Re: [ccp4bb] molprobity clashscore, symmetry-related molecules?
MolProbity works on chains present in the pdb file. Therefore, I would predict that if the pdb file can be made to consist of several chains (built by symmetry operations) and bearing each a distinct chain name, then MolProbity would (artificially) work on symmetry-related molecules as well. Nadir Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 23/10/2014 13:06, "Oliver Smart" wrote: on 23/10/14 11:52 AM, Tim wrote: Hi everybody, Molprobity does not take into account contacts/clashes from symmetry-related molecules, or does it? Thanks in advance, Tim Tim, I am not sure. In my experience MolProbity reduce does not take crystal contacts into account (but reduce does a great job otherwise). But this might have been improved. Oliver - Dr Oliver Smart Director SmartSci Limited http://www.smartsci.uk/ & Consultant Global Phasing Ltd http://www.globalphasing.com/
Re: [ccp4bb] [Proteopedia] announcement: course on Proteopedia with JSmol
Dear Angel, Jaime and Joel, Proteopedia is great work, but I am unlucky as I won't be able to visit you at Alcalà. This makes me think it would be great to have a web seminar on the topic. Thanks, Jaime! Enjoy your meeting in Madrid and around. Best regards, Nadir Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 23/09/2014 19:17, Joel Sussman wrote: Dear Jmol aficionados, For those to happen to be in Spain (or able to come, you will be most welcome), I'd like to share the information about a 2-day course that we will held in Alcalá thanks to a visit of Jaime Prilusky. Dates are October 2nd and 3rd (Thu-Fri), in my University (Alcalá de Henares, in Madrid province) The course will focus on Proteopedia and its uses to study, display and teach macromolecules. It will be run in English and/or Spanish, according to the audience. All information is available at http://bit.ly/P14UAH Hope to see some of you! Dr. Angel Herráez Biochemistry and Molecular Biology, Dept. of Systems Biology, University of Alcalá E-28871 Alcalá de Henares (Madrid), Spain
Re: [ccp4bb] Guard columns from FPLC
Well, Pharmacia (now, GE) used to sell such a guard column for FPLC prior to Akta. And I remember buying adaptors to be able to connect the guard column and FPLC columns to an HPLC system. So all the tools exist. But keep in mind that a guard column volume must in all cases be kept minimal to avoid sample dilution which can be deleterious to resolution in size-exclusion chromatography. Prior to FPLC with (or without) a guard column, I have always used an Eppendorf microcentrifuge followed by 2u-filtering prior to injecting the sample and have consistently observed that these remove most aggregates if not all. HTH, Nadir Mrabet Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 16/09/2014 19:01, Christian Roth wrote: Hi Anita, never heared about Guard columns for Superdex columns. These guard columns are usually for HPLC columns (RP) or Silica High pressure gelfiltration columns like from Tosoh Bioscience and part of the acutal column itself, though replaceable. Christian Am 16.09.2014 09:29, schrieb Anita P: Hi All, Sorry for this off topic. I have heard that there are these little columns called guard columns which can be attached to AKTA purifiers. These columns prevent the incoming huge aggregates to be deposited and blocking of the gel filtration columns. Can any one advice me regarding where to purchase these columns. I could not find them in GE website. We have Superdex 16/60 on AKTA purifier. Thanks in advance. Have a good day Anita
Re: [ccp4bb] areaimol and hydrogens
Jose, Your are most probably right. Atoms used for ASA calculations are "unified atoms" as their vdW radii incorporate light atoms (hydrogens) which, by and large, crystallographers don't see. Adding extra H atoms is likely to end up in miscalculations. Nadir Mrabet Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 28/07/2014 14:20, Jose Manuel Duarte wrote: I believe that ignoring hydrogen atoms is the default behaviour of any software calculating ASA values. Normally the VdW radii values of the heavy atoms already include the hydrogens implicitly. If your input structure has hydrogens and they were included in the calculation it would result in over-estimating the ASA values. I'm not familiar with AREAIMOL but I guess it must behave in a similar way. Jose On 28/07/14 14:02, Harry Mark Greenblatt wrote: BS"D Dear All, I understood from the areaimol documentation that hydrogens are not included as one of the default atoms. But one can add atoms, and so I added an "ATOM" line for hydrogen. The program quite happily accepted my input line, but later on stated explicitly that it was ignoring the hydrogens. Is there no way to include hydrogens? Thanks Harry - Harry M. Greenblatt Associate Staff Scientist Dept of Structural Biology Weizmann Institute of SciencePhone:972-8-934-3625 234 Herzl St.Facsimile: 972-8-934-4159 Rehovot, 76100 Israel harry.greenbl...@weizmann.ac.il <mailto:harry.greenbl...@weizmann.ac.il>
Re: [ccp4bb] metal chelation
Hi Adam, I have not read all the thread as it came all at once and late (9:00pm here). I believe the best way to strip a protein of metals is to first adsorb it onto a solid support (e.g. IEX) and then use a sufficiently low-pH (say equal or below 6) buffer that contains also EDTA. You will probably need several washes but it works! Also be aware that EDTA binds well to several proteins. HTH, Nadir Mrabet Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 19/05/2014 20:21, Adam Brummett wrote: Thank you everyone for the comments and suggestions. To answer a few questions: -I do not use a treated buffer system. I have just used the nano-pure water. I have looked into Chelex, but before I bought it I wanted to see if you all recommended it. I was trying to avoid this, but it may not be possible now. -the active site does bind metals and is promiscuous in binding, so it is not know if the His tag or active is the source of contamination, but cleavage is not an option for us. The biding of metal is going to be needed for phasing, so good point Tim, hopefully just not in the His site. -thank you Vivoli for the protocol, seems very thorough. Have you had success with it? I anticipate I'll need to go down this road. -Roger, the metals you mentioned (Zn and Fe) are the problem and I expect to have to go to heroic measures to get an apo enzyme . But you did mention easier ways of getting metal substituted. I have some evidence that I can do this. Do you have any other thoughts on this matter? Maybe a reference to something similar (non-apo but could substitute? Thank you all so much for the help and advice. -Adam On May 19, 2014, at 12:55 PM, Roger Rowlett <mailto:rrowl...@colgate.edu>> wrote: The answer depends on a number of questions: * What metal ion are you trying to eliminate? * What kind of metal-binding site is involved? o A peripheral or loose binding site? (e.g. surface calcium ions)--these may respond to chelators o An active site coordinated metal? (e.g., metalloenzyme)--these can be refractory Many metalloenzymes are not going to give up their metal to chelators, or just any chelator, or at all. Denaturation, dialysis, and refolding is an extreme way of removing metal ions to make apoprotein. Won't work for every protein. Chelation can be highly specific, that is one chelator may work, while another, similar one, will not. Some metal ions are notoriously difficult to eliminate, because they are adventitious trace contaminants in nearly everything, e.g. zinc and maybe even iron. (Plastic-ware seems to be often loaded with trace iron, and also is capable of adsorbing metal ions form solution.) To make apo-enzymes from zinc proteins, you have to go to heroic efforts to ensure that glassware, water, buffers, and reagents are zinc-free, especially if you don't have high (mM) concentrations of protein to work with. A His-tag is very likely to snag adventitious metals from solution, and can often mess up metal analysis for metalloproteins by providing "extra" metal. If this is a problem for your application, you may want to consider removing the His-tag. If you are making apoenzyme to get a different metal installed (metallosubstitution), there are slightly easier ways to do that than going through the apoenzyme route. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 5/19/2014 1:20 PM, SUBSCRIBE CCP4BB Adam Brummett wrote: Hello All, I apologize for the non-crystal related question. I am trying to get a fully metal-free apo enzyme. The 6x His construct is consistently purified with some metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA and DFO and then dialyzing it away, but this has shown little to no effect. Any thoughts or recommendations would be greatly appreciated. Thanks. Adam
Re: [ccp4bb] repulsive effects of arginine
Resonance was to be understood exactly as meaning all the bonds are averaged between both types (single and double bonds). Furthermore, fluctuations in the immediate environment will affect electron distribution with time, as proteins exist in a dynamic state. Nadir Mrabet Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr On 08/10/2013 19:27, Andrew Purkiss wrote: An example of pi-pi stacking of the guanidinium groups, can be seen on a structure which I worked on; pdb-code: 2x2u. Look at the interactions between Arg 77 and its symmetry mate, with Arg 144 (and symmetry copy) flanking, giving rise to a stack of 4 Arginine guanidinium groups, with a sulphate ion neutralising the environment. Such pi-pi stacking is also commonly seen with Tyrosine and Phenylalanine, with cation-pi interactions also common (e.g. Lys NZ to the planar side of Phe). Resonance is not really a correct description of these delocalised pi-orbitals; as there are no single and double bonds, but all the bonds are an average of both types. Additionally, remember that the Arginine(s) may not actually be charged, as the local environment of ionic sidechains can move the pKa value(s) a long way from the expected value for an isolated sidechain, with the pH of the crystallisation condition also potentially affecting what is charged. Andrew Purkiss. On Tue, 2013-10-08 at 18:34 +0200, Nadir T. Mrabet wrote: Yes, indeed Andrey. And this results from resonance (tautomerization) of the guanidinium group. Regards, Nadir Mrabet Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr On 08/10/2013 18:01, Andrey Feklistov wrote: Hi Jan, please note, Arg-Arg proximity is not always repulsive: guanidinium groups can associate bridged by H-bonds and interactions with water molecules or neighboring amino acids. There are many examples of these unusual Arg formations, see for reference: Neves, Yeager and Abagyan (2012) "Unusual Arginine Formations in Protein Function and Assembly: Rings, Strings and Stacks", J. Phys. Chem. B 116, 7006−7013 Hope this is helpful, Andrey
Re: [ccp4bb] repulsive effects of arginine
Jan, Ionic interaction does reduce the charges born by the partners in isolation. This is why charged residues found in the protein core are always paired. Furthermore, concerning arg, beyond the fact that they are found to autointeract as Andrey pointed out, the charge they bear is spread over all the guanidinium group so that it is not punctual as is the case for lys NZ. H-bonds also solvate charges. Are you stating that asp can in no way be entrapped between the arginines? Recall arg-asp interactions in proteins are "hot spots". Cheers, Nadir Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr On 08/10/2013 18:33, Jan van Agthoven wrote: Thanks for your responses, Andrey, I had no idea about these arginine associations. In this case the arginines are facing each other guanidinium to guanidinium. I guess they wouldn't attract. Nadir, the asp is not entrapped between the two arginines. But Hermann is probably right by saying that the asp is too close to the antibody arginine. Our binding essays were done by fluorescent labeling of the ligand. Clustering shouldn't play a role. It's hard to explain, there is no steric hindrance. I guess I'll have to drop this idea of repulsion. 2013/10/8, Andrey Feklistov : Hi Jan, please note, Arg-Arg proximity is not always repulsive: guanidinium groups can associate bridged by H-bonds and interactions with water molecules or neighboring amino acids. There are many examples of these unusual Arg formations, see for reference: Neves, Yeager and Abagyan (2012) "Unusual Arginine Formations in Protein Function and Assembly: Rings, Strings and Stacks", J. Phys. Chem. B 116, 7006-7013 Hope this is helpful, Andrey
