Re: [ccp4bb] Peptide solubility issues
Hello Mo Wong, Some points below are good, but don't underestimate custom peptides sometimes... can be much harder/expensive than recombinant proteins. If you ordered the peptides it is good to know how they synthesized them, and how the elution profiles during purification (RP HPLC..) looked like, particularly that they are very hydrophobic. Did they use formic acid, or maybe they already dissolved them in DMSO.. Did they verify the product/bonds with MALDI-TOF for example and NMR? Furthermore, you can try 0.1-0.5 % Triton X-100. My vote would be for a final concentration of 5-25 uM (containing up to 5 % solvent), from a stock of 50-100 % solvent at 1 mM.. Btw I forgot.. for SPR (depends on the system, and especially for a 12mer), some detergents and even a minor DMSO/ethanol can affect the measurements drastically. Best wishes toufic el arnaout On Wed, Mar 5, 2014 at 12:19 AM, Mo Wong mowon...@gmail.com wrote: Hi all, Slightly off topic - but I'm having trouble solubilizing some peptides for SPR and hoped someone on the BB might have some other suggestions. The peptides are intra-chain S-S cross-linked 12mers with pIs of ~3. 10 residues are hydrophobic and 2 are acidic. Peptides have been tested with and without N- and C-terminal modifications (amidation/acetylation). I have tried: ddH2O raising (and lowering) pH (tested up to 8.5) with different buffers Including DMF as a co-solvent (I'm avoiding DMSO because of a methionine present in the peptide) - peptide still visibly precipitates out at 100uM in 5% DMF (also a 1mM stock in 50% DMF shows significant amounts of ppt) Adding a trace amount of detergent (0.005% Tween 20) I'm guessing I could try other co-solvents such as ethanol or initially solubilizing peptide in dilute NaOH before bringing the pH down with addition of a buffer (though I'm concerned about alkaline hydrolysis). Anyway, I'd rather have some insight from people before I waste any further peptide. Thanks for any suggestions.
Re: [ccp4bb] delete subject
http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0053374 On Fri, Mar 29, 2013 at 4:09 AM, Eric Bennett er...@pobox.com wrote: Scott, I'm not sure I understand your last paragraph. Once researchers have had their data pass peer review (which I interpret as meaning a journal has accepted it), how often do you think it happens that it does not immediately get published? Just depositing data in the PDB, or posting it on a public web site, is not meet[ing] the veracity of peer review. There is something to be said for giving credit to the first people who have subjected their data to peer review and had the data pass that step, otherwise people will be tempted to just post data of dubious quality to stake a public claim before the quality of the data has been independently checked. In a case where this initial public non-peer-reviewed posting is of unacceptable data quality, that would dilute credit granted to another person who later obtained good data. An unfortunate number of problematic structures still sneak through peer review. Relaxing quality review standards that must be passed before a scientist gets to claim credit for a discovery is a step backwards IMO. Cheers, Eric On Mar 28, 2013, at 5:06 PM, Scott Pegan wrote: Hey everyone, Both Mark and Fred make some good points. I totally agree with Nat (beat me to the send button). Although in an ideal world with all the advancements in crowd sourcing and electronic media, one might think that posting data on a bulletin board might be considered marking one's turf and protect the scientist place in that pathway towards discoveries. Regrettably, the current reality doesn't' support this case. As structural biologists, we are still in the mode of first to publish gets the bulk of the glory and potentially future funding on the topic. For instance, when I was in graduate school, the lab I was in had KcsA crystals at the same time as a couple of competing groups. Several groups including the one I belong to had initial diffraction data. One group was able to solve KcsA, the first K channel trans-membrane protein structure, first. That group was led by Roderick Mackinnon, now a Noble Laureate partly because of this work. Now imagine if one of Mackinnon's student would have put up the web their initial diffraction data and another group would have used it to assist in their interpretation of their own data and either solved the structure before Mackinnon, or at least published it prior. Even if they acknowledged Mackinnion for the assistance of his data (as they should), Mackinnion and the other scientists in his lab would likely not have received the broad acclaim that they received and justly deserved. Also, ask Rosalind Franklin how data sharing worked out for her. Times haven't changed that much since ~10 years ago. Actually, as many have mentioned, things have potentially gotten worse. Worse in the respect that the scientific impact of structure is increasingly largely tide to the biochemical/biological studies that accompany the structure. In other words, the discoveries based on the insights the structure provides. Understandably, this increasing emphasis on follow up experiments to get into high impact journals in many cases increases the time between solving the structure and publishing it. During this gap, the group who solved the structure first is vulnerable to being scoped. Once scoped unless the interpretation of the structure and the conclusion of the follow up experiments are largely and justifiably divergent from the initial publications, there is usually a significant difficulty getting the article published in a top tier journal. Many might argue that they deposited