Re: [ccp4bb] off topic: Fluorescence labeled protein, neutral at pH=7
Hi Xiang. If you are doing in vitro experiments like binding studies then try BODIPY Fluorophores. It conjugates the protein. Advantage is molecular weight of these BODIPY Fluorophores is few hundreds Da. Also, these dyes have well separable absorption and emission spectrums. Good luck Raj On Mon, Sep 16, 2013 at 9:24 PM, 李翔 lixiang1...@gmail.com wrote: Hi everyone, I want to get some fluorescence labeled protein for my experiment. I wish it is under 40K Da and have isoelectric point of 7 or slightly lower. Do you have any suggestions on the commercially available protein please? Thanks very much for your help! Sincerely, Xiang -- Li Xiang Department of chemistry, Purdue University Email：lixiang1...@gmail.com
Re: [ccp4bb] Data Fitting for protein-ligand interaction.
Hi Monica If protein is Homo-tetramer then one can expect the identical binding sites. I am also working on homo-dimeric protein which binds to DNA. I used PRISM to estimate the binding affinity through flourescence bindingmethod using “SATURATION and NON-LINEAR REGRESSION and ONE SITE binding Model’ considering protein concentration as dimer or monomer. Then I looked for the sensibility of the model by analyzing (comparing) best fit values (like SD, Bmax) of the parameters with reasonable certainty. Unlike ITC binding model fitting (gives good estimate of stoichiometry, cooperativity and also binding sites (one, two or sequential binding sites)), flourescence binding models do not give very good estimate of these parameters. Thus, based on protein you should assume the model which fits to the properties of the protein. Good luck Raj On Thu, Sep 12, 2013 at 12:59 PM, MONICA MITTAL monica.mitta...@gmail.comwrote: Dear All, Firstly sorry for asking a non-crystallography question, but i want help in understanding the data analysis for fitting a protein-ligand binding data. Actually i have a protein which is a tetramer in solution and i have done its flourescence binding with a ligand. I am trying to fit the data to a 4-site binding model in scientist. But i donot have a correct model to fit in the data for identical or non-identical, co-operative or sequential binding. Can anyone help me in analysing the binding data. Any help will be highly appreciated. Thankyou !
Re: [ccp4bb] pseudotranslation issues
Dear Fulvio Saccoccia As you suspected it might be I4 instead of P4. Since fractional coordinates are (0.5, 0.5, 0.479) and, 0.479 is almost 0.5. Try to integrate and scale in I4 and see how chi-square behaves. Raj On Fri, Jun 7, 2013 at 11:17 AM, Fulvio Saccoccia fulvio.saccoc...@uniroma1.it wrote: Dear ccp4bb users, I have a data set collected at 2.2A resolution that I can integrate in P4. Aimless, Phaser and Xtriage recognized a pseudotranslation (34.6% of origin peak) at fractional coordinates 0.500 0.500 0.479 (ORTH 46.7 46.7 37.4). However labelit.index run with sublattice_allow=true search did not detect the pseudotranslation vector. So far, any attempts to phase by molecular replacement was unsuccesfull and I suspect that it could be due to incorrect assignement of space group: Now, some questions arise: 1) does the pseudo translation exist or do not? 2) if translational NCS is present, could it mimic a P41/P42/P43 space group? 3) in any case, any advices in order to properly deal with tNCS will be of great help? I thank you in advance Best wishes -- Fulvio Saccoccia, PhD Sapienza University of Rome Dept. of Biochemical Sciences A. Rossi Fanelli Piazzale Aldo Moro, 5 00185 Rome (Italy)