Re: [ccp4bb] repulsive effects of arginine
Yes, indeed Andrey. And this results from resonance (tautomerization) of the guanidinium group. Regards, Nadir Mrabet Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr On 08/10/2013 18:01, Andrey Feklistov wrote: Hi Jan, please note, Arg-Arg proximity is not always repulsive: guanidinium groups can associate bridged by H-bonds and interactions with water molecules or neighboring amino acids. There are many examples of these unusual Arg formations, see for reference: Neves, Yeager and Abagyan (2012) "Unusual Arginine Formations in Protein Function and Assembly: Rings, Strings and Stacks", J. Phys. Chem. B 116, 7006−7013 Hope this is helpful, Andrey
Re: [ccp4bb] AW: [ccp4bb] repulsive effects of arginine
Dear Jan, Is there a possibility for bifurcated salt bridges/H-bonds where the Asp would be entrapped between the two arginines, in which case, repulsive effects would not take place ? Cheers, Nadir Mrabet Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr On 08/10/2013 17:00, Jan van Agthoven wrote: Dear Herman, The arginine of the antibody is approximately at same distance from the arginine of the ligand then the aspartic acid of the receptor, respectively 3.3 and 3.25 A. But you're right! This same aspartic acid makes a salt bridge with the arginine of the ligand, when this one is bound to the receptor. So presumably, the ligand loses one salt bridge when the antibody binds to the ligand receptor complex. What is troubling is that the ligand is bound to the receptor by many other hydrogen bonds and salt bridges. Also, mutation of this aspartic acid to alanine hasn't shown a dramatic effect on ligand binding. So I was thinking about a double effect: repulsion + loss of one salt bridge. Best, Jan 2013/10/8, herman.schreu...@sanofi.com : Dear Jan, since electrostatics go with one over distance-square, there may still be some electrostatic repulsion if the aspartic acid is further away as the arginine. Another question is, what happens with the arginine of the ligand in absence of the antibody? Does it then make a salt bridge with the aspartic acid of the receptor? I expect that losing an a salt-bridge interaction between ligand and receptor will cause a significant drop in affinity which may explain the effect of the antibody. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Jan van Agthoven Gesendet: Montag, 7. Oktober 2013 22:47 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] repulsive effects of arginine Hi everyone, I'm working on structure of an antibody that inhibits a receptor. The antibody doesn't induce any conformational change in the receptor and doesn't bind the ligand binding site. If we superimpose the receptor with antibody and ligand the only hindrance we find is a electrostatic repulsion between two arginines (3.3A): one is part of the antibody and one is part of the ligand and involved in ligand binding. However the arginine coming from the antibody makes a salt bridge with an aspartic acid from the receptor. Does this neutralize it's charge? Can we still say that it has a repulsive effect? Thanks
Re: [ccp4bb] Modelling Software for beta turn design
Hi Wenzong, I would certainly try it also another way. Choose you beta turn sequence. Insert it between the sequences of both beta stands and submit the whole sequence (beta-turn-beta) to homology modeling. Try every beta turn sequence to select top results. You could download MolIde (http://dunbrack.fccc.edu/molide/) and do the exercise in local mode. Best of luck, Nadir Mrabet Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 23/04/2013 00:48, Wenzong Li wrote: Dear All, I want to use modelling software to design a tight beta turn replacing a loop between two beta strands. Can anyone suggest a program to do this sort of modelling? Thank you Wenzong
[ccp4bb] Recent IUPAC definition of the hydrogen bond
Hi, I wonder how much of an impact should there be on structure refinement, validation and, last but not least, molecular modeling, if we take into account the recent (2011) re-definition of the hydrogen bond made by the IUPAC. Ref: "Elangannan Arunan, Gautam R. Desiraju, Roger A. Klein, Joanna Sadlej, Steve Scheiner, Ibon Alkorta, David C. Clary, Robert H. Crabtree, Joseph J. Dannenberg, Pavel Hobza, Henrik G. Kjaergaard, Anthony C. Legon, Benedetta Mennucci, and David J. Nesbitt Pure Appl. Chem., Vol. 83, No. 8, pp. 1619–1636, 2011. doi:10.1351/PAC-REP-10-01-01 © 2011 IUPAC, Publication date (Web): 8 July 2011 Defining the hydrogen bond: An account (IUPAC Technical Report)*" Thanks for your input and interest. Greetings, Nadir Mrabet -- Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr
Re: [ccp4bb] Off Topic- Cystine Detection
Perhaps you should have a look at http://193.146.160.29/gtb/sod/usu/$UBUG/repositorio/10320076_Hawkins.pdf. and at "An introduction to methods for analyzing thiols and disulfides: Reactions, reagents, and practical considerations. Rosa E. Hansen 1, Jakob R. Winther Analytical Biochemistry 394 (2009) 147–158. HTH, Nadir Mrabet Pr. Nadir T. Mrabet Structural & Molecular Biochemistry N-gere - INSERM U-954 University of Lorraine, Nancy School of Sciences and Technologies & School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet univ-lorraine.fr On 07/02/2013 04:17, Dr. Anthony Addlagatta wrote: *** This message has been scanned by the InterScan for CSC SSM by IICT security policy and found to be free of known security risks. *** Dear Yuri, If you have access to mass spec, this should be a straight forward experiment. Find the reference here. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291582/pdf/v010p00017.pdf What was the result in the Ellman´s reaction? Unless you have other reactive cysteines in your protein protein, you should not see any color if the pair of cysteines on surface form disulfide. Anthony - Dr. Anthony Addlagatta Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad AP-500 607, INDIA Tel:91-40-27191812 Web: https://sites.google.com/site/chembioliict/home/dr-anthony-addlagatta-1 -- Original Message --- From: Yuri Pompeu To: CCP4BB@JISCMAIL.AC.UK Sent: Wed, 6 Feb 2013 16:10:35 + Subject: [ccp4bb] Off Topic- Cystine Detection *** This message has been scanned by the InterScan for CSC SSM at IICT and found to be free of known security risks. *** Dear All, I am trying to probe the existence of a disulfide bond on the surface of my protein. I have attempted Ellman´s and my results were not as clear as I would have hoped for. I am not a sulfur/cysteine chemist and would appreciate the advice on what experiments to try! Thanks a bunch YAP --- End of Original Message --- This Mail Scanned by ClamAV and Spammassassin
Re: [ccp4bb] X-PLOR
Thanks Francisco. Nicolas, thanks also for your feedback. In fact, I had already downloaded the X-PLOR manual, but I needed some more details on the H-atoms optimization procedure. That was for the purpose of a teaching work on a 1995 paper. Best regards, Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 27/04/2012 16:59, Francisco Hernandez-Guzman wrote: Nadir, There is an explicit bulletin board for questions regarding CNS and XPLOR. I would suggest posting your question there. http://tech.dir.groups.yahoo.com/group/cnsbb/ Cheers, Francisco -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nadir T. Mrabet Sent: Friday, April 27, 2012 6:34 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] X-PLOR Hi, Could someone explain to me the scientific details of the protocols used in X-PLOR to (1) build explicit hydrogen atoms onto X-ray structures and (2) optimize their positions? Many thanks in advance. Greetings, Nadir
[ccp4bb] X-PLOR
Hi, Could someone explain to me the scientific details of the protocols used in X-PLOR to (1) build explicit hydrogen atoms onto X-ray structures and (2) optimize their positions? Many thanks in advance. Greetings, Nadir -- Pr. Nadir T. Mrabet Structural& Molecular Biochemistry INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr
Re: [ccp4bb] protein structure for high schoolers
I would suggest MolProbity along with KiNG (on Firefox!) would be most appropriate (http://kinemage.biochem.duke.edu/). HTH, Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry N-gere - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 20/01/2012 22:41, James Whittle wrote: Hi- I am trying to help my former chemistry teacher set up a demonstration of protein structure for her class. I'd like to include electron density maps, and maybe show an enzyme active site. Are there suggestions from the BB on the easiest way to do this? Would pymol be the program of choice, or is there a simpler program that could show electron density? Has anyone already created such a demonstration they could and have advice on it? James
Re: [ccp4bb] Metal won't strip from IMAC
Have run into a similar problem. Cleared the background color by running 2M NaOH together with 0.2M EDTA. Better replace BMT with TCEP (1 mM). Also keep in mind that adsorption is pH dependent, that is the higher the pH, the better is adsorption. Many proteins adsorb irreversibly above pH 7.0. If you reduce the pH, say to 5.2-5.5, not only you make adsorption less stronger (hence, column capacity may drop down), but you will at the same time prevent cysteine oxidation. You can also increase [imidazole] in the equilibration buffer to reduce adsorption. HTH, Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 13/01/2012 01:42, Michael Thompson wrote: Katherine, You are not alone. I have inadvertently destroyed a GE HisTrap column with high concentrations of proteins that contain many exposed cysteines. In my case the Co2+ resin turned a very dark purplish-brown and the protein appeared to have crashed out on the column. I didn't try to strip it, because I figured it was done for anyway, so I can't tell you any more about the problem. Here's how I explained it to myself (whether or not this is actually right I'm not 100% sure, but it makes sense in my head). The columns I was using have a maximum concentration of 5mM for DTT and 10mM for B-mercaptoethanol. So that seems like the column can handle 10mM thiol groups. If you have a protein with many cysteines and it is very highly concentrated (as was the case for me) then you are adding considerably more thiol groups to the solution. This abundance of thiols reduces the metal on the column, and disaster ensues. For me, repeating the same prep with less DTT (3mM vs. 5mM) in the buffer fixed the issue. If you are concerned about your protein oxidizing at lower concentrations of DTT or BME, the other alternative is to switch to TCEP. The IMAC columns can tolerate higher concentrations of TCEP, and it is a far superior reducing agent (more stable, more reductive, etc.)...but also a lot more expensive (although you can get away with using much less because it works so much better). HTH, Mike - Original Message - From: "Katherine Sippel" To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, January 12, 2012 4:01:10 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Metal won't strip from IMAC Hi all, I've run into a bit of a protein purification conundrum and wondered if anyone had encountered a similar situation. I've exercised all of my google-fu and can't find anything. It's a fairly straightforward setup; His-tagged protein and Talon Co2+ resin, load lysate, wash with 5 mM imidazole, elute with 150 mM imidazole. There is protein in the elution fractions as would be expected. The strangeness occurs when I try to regenerate the column. Using the standard protocol of 25 mM MES, 100 mM NaCl pH 5 doesn't change the color of the resin back to light pink the way it should with a regenerated column. I try stripping with the suggested 0.2M EDTA, still pink, 0.5M EDTA, still pink, 8 M urea plus 4% CHAPS and then EDTA, still pink, 1 M NaOH then EDTA, still pink. I've checked the resin using a Western (with a really specific monoclonal Ab) and it seems that my protein has somehow irreversibly bound to the column and is preventing the metal from releasing the sepharose. I've even tried competing the protein off with excess Co2+ and Mg2+ (the endogenous divalent bound cation). Clearly the solution is swapping to a Ni column, but this is really bugging me now. Has anyone run into this problem with IMAC before? Background: The protein does bind divalent cations (Mg and Mn) with low affinity (~1 mM) and has a ridiculous number of cysteines (10 in 416 residues total). There is 1 mM BME and 1 mM MgCl2 in all of the buffers. Thanks, Katherine
Re: [ccp4bb] How to assess geometry in a model?
