Re: [ccp4bb] Binding constants/kinetics for crystallisation
Roger, Very low values of Kd (nM) may mean that you have a good chance of finding both the proteins (or protein-nucleic acid) together if you get crystals of the mixture. I therefore think that the measurements are useful in that sense. However, low Kd does not necessarily mean that you get the complex to crystallize. It is possible that one of the components packs better, therefore would crystallize on its own. Also, there could be cases where the proteins have very low affinity for each other (Kd in 100's of micromolar to millimolar range), yet would readily crystallize as a complex. If you want to know some specific cases in this category, look for examples of some ubiquitin receptors co-crystallized with ubiquitin. Gel filtration of the mixture is not the only answer. You could also mix A and B with one of the components in slight excess (say B is 1.5-fold molar excess than A) and then set up trays without any chromatography. I have spoken to a few crystallographers working on protein-protein complex and they all seem to agree that the the mixing approach works. I am not sure if anyone has studied the relationship between thermodynamics of protein-protein or protein-nucleic acid complex and their crystallizability. I would like to know, if you find any. Chitta - Original Message - From: Roger Pickman rpick...@googlemail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, December 7, 2012 10:11:15 AM Subject: [ccp4bb] Binding constants/kinetics for crystallisation Dear all - is there a rule of thumb for favourable values of Kd, kon and koff of protein-protein or protein-dna complexes for protein crystallisation? Are these measurements useful in crystallisation, or should one just put it down a gel filtration column, hope for a complex and not worry? If anyone can point toward a reference, i'd appreciate it. Roger
Re: [ccp4bb] seleno methionine labelling of protein
Tim, My understanding is that the D-isomer does not get incorporated. I could be wrong though. Bacteria may convert the D to the L-form. Chitta - Original Message - From: Tim Gruene t...@shelx.uni-ac.gwdg.de To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, November 12, 2012 4:25:34 AM Subject: Re: [ccp4bb] seleno methionine labelling of protein -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear , I am not up to date, but is there an expression system that allows you to incorporate D-amino acids into proteins? In terms of phasing only the Se-atom is a point or sphere, so the signal won't be altered provided the position of that atom is the same in either case. Best, Tim On 11/12/2012 10:06 AM, Faisal Tarique wrote: Dear All just want to ask the difference between labelling your protein with D L selenomethionine mixture and L selenomethionine alone..will it make difference in anomalous diffraction,detection of Se signal and extraction of phases. Or will be the same for both..? - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQoMCOUxlJ7aRr7hoRAopyAKD4kBksPb4zIDYDgok5r5BuuefndgCgrf3S r+YIxyjC50XywK1Tgc+fglk= =3GcK -END PGP SIGNATURE-
Re: [ccp4bb] Intra-molecular interactions
Dee, Intramolecular electrostatic interactions are common in protein structures, for example between i and i+4 pairs on the same face of a helix. Robert Baldwin (Stanford) and others did a lot of work on the contribution of intramolecular salt bridges to helix stabilization in model helices. If I remember correctly, some of his studies indicated that disrupting the ionic interaction could completely destabilize the helix. Your real question is if they play a role in disordered regions. The way I think about this is that an intramolecular salt bridge may be able to induce structure in an otherwise disordered region (if the pairs are strategically located). Alternatively, the entropic penally of folding may be too much to pay for by the enthalpy gain due to such an interaction (remember solvation). The bottom line is that the fact that it is disordered means the interaction is not contributing significantly. Chitta - Original Message - From: Xiaodi Yu uppsala@hotmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Saturday, November 3, 2012 12:06:32 PM Subject: [ccp4bb] Intra-molecular interactions Dear All: I have a quick question: how common it is that electrostatic interactions are involved in intra-molecular interactions, particularly in intrinsically disordered proteins? Is this interaction specific and any example? Thanks, Dee Xiaodi Yu, Ph.D. Boston Children's Hospital Dana-Farber Cancer Institute Harvard Medical School 3 Blackfan Boston, MA 02115
[ccp4bb]