it first, but I haven't seen anyone win that argument either. Because follow up articles will cite the publication describing the structure, not the PDB entry. Naturally, many could and should argue that this isn't they way it should be. We could rapidly move science ahead in many cases if research groups were entirely transparent and made available their discovers as soon as they could meet the veracity of peer-review. However, this is not the current reality or model we operate in. So, until this changes, one might be cautious about tipping your competition off whether they be another structural biology group looking to publish their already solved structure, or biology group that could use insights gathered by your structure information for a publication that might limit your own ability to publish. Fortunately, for Tom his structure sounds like it is only important to a pretty specific scientific question that many folks might not be working on exactly. Scott -- ** Toufic El Arnaout Trinity Biomedical Science Institute (TCD) 152-160 Pearse Street, Dublin 2 **
Re: [ccp4bb] Oligomerization state
Hello Theresa, Maybe a functional assay (if possible) is better than determining the oligomeric state? Why is finding out the oligomeric state (which is unknown anyway and the answer can be compared to nothing), the answer to is having a tag ok? will the protein crystallize? What if the analysis shows a mixture of different protein oligomeric states in solution? For the general analysis, try SEC-MALLS. I guess the accuracy is 90-95 % for membrane proteins. You might have however to try different detergents or try different columns depending on the empty micelle size and how close it is to the protein-micelle peak etc (please ask if you need a detailed explanation). You will still need to do a func assay, because again you don't know if a particular detergent is harsh from the beginning affecting the oligomeric state, and it is not the tag. Best wishes toufic PS: (1) 100 % accuracy is a complicated term (even crystallography has a R-factor). The error value might be a different oligomeric state or simply an error of the method or experiment. (2) there is always a problem with detergents and lipids with memb proteins :) (3) Thermofluor is just a method, might need fluorescence background optimization, also trying different hydrophobic compounds/detergents etc ** Toufic El Arnaout Trinity Biomedical Science Institute (TCD) 152-160 Pearse Street, Dublin 2, Ireland ** On Thu, Mar 21, 2013 at 6:10 PM, Theresa Hsu theresah...@live.com wrote: Dear all I have a His-tagged membrane protein with unknown oligomerization state. But I am worried that tag addition may induce different state than in native and affect its crystallizability. Is there a single method that can determine the oligomerization state with nearly 100% accuracy? I have use of AUC and SAXS but there seems to be ambiguity about detergent and lipid effects. Is Thermofluor a right method? Does oligomerization require special assembly proteins, which will mean that tag cleavage is not useful to obtain native state? Thank you. Theresa
Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag
Hi Raji, I addition to the tips from Pascal, I would like to say that for a memb protein I worked on with a his-tag separated by a thrombin site, I used thrombin cross linked to agarose from Sigma (1 mL). The beads can be collected, washed and reequilibrated, making it ready for use so many times. In my case 1 mL of this thrombin was enough to cleave up to 5-10 mg of target protein, at 4 °C for 16 h (with slight rotation in a 15 mL tube, total volume up to 10 mL). In FC-12 there was a little better cleavage than in DDM. My opinion is that how thrombin (or most other proteases) will cleave may mostly depend on your protein/fusion type/protein-micelle complex structure/access to the site... You just have to try. Best wishes. toufic On Wed, Feb 20, 2013 at 5:15 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Folks, Sorry this isn't a non-ccp4 post. I am working with a membrane protein for which I am finally able to scale up expression. I am now also able to partially purify my protein from a medium-scale (12-18L) bacterial culture using a two-step tandem affinity purification protocol (Talon followed by amylose affinity steps). As the next purification step, I am about to set up a pilot thrombin cleavage experiment to separate my protein from the His.MBP fusion tag (see below). The construct that I am working with is as follows: His.MBP--ThrombinSite--Membrane Protein There is only one theoretical thrombin cleavage site in the entire fusion protein i.e., at the desired cleavage site with no theoretical secondary sites. I would like to try cleavage both at 4C and around 25C from 4h to overnight but I also have to balance the trials with the material I must generate for the endless permutations and combinations one can try. Each sensible pilot experiment is going to use up partially purified protein from 6-12L preps. FYI. All purification buffers contain DDM and I haven't yet done extensive detergent screens. Please could I ask the community to share tips/suggestions about large-scale thrombin cleavage experiments with their favorite membrane proteins. Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- ** Toufic El Arnaout Trinity Biomedical Science Institute (TCD) 152-160 Pearse Street, Dublin 2 Tel.: +353 85 83 40 157 **
Re: [ccp4bb] CCP4MG and python.exe