Along you can consider also using MolProbity online. Best, Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr Cell.: +33 (0)6.11.35.69.09 On 09/12/2011 06:52, Robbie Joosten wrote: Hi Matt, WHAT_CHECK writes out a file called check.db that contains per-residue scores for several quality metrics. It is fairly easy to parse. Cheers, Robbie Date: Thu, 8 Dec 2011 23:08:45 -0500 From: mattw...@gmail.com Subject: [ccp4bb] How to assess geometry in a model? To: CCP4BB@JISCMAIL.AC.UK Hi Folks I'm looking for a way to score each atom (or residue) in a model based on it's geometry. I know these scores exists because various software packages speak of outliers, even including a sigma value in some cases. So I'm looking for a simple way to get a complete list (not just outliers). Does anyone know of a package that can be made to output these scores? Thanks, Matt
Re: [ccp4bb] Description of a newly solved structure
I would also suggest using MolProbity to assess structure quality. Best, Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 01/12/2011 16:34, Katherine Sippel wrote: For drawing a 2D fold topology I'd suggest TopDraw (http://www.ccp4.ac.uk/html/topdraw.html). To see if you structure looks like other structures I'd look at the DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/). Dali is pretty good for digging out results/discussion fodder because it gives you another structure(s) to compare. PISA would be my suggestion for roughly identifying any oligimerization though it's best to back those claims up with experimental data. If you've got ligands bound Gerard Kleywegt and the PDBe staff have put together a lovely means of searching and representing motifs which is also useful for comparative purposes (http://www.ebi.ac.uk/pdbe-site/pdbemotif/). There are a panoply of good figure generating softwares available but my personal preference is Pymol for it's flexibility, comprehensive wiki, and supportive users groups (http://www.pymol.org/). If your just looking for a description of what to put into a results and discussion section for a new structure you might try browsing through the Acta Cryst D archives for some good examples. Hope this helps, Katherine On Wed, Nov 30, 2011 at 10:49 PM, sadaf iqbal <mailto:sadaf_che...@yahoo.com>> wrote: Hello everyone, I have submitted one cysteine protease structure in PDB and now i have to describe the structure completely for my PhD thesis. I know some web servers who are good to provide knowledge about protein but i would like to ask expert persons of this field as i am quite new in describing a structure. Which softwares/web servers you prefer when you are going to describe one complete native structure? I am a chemist basically. Thanks in advance Sadaf Iqbal PhD Scholar ICCBS, University of Karachi, Pakistan. & Visiting Scientist University of Hamburg, Germany.
[ccp4bb]
Hi, We have been receiving several spam mail from this person. Any way to stop that quickly? Thanks, Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 25/11/2011 09:22, Sampath Natarajan wrote: http://zabara.com.br/jogosonline/include/libs/smarty/templates_c/rpdmgla.htm
Re: [ccp4bb] projecting SC value on protein surface
Hi Pavlina, I suggest you have a look at http://wiki.c2b2.columbia.edu/honiglab_public/index.php/Software:GRASP2. Runs on Windows. HTH Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 07/09/2011 15:57, Pavlina Rezacova wrote: Dear colleagues, we would like to project shape complementarity value (calulated in SC in ccp4) onto a protein surface. By reading the manual I learned that it can be done with GRASP surface. However we have no acess to GRASP (it only runs on Silicon Graphics right?). Is there any alternative way to do it? Thanks for any advices and suggestions. Pavlina
Re: [ccp4bb] off-topic: 2 peaks on Cation
In your original mail (2011-02-18; 18:45 GMT), you wrote: "Possibly deamidation of the protein, in particluar one or more lysines" Hence, my comment was based on your own writing, that is deamiDation. Wishing you a better week. Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 18/02/2011 21:07, Soisson, Stephen M wrote: I'll swap you for the deamination, as that pertains to lysines...it's been a long week :) Steve -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nadir T. Mrabet Sent: Friday, February 18, 2011 2:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off-topic: 2 peaks on Cation Typo! I actually meant deamidation. Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 18/02/2011 20:36, Christian Roth wrote: Hi, you did not mention anything about your protein, but if it shows a metal dependency, than different amounts of the metal ion might changen the overall charge and influence the interactions with the ion exchanger. There might be also a modification of an aminoacid f.e. decarboxylation of an aspartate or glutamate. Christian Am Freitag 18 Februar 2011 18:13:36 schrieb Ulli Hain: Hi, I was wondering if anyone had possible explanations for a recombinantly expressed soluble protein that runs as 2 equal, slightly overlapping peaks on a cation exhanger but as one peak on a size exclusion column and same electrophoretic mobility on SDS-PAGE. -Ulli Adelaide Ulricke Hain PhD Candidate Johns Hopkins Bloomberg School of Public Health Department of Biochemistry and Molecular Biology 615 North Wolfe Street Baltimore, MD 21205 Notice: This e-mail message, together with any attachments, contains information of Merck& Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] off-topic: 2 peaks on Cation
Typo! I actually meant deamidation. Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 18/02/2011 20:36, Christian Roth wrote: Hi, you did not mention anything about your protein, but if it shows a metal dependency, than different amounts of the metal ion might changen the overall charge and influence the interactions with the ion exchanger. There might be also a modification of an aminoacid f.e. decarboxylation of an aspartate or glutamate. Christian Am Freitag 18 Februar 2011 18:13:36 schrieb Ulli Hain: Hi, I was wondering if anyone had possible explanations for a recombinantly expressed soluble protein that runs as 2 equal, slightly overlapping peaks on a cation exhanger but as one peak on a size exclusion column and same electrophoretic mobility on SDS-PAGE. -Ulli Adelaide Ulricke Hain PhD Candidate Johns Hopkins Bloomberg School of Public Health Department of Biochemistry and Molecular Biology 615 North Wolfe Street Baltimore, MD 21205
Re: [ccp4bb] off-topic: 2 peaks on Cation
Given no info on the protein, it can be anything. Is it recombinant? Which host? etc. Oxydation (cys, met) is also a possibility By the way, deamination concerns asn and gln, not lys. Best, Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 18/02/2011 19:45, Soisson, Stephen M wrote: Possibly deamidation of the protein, in particluar one or more lysines. What does the Mass spec look like? Cheers, Steve *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Ulli Hain *Sent:* Friday, February 18, 2011 12:14 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] off-topic: 2 peaks on Cation Hi, I was wondering if anyone had possible explanations for a recombinantly expressed soluble protein that runs as 2 equal, slightly overlapping peaks on a cation exhanger but as one peak on a size exclusion column and same electrophoretic mobility on SDS-PAGE. -Ulli Adelaide Ulricke Hain PhD Candidate Johns Hopkins Bloomberg School of Public Health Department of Biochemistry and Molecular Biology 615 North Wolfe Street Baltimore, MD 21205 Notice: This e-mail message, together with any attachments, contains information of Merck& Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] What is the simplest method to analytically compute the Solvent-Accessible Surface Area of a given atom in a protein?
It can go both ways: If you increase atom radii by adding that of the probe (e.g. 1.4 A° for a water probe) and calculate the "molecular surface" using a zero probe with atom radii as previously defined (Ri +1.4 A°), the area you get is that of the accessible surface. Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr Cell.: +33 (0)6.11.35.69.09 On 19/01/2011 16:46, Richard Edward Gillilan wrote: Since several people have asked me for code, I now realize that I've contributed my bit to the confusion over names of various surface definitions. To clear things up there is an excellent online article which covers the history of various molecular surface definitions that I highly recommend: www.netsci.org/Science/Compchem/feature14e.html Mainly there are two related surface definitions: (a) solvent-accessible surface (b) molecular surface = contact surface + reentrant surface "solvent-excluded volume" has been defined using both of the above. Both (a) and (b) reduce to the same VDW surface when the solvent probe is zero. There are also a number of smooth approximations to these definitions and others. Richard Gillilan MacCHESS
Re: [ccp4bb] What is the simplest method to analytically compute the Solvent-Accessible Surface Area of a given atom in a protein?
Good points Richard! The ambiguity with surface definition starts with the assumption that atoms are (i) spheres and (ii) with fixed radii. I am not sure Connolly was able to sell his original algorithm due to conflicts of interest with the Scripps, where it had been actually developped first. How could I get the Varshney code? Best regards, Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr Cell.: +33 (0)6.11.35.69.09 On 13/01/2011 13:40, Richard Edward Gillilan wrote: : *Subject: **Re: [ccp4bb] What is the simplest method to analytically compute the Solvent-Accessible Surface Area of a given atom in a protein?* My knowledge on this is probably quite out of date by now, but some years ago there was a lot of research on this topic because such surfaces are important in electrostatics and implicit solvation models (calculating surface area) as well as molecular graphics. I think the most widely-used definition of a solvent-accessible surface is Lee-Richards surface in which a solvent-sized sphere is rolled along the surface of the protein. Surface is therefore rigorously defined as a piecewise collection of convex and concave patches of spheres and tori. It was Connolly who implemented (and sold) a practical algorithm for computing these surfaces. They were even known as Connolly surfaces and rendered as dots before modern computing hardware allowed for rendering surfaces. Several groups have developed high-efficiency versions of the calculation. Harold Scheraga's group, for example, has some FORTRAN code for this. Fred Brook's virtual reality group also developed a high-effeciency parallel version (Varshney was the guy's name I think) in C. There have been many approximations over the years I think ... but you asked about analytical models. The these algorithms are non trivial. That's a understatement. And there is actually a mathematical ambiguity in the surface definition itself. The Varshney code is freely available ... I received email permission from both Varshney and his thesis advisor to freely distribute the code. I even offered it to Warren Delano years ago when he was writing Pymol, but he refused to include it because he felt there still might be legal issues that would effect Pymol. So ... Pymol contains only a somewhat improvised an non-rigorous surface algorithm (last time I looked). Fine for graphics of course. en.wikipedia.org/wiki/Accessible_surface_area <http://en.wikipedia.org/wiki/Accessible_surface_area> Richard On Jan 13, 2011, at 1:00 AM, Francois Berenger wrote: Hello, Does someone know some good articles on this particular topic? I'd like to implement the thing myself, however if there is a good software doing the job (with readable source code), I might use and cite it. Best regards, Francois.
