[ccp4bb] rigid flexible analysis
Hi everyone! I would like to know the current online servers available for analyzing the rigid and flexible regions of proteins.Looking forward for precious inputs from the community. Sincerely, Dr. Gauri Misra Assistant Professor Amity Institute of Biotechnology Amity University Noida (U.P.) India
Re: [ccp4bb] Protein thermostability
Hi Hsu, You can check my paper. We have done a similar analysis of several factors responsible for thermal stability, analysis done using various mesophiles and thermophiles, Crystal structure of the *Bacillus anthracis* nucleoside diphosphate kinase and its characterization reveals an enzyme adapted to perform under stress conditions. Gauri Misra et al. Proteins: Structure, Function, and Bioinformatics Volume 76, Issue 2, http://onlinelibrary.wiley.com/doi/10.1002/prot.v76:2/issuetoc pages 496–506, 1 August 2009 Hope it gives you some help. Best wishes, Gauri On Sat, Jun 30, 2012 at 7:45 AM, Theresa Hsu theresah...@live.com wrote: Dear all I have two homologues structures, from a mesophilic bacterium and a thermophilic bacterium. Is there any software or server that can calculate the differences that contribute to thermostability, e.g. proportion of amino acids that form Hydrogen bonds and number of prolines? Thank you.
[ccp4bb] off topic question
Hi, A nuclear receptor is purified only in the presence of strong affinity bound ligand. Is there some method to study and quantitate binding affinities of this protein with other ligands (it is bound to the high affinity ligand after purification)? Attempts to purify in presence of low affinity ligands and subsequent substitution trials were not successful. I therefore seek suggestions from the expert community. Gauri
Re: [ccp4bb] mutation and minimization
You can try Swiss-PDB viewer.. 2011/5/12 Thomas Holder thomas.hol...@tuebingen.mpg.de Hi Andreas, I would like to introduce point mutations in a structure and quickly (and dirtily) minimize the new residue. (Best rotamer dependent on local environment, or the like.) What are simple approaches that don't involve VMD/NAMD or some such overkill. PyMOL has a Mutagenesis wizard but you have to pick the best rotamer yourself. Cheers, Thomas -- Thomas Holder MPI for Developmental Biology Spemannstr. 35 D-72076 Tübingen
[ccp4bb] GST tag digestion conditions
Hi everyone, This is although an off topic question but I would certainly seek expert advices on the following query: I have a GST-tagged protein that is purified in presence of the steroids. I carry out an in column digestion using thrombin. Digestion buffer compositin: 50mM Tris pH: 8.0, 150mM NaCl, 10% glycerol, 100micro Molar steroid, 0.15 n-octyl-beta glucoside, 1mM DTT, 3mM CaCl2, 12units thrombin/ml of protein Problem: Protein when purified in presence of steroid like testosterone gets around 85% digested on incubating at 4 degree C for 24 hours with the above specified thrombin quantity. However, when the steroid is replaced by some plant steroids like phytoestrogen there is no GST digestion under the same conditions? Can someone provide some inputs for achieving the perfect GST-digestion? Whether a change in some of the constituents of the buffer or some other factor could be effective? Thanks in advance for any suggestions. Cheers Gauri
Re: [ccp4bb] Methods to calculate the importance of mutated residue on the stability of protein
Hi Keat, Check this http://cupsat.tu-bs.de/ (CUPSAT: Cologne University Protein Stability Analysis Tool) Hope it serves your purpose. Gauri On Wed, Apr 13, 2011 at 9:50 AM, Heng Keat Tam t...@bio.chemie.uni-freiburg.de wrote: Dear all, Do anyone know the way to estimate the importance of mutation contributing to the stability of protein? Thanks for the help. Heng-Keat
Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
Hi, You can read the spectrophotometric absorption at 280 nm and 200nm in UV range. It should serve your purpose and provide a decent idea for the amount of protein in the sample. Provided that absorbance at 280nm is given by aromatic rings but at the same time absorbance at 200nm is contributed by peptide bonds. A simultaneous reading at 260 nm which can be deducted later will substract the effect of nucleic acids. The relation between the readings at these values and protein concentration you can easily find anywhere on the web. Best wishes Gauri
[ccp4bb] Question about GST cleavage
Just an offshoot of the same Question.. I would like to ask whether the same applies for GST-tag digestion using thrombin.. No agitation gives better results in the above case too... Any personal experiences On Thu, Mar 31, 2011 at 11:29 AM, Klaus Piontek klaus.pion...@ocbc.uni-freiburg.de wrote: And not at full moon! Klaus Am 31.03.2011 um 16:23 schrieb Xiaopeng Hu: Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng Dr. Klaus Piontek Albert-Ludwigs-University Freiburg Institute of Organic Chemistry and Biochemistry, Room 401 H Albertstrasse 21 D-79104 Freiburg Germany Phone: ++49-761-203-6036 Fax: ++49-761-203-8714 Email: klaus.pion...@ocbc.uni-freiburg.de Web: http://www.chemie.uni-freiburg.de/orgbio/w3platt/
Re: [ccp4bb] kinase purification
You can use ATP agarose for purification and include the cofactor required right from expression till purification steps. On Tue, Mar 29, 2011 at 8:10 PM, Neeraj Kapoor nkap...@mail.rockefeller.edu wrote: Hi All, I am trying to express a kinase but unfortunately there is aggregation happening as the protein is purified over a column. SInce I am new to the field of kinase expression and purification, I was wondering if someone could provide me with a couple of good references that can hit the ground running for me. I would also very much appreciate any helpful suggestions that anyone might have. thanks Neeraj
[ccp4bb] protein aggregation
Hi, What are the different methods to prevent protein aggregation while concentrating so as to increase the concentration of the protein? I have some idea of adding EDTA and charged amino acids like L-Arg and L-Glu. I would appreciate if the readers share their experiences. Thanks! Gauri
Re: [ccp4bb] protein aggregation
The protein purifies nicely there is no problem in that. Just at the last step when it is concentrated it starts precipitating beyond a concentration of 1mg/ml. Already the purification buffers have the detergent. On Wed, Mar 23, 2011 at 1:44 PM, Kornelius Zeth kornelius.z...@tuebingen.mpg.de wrote: 2 M urea and detergents On Wed, 23 Mar 2011 13:41:55 -0400 gauri misra kamga...@gmail.com wrote: Hi, What are the different methods to prevent protein aggregation while concentrating so as to increase the concentration of the protein? I have some idea of adding EDTA and charged amino acids like L-Arg and L-Glu. I would appreciate if the readers share their experiences. Thanks! Gauri -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany kornelius.z...@tuebingen.mpg.de Tel -49 7071 601 323 Fax -49 7071 601 349
Re: [ccp4bb] Can procheck or other tools report bad geometry for ligand?