I agree with Garib that we should stop this, because nothing productive seems to be coming out of this discussion. I however like to clarify one thing about the comment I made about agism. I merely intended to interpret what the age limit preference means in the context of Government of India's policy (therefore, I used the quotation mark). I DO NOT endorse the policy. The government stipulates an age limit for any government job, including the job at MBU, IISC (last time I knew, it used to be controlled by the Government, at least in the recruitment and retirement part). Clearly, it does not confirm to the Equal Employment Opportunity (EEO) in the US, very much like many regulations in China or in Europe do not mean anything in the US. What works for US does not necessarily work for India. I always wondered what the age limit means. Why should there be one? Maybe the answer lies in the socio-economic structure of the country. Here is a country with nearly a billion people with much less opportunity (the GDP is nowhere comparable to that of US). The question for the law makers is how to distribute a very few job among the most qualified people. If we are looking at two candidates that have very similar credentials, maybe the one who accomplished them at an earlier age is better?. I think that's what the word 'preferably' signifies. I also think that it is somehow related to some grant agencies in the US determining who is a 'young investigator'. Again, because the opportunities are so few. Am I wrong? Once again, I love the Equal Employment Opportunity (EEO) in US and that's one of the reasons I came to US. But, do I think a discussion over CCP4bb can change government of India' policy and India's socio-economic status? I think not, but then, I may be a pessimist. Best Chitta - Original Message - From: Garib N Murshudov ga...@mrc-lmb.cam.ac.uk To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, October 30, 2012 5:40:23 AM Subject: [ccp4bb] Dear all Could we stop at this point. regards Garib On 30 Oct 2012, at 18:35, Kavyashree Manjunath wrote: Dear Sir, I agree to that. But I presume that this is a platform to discuss scientific problems and not a forum to discuss or pour out personal frustrations. There may be other channels for such grievances but not this. I was just hoping this does not become a social network wherein everyone are free to express anything. Thank you kavya -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Kavya, given the test is written in proper English with proper grammer etc., I think you are actually asking for censorship. I am happy ccp4bb does not censor such emails, be they in accordance with one's opinion or not! Best, Tim On 10/30/2012 06:15 AM, Kavyashree Manjunath wrote: Dear CCP4 users, It is extremely sad that CCP4BB has failed to moderate/screen for such spam mails! Thanks kavya Dear Friends, There is no need to apply to this position, we suggest. It is a PREDETERMINED SELECTION, i.e. candidate is fixed and this (advertisement, screening, selection board, selection and approval) is just the procedure. It does not matter whether you apply or not. If you apply and called for interview, then you have to waste your valuable time as well as huge travel money unless some Big Boss is fixing you to the post. Interestingly Indian Institute of Science recruits and carries faculties and trains them in such a way that it has become a epicentre of recruitment scams across India and it make rest of Indian Scientists/Faculties in their path of scams and CRIME. Students also inherit the character of their boss. They do not participate in any form of fair selection in the country. Almost all cases they select and load many times inferior candidates even though candidate was not seen by anybody or interviewed. Similarly they distribute various national awards among themselves and within their group. THEY ARE NOT ASHAMED AT ALL. This is just an attempt of WASTING HUGE PUBLIC MONEY by a bunch of crooks who are good for nothing but worst for everything. Sham - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQj5K5UxlJ7aRr7hoRAum6AKDzlXQSoX827+OrPJOiWy1zF24pVgCgymMq Hgv5aAxCqVjSnONml1GSfx0= =KVPK -END PGP SIGNATURE- -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed
Re: [ccp4bb] oof topic: pH effect on substrate analog
Peter, I think it would depend if the substrate analog have ionizable groups? If the analog does not have ionizable groups, it is hard to imagine how the the titration of ionizable groups on the protein would impair the binding. Chitta - Original Message - From: Peter Hsu hsuu...@u.washington.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, October 30, 2012 12:12:58 PM Subject: [ccp4bb] oof topic: pH effect on substrate analog Hi all, I'm working on a protein that I recently got crystals of. My functional studies show that the protein has optimal activity at lower pHs, while losing 90% activity at about pH8. I've been trying to soak/cocrystallize a substrate analog (small molecule) into my crystals (grown at ~pH8) with no real luck. I'm wondering, since I lack activity at this pH point, would it lead to no binding of a substrate analog? Thanks for any insights Peter
Re: [ccp4bb] Ca or Zn
Veerendra, You can rule out if zinc has replaced calcium ions (although I agree with Nat and others that looking at the coordination sphere should give a big clue) by taking a few crystals, washing them a couple of times and subjecting them to ICP-MS analysis, if you have access to this technique. You can learn how many zinc, if any, have bound per one protein molecule in the dissolved crystal. Best Chitta - Original Message - From: Veerendra Kumar veerendra.ku...@uconn.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, October 30, 2012 2:55:33 PM Subject: [ccp4bb] Ca or Zn Dear CCP4bb users, I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites. I purified the protein in presence of Ca2+ and crystallized the Ca2+ bound protein. I got crystal and solved the structure by SAD phasing at 2.1A resolution. I can see the clear density in the difference map for metals at the expected binding sites. However I had ZnCl2 in the crystallization conditions. Now i am not sure whether the observed density is for Ca or Zn or how many of them are ca or zn? Since Ca (20 elctron) and Zn (30 electron), is this value difference can be used to make a guess about different ions? is there any way we can find the electron density value at different peaks? Thank you Veerendra
Re: [ccp4bb] Faculty positions at Molecular Biophysics Unit, IISc, Bangalore
Please allow me to share with you some history here. Late Prof. GN Ramachandran (of Ramachandran Plot) was a faculty of MBU, IISC. If you want to find out yourself how good/bad it is, please register for the upcoming conference on International Conference on Biomolecular Forms and Functions: celebrating 50 years of the Ramachandran Map. Chitta P.S. The agism means: 'we prefer assistant professors who are not too old' (35 years according to Indian life expectancy is 45 years in the US). Chitta Das Assistant Professor of Chemistry Department of Chemistry Purdue University 560 Oval Drive West Lafayette IN 47907 Ph. 765 494 5478 http://www.chem.purdue.edu/das/ - Original Message - From: James Stroud xtald...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, October 29, 2012 3:21:51 PM Subject: Re: [ccp4bb] Faculty positions at Molecular Biophysics Unit, IISc, Bangalore The agism in the advertisement doesn't do the institute much credit. I'm inclined to believe Sham, given the Institutes stated policies: Applicants, preferably below 35 years James On Oct 29, 2012, at 11:28 AM, Narayana VL Sthanam wrote: Sham, who so ever you are, if you have such a long list of complaints, why don’t you put your name clearly and complain openly, instead of hiding behind some anonymous ‘SHAM’ name. What you write about IISc may be all true or it may be reflection of your frustration for not getting a job at IISc! How do we know? So, grow a ‘spine’, if you have a complaint, say it like a man and do not hide behind and bad mouth others anonymously like spoiled child. You are not only throwing dirt on such a prestigious Indian institution, and also on many decent and capable scientists who do outstanding work and produce brilliant graduate students, some of which I was fortunate to have in my lab. Best Narayana Sthanam -- Narayana Sthanam,Ph D Professor of Structural Biology 244 CBSE 1025 18th Street South Center for Biophysical Sciences and Engineering University of Alabama at Birmingham Birmingham, Al 35294 Phone: 205 934 0119 URL: http://www.opt.uab.edu/narayanalab “ Never let success go to your head, nor failure to your heart ” From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of U US Sent: Monday, October 29, 2012 12:03 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Faculty positions at Molecular Biophysics Unit, IISc, Bangalore Dear Friends, There is no need to apply to this position, we suggest. It is a PREDETERMINED SELECTION, i.e. candidate is fixed and this (advertisement, screening, selection board, selection and approval) is just the procedure. It does not matter whether you apply or not. If you apply and called for interview, then you have to waste your valuable time as well as huge travel money unless some Big Boss is fixing you to the post. Interestingly Indian Institute of Science recruits and carries faculties and trains them in such a way that it has become a epicentre of recruitment scams across India and it make rest of Indian Scientists/Faculties in their path of scams and CRIME. Students also inherit the character of their boss. They do not participate in any form of fair selection in the country. Almost all cases they select and load many times inferior candidates even though candidate was not seen by anybody or interviewed. Similarly they distribute various national awards among themselves and within their group. THEY ARE NOT ASHAMED AT ALL. This is just an attempt of WASTING HUGE PUBLIC MONEY by a bunch of crooks who are good for nothing but worst for everything. Sham Date: Mon, 29 Oct 2012 11:47:06 +0530 From: vikasnavra...@gmail.com Subject: [ccp4bb] Faculty positions at Molecular Biophysics Unit, IISc, Bangalore To: CCP4BB@JISCMAIL.AC.UK Dear all Kindly make a note. Indian Institute of Science, Bangalore. Molecular Biophysics Unit ( http://mbu.iisc.ernet.in/ ) Opening for Assistant Professor Positions. Applicants, preferably below 35 years, should have a Ph.D. with postdoctoral experience with an excellent publication record. We seek candidates in the general area of structural biology with an emphasis on understanding macromolecular systems at the molecular level. Applications with a detailed CV, research plan (not exceeding 3 pages) and names of at least 4 referees should be sent to chair...@mbu.iisc.ernet.in Best