Re: [ccp4bb] Removing a tight binding ligand
Hi, It could help if you said what your ligand is. Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 08/10/2010 03:05, SIPPEL,KATHERINE H wrote: Hi all, I am working with a substrate binding protein. The protein scavenges its endogenous ligand out of the E. coli used for expression. I need to get this ligand out for both crystallographic and kinetic studies. I have tried denaturing in urea and refolding the protein with limited success. It refolds properly according to the CD spectra but it some how manages to hold on to trace amounts of ligand despite serial dialysis (500ml to 5ml of sample) in 8M, 6M, 4M, 2M 1M urea followed by 50mM Tris. I also have a homolog that abjectly refuses to refold in either urea or guanidine, though it does turn the dialysis tubing into a lovely snow globe. There are alternative methods of performing the kinetics, but those will require destroying the protein which doesn't help on the crystallography front. I was wondering if any of you out there had experience successfully removing very tightly bound ligands by an alternative method. I didn't see any mention on the subject in the archives. I had hoped you might be able to point me in the right direction. Thanks for your time, Katherine Ph. D. candidate Department of Biochemistry and Molecular Biology College of Medicine University of Florida
Re: [ccp4bb] Native Gel Theory and Practice
Thanks Jürgen. Yes, you may show dissociation. However, especially if you deal with assembly, then it might be difficult, if not impossible, to tell your exact subunit composition if you runBNP only in the first direction. Jacob mentions the possible occurrence of complex assemblies (AB, BB, ABB, AAB). Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 19/05/2010 16:05, Jürgen Bosch wrote: You don't necessarily need the second dimension. BN-PAGE gel: protein A alone| some proteins you know the size as reference|protein B alone| your mixture You will be able to see in the mixture a) one or b) multiple bands, since the Coomassie is equally distributed and attached to your protein in a non-denaturing way it is directly proportional to the molecular mass of your protein complex. I'm searching for the protocol which was I believe either from the Görg group or a group in Berlin, perhaps also Mann demonstrating dissociation of a multimeric complex in dependence of reducing agent. If I find it I will post it tonight. Jürgen On May 19, 2010, at 10:01 AM, Nadir T. Mrabet wrote: Maia speaks about native PAGE for which protein mobility (migration) depends on 3 different parameters as she states: charge, mass and shape. Blue native PAGE, which might be the answer to Jacob's question, is a 2D gel: Native in the first direction, then SDS-PAGE in the second one. You actually need both data to infer stoechiometry and subunit composition. Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr <http://medecine.uhp-nancy.fr> On 19/05/2010 13:01, Jürgen Bosch wrote: Not quite correct, look into Blue Native PAGE. There you can seperate natively by mass. 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 19, 2010, at 1:31, Maia Cherney <mailto:ch...@ualberta.ca>> wrote: Dear Jacob, I offer you my opinion. Are you talking about electrophoresis? As far as I know it does not work for the mass. The velocity of a protein depends on the charge at a particular pH, the mass and shape of molecules etc. It's very difficult to take all these things into consideration. Otherwise this would be a very convenient method, much easier than the analytical centrifugation or gel-filtration that are usually used. However, electrophoresis does not work for mass determination. Besides, complex formation hugely depends on the protein concentration. If you dilute your mixture, your complexes might dissociate. There is equilibrium constant between different types of complexes. Maia Jacob Keller wrote: Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MWCharge MW/Charge A 22 -5-4308 B 17-24 -702 This experiment is partly to determine stoichiometry, but also to determine roughly the strength of the interaction. B definitely runs much faster than A alone, as predicted, but I am wondering what to expect with various oligomers. Should ABB run faster or slower than AB? What about AABB? Theoretically, AA should certainly run slower than A, and BB slower than B, simply because the mass/charge ratio is the same, but the overall mass is greater. But what happens when you have AAB, for example? There must be an equation relating the mass/charge and mass (and perhaps gel percentage) to the speed traveled in the gel--but what is the equation? Thanks for your consideration, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu <mailto:j-kell...@northwestern.edu> *** - 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-
Re: [ccp4bb] Native Gel Theory and Practice
Maia speaks about native PAGE for which protein mobility (migration) depends on 3 different parameters as she states: charge, mass and shape. Blue native PAGE, which might be the answer to Jacob's question, is a 2D gel: Native in the first direction, then SDS-PAGE in the second one. You actually need both data to infer stoechiometry and subunit composition. Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 19/05/2010 13:01, Jürgen Bosch wrote: Not quite correct, look into Blue Native PAGE. There you can seperate natively by mass. 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 19, 2010, at 1:31, Maia Cherney wrote: Dear Jacob, I offer you my opinion. Are you talking about electrophoresis? As far as I know it does not work for the mass. The velocity of a protein depends on the charge at a particular pH, the mass and shape of molecules etc. It's very difficult to take all these things into consideration. Otherwise this would be a very convenient method, much easier than the analytical centrifugation or gel-filtration that are usually used. However, electrophoresis does not work for mass determination. Besides, complex formation hugely depends on the protein concentration. If you dilute your mixture, your complexes might dissociate. There is equilibrium constant between different types of complexes. Maia Jacob Keller wrote: Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MWCharge MW/Charge A 22 -5-4308 B 17-24 -702 This experiment is partly to determine stoichiometry, but also to determine roughly the strength of the interaction. B definitely runs much faster than A alone, as predicted, but I am wondering what to expect with various oligomers. Should ABB run faster or slower than AB? What about AABB? Theoretically, AA should certainly run slower than A, and BB slower than B, simply because the mass/charge ratio is the same, but the overall mass is greater. But what happens when you have AAB, for example? There must be an equation relating the mass/charge and mass (and perhaps gel percentage) to the speed traveled in the gel--but what is the equation? Thanks for your consideration, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Query - inhibitor screening by fluorescence based assay.
You are measuring fluorescence changes which are likely to be due to compound binding by your enzyme. In this case your blank must be your "compound" blank and no other. Then you measure changes in fluorescence intensity and lambda max of emission as you add your enzyme. I do not know you system. Note however that binding (1) might be non specific and (2) need not correlate with enzyme inhibition. Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 19/05/2010 08:48, suresh mattegunta wrote: Dear All, Sorry for the non crystallography related question We are performing a fluorescence based assay to screen for inhibitor compounds of our enzyme and ultimately crystallize the enzyme along with inhibitor. We see that some of our compounds are autofluorescent and thus are effecting fluorescence. In such a case we make a compound blank (assay buffer+ compound) and subtract it from test (enzyme + compound) but still we see the values of test much higher compared to control (only enzyme). In this case can we bring down the values of compound balnk and test by a factor which brings the value of compound blank to the level of blank and compare the values of resultant test value with that of control after subtracting the respective blanks. For example we have the following arbitrary fluorescent readings (AFU) for one compound: Blank (70342) Control (163243) Compound balnk (1865830) Test (2020455) Since compound blank / blank ie (1865830)/(70342) = 26.52 can we do a normalization of compound blank and test values by a factor 26.52, which brings teh value of compound blank to teh level of blank and get the values (1865830)/26.52 = 70355 and 2020455/26.52 = 76186 and say the compound is inhibitor by comparing this test value with the value of control after subtracting teh values of respective blank. Thank you in advance, Suresh -- Suresh V Mattegunta MS Senior Research Associate Discovery Biology Division Aptuit Laurus PVT LTD. ICICI Knowledge Park Hyderabad 500078 India
Re: [ccp4bb] Ion exchange protein lost
Human gcsf has a pI ~ 6. As Ursula suggests you might be better off with anion exchange. There are protocols that show you can both refold and purify onto such column type. Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 12/05/2010 19:17, Ursula Schulze-Gahmen wrote: Like Juergen suggested, your protein is most likely stuck to the column, either because it never was really was refolded or because it doesn't like to be at pH 4.5. Do you really need such a low pH to have it bind to source 15S. Check the pI of the protein and perhaps some different pH buffer or try an anion exchange column if possible. Ursula On 5/11/10 7:53 PM, megha goyal wrote: Hi all, Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. Then we perform concentration using proflux M12 [just concentration and not diafiltration]. Adjust the pH of concentrate to 4.5 and load it to source 15S resin [strong cation exchange]. The problem is we do not recover our protein on performing IEX. HPLC and absorbance reading on concentrate show the presence of our protein. Buffer for loading is 25 mM na acetate pH 4.5 and elution is same buffer with 0.5 M NaCl. No protein is lost in flow thru and even 2M Nacl washing does not show our protein. . Only when we perform NaOH wash we do see some peak but could not analyse it as it is too alkaline and cant run on SDS PAGE or HPLC. What could be the reason. Where do we lose our protein. Kindly shed some light on this on where shall I be going wrong. thanks and regards, meg -- Ursula Schulze-Gahmen, PhD. QB3, Tjian Lab MCB, 16 Barker Hall #3204 University of California Berkeley Berkeley, CA 94720-3204 Phone: (510) 642 8258 uschulze-gah...@lbl.gov
Re: [ccp4bb] How could I Extract Iron from Protein?
I would go for DTPA and acid pH. Better than dialysis, I would use IEX and flush le bound protein with a buffer with as low a pH as possible with 10-100 mM DTPA, then wash with no DTPA, and elute le protein with clean (e.g. treated with Chelex) salt. HTH, Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 11/05/2010 19:12, Javier Gonzalez wrote: Since you have only Asp and His ligands coordinating the Fe ion, dialysis against, say, 0.1 M Citrate at pH 5 will do the trick. Citrate will chelate the Fe(III) (if the protein has Fe(II) it will be oxidized during dialysis due to air oxygen) avoiding precipitation of Fe(OH)3 and the acid will protonate the protein ligands. If the affinity is really high, adding also some guanidinium chloride (0.5-1M) in the first dialysis step to loosen the protein a little bit will also help. Best, Javier M. Gonzalez, PhD. University of Maryland Baltimore Department of Pharmaceutical Sciences X-Ray Crystallography Shared Service (UMXSS) 20 Penn St., HSFII, Rm 514 Phone/Fax: 410-7061124/410-7060886 21201 Baltimore, MD http://www2.pharmacy.umaryland.edu/psc/xray/ On Tue, May 11, 2010 at 12:26 PM, Roger Rowlett <mailto:rrowl...@colgate.edu>> wrote: I would try dialyzing against a solution with 1,10-phenanthroline, 1,10-phenanthroline-2-carboxylate, or pyridine-2,6-dicarboxylate. Removal of metals from proteins is often not just dissociative, but requires the associative interaction of a chelating agent. Which one works is often empirical. Cheers. On 5/11/2010 11:11 AM, Wenguang LIANG wrote: Dear all, I have a protein which binds iron with two D and two H as active site. I have tried to extract the iron by dialysis. First, 20mM tris, pH7.5, 150mM NaCl, 20EDTA, 10mM Na2S2O3, O/N. Then, followed by dialysis with 20mM tris, pH7.5, 150mM NaCl, 1EDTA O/N to remove the EDTA and Na2S2O3. However, after dialysis, the density of the Iron is still in the protein. Could anybody please give me some suggestion on how to extract Iron out of the protein? Or I can just use Chelex-100 to extract it instead of Iron. Thank you very much for help, Best wishes, Vinson Liang -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu <mailto:rrowl...@colgate.edu>
Re: [ccp4bb] pdb-l: [mockeldri...@yahoo.com: Retraction of 12 Structures]
Kevin, 1hps and 1hos do no appear to include Murthy as an author. Since I need good as well as (very) bad examples for my molecular modeling teaching, I spent some time analyzing the structures which pdb codes you provided with MolProbity. All score very bad: high B factors, lots of clashes, many Ramachandran outliers, unfavorable rotamers and bad bond angles. The lesser bad structures in your list come (surprisingly?) as 1hos and 1hps, but still they are far from optimal. I have those data available in the case you need them. I would strongly advise the use of MolProbity (at least) by both crystallographers and modelers, but also by curators at the rcsb.. Regards, Nadir Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr Kevin Karplus wrote: Firas Khatib pointed out that several of the PDB files by Krishna Murthy's group were identified as problematic in the RosettaHoles paper: http://www.proteinscience.org/details/journalArticle/110975/RosettaHoles_Rapid_assessment_of_protein_core_packing_for_structure_prediction_r.html I notice that the RosettaHoles paper also identifies 1BGX as a problematic file, but that was not listed on the UAB site http://main.uab.edu/Sites/reporter/articles/71570/ I also notice that most of the identified files have not yet been marked as obsolete in PDB's "obsolete" file. In fact, only 1BEF was so marked when I just checked ftp://ftp.wwpdb.org/pub/pdb/data/status/obsolete.dat Question for the community: should we remove ALL the PDB files from Krishna Murthy's group as suspect? I find the following in the current PDB: listed in article: 1cmw 1df9 2qid 1g40 1g44 1l6l 2ou1 1rid 1y8e 2a01 2hr0 others probably from same author: 1bgx 1ay1 1hef 1heg 1sbg 1hps 1hos (Incidentally, variations in name abbreviations make it difficult to be sure that I have found all of the files, or that all of the files involve the same person.) Has anyone examined 1bgx, 1ay1, 1hef, 1heg, 1sbg, 1hps, and 1hos to see if there is any evidence of fraud on those files? I'm strongly tempted to remove them from my template libraries, unless I hear from someone who was involved in the investigation that these files were carefully examined and determined to be ok. The fact that 1bgx was identified by RosettaHoles as suspect makes me wary of just trusting that silence means they were cleared. Kevin Karplus karp...@soe.ucsc.eduhttp://www.soe.ucsc.edu/~karplus Professor of Biomolecular Engineering, University of California, Santa Cruz Graduate Director, Bioinformatics (Senior member, IEEE) (Board of Directors, ISCB) Editorial Board, Bioinformatics (Oxford University Press) life member (LAB, Adventure Cycling, American Youth Hostels) Effective Cycling Instructor #218-ck (lapsed) Affiliations for identification only. TO UNSUBSCRIBE OR CHANGE YOUR SUBSCRIPTION OPTIONS, please see https://lists.sdsc.edu/mailman/listinfo.cgi/pdb-l .
Re: [ccp4bb] Van der Waals contacts
Totally agree with what you write. Yet, I was only answering the question asked (that dealt mostly with "van Der Waals contacts and effective contacts"). At a time of crisis (DeltaG = only a few kcal/mol), I would say 0.6 kcal/mol would be a little more than negligible. Question of balance. Nadir Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr Bernhard Rupp wrote: It might be worthwhile to consider the energy column in the pdf: At RT we have about 0.6 kcal/mol thermal energy, so a *single attractive* vdW interaction has little impact - it is generally the sum of many of those contributing to notable and important attractive forces. For a *single repulsive* (steep branch) vdW this is quite different - your quickly get energetically significant repulsions even for one bad contact. So the vdW repulsion are both useful (nearly independent) stereochemical restraints and a good indicator for improbable conformations (see molprobity clash score, rama, etc). BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Nadir T. Mrabet Sent: Wednesday, July 15, 2009 7:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Van der Waals contacts This issue is far from being trivial. To address the question, I recommend looking at this simple example (attached pdf file) which shows that even when the atom distance is twice the vdw diameter, there is still attraction between atoms (unified type, with implicit H atoms). Some, but not all, consider that water screens out interactions, that is, the vdw interaction is attractive provided that R < Rm + 2 x R(H2O). But then, one may also need to consider the special case of water-mediated H-bonds, where the heavy atoms being considered are polar. HTH, Nadir Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. Clayton, Gina Martyn wrote: Hi CCP4ers Perhaps I am hashing over old news...but We are having a discussion about Van Der Waals contacts and effective contacts i.e. the "real distance" of a VDW bump between say a CH and a CH group which sometimes is described as between a C and a C as i.e. 2x 1.6A and ending about 4A but not including hydrogen. Some programs list contacts, to say a ligand, as far as 6A apart and some of the simulation programs use that distance too for contacts for protein protein interactions. Does anyone know of a good paper that discusses the effective distance or has a comment on where a VDW force may begin and end or it's effective distance - though some say it never truly ends just approaches zero... G Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] structure <-> function
Ok, now we can perhaps debate of another problem. With a multiple choice question that has more than one acceptably good answer, is it "convergent", or rather "independent", evolution? This multiple choice question is open for discussion. Greetings, Nadir Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr Justin Lecher wrote: Many thanks to everyone who replied. Once again a proof of this community! I am sure I will find what I am looking for in the cited publications. Thanks, justin
Re: [ccp4bb] Glycerol as metal chelator?
I wouldn't use a pH of 9 when dealing with metal cations in solution. By and large, metal hydroxides precipitate at alkaline pH. HTH Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr Guenter Fritz wrote: Ho, interesting case. Glycerol actually forms weak complexes with metal ions: Journal of Inorganic Biochemistry,60 299-302 (1995) and some references therein. But to be true, I am surprised that glycerol at concentrations of 10-15% (?) as used in cryo conditions can compete with a metal ion binding site in a protein. I remember I myself did titrations with Zn2+ in the presence/absence of 5% glycerol and did not observe any effect on the binding. There might be another effect (?) although I have no idea what it might be. I am working with a metalloprotein that binds cobalt and iron. I was surprised that the solved structures showed the crystals cryoprotected with glycerol are metal free while crystals cryoprotected with ethylene glycol had the metals present. Both cryoprotectant solutions contained metal in the 10 mM range and are buffered at pH 9. I assume glycerol must be a weak chelator otherwise it wouldn't be so ubiquitous in protein biochemistry. Has anyone else experienced this before with glycerol? Ho UC Berkeley
Re: [ccp4bb] Van der Waals Interaction Distances
Hi, This is one table (below) I use to convince my students that Evdw is still negative when R/Rm = 2, where Rm = Ri + Rj. To take into account water screening effect, however, one should consider vdw attraction only while R < Rm + 2 x Rw, where Rw is the radius of water. hth, Nadir ** ** ** ** ** *1.00* *1.000* *1.000* *-0.130* *1.05* *0.936* *1.069* *-0.122* *1.10* *0.810* *1.234* *-0.105* *1.15* *0.678* *1.475* *-0.088* *1.20* *0.558* *1.793* *-0.072* *1.25* *0.456* *2.195* *-0.059* *1.30* *0.371* *2.692* *-0.048* *1.35* *0.303* *3.299* *-0.039* *1.40* *0.248* *4.033* *-0.032* *1.45* *0.204* *4.911* *-0.026* *1.50* *0.168* *5.957* *-0.022* *1.55* *0.139* *7.193* *-0.018* *1.60* *0.116* *8.646* *-0.015* *1.65* *0.097* *10.346* *-0.013* *1.70* *0.081* *12.324* *-0.011* *1.75* *0.068* *14.616* *-0.009* *1.80* *0.058* *17.260* *-0.008* *1.85* *0.049* *20.298* *-0.006* *1.90* *0.042* *23.776* *-0.005* *1.95* *0.036* *27.742* *-0.005* *2.00* *0.031* *32.252* *-0.004* -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr Jim Fairman wrote: Fellow CCP4 Board Members, What is the general consensus of the structural biology community for a range of distances that would be considered a Van der Waals contact/interaction (eg: hydrogen bonds are usually considered to be 2.5-3.5 angstroms not including the hydrogen atoms)? Cheers, Jim -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 jfair...@utk.edu <mailto:jfair...@utk.edu> james.fair...@case.edu <mailto:james.fair...@case.edu>
Re: [ccp4bb] PDB protein strucutrues as screen saver
Hi, You may want to have a look at http://www.luminorum.com/html/luminorum_ltd___extras.html. hth Nadir -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr Jayashankar wrote: Dear Scientists, It may be too much... But as a biophysics student I would like to appreciate and feel happy to have pdb structures as my computers screen savers than to have some funny and fancy stuffs. And it may help me as a motivator to solve my own structures in future I want to ask is there any existing script that grep strucutres one by one with one line definition of that structure. S.Jayashankar Research Student Institute for Biophysical Chemistry Hannover Medical School Germany.