Hi, Swiss-pdb viewer works well for small peptides, you can check if it serves your objective too... Even WHAT IF provides clues to bond angles, bond length and torsion angles. Gauri On Thu, Mar 10, 2011 at 3:56 PM, Robert Immormino immorm...@gmail.comwrote: Hi Halliang, If the ligands are in the pdb het dictionary I think MolProbity will look at bonds and angles...maybe even dihedrals. Cheers, -bob On Thu, Mar 10, 2011 at 12:23 PM, Hailiang Zhang zhan...@umbc.edu wrote: Hi there, I want to found some bad geometry for my ligand (sugar rings). The procheck .out file seems only shows the bad bond length or angles for protein. Is there any way we can get these information for sugar rings? Thanks in advance! Hailiang
Re: [ccp4bb] determining the domain for overexpression and crystallization
Hi, To start with it would be great if you look in to the secondary structure prediction of the sequence using any of the standard servers like PSIPRED, JPRED etc. Many more available at expasy site http://ca.expasy.org/tools/. Whatever construct you finally choose to make just remember the standard rule that we generally follow is to avoid deleting the alpha helices and beta sheets. You can design your initial primers so as to obtain the complete amplification of these secondary structures from any part within the protein. You can even use the various modules of the following online available server http://scratch.proteomics.ics.uci.edu/ to have an idea of the intrinsically disordered regions in the protein, transmemebrane regions and disulfide bonds that would certainly help you in initiating in the right direction. Best wishes Gauri On Mon, Mar 7, 2011 at 4:10 AM, Xianhui Wu wuxian...@gmail.com wrote: Dear all, Before we try to study the crystal structure of an unknown protein, we need to determine the sequence that can fold into a compact and stable 3D domain. What kinds of methods can we choose? -- Best regards, XH Wu
Re: [ccp4bb] creat a model with insertion
Hi Chen, Check SPDBViewer if it is of some help to you! Gauri On Thu, Jan 13, 2011 at 5:57 AM, Qing Chen qchen.c...@googlemail.comwrote: Dear all, I have the wt protein structure that contains two domains. I want to creat a model with two glycerin inserted between the two domains. Which software or webserver could do this? Pls help. Qing Chen
Re: [ccp4bb] Looking for the following values...
Hi, Generally if we use CCP4i we can find these details easily in scala log files. Gauri On Thu, Jan 13, 2011 at 2:48 PM, J. Fleming jonathan...@hotmail.com wrote: Hi All, I'm about ready to deposit my structure and have used pdb_extract to aid in the process. Unfortunately the following values were not found and are required by ADIT: 1) Under Data Collection, Reflections section: Observed criterion sigma(F) and Observed criterion sigma(I) 2) Under Refinement, Refinement Statistics section: Number unique reflections (all) I looked in my log files for HKL2000, PHASER, and PHENIX but am confused on where to find the required values above. I tried searching the logs for the mmCIF items but that didn't help. Could someone point me the right direction so I can deposit my structure with the correct values? Thanks in advance, -Jon
Re: [ccp4bb] superpositioning two ligands
Dear Rex, You can try SPDB viewer if it serves your purpose. Gauri
Re: [ccp4bb] dissolving peptides !
Rashmi, You can try 1%DMSO and also acetonitrile but the former is preferable. Gauri On Mon, Jun 7, 2010 at 3:04 AM, rashmi panigrahi rashmi.panigrah...@gmail.com wrote: Hello everybody, I have a 16 mer peptide which is predicted to be positively charged alpha helix and has 50% of its sequence hydrophobic. Could any one recommend the best way to dissolve it, and then it can be used for crystallization trials. thanks -- rashmi
Re: [ccp4bb] Alignment software
Try ESPript.. Gauri
[ccp4bb] refines Query for non covalent interactions
Thanks to all for responding.. Infact precisely i am looking for some server that can calculate hydrogen bonds, electrostatic interactions, and hydrophobic interactions present in various pdbs...simultaneously Cheers GM
[ccp4bb] noncovalent interactions
Hi, Which free online servers are