found two crystals in a condition from a screen (0.05M Calcium chloride dihydrate, 0.1M M Bis-Tris pH6.5 and 30% PEG MME 550). The small crystals appeared after a month and started to grow over the next 5 days after I first saw them (see pictures attached). I just check the same drop today and now the crystals are gone. So I was wondering what happened and if anyone experienced this before. Any insight or advice on what to do would be greatly appreciated. Thanks Christine Small 5 days later Hi Christine, I had similar experience. In my case, another crystal showed again with different size a few days later. Sometimes, it seems like it is a common event to others as well as I heard although my case only takes about a week to be crystallized. I'd rather wait or just set up again or in a slightly different way. Wish you well. Young-Jin
[ccp4bb] (off topic) effective chemical lysis of E. coli under anaerobic conditions
Dear All, Apologies for the off-topic question. I'm seeking suggestions on the best way to achieve an effective, but not over-harsh, chemical lysis of E coli expressing an oxygen-sensitive Fe-S protein. We need to lyse anaerobically but do not have access to a sonicator that we can use in the glove box. We have used a proprietary detergent mix in previous attempts but have not been overly impressed with the results- lots of protein (more than I would expect based on past experiences with related enzymes) remains in the pellet. I've seen literature protocols based on Triton (up to 1.2%) but am worried (perhaps without basis) that this might interfere with downstream steps (reconstituting the Fe-S cluster and possibly crystallisation). The protein is His-tagged. Does anyone know a better way? Thanks in advance. Best wishes Jim -- Dr. James Spencer, Lecturer in Microbiology School of Cellular and Molecular Medicine Medical Sciences Building University of Bristol University Walk Bristol BS8 1TD jim.spen...@bristol.ac.uk http://www.bristol.ac.uk/cellmolmed/staff/spencer.html -- Tel: (44) (0) 117 331 2084 Fax: (44) (0) 117 331 2091
Re: [ccp4bb] (off topic) effective chemical lysis of E. coli under anaerobic conditions
0.5% octyl-thioglucopyranoside + lysozyme + DNAse in 30 mM TRIS pH 8.0 - degassed/sparged and supplemented with whatever reducing agent you need. Stir in cold for 30-40 minutes at a ratio of 3ml of solution to 1g of cell pellet (wet). Artem On Tue, Nov 29, 2011 at 8:49 AM, Jim Spencer, Cellular and Molecular Medicine jim.spen...@bristol.ac.uk wrote: Dear All, Apologies for the off-topic question. I'm seeking suggestions on the best way to achieve an effective, but not over-harsh, chemical lysis of E coli expressing an oxygen-sensitive Fe-S protein. We need to lyse anaerobically but do not have access to a sonicator that we can use in the glove box. We have used a proprietary detergent mix in previous attempts but have not been overly impressed with the results- lots of protein (more than I would expect based on past experiences with related enzymes) remains in the pellet. I've seen literature protocols based on Triton (up to 1.2%) but am worried (perhaps without basis) that this might interfere with downstream steps (reconstituting the Fe-S cluster and possibly crystallisation). The protein is His-tagged. Does anyone know a better way? Thanks in advance. Best wishes Jim -- Dr. James Spencer, Lecturer in Microbiology School of Cellular and Molecular Medicine Medical Sciences Building University of Bristol University Walk Bristol BS8 1TD jim.spen...@bristol.ac.uk http://www.bristol.ac.uk/**cellmolmed/staff/spencer.htmlhttp://www.bristol.ac.uk/cellmolmed/staff/spencer.html -- Tel: (44) (0) 117 331 2084 Fax: (44) (0) 117 331 2091
Re: [ccp4bb] Superpositions: Deviation by Residue
Dear Crystallographers, Thanks very much for all who responded to my request. Below is a compiled list of the various ways to skin this crystallographic cat! Jacob -Moleman ca plot distance-CCP4's superpose-distance matrix analysis, e.g. http://www.csb.yale.edu/userguides/datamanip/ddmp/ddmp_descrip.html, or other programs-Lsqkab, with the deltas card-If you use the MatchMaker tool in UCSF Chimera to make the superposition, it has an option to show the corresponding sequence alignment. The sequence alignment will have an RMSD header running across the top, which is a bar graph of the RMSD values. You can the alignment's Headers-Save... menu item to save the numerical values to a file if you want. OR If you already have the structures superimposed on your own, you can use Chimera's Match-Align tool to create a superposition-based sequence alignment, and do the same thing with its RMSD header.-Coot prints this information to the terminal, so you can start coot and 'tee' its output into a file.-Various fortran/homebrew programs available from individuals-LGA server: http://proteinmodel.org/AS2TS/LGA/lga.html On Tue, Nov 29, 2011 at 3:44 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Jacob, Coot prints this information to the terminal, so you can start coot and 'tee' its output into a file. Tim On 11/28/2011 11:53 PM, Jacob Keller wrote: Let me refine my question (sorry for my lack of clarity): is there a program that will output the distances between the corresponding ca's of a superposition on a residue-by-residue basis, and not just a global RMSD value (doubtless these numbers are part of the superposition algorithm itself)? I want to plot these values as a function of residue number to show which parts of the structures deviate more or less from each other. Jacob - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO1KmAUxlJ7aRr7hoRAjy0AJ0WepnWXxMHVFfAE1oSX0rQ5BV4zQCggo/N OQ67cZNE7jMPZyXL4v1zOjE= =JXFJ -END PGP SIGNATURE- -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Crystallographic and Physiologic but not Solution Oligomers
Dear Crystallographers, does anyone have any nice examples/references of proteins which form demonstrably physiologically-relevant oligomers in crystals, but which do not appear to do so in solution? I am thinking particularly that domains of membrane proteins which oligomerize primarily through their TM domains would be subject to this, but all comers are certainly welcome... Jacob -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Withdrawal of subscription
To whom it may concern, I am writing this in request to the cancellation of my subscription to CCP4BB mail. It was indeed a very rewarding and a knowledgeable experience to be a part of this family. With regards Debanjan Choudhuri
Re: [ccp4bb] Withdrawal of subscription
With such a nice parting letter, we find it disheartening to let you go. James On Nov 29, 2011, at 7:44 PM, debanjan choudhuri wrote: To whom it may concern, I am writing this in request to the cancellation of my subscription to CCP4BB mail. It was indeed a very rewarding and a knowledgeable experience to be a part of this family. With regards Debanjan Choudhuri
Re: [ccp4bb] Withdrawal of subscription
Just you wait--he'll be back...won't he? On Tue, Nov 29, 2011 at 11:11 PM, James Stroud xtald...@gmail.com wrote: With such a nice parting letter, we find it disheartening to let you go. James On Nov 29, 2011, at 7:44 PM, debanjan choudhuri wrote: To whom it may concern, I am writing this in request to the cancellation of my subscription to CCP4BB mail. It was indeed a very rewarding and a knowledgeable experience to be a part of this family. With regards Debanjan Choudhuri -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