Re: [ccp4bb] protocol for harvesting proteins from bacterial periplasmic space
Artem, Artem Evdokimov wrote: Please note that osmotic shock extraction typically employs EDTA which is obviously bad for IMAC. This is not entirely correct. I have used extracts with 5 mM EDTA for IMAC in the past. If your IMAC column volume is large enough, only the top 1-2 mm will be depleted of Me2+ (easily seen with Cu2+). Moreover, it is always possible to add some Me2+ to your extract prior to IMAC. All this was published long ago (Biochemistry. 31: 2690-2702, 1992). Nadir -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
Two things to be mentioned. * IDA columns bear an overall negative charge. I expect this behavior holds true with NTA gels. Hence salt, (>= 0.5 M NaCl) must be present in your adsoprtion buffer to quench possible repulsive electrostatic interactions. * You are dealing with protein adsoption by coordination bond formation to a metal-chelate. Coordination bond lentghs decrease (and binding improves) as ionic strength increases, so a 1-2 M salt concentration in you buffer may turn out to be appropriate. HTH, Nadir Mrabet -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr Fred wrote: Hi everyone, Thanks for answer my question. Just to add some more notes regarding to my expression system. The insert-vector (pET28) has been sequenced and the his-tag is N-terminal. The anti his-tag WB is positive and the binding buffer's pH is 8.2 (double-checked). I had already experienced the same problem before, which I solved just increasing urea from 6M to 8M. Now, I have reached GndHCl 6M and no binding at all. I'm currently running a SDS-PAGE with samples eluted from Talon and let you know the results. All the Best, Fred --- Fred // schrieb am *Di, 27.1.2009: * *Von: Fred Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind An: CCP4BB@JISCMAIL.AC.UK Datum: Dienstag, 27. Januar 2009, 22:00 * *Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is appreciated. Fred *
Re: [ccp4bb] Definition of salt bridge
The problem lies with your definition of "significant". If it is non null, then any interaction is significant (dual-pan balance concept). Coulomb's energy is a function of 1/r^2, therefore at 8 Angs, it is still 15% of Emax. Even H-bonds are sometimes considered relevant up to 5 Angs. Nadir Mrabet Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] Ibrahim Moustafa wrote: Yes, it is electrostatic interaction. But when searching for a salt-bridge in a protein structure it won't be considered a significant non-bonded interactions at 8 A distance. Also, the electrostatic interaction extends beyond 8 A. For a significant interaction the distance need to be < 8A. Ibrahim On 10/16/08 12:10 PM, "Nadir T. Mrabet" <[EMAIL PROTECTED]> wrote: -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] Hi, Salt bridges (or ion pairs) can be long-range (up to 7-8 Ang). They obey Coulomb's law. In contrast, H-bonds are short-range and are further anisotropic. For those with general interest in electrostatics, I suggest to go back to the 1978 paper of Max Perutz: Electrostatic Effects in Proteins Science (1978) 201 (4362), 1187-1191. Nadir Mrabet Jayashankar wrote: Dear Fransico, *Salt bridges are close range electrostatic interaction which depend on conformer population. *S.Jayashankar Research Student Institute for Biophysical Chemistry Hannover Medical School Germany. On Thu, Oct 16, 2008 at 8:21 AM, Chavas Leo <[EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>> wrote: Dear Francisco -- On 15 Oct 2008, at 17:05, Francisco J. Enguita wrote: how can you define a salt-bridge within a protein structure ? According to Wikipedia: a salt bridge in proteins is "a relatively weak ionic bond between positively and negatively charged side-chains of proteins." Now, at far as I understand (based on "Structure and Mechanism in Protein Science - Alan Fersht), you have a salt bridge when two groups are making an hydrogen bond that is favored by electrostatic interaction, electrostatic energies being weak in water. To quote the author of the book, let say you have the following equilibrium: E-NH3+ --- OH2 + OH2 --- -O2C-S <==> E-NH3+ --- -O2C-S + H2O --- H2O The right-hand side equation would be more "favorable", as the electrostatic interaction will be more stable than in the left-hand side where both ions would be in contact with water molecules. HTH Kind regards. -- Leo -- Chavas Leonard, Ph.D. @ home Research Associate Marie Curie Actions Fellow Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]> http://personalpages.manchester.ac.uk/staff/leonard.chavas/
Re: [ccp4bb] Definition of salt bridge
-- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] Hi, Salt bridges (or ion pairs) can be long-range (up to 7-8 Ang). They obey Coulomb's law. In contrast, H-bonds are short-range and are further anisotropic. For those with general interest in electrostatics, I suggest to go back to the 1978 paper of Max Perutz: Electrostatic Effects in Proteins Science (1978) 201 (4362), 1187-1191. Nadir Mrabet Jayashankar wrote: Dear Fransico, *Salt bridges are close range electrostatic interaction which depend on conformer population. *S.Jayashankar Research Student Institute for Biophysical Chemistry Hannover Medical School Germany. On Thu, Oct 16, 2008 at 8:21 AM, Chavas Leo <[EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>> wrote: Dear Francisco -- On 15 Oct 2008, at 17:05, Francisco J. Enguita wrote: how can you define a salt-bridge within a protein structure ? According to Wikipedia: a salt bridge in proteins is "a relatively weak ionic bond between positively and negatively charged side-chains of proteins." Now, at far as I understand (based on "Structure and Mechanism in Protein Science - Alan Fersht), you have a salt bridge when two groups are making an hydrogen bond that is favored by electrostatic interaction, electrostatic energies being weak in water. To quote the author of the book, let say you have the following equilibrium: E-NH3+ --- OH2 + OH2 --- -O2C-S <==> E-NH3+ --- -O2C-S + H2O --- H2O The right-hand side equation would be more "favorable", as the electrostatic interaction will be more stable than in the left-hand side where both ions would be in contact with water molecules. HTH Kind regards. -- Leo -- Chavas Leonard, Ph.D. @ home Research Associate Marie Curie Actions Fellow Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]> http://personalpages.manchester.ac.uk/staff/leonard.chavas/
Re: [ccp4bb] off-topic: Enzymology textbook recommendations?
Alan Fersht: Enzyme structure and mechanism. Definitely! Nadir Mrabet -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] Donnie Berkholz wrote: On 13:37 Thu 11 Sep , Tim Fenn wrote: On Thu, 11 Sep 2008 13:24:12 -0700 William Scott <[EMAIL PROTECTED]> wrote: I just found out that in a couple of weeks I am going to be teaching a graduate student-level enzymology course. Can anyone recommend a good text, and possibly good websites authored by people who are predisposed to consider plagiarism the highest form of complement? A few classics I wouldn't be caught without: "Catalysis in Chemistry and Enzymology" William P. Jencks "Enzymatic Reaction Mechanisms" Christopher Walsh Our resident enzymologist (who just retired) recently picked up a copy of a current book that claims to update Jencks and Walsh, since they were both published before 1980. It's by Frey & Hegeman: http://www.oup.com/us/catalog/he/subject/Chemistry/Biochemistry/ProteinsandEnzymes/?view=usa&ci=9780195122589 If you try it out, I'd love to hear how it goes.
Re: [ccp4bb] Na Acetate Buffer
Michael's comments are correct and follow indeed appropriate chemistry theory. In practice however, as said earlier, it is less likely to read a correct pH value with a pH-meter (unless extensive care and adjustments are taken beforehand) than to predict amounts of acid and base to use reliably based on the HH equation. I take this opportunity to correct my mistyping in my previous mail: "In the second case, the HH is written 4.5 = 4.76 + log([NaOH])/(25 - [NaOH])" should be rewritten into "In the second case, the HH is written 4.5 = 4.76 + log([NaOH]/(25 - [NaOH]))". Greetings, Nadir -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] R.M. Garavito wrote: Buffer making is very much an empirical process, but there is a comment that needs to be made about the use of the H-H equation and pKa values. I have to teach our department's biochemistry laboratory, and I sadly would have to take off points from all the discussions as the H-H equation and pKa won't give the right answer as pKa is for an ideal (i.e., infinitely dilute) solution. A 25 mM Na acetate solution is not dilute. You need to use the pKa' which sadly changes as the concentration of the buffer increases or decreases: while the pKa of acetic acid is 4.76 (@25˚C), at 100mM, the pKa' is 4.60. The National Bureau of Standards (now NIST) has a detailed list of standard buffer recipes and pKa' values for most of the common buffers (e.g., see Bates, J. Natl. Bur. Stand. 66A, 179, 1962). That said, Nadir's method is a fine way to make a buffer of a known pH (using a well calibrated pH meter) at a known temperature, and it will allow you to make a buffer with the same pH value almost every time (depending on how your room temperature changes throughout the year). Being able to consistently and reliably repeat the buffer formulation is the most important point. 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) 353-9334 Email: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>/ /************/ On Jul 22, 2008, at 11:20 AM, Nadir T. Mrabet wrote: I bet it is more difficult to adjust a pH-meter than to use the Henderson-Hasselbalch equation and still get the expected pH with a pretty good accuracy especially if your work near the pKa. There are actually two ways to prepare this 25 mM buffer, pH 4.5. The pKa of acetate is 4.76 at 25 °C (with dpKa/° C = +0.0002, so don't worry too much about this). Reference is "Buffers for pH and Metal Ion Control", Perrin & Dempsey, Chapman & Hall, NY, ISBN 0 412 21890 9. High-grade glacial acetic acid (99-100%) is 18 N. Make a stock solution of 250 mM (eg 3.472 mL for 1.0 L final). Keep is a dark, tightly closed bottle. Make a stock solution of 250 mM sodium acetate (if you use FW, not MW, to calculate mass to use, then no worry about anhydrous or not since water is also taken into account if present) or make a stock solution of 5N NaOH. Keep is a dark, tightly closed bottle. Use then the Henderson-Hasselbalch equation (HH), pH = pKa + log ([A-]/[AH]). In the first case, you write it : 4.5 = 4.76 + log ([sodium acetate]/[acetic acid]) Second equation is [sodium acetate] + [acetic acid] = 25 mM which gives [sodium acetate] = 8.886 mM and [acetic acid] = 16.134 mM. For 1.0 L buffer, mix adequate volumes of stock solutions of sodium acetate and acetic acid and complete with water (add acid after un first fill with water to ~ 800 mL). In the second case, the HH is written 4.5 = 4.76 + log([NaOH])/(25 - [NaOH]), which gives [NaOH] = 8.886 mM (same result as above for sodium acetate which was then the base). The added advantage of using HH and stock solutions is that even if your pH is not exactly 4.5, say 4.55, if you make a new buffer the next day or even the next month, your buffer will have the same pH value. I don't expect you can ever achieve such a repeatability using a pH-meter. HTH, Nadir Mrabet -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]> William G. Scott wrote: So what, then, will be the concentration of the acetate ion in your stock so
Re: [ccp4bb] Na Acetate Buffer
I bet it is more difficult to adjust a pH-meter than to use the Henderson-Hasselbalch equation and still get the expected pH with a pretty good accuracy especially if your work near the pKa. There are actually two ways to prepare this 25 mM buffer, pH 4.5. The pKa of acetate is 4.76 at 25 °C (with dpKa/° C = +0.0002, so don't worry too much about this). Reference is "Buffers for pH and Metal Ion Control", Perrin & Dempsey, Chapman & Hall, NY, ISBN 0 412 21890 9. High-grade glacial acetic acid (99-100%) is 18 N. Make a stock solution of 250 mM (eg 3.472 mL for 1.0 L final). Keep is a dark, tightly closed bottle. Make a stock solution of 250 mM sodium acetate (if you use FW, not MW, to calculate mass to use, then no worry about anhydrous or not since water is also taken into account if present) or make a stock solution of 5N NaOH. Keep is a dark, tightly closed bottle. Use then the Henderson-Hasselbalch equation (HH), pH = pKa + log ([A-]/[AH]). In the first case, you write it : 4.5 = 4.76 + log ([sodium acetate]/[acetic acid]) Second equation is [sodium acetate] + [acetic acid] = 25 mM which gives [sodium acetate] = 8.886 mM and [acetic acid] = 16.134 mM. For 1.0 L buffer, mix adequate volumes of stock solutions of sodium acetate and acetic acid and complete with water (add acid after un first fill with water to ~ 800 mL). In the second case, the HH is written 4.5 = 4.76 + log([NaOH])/(25 - [NaOH]), which gives [NaOH] = 8.886 mM (same result as above for sodium acetate which was then the base). The added advantage of using HH and stock solutions is that even if your pH is not exactly 4.5, say 4.55, if you make a new buffer the next day or even the next month, your buffer will have the same pH value. I don't expect you can ever achieve such a repeatability using a pH-meter. HTH, Nadir Mrabet -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] William G. Scott wrote: So what, then, will be the concentration of the acetate ion in your stock solution when you have finished? (Disclaimer: I get to teach this stuff periodically in remedial chemistry as a punishment for deployment of excessive sarcasm during faculty meetings.) On Jul 22, 2008, at 6:10 AM, Santosh wrote: Hi, Make a 1M Na-Acetate do not make up to the 1 Ltr volume. Leave some extra volume and now start adding Acetic acid till you get pH 4.5 (Glacial Acetic Acid). Now make up the volume to 1ltr or how much ever you are deciding to make the 50X stock solution. Best, Santosh On Mon, Jul 21, 2008 at 11:20 PM, William G. Scott < [EMAIL PROTECTED]> wrote: This is a job for the trusty Henderson-Hasselbalch equation: http://en.wikipedia.org/wiki/Henderson-Hasselbalch_equation On Jul 21, 2008, at 8:12 PM, Meg wrote: Dear All, I want to prepare 25 mM sodium acetate buffer pH 4.5. can anyone give the exact composition of how to prepare it. we prepare it using sodium acetate and acetic acid combination. i am not able to arrive at the calculatation correctly, so if anyone can explain me with the above buffer how to calculate. and what sodium acetate [Anhydrous / trihydrate] and acetic acid [glacial/ plain] to use. thanks n regards Meg goyal, M.SC Biotechnology [Research] Institute of science, Fort Mumbai, INDIA
Re: [ccp4bb] Imidazole's ability to chelate metal ions
Well, I happen to lecture on this... The bible is "Data for biochemical research", Dawson et al., Oxford University Press. Should be available in most biochemistry labs, if not in the library. Cheers, Nadir -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] Jacob Keller wrote: Could those who responded with numbers for affinities of imidazole for metal ions please divulge their sources? It is not that I doubt their veracities, but it would be a nice reference to have on hand. For those wondering about why I was asking about imidazole's affinity for metal ions, I was wondering whether the presence of imidazole would affect a metal-ion-dependent reaction. With, for example, 200 mM imidazole and 10 mM Ca++ or Mg++, what would be the amount of free metal? This can of course be calculated from imidazole's binding constant for these ions, which is another reason I ask for the sources of the numbers quoted in a couple of the responses. Thanks for all of the helpful responses so far, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: "Nadir T. Mrabet" <[EMAIL PROTECTED]> To: "Jacob Keller" <[EMAIL PROTECTED]> Cc: Sent: Friday, July 18, 2008 8:55 AM Subject: Re: [ccp4bb] Imidazole's ability to chelate metal ions Imidazole can indeed complex (monodentate) metal ions but not chelate them (bidendate, at least). However, the stability constant, K, of such complexes is rather low, eg log K = 0.1 for Mg, 3.3 for Fe and 4.2 for Cu. In comparison, metal chelates are formed with EDTA, for which log K = 10.6 for Mg, 14.2 for Fe and 18.8 for Cu. So the difference amounts to several orders of magnitude. It should also be pointed out that the competitive effect of imidazole in IMAC does not involve binding to free metal ions, but instead coordination to immobilized metal chelates, eg Ni(II)-nitrilotriacetate (Ni-NTA, where NTA is the chelator). In any situation where one assays a protein whose activity and/or stability and/or else is/are metal dependent, one should rather use buffers (see below) that do not interfere (eg Good's buffers). I suspect the imidazole in your case is either a buffer (pKa 7.0) or else results from competitive elution from an IMAC column. What should be done depends on your exact conditions. hth, Nadir -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] Jacob Keller wrote: Dear Crystallographers, Does anybody happen to know whether imidazole is able to chelate metal ions in solution? It seems reasonable that since it can compete for binding to IMAC resins, it should have some affinity for at least Ni++ and Co++, but what about metal ions like Ca++ and Mg++? I assume that the affinity is weak, but at the concentrations at which we are wont to use it in our elutions (~100-500 mM), does it not seem likely that other metal ions are being competed away from our proteins as well? Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]> ***
Re: [ccp4bb] Imidazole's ability to chelate metal ions
Imidazole can indeed complex (monodentate) metal ions but not chelate them (bidendate, at least). However, the stability constant, K, of such complexes is rather low, eg log K = 0.1 for Mg, 3.3 for Fe and 4.2 for Cu. In comparison, metal chelates are formed with EDTA, for which log K = 10.6 for Mg, 14.2 for Fe and 18.8 for Cu. So the difference amounts to several orders of magnitude. It should also be pointed out that the competitive effect of imidazole in IMAC does not involve binding to free metal ions, but instead coordination to immobilized metal chelates, eg Ni(II)-nitrilotriacetate (Ni-NTA, where NTA is the chelator). In any situation where one assays a protein whose activity and/or stability and/or else is/are metal dependent, one should rather use buffers (see below) that do not interfere (eg Good's buffers). I suspect the imidazole in your case is either a buffer (pKa 7.0) or else results from competitive elution from an IMAC column. What should be done depends on your exact conditions. hth, Nadir -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] Jacob Keller wrote: Dear Crystallographers, Does anybody happen to know whether imidazole is able to chelate metal ions in solution? It seems reasonable that since it can compete for binding to IMAC resins, it should have some affinity for at least Ni++ and Co++, but what about metal ions like Ca++ and Mg++? I assume that the affinity is weak, but at the concentrations at which we are wont to use it in our elutions (~100-500 mM), does it not seem likely that other metal ions are being competed away from our proteins as well? Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]> ***
Re: [ccp4bb] pH gradient in Mono Q
John, You can adjust your ionic strength not only with NaCl/KCl/etc but also by increasing the concentration of the components in your buffer mixture. If you do it right, I can assure you can get a linear pH gradient. And, as mentioned earlier by Tom, you can use both pH and salt gradients to refine your separation, even batchwise (no linear gradient required... if it works). The problem working with pH gradients is re-equilibrating the resin, since what you are actually doing is titrate a supposedly high-capacity buffer with another high-capacity buffer. Therefore, patience is required and sufficient volumes of buffer A. Again, I ask the question: Did Matthew ever mentioned before he intended to perform a CHROMATOFOCUSING experiment? Nadir -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] John A. Newitt wrote: At 1:50 PM -0400 6/24/08, R.M. Garavito wrote: Matthew, You're not going to ruin your column, but you won't get great performance either. Elution by pH change is a very common method, but getting a really linear pH gradient is very hard. The Mono Q matrix is a strong anion exchanger, meaning that it is insensitive to pH changes, i.e., you can't titrate it smoothly with acid or base. DEAE resins, which are weak anion exchangers, have a nice pH titration curve and lend themselves better to elution by pH change. This is the reason chromatofocusing is not a commonly used method, and its expensive. There is a company the sells a proprietary buffer system and gradient programming calculator to create a stable pH gradient for separation on a MonoQ column or other strong ion exchanger. <http://www.cryobiophysica.com/> My problem with pH gradient techniques is that they don't work very well unless your protein is happy in low ionic strength buffers, which is almost never the case with my projects. This company now claims that it can create the pH gradient with NaCl present, but I haven't tried this yet. - John
Re: [ccp4bb] pH gradient in Mono Q
Michael, Well, why do you need to titrate the exchanger rather then the proteins themselves? MonoQ is a lot simpler to adjust pHwise, as with DEAE you actually titrate both the matrix and the proteins. Recommended buffers to use are Goods' (pKa from 8 to 6.15 at 20 °C) + acetic acid (pKa 4.76). An equimolar (50mM) mixture of these with Buffer A titrated to 8.0 and Buffer titrated to 4.0 has been shown (in my hands) to yield a very linear gradient (must not be too steep, though). Matthew's question does not seem to concern chromatofocusing. Hth, Nadir -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] R.M. Garavito wrote: Matthew, You're not going to ruin your column, but you won't get great performance either. Elution by pH change is a very common method, but getting a really linear pH gradient is very hard. The Mono Q matrix is a strong anion exchanger, meaning that it is insensitive to pH changes, i.e., you can't titrate it smoothly with acid or base. DEAE resins, which are weak anion exchangers, have a nice pH titration curve and lend themselves better to elution by pH change. This is the reason chromatofocusing is not a commonly used method, and its expensive. Andreas has pointed you in the general direction for chromatofocusing, but there is a "poor man's" way to do it. We use this method a lot, and the key is using a weak ion exchanger (like DEAE or CM) and a mix of buffers with pKas that span the titration range you want to exploit. Remember, you actually want to titrate the resin with the buffer: as the pH shifts away from the pKa of one buffer component, it moves into the buffering range of the other. If you do it correctly, you get a nice, flatter titration curve from the resin, which spreads out the release of the proteins. We have used a mixture of Tris and Bis-Tris-Propane with a HiTrap-DEAE or Sepharose-DEAE FF columns. Hope this helps, 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: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>/ // On Jun 24, 2008, at 12:53 PM, Matthew Chu wrote: Dear All, Sorry for off-topic question. Does anyone have any experience in purifying protein using pH gradient in Mono Q column? I have been googling for a whole day, only one paper was found to mention performing pH gradient in Mono Q, but in a mixture of amine buffering species, which is a bit too complicated (J. Chromatogr. A 1164 (2007) 181 - 188. Can Tris-Cl/Tris-base or phosphate buffer give a linear pH gradient from pH 8.0 to 4.0? Is it usual to perform pH gradient in Mono Q as I don't want to ruin my Mono Q column... Any suggestions are welcome. Thanks in advance! Kind regards, Matt Matthew LH Chu PhD Student School of Pharmacy and Pharmaceutical Sciences University of Manchester