Hi Rex, Maybe it is related to a 3rd party package then.. Are you using Windows? Check C++ (https://www.microsoft.com/en-us/download/details.aspx?id=) or mySQL (https://www.mysql.com/downloads/) Try to reinstall these, probably might help, but be sure about the 64 or 32 bit version. toufic On Sat, Feb 16, 2013 at 8:15 PM, Rex Palmer rex.pal...@btinternet.comwrote: I have developed a problem with CCP4MG on my pc. I get the message python.exe has stopped working. I have tried recompiling the program to no avail. Any ideas please. Rex Palmer http://www.bbk.ac.uk/biology/our-staff/emeritus-staff http://rexpalmer2010.homestead.com
Re: [ccp4bb] Faculty positions at Molecular Biophysics Unit, IISc, Bangalore
There is a big difference between reading does not discriminate on the basis of race, color, religion, sex, age, sexual orientation, marital status, national origin, disability, or status as a disabled, Vietnam-era, or other eligible veteran and below 35 years. Then regarding Prof. Chitta's reply, comparing 35 years (india) and 45 years (US) extends it not just to the age, but also to the origin maybe :) toufic On Mon, Oct 29, 2012 at 7:21 PM, James Stroud xtald...@gmail.com wrote: 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*https://mail.ad.uab.edu/owa/redir.aspx?C=80a7a9ef03ea46b58d648b78f13d4272URL=http%3a%2f%2fwww.opt.uab.edu%2fnarayanalab “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
Re: [ccp4bb] inflluence of pH for crystallization on protein 3-D structure
Hi Acoot, Here are some examples you can look at: PDB: 1X0I (crystal structure of bacteriorhodopsin (acid blue form) at pH 2) PDB: 1X0K (crystal structure of bacteriorhodopsin at pH 10) PDB: 2W2E (crystal structure of aquaporin at pH 3.5) PDB: 1YMG (crystal structure of aquaporin at pH 10) People are aware of course of the effect of pH on the structure and they always deposit many PDBs when they have different crystal forms/bound molecules, or pHs. The difference sometimes can be large that they will do another publication/study like when getting the different structure at a different pH. I can't remember the details but a virus protein crystal structure was once crystallized at both pH 4 and pH 7 which gave totally unrelated structures. Functional studies are important before the interpretation of the structure. Regards Toufic El Arnaout Membrane Structural and Functional Biology Group Trinity College Dublin On Sun, Oct 28, 2012 at 3:00 AM, Acoot Brett acootbr...@yahoo.com wrote: Dear All, A protein crystal can be got at pH 5 or 8, or a pH with much extreme value. What will be the relatively extreme pH value to get the crystal on the protein structure solved based on the crystal got? I mean usually we regard the physiological pH as 7. If a crystal was got at pH 5, the structure solved may be different from the protein structure at pH 7. But it seems there is rarely analysis on the discrepancy of the protein structures when publishing 3-D structure with the protein crystal got at relatively extreme pH. I am looking forward to getting your comment on it. Cheers, Acoot
[ccp4bb] Nobel Prize 2012
Congratulations to Robert Lefkowitz and Brian Kobilka: Nobel Prize in Chemistry, 2012! Congrats to the protein structure community. toufic el arnaout
Re: [ccp4bb] [OT]: Purification in ArcticExpress
Hi Katherine, Maybe you can try some useful ideas for your case listed in this study: Protein Expr Purif. 2007 April; 52(2): 280–285. Effect of Osmotic Stress and Heat Shock in Recombinant Protein Overexpression and Crystallization. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1865119/ Best wishes Toufic El Arnaout Membrane Structural and Functional Biology Group Trinity College Dublin On Mon, Sep 24, 2012 at 3:38 PM, Katherine Sippel katherine.sip...@gmail.com wrote: Hi All, I've recently swapped over to expression in the ArcticExpress(DE3) cells for a particularly rock-like protein. I've got soluble expression but I'm having the issue of Cpn60 (the overexpressed chaperonin) piggybacking along with the tagged protein. Google-fu and the CCP4bb archives indicate that this is a known issue but I haven't seen a solution thus far. Does anyone out there have any tricks up their sleeve? Also in case you were wondering in the various BL21 lines, even at extremely low temperatures and 10 uM IPTG, it expresses well but produces inclusion bodies that are completely insoluble in 8M urea, 6M guanidine, high temperature, high pH, high reducing agent, and a number of detergents so swapping back to another cell line isn't in the cards. Any help would be appreciated. Cheers, Katherine
Re: [ccp4bb] the lysozyme of membrane proteins?
Hi, Just for info if you were to use the LCP method (a course by itself), check this about OmpF (membrane lysozyme): http://www.sciencedirect.com/science/article/pii/S1047847712000834 bR protein sometimes takes weeks to give crystals and people prefer the dark (depends on the conf state).. but good idea for spectro assays (check reaction centres/light-harvesting complexes too). Beta barrels are very stable too. If you want to use the GFP fusion and you want to cleave it, it might add extra time and steps to the students than going directly with a GFP free his-tagged protein to trials. Regards Toufic El Arnaout Membrane Structural and Functional Biology Group Trinity College Dublin On Tue, Sep 11, 2012 at 10:18 PM, Ho Leung Ng h...@hawaii.edu wrote: Hello, I am developing an undergraduate biochemistry lab class and would like to incorporate experiments with membrane proteins. Does anyone have suggestions on membrane proteins that are relatively easy to express, purify, and assay? Bonus points for crystallizable! At the moment, my leading candidate is aquaporin AqpZ from E. coli. I am planning to express the membrane protein as a GFP fusion so students can easily follow it through the course of the labs. Thank you, Ho Ho Leung Ng University of Hawaii at Manoa Assistant Professor, Department of Chemistry h...@hawaii.edu
