[ccp4bb] screen kit
hello all, is there any screen kit that is highly effective for the crystallization of protein-nucleic acids complexes? deng.
Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo
Hi, I would like to get recommendations for a proper cryo solution for a crystallization hit from the Hampton crystal screen Ammonium di- hydrogen phosphate 0.4M. I tried increasing glycerol up to 30% with the same ammonium phosphate concentration or increasing glycerol up to 30% in the presence of 1.3M ammonium phosphate. Both gave iced up drops (I only tried quick and dirty tests by dipping a cover glass in liquid nitrogen). Thank you. -Chris
Re: [ccp4bb] Pymol question
Dear Maher, as far as I know pymol uses the APBS (http://www.poissonboltzmann.org) for the calculations and your question is answered in the FAQ: http://www.poissonboltzmann.org/apbs/frequently-asked-questions/what-are-the-units-of-electrostatic-potential (kT/e) I don't know if this also answers your second question, but you could let us know in case it does. Tim On Wed, May 25, 2011 at 03:58:17PM -0700, Maher Alayyoubi wrote: Hi everyone, I have two questions: 1- Does anybody know what are the units on the display ruler after you calculate the vaccum electrostatics using pymol? 2- What are the default kT/e values used by pymol? Thank you, Maher -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo
Dear Chris, I have not used this particular condition, but as a rule of thumb I use like with like, e.g. glycerol, ethylene glycol, PEG400 etc. with PEG conditions, and saltlike compounds (sucrose, xylitol, salts) for salt conditions. In your case I would try glucose or xylitol or just look what happens if you increase the ammonium phosphate concentration to say 2M without adding glycerol. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris Ulens Sent: Thursday, May 26, 2011 12:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo Hi, I would like to get recommendations for a proper cryo solution for a crystallization hit from the Hampton crystal screen Ammonium di- hydrogen phosphate 0.4M. I tried increasing glycerol up to 30% with the same ammonium phosphate concentration or increasing glycerol up to 30% in the presence of 1.3M ammonium phosphate. Both gave iced up drops (I only tried quick and dirty tests by dipping a cover glass in liquid nitrogen). Thank you. -Chris
Re: [ccp4bb] Problems in refinement
How very odd! I have no ideas on the Zn phenonema - what do the R factor plots look like against resolution - is there some aberrant reflection which was part of the FreeR set? The theory is that excluding 5% of the data should not affect the model seriously at all.. Re the 2nd point. Two things - I would first check that the occupancy is actually correct in the file - I have messed that up at times.. And then maps do sometimes have weird pits and peaks - presumably related to missing data or imperfect scaling - I guess that could cause such a problem in one region of the map.. Eleanor On 05/26/2011 11:11 AM, Petr Kolenko wrote: Dear colleagues, I have two problems in two structure refinements using REFMAC5. 1) 1.8A resolution, zinc in the active site. Refinement using work reflections - ADP for Zinc was about 14. Final refinement including all reflections increase ADP to 20 or even higher values - followed by very high positive difference density in position of the zinc. I have tried also PHENIX, the same thing. I changed ADP manually to 14 and only calculated maps (no refinement) look good. May I deposit the structure using manually fixed ADP according to the best agreement to the observed and difference electron density? By the way, it is clear that this is zinc. 2) 1.9A resolution, about 600AA, all of them OK in electron density. But, somehow, about ten atoms give very strong positive electron density suggesting they are not taken into account in refinement. On the other hand, ADPs are reasonable and seem to be refined. All of these atoms are fully occupied. I tried to omit whole residues and build them again, but the maxima appeared again. Using of PHENIX resulted in no difference electron density for these atoms. I have also tried to take PHENIX output to REFMAC, but the maxima are there again. It is always one or two atoms from the same residues - sometimes Calpha, sometimes Cbeta, sometimes C, sometimes NH1, but still on the same five residues. Does anyone have any idea how to solve this problem? Many thanks for any response. Petr
Re: [ccp4bb] Histogram/Plot of Buried Surface Areas
Dear Jacob, I do not have exactly what you are asking about but some clues. I am not fond of using buried surface area as an indicator of anything in particular and always use energy estimates, however unreliable they may be. In J.Comp.Chem. 31(1),133-143, I put a probability plot for PISA to give erroneous oligomeric states as a function of complex's free energy (page 141, long-dashed line). In page 138, there's a plot showing correlation between free energy (as estimated by PISA) and buried surface energy. Combining the two, you may get a rough answer to your question :) Hope this helps, Eugene. On 26 May 2011, at 03:41, Jacob Keller wrote: Dear Crystallographers, is anyone aware of a reference or plot addressing buried surface area (or PISA output values) versus veracity of a complex? I am trying to determine the physiological relevance of a crystallographically-observed assembly, and would love to put my PISA output in the context of verified complexes versus crystal contacts. Thanks, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Problems in refinement
60% occupied Zn site perhaps ? Q2 do you have leftover atoms from a previous dual conformation refinement ? Try deleting the corresponding residues in a texteditor and not coot to ensure they are really gone, then rebuild the section into the new diff- map. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On May 26, 2011, at 6:11, Petr Kolenko kole...@imc.cas.cz wrote: Dear colleagues, I have two problems in two structure refinements using REFMAC5. 1) 1.8A resolution, zinc in the active site. Refinement using work reflections - ADP for Zinc was about 14. Final refinement including all reflections increase ADP to 20 or even higher values - followed by very high positive difference density in position of the zinc. I have tried also PHENIX, the same thing. I changed ADP manually to 14 and only calculated maps (no refinement) look good. May I deposit the structure using manually fixed ADP according to the best agreement to the observed and difference electron density? By the way, it is clear that this is zinc. 2) 1.9A resolution, about 600AA, all of them OK in electron density. But, somehow, about ten atoms give very strong positive electron density suggesting they are not taken into account in refinement. On the other hand, ADPs are reasonable and seem to be refined. All of these atoms are fully occupied. I tried to omit whole residues and build them again, but the maxima appeared again. Using of PHENIX resulted in no difference electron density for these atoms. I have also tried to take PHENIX output to REFMAC, but the maxima are there again. It is always one or two atoms from the same residues - sometimes Calpha, sometimes Cbeta, sometimes C, sometimes NH1, but still on the same five residues. Does anyone have any idea how to solve this problem? Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz
Re: [ccp4bb] Problems in refinement
Dear colleagues, Thanks for the responses. But, Zn occupancy is not an issue. There is positive maximum, not negative. And Q2, I tried to do it in coot and also in text editor. Both results were the same. And I double-checked the line for the atoms in the pdb file for ADP and occupancy. Regarding to Eleanor Dodson response and imperfect scaling, these weird peaks would be there after PHENIX refinement as well, or not? Thanks again. Petr 2011/5/26 Jürgen Bosch jubo...@jhsph.edu: 60% occupied Zn site perhaps ? Q2 do you have leftover atoms from a previous dual conformation refinement ? Try deleting the corresponding residues in a texteditor and not coot to ensure they are really gone, then rebuild the section into the new diff- map. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On May 26, 2011, at 6:11, Petr Kolenko kole...@imc.cas.cz wrote: Dear colleagues, I have two problems in two structure refinements using REFMAC5. 1) 1.8A resolution, zinc in the active site. Refinement using work reflections - ADP for Zinc was about 14. Final refinement including all reflections increase ADP to 20 or even higher values - followed by very high positive difference density in position of the zinc. I have tried also PHENIX, the same thing. I changed ADP manually to 14 and only calculated maps (no refinement) look good. May I deposit the structure using manually fixed ADP according to the best agreement to the observed and difference electron density? By the way, it is clear that this is zinc. 2) 1.9A resolution, about 600AA, all of them OK in electron density. But, somehow, about ten atoms give very strong positive electron density suggesting they are not taken into account in refinement. On the other hand, ADPs are reasonable and seem to be refined. All of these atoms are fully occupied. I tried to omit whole residues and build them again, but the maxima appeared again. Using of PHENIX resulted in no difference electron density for these atoms. I have also tried to take PHENIX output to REFMAC, but the maxima are there again. It is always one or two atoms from the same residues - sometimes Calpha, sometimes Cbeta, sometimes C, sometimes NH1, but still on the same five residues. Does anyone have any idea how to solve this problem? Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz -- Petr Kolenko kole...@imc.cas.cz http://kolda.webz.cz
Re: [ccp4bb] Problems in refinement
regarding the first question - is it now not much more common to refine only against work reflections, and not do a final refinement agains all reflections? (avoids the problem rather than solve it, I admit) regarding the second question - if you take the model from refmac and calculate a map using that and the original processed dataset (i.e. before it has seen refmac), do the peaks also show up? (I guess you have checked the pdb-file output from refmac carefully in a text editor?) Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 26 May 2011, at 12:11, Petr Kolenko wrote: Dear colleagues, I have two problems in two structure refinements using REFMAC5. 1) 1.8A resolution, zinc in the active site. Refinement using work reflections - ADP for Zinc was about 14. Final refinement including all reflections increase ADP to 20 or even higher values - followed by very high positive difference density in position of the zinc. I have tried also PHENIX, the same thing. I changed ADP manually to 14 and only calculated maps (no refinement) look good. May I deposit the structure using manually fixed ADP according to the best agreement to the observed and difference electron density? By the way, it is clear that this is zinc. 2) 1.9A resolution, about 600AA, all of them OK in electron density. But, somehow, about ten atoms give very strong positive electron density suggesting they are not taken into account in refinement. On the other hand, ADPs are reasonable and seem to be refined. All of these atoms are fully occupied. I tried to omit whole residues and build them again, but the maxima appeared again. Using of PHENIX resulted in no difference electron density for these atoms. I have also tried to take PHENIX output to REFMAC, but the maxima are there again. It is always one or two atoms from the same residues - sometimes Calpha, sometimes Cbeta, sometimes C, sometimes NH1, but still on the same five residues. Does anyone have any idea how to solve this problem? Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz
Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo
Dear Chris, Indeed,. according to McFerrin and Snell, 2002, Appl. Crystallogr., they recommend 30%(v/v) PEG400, or 35% EG (ethlylene glycol) or 30% PG (propylene glycol) However, they also mention the use of 35% (v/v) glycerol. regards Kristof On 26 May 2011, at 13:01, herman.schreu...@sanofi-aventis.com wrote: Dear Chris, I have not used this particular condition, but as a rule of thumb I use like with like, e.g. glycerol, ethylene glycol, PEG400 etc. with PEG conditions, and saltlike compounds (sucrose, xylitol, salts) for salt conditions. In your case I would try glucose or xylitol or just look what happens if you increase the ammonium phosphate concentration to say 2M without adding glycerol. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris Ulens Sent: Thursday, May 26, 2011 12:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo Hi, I would like to get recommendations for a proper cryo solution for a crystallization hit from the Hampton crystal screen Ammonium di- hydrogen phosphate 0.4M. I tried increasing glycerol up to 30% with the same ammonium phosphate concentration or increasing glycerol up to 30% in the presence of 1.3M ammonium phosphate. Both gave iced up drops (I only tried quick and dirty tests by dipping a cover glass in liquid nitrogen). Thank you. -Chris -- Kristof Van Hecke, PhD Biomoleculaire Architectuur Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 --
Re: [ccp4bb] Problems in refinement
If you are using TLS refinement the please check TLS definitions.It may be that atoms for which you have positive density are not in TLS definitions. Try to use without TLS. regards Garib On 26 May 2011, at 11:11, Petr Kolenko wrote: Dear colleagues, I have two problems in two structure refinements using REFMAC5. 1) 1.8A resolution, zinc in the active site. Refinement using work reflections - ADP for Zinc was about 14. Final refinement including all reflections increase ADP to 20 or even higher values - followed by very high positive difference density in position of the zinc. I have tried also PHENIX, the same thing. I changed ADP manually to 14 and only calculated maps (no refinement) look good. May I deposit the structure using manually fixed ADP according to the best agreement to the observed and difference electron density? By the way, it is clear that this is zinc. 2) 1.9A resolution, about 600AA, all of them OK in electron density. But, somehow, about ten atoms give very strong positive electron density suggesting they are not taken into account in refinement. On the other hand, ADPs are reasonable and seem to be refined. All of these atoms are fully occupied. I tried to omit whole residues and build them again, but the maxima appeared again. Using of PHENIX resulted in no difference electron density for these atoms. I have also tried to take PHENIX output to REFMAC, but the maxima are there again. It is always one or two atoms from the same residues - sometimes Calpha, sometimes Cbeta, sometimes C, sometimes NH1, but still on the same five residues. Does anyone have any idea how to solve this problem? Many thanks for any response. Petr -- Petr Kolenko petr.kole...@biochemtech.uni-halle.de http://kolda.webz.cz
Re: [ccp4bb] {**SPAM**} [ccp4bb] screen kit
The screen described here might be worth checking out as well:
Crystallization of bFGF-DNA aptamer complexes using a Sparse Matrix designed
for protein-nucleic acid complexes
Jamie J. Cannone, , Cindy L. Barnes, , Aniruddha Achari, and Craig E.
Kundrot ,
Journal of Crystal Growth
Volume 232, Issues 1-4, November 2001, Pages 409-417
Kushol
Kushol Gupta, Ph.D.
Research Associate
Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine
kgu...@mail.med.upenn.edu
215-573-7260 / 267-259-0082
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Vellieux Frederic
Sent: Thursday, May 26, 2011 2:55 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] {**SPAM**} [ccp4bb] screen kit
Hi,
Hampton research claims that their Natrix series of screening kits is
designed for nucleid acid and nucleic acid/protein complexes.
http://hamptonresearch.com/product_detail.aspx?cid=1sid=27pid=8
Fred.
dengzq1987 wrote:
hello all,
is there any screen kit that is highly effective for the
crystallization of protein-nucleic acids complexes?
deng.
Re: [ccp4bb] Pymol question
Firstly, I think in Pymol there is no true electrostatic potential calculator, but only a charge-smoothed surface presentation ( http://www.pymolwiki.org/index.php/Protein_contact_potential). So, If you want to calculate the real electrostatic potential in Pymol (by Possion-Boltzman method), you have to install a plugin such as APBS or Delphi. Personally, I prefer Delphi, because there is a good and simple tutorial written by James Stroud from USC ( http://structure.usc.edu/howto/delphi-surface-pymol.html). And you also can read the man page of Delphi where describe the details how it works ( http://wiki.c2b2.columbia.edu/honiglab_public/index.php/Software:DelPhi). On Thu, May 26, 2011 at 12:51 PM, Tim Gruene t...@shelx.uni-ac.gwdg.dewrote: Dear Maher, as far as I know pymol uses the APBS (http://www.poissonboltzmann.org) for the calculations and your question is answered in the FAQ: http://www.poissonboltzmann.org/apbs/frequently-asked-questions/what-are-the-units-of-electrostatic-potential (kT/e) I don't know if this also answers your second question, but you could let us know in case it does. Tim On Wed, May 25, 2011 at 03:58:17PM -0700, Maher Alayyoubi wrote: Hi everyone, I have two questions: 1- Does anybody know what are the units on the display ruler after you calculate the vaccum electrostatics using pymol? 2- What are the default kT/e values used by pymol? Thank you, Maher -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) iD8DBQFN3jDOUxlJ7aRr7hoRAuONAJ9/PlFL6WgLhiw44GrofkSibUw8IgCfRQxh YhReBu+gIxwpBm/7PHQwW4w= =dLaZ -END PGP SIGNATURE- -- Xiaoguang Xue, PhD student Utrecht University Crystal Structural Chemistry Padualaan 8. Room N807 3584 CH Utrecht The Netherlands Tel. +31-30-253-2383
Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo
I always try sugars for everything. There is a cryocrystallography webinar at rigaku.com with embedded videos on how to do this. 50% to 100% saturated sugar (sucrose, glucose, trehalose, sorbitol, et al.) in reservoir buffer is usually what I try. :) -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris Ulens Sent: Thursday, May 26, 2011 5:27 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo Hi, I would like to get recommendations for a proper cryo solution for a crystallization hit from the Hampton crystal screen Ammonium di- hydrogen phosphate 0.4M. I tried increasing glycerol up to 30% with the same ammonium phosphate concentration or increasing glycerol up to 30% in the presence of 1.3M ammonium phosphate. Both gave iced up drops (I only tried quick and dirty tests by dipping a cover glass in liquid nitrogen). Thank you. -Chris
Re: [ccp4bb] Histogram/Plot of Buried Surface Areas
Hi Jacob - These references might(?) be helpful: Proteins. 1995 Dec;23(4):580-7. Protein-protein interaction at crystal contacts. Janin J, Rodier F. J Mol Biol. 2004 Feb 27;336(4):943-55. A dissection of specific and non-specific protein-protein interfaces. Bahadur RP, Chakrabarti P, Rodier F, Janin J. Proteins. 2005 Jul 1;60(1):36-45. Hydration of protein-protein interfaces. Rodier F, Bahadur RP, Chakrabarti P, Janin J. The problem you describe is a tricky one - crystal packing can be deceiving. Complementing your structural studies with some biophysical measurements (ie: comparing computed hydrodynamic properties to those observed in gel filtration or centrifugation or SAXS or the like) could help flesh out these types of questions. Hope this helps, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Wednesday, May 25, 2011 10:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Histogram/Plot of Buried Surface Areas Dear Crystallographers, is anyone aware of a reference or plot addressing buried surface area (or PISA output values) versus veracity of a complex? I am trying to determine the physiological relevance of a crystallographically-observed assembly, and would love to put my PISA output in the context of verified complexes versus crystal contacts. Thanks, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo
Chris, As others have said, using sugars as cryoprotectants is a good first choice. However, we have run into problems freezing crystals with sugars when the primarily crystallization reagent is a salt at high concentrations (0.8-2M). Although 0.4M ammonium phosphate, is not particularly high, you might try ammonium formate or sodium malonate, sometimes even sodium citrate works (1.0-1.2 M). My worry about any crystals grown in phosphate is that the phosphate anion may be crucial to crystals growth, and its displacement by like ions (sulfate) may be detrimental. If it is not in your case, you might also try lithium sulfate. We have used mixtures of sodium citrate and lithium sulfate to freeze crystals grown in 0.8 M sodium citrate. One other point is that sometimes the crystals need to have a bit of the cryoprotectant as a component of crystallization. I have seen cases where crystals could not be transferred into glycerol, but adding 1-3% glycerol to the crystallization mix yielded crystals that could be transferred into glycerol for freezing. You have a lot more options to try, but a drop on a coverslip may not be the best way to test freezing. Proper freezing depends on having maximal heat transfer, sometimes that can be defeated by having large heat reservoirs (i.e., big drops and the big coverslip) and insulators (i.e., glass coverslips). Freezing works because the objects size (drop and crystal) is small enough to allow rapid and relatively isotropic heat transfer. We alway use a slightly larger loop to test our cryoprotectants. When using ammonium phosphate, watch out for the formation of struvite (NH4MgPO4), a type of kidney stone. They are lovely looking (often octahedral) crystals that easily grow in ammonium phosphate; although magnesium phosphate can be soluble up to ~12 mM, the presence of ammonium markedly increases the formation of struvite, even with micromolar (contaminating) concentrations of magnesium. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris Ulens Sent: Thursday, May 26, 2011 5:27 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo Hi, I would like to get recommendations for a proper cryo solution for a crystallization hit from the Hampton crystal screen Ammonium di- hydrogen phosphate 0.4M. I tried increasing glycerol up to 30% with the same ammonium phosphate concentration or increasing glycerol up to 30% in the presence of 1.3M ammonium phosphate. Both gave iced up drops (I only tried quick and dirty tests by dipping a cover glass in liquid nitrogen). Thank you. -Chris
Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo
Chris, Crystals can be very tollerant of cryo-solutions that respect the degree of hydration of the lattice. You can use any cryo-solution you want, including oil. Try 10% Di-ethylene glycol, 10% 1.2-propanediol, 10% glycerol, 10% PEG 10K, 10% buffer solution, 1M NaCl (pH close to the one you use now; slightly higher or lower may help). 1M NaCl should provide enough ionic strength for the duration of the cryo-soak to prevent the crystals from dissolving. Test one crystal in this solution. If the crystals crack reduce the PEG 10K concentration if they dissolve increase the NaCl concentration or try: Try 8% Di-ethylene glycol, 8% 1.2-propanediol, 8% glycerol, 8% PEG 10K, 8% MPD 10% buffer solution, 1M NaCl. As already suggested cryosalts may work, but Ammonium di-hydrogen phosphate 0.4M is on the low side as far as ionic strength is concerned and if the crystals are very big they are likely to crack. No problems if you have needles or smallish crystals. Enrico. On Thu, 26 May 2011 13:01:08 +0200, herman.schreu...@sanofi-aventis.com wrote: Dear Chris, I have not used this particular condition, but as a rule of thumb I use like with like, e.g. glycerol, ethylene glycol, PEG400 etc. with PEG conditions, and saltlike compounds (sucrose, xylitol, salts) for salt conditions. In your case I would try glucose or xylitol or just look what happens if you increase the ammonium phosphate concentration to say 2M without adding glycerol. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris Ulens Sent: Thursday, May 26, 2011 12:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo Hi, I would like to get recommendations for a proper cryo solution for a crystallization hit from the Hampton crystal screen Ammonium di- hydrogen phosphate 0.4M. I tried increasing glycerol up to 30% with the same ammonium phosphate concentration or increasing glycerol up to 30% in the presence of 1.3M ammonium phosphate. Both gave iced up drops (I only tried quick and dirty tests by dipping a cover glass in liquid nitrogen). Thank you. -Chris -- 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] Hydrophobic protein surface and SDS page
Dear All, Sorry for the off topic question. I am purifying one protein which is showing increased molecular weight +5kDa (more than adding up the Hexa His and cloning artifact) in normal 12% SDS PAGE. The DNA sequence is as it is. The gel runs without blurring the lanes and without any difficulties. My protein may contains hydrophobic patches, does it may cause retard migration. Please help me suggesting the possible reason of increased molecular weight. Thank you for your kind suggestions. Sincerely, Debajyoti
Re: [ccp4bb] Hydrophobic protein surface and SDS page
Hi Debajyoti, Migration of proteins in SDS containing gels is dependent on hydrophobicity, the amount of SDS that your protein binds (despite the fact that in theory all proteins should behave the same way under these conditions) and charge. Highly basic or acidic proteins will migrate anomalously, thermophilic proteins (which are usually more highly charged and hydrophobic) also tend to migrate anomalously..membrane proteins also because they usually tend to interact differently with the detergent; it is not unusual to observe stable non-covalent homo-oligomers of membrane proteins even in an SDS-PAGE gel. Phosphorylation and glycosylation will also affect the apparent MW as estimated from the SDS-PAGE experiment. What you observe is not unusual at all. If you want to be sure of your MW, Mass Spec will tell you what you want to know. Hope this helps, good luck -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
[ccp4bb] Structural Biologist/Protein Biochemist position at Heptares Therapeutics Ltd.
We are looking for an outstanding Structural Biologist/Protein Biochemist for our group at Heptares Therapeutics Ltd. Heptares is a Drug Discovery Company located in Hertfordshire UK, working on G protein coupled receptors involved in CNS and metabolic disease. We are using a unique structure based approach, which originated from work at the MRC Laboratory of Molecular Biology in Cambridge, to characterise this important family of receptors and develop novel innovative therapeutics for these diseases. We are now seeking enthusiastic and motivated scientists with experience in the expression, purification and crystallisation of proteins to assist Heptares in obtaining unique structures of GPCRs for drug discovery. This is an exceptional opportunity to participate in pioneering science whilst helping to grow an Industry-leading Drug Discovery company. Applicants should have a PhD and post-graduate experience in a research environment. Candidates should be familiar with protein expression and purification in a variety of expression systems such as E.coli, baculovirus and mammalian cells. We also wish to recruit candidates with experience in protein crystallisation who are interested in following proteins through purification to structural studies. Previous experience of working with GPCRs or other membrane proteins would be an advantage. Experience of working in a drug discovery environment where structural studies are used to drive medicinal chemistry would also be of benefit. Further information can be found on our website (www.heptares.com). To apply for this position, please send your application to h...@heptares.com, quoting reference number 2011/1B Best wishes, Joao Dias, Ph.D. Senior Scientist Heptares Therapeutics Ltd BioPark, Broadwater Road, Welwyn Garden City, Herts, AL7 3AX UK This document contains company confidential and/or proprietary information. It is intended for the exclusive attention of the addressee(s) above and should not be copied or disclosed to any other. If you have received this transmission in error, please make no use of its contents and contact the sender.
[ccp4bb] Postdoctoral Position in Structural Biology Computing, Boston, MA
Hi! There is an immediate opening for a computationally-minded structural biology post-doc in the Sliz Group at Harvard Medical School in Boston, MA working in support of the SBGrid Consortium. The SBGrid Consortium is a non-profit service center of Harvard Medical School developed to support the computing needs of structural biology labs across the world. The software distribution we support contains more than 225 scientific software applications for Linux and OS X and is integrated into an easy to use shell environment. It is used by more than 1000 researchers in our 150+ member labs. This sort of scientific software is mostly written by academics to scratch their own itch, and it often comes as source code with little documentation on how to build and configure it to do something useful[1]. The software is written in a polyglot of languages (Fortran, C, C++, Python, Java, Perl, Tcl/Tk and many sh/csh scripts) and built with any one of a number of different build systems (autotools, cmake, scons, homebrew shell scripts/makefiles, setuptools, etc). The primary responsibilities of the position will be installing and configuring structural biology software for use in the Consortium laboratories as well as providing first level support for software users. There may also be opportunities to work on our NSF-funded project to develop grid portals for computationally demanding structural biology work flows. The exact duties of the position will be dependent on your qualifications. The ideal candidate will have a background in structural biology and a particular interest in the software, methods and computational requirements of structural biology computing. You should have some knowledge of Python and shell scripting or a strong desire to learn. Interested candidates should email a cover letter, CV, publications list, and names and contact information for three references to Dr. Piotr Sliz. ps...@hkl.hms.harvard.edu -ben [1] CCP4 being the obvious exception! -- | Ben Eisenbraun | SBGrid Consortium | http://sbgrid.org| | Harvard Medical School | http://hms.harvard.edu | | | | Einstein argued that there must be simplified explanations of nature, | | because God is not capricious or arbitrary. No such faith comforts| | the software engineer. Frederick P. Brooks |
Re: [ccp4bb] CCP4BB Digest - 25 May 2011 to 26 May 2011 (#2011-146)
Two topics today... 1) Problem with LIBCHECK. Thanks to all who replied to my issue. I was able to resolve the issue before the flurry of responses came in. Basically, I took my CSA modified amino acid, aligned it as best I could to the CYS (especially at the CA), and then manually edited the PDB file to include the new CSA atoms from CB onward, leaving the backbone as the CYS atoms, and changing all the residue names to CSA. Of course, this meant that the CSA sidechain was in no-man's land, but anyway, refined it in PHENIX restraining the distance from the terminal carbon and the sulfur of the crosslink partner to 1.83 +/- 0.05 A, and let 'er rip, including the .cif file for CSA in the monomer library in my .def file. The output was good enough to refine manually, and I'm happy with the results. 2) re: problems in refinement Petr, I second Pavel's recommendations. Furthermore, in your second case with the positive difference density for residues that seem to be refined well, I had something like this happen for me, although at much lower (3.1 A) resolution. I fixed it by making those trouble residues a TLS group and refining with TLS in PHENIX. After one round of refinement, the problem was fixed and I was able to remove TLS from the refinement strategy. Hope it helps, Geoff -- Geoffrey K. Feld Department of Chemistry 492 Stanley Hall University of California, Berkeley Vigilia pretium libertatis