Re: [ccp4bb] 3D model building without CRT monitor
Hi, I bought myself a brand-new Viewsonic P227f crt less than a year ago (in France). So I suspected this item to be still available. But no longer!. I however found something that might still be of interest to you (http://www.viewsonic.com/products/crtmonitors/graphicseries/g90fB/). Seems to be the one and only crt from Viewsonic (but only 19"). Cheers, Nadir -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] Cell.: +33 (0)6.11.35.69.09 Krojer,Tobias wrote: Dear all, recently some of our CRT monitors broke down and we realized that these monitors are no longer produced. However, we would still like to continue model building in 3D which is apparently not possible with current TFT displays. Beside the possibility of getting old CRT monitors at ebay, I was wondering how other groups solved this problem. Thank you very much for suggestions! best wishes, Tobias Tobias Krojer, PhD IMP (Research Institute of Molecular Pathology) Dr. Bohr-Gasse 7 1030 Vienna Austria Tel.: +43-1-797303358 Fax.: +43-1-7987153
Re: [ccp4bb] crashing-out protein eluted from Nickel column
Hi, One has to bear in mind that adsorption at high pH (above 6.5 is already high for Imac; pKa of exposed His is 6.2) leads to stronger interactions which, in turn, require stronger eluting conditions, e.g. > 200 mM imidazole (pKa ~ 7.0). I suggest lowering the pH of the adsortion buffer, e.g. 6.5 or even 6.0 (try and see) so that you dont need to go beyond 100 mM imidazole in your elution buffer (as mentioned earlier, you must ajust the pH of the latter as required with extra HCl). Best, Nadir Mrabet -- Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] Ronnie Berntsson wrote: Hi, In addition to the tips already suggested, you could also try to elute your protein with histidine.. I've got a protein that crashes out with imidazole as well, and I have succesfully used 200mM histidine for elution (using either KPi or MES buffer in my case). Cheers, Ronnie On Feb 15, 2008, at 5:47 PM, Juliana Barbosa Coitinho wrote: Dear all, I am with the same problem of Jacob. My protein is precipitating, especially when I 'freeze it'. I am using a His Trap HP column coupled with a desalting column. Already tried to elute with gradient of imidazole (that allowed an elution with less amount of imidazole), but even then, the protein still crashes-out. I am trying to use the protein immediately after purified it, but this is not always possible and I am losing much protein with that. I will try to use the tips that have already been said. But if someone can help me more, I will thank!!! Thanks a lot! Juliana Abra sua conta no Yahoo! Mail <http://br.rd.yahoo.com/mail/taglines/mail/*http://br..mail.yahoo.com/>, o único sem limite de espaço para armazenamento! Ronnie Berntsson -- Ph.D. Student Department of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials University of Groningen Nijenborgh 4, 9747 AG Groningen, The Netherlands telephone: +31 50 363 4195 telefax: +31 50 363 4165 e-mail: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]> homepage: http://www.rug.nl/gbb/research/researchgroups/enzymology/index
Re: [ccp4bb] Primary source for detergent properties
Hi, The only published compendium on detergents still available (to my knowledge) is http://www.merckbiosciences.co.uk/docs/docs/LIT/CB0068_M.pdf. No phase diagrams though. Regards, Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 UHP - Nancy 1, School of Medicine Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] Cell.: +33 (0)6.11.35.69.09 R.M. Garavito wrote: Jacob, Actually, there is no real definitive compendium of detergents and their properties (solubility, CMC, aggregation number, etc.). Because of their importance to industry, commercial compendia, as Anatrace's catalog, are often the most complete. However, most commercial compendia are not focused on the detergents we are interested in; they want to make dishes cleaner while protecting your hands (i.e., SDS is good). One of the best sources for detergent information of all sorts is Surfactants and Interfacial Phenomena by M. J. Rosen, but is is dated and out of print, I believe. Academic sources are few and often have circular citations. I can name several cases in the more popular reviews that cannot be traced back to a original reference. All the real biochemists and chemists who really brought detergents into biochemistry are retired (e.g., the Reynolds) or moved on to other things (e.g., Helenius). Lastly, even when academics measure the properties of a detergent, the work is not generally published, aside from being buried deeply in a thesis or is so specific to certain conditions that the information may not be very useful. One alternative is to enlist Anatrace and its parent USB, for example, to set up a Wiki for depositing detergent information and notes. Thus, anyone who has primary data on detergents could place it there with relevant experimental details. In the spirit of full disclosure, I still consult with Anatrace and have provided them with some of my group's primary data on detergents. Best regards, 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: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]>/ // On Oct 22, 2007, at 5:48 PM, Jacob Keller wrote: Dear CCP4BB, Although this is not exactly CCP4-related, I thought somebody here might know whether there is somewhere a definitive list or tabulation of detergent properties which are not simply copied out of catalogs, but have been traceably experimentally determined. In particular, it would be great to have phase diagrams for common detergents with detergent concentration versus pH, salt concentration, temperature, etc. Would this not be incredibly helpful for the scientific community? And yet, this is not so easy to find Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.467.4049 cel: 773.608.9185 email: [EMAIL PROTECTED] <mailto:[EMAIL PROTECTED]> ***
Re: [ccp4bb] questions about hydrophobic core
Hi, There exists a formal analytical way in doing this using the "survol" command in BRUGEL package. In short, this command define the accessible surface (external and cavities) to the probe you choose and creates separate masks (ensembles) of all atoms/residues that define these surfaces. The way to go, then, would be to create a collection mask of all accessible atoms/residues and subtract this from your protein mask to be left with the mask that contains the core residues. You could also use a cutoff of 5-10% ASA max (there are different ways of calculating these !). On the other hand, taking away even residues that display very little ASA (< 5%) would certainly leave you with genuine core residues. Hope this helps. Greetings, Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 UHP - Nancy 1, School of Medicine Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. Sebastiano Pasqualato wrote: Hi all, a few days ago I sent a post in which I was asking if anybody knew a program to automatically define the hydrophobic core of a protein, given the pdb. Unfortunately I got no answers, and indeed a more thorough googling around revealed that such a program might not exist. So it seems I have to define my hydrophobic core residues by hand... So now my question would be: how to define the hydrophobic core residues? I would tend to say that those that bury more than ## % (say 70%, 80% ??) of their otherwise solvent accessible surface area could be defined as such, but how can I get such a /per residue/ percentage? (NB: this is not the asa buried upon interaction, so I don't know how to get the asa of the "free" amino acid) Alternatively, are there other simple and defined rules to state which are the hydrophobic core residues? Any help appreciated, thanks in advance, ciao s -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 Milano Italy tel +39 02 9437 5094 fax +39 02 574 303 310 No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.472 / Virus Database: 269.9.0/853 - Release Date: 6/18/2007 3:02 PM
Re: [ccp4bb] Nature policy update regarding source code
Hi, I believe such requirements concern only "Nature Methods" rather than "Nature" by and large. Regards, Nadir Mrabet Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 UHP - Nancy 1, School of Medicine Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] [EMAIL PROTECTED] wrote: I thought that some of you might be interested that the journal Nature has clarified the publication requirements regarding source code accessibility. It is likely that some of you deserve congrats for this. Cheers! http://www.nature.com/nmeth/journal/v4/n3/full/nmeth0307-189.html Although there are still some small problems, I think that this is a big step forward, and certainly an interesting read, if you are interested in FOSS and science. Regards, Michael L. Love Ph.D Department of Biophysics and Biophysical Chemistry School of Medicine Johns Hopkins University 725 N. Wolfe Street Room 608B WBSB Baltimore MD 21205-2185 Interoffice Mail: 608B WBSB, SoM office: 410-614-2267 lab:410-614-3179 fax:410-502-6910 cell: 443-824-3451 http://www.gnu-darwin.org/
Re: [ccp4bb] PCB buffer and MMT buffer
Hi all, I only wish to draw your attention to the fact that the Henderson-Hasselbach equation (as used in the paper you cite) is only valid if you use zwitterionic buffers. Otherwise, with other buffers, especially phosphate and multi-acids (e.g. succinate, citrate and so on), any calculation using the Henderson-Hasselbach equation must refer to activities and not concentrations. Given this, expect a phosphate buffer pH to increase by 0.3-0.4 units upon a 10-fold dilution (That is from 1.0M to 0.1M in the referenced paper). Regards, Pr. Nadir T. Mrabet Cellular & Molecular Biochemistry INSERM U-724 UHP - Nancy 1, School of Medicine Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: [EMAIL PROTECTED] Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. Jose M de Pereda wrote: Hi Jenny (and others), The buffer systems used in the PACT screen are described in the following paper by Janet Newman. /Acta Cryst./ (2004). D*60*, 610-612[ doi:10.1107/S0907444903029640 <http://dx.doi.org/10.1107/S0907444903029640> ] Novel buffer systems for macromolecular crystallization J. Newman <http://scripts.iucr.org/cgi-bin/citedin?search_on=name&author_name=Newman,%20J.> I am not aware of any commercial source of these buffer systems. But the paper describes the recipes to make them, which is quite easy. HTH Jose On 30/01/2007 5:19 Jenny wrote: Hi, Sorry to bother you all.I'm going to try to reproduce some results from the initial screening from PACT kit.The conditions are something like 0.1M PCB buffer, 25% w/v PEG 1500 and 0.1 MMT buffer, 25%w/v PEG 1500. I was just wondering is there any easy way to get these buffers?Do you happen to know where to order?Or I have to order the component formula separately and then make buffers by myself?Thanks. Jenny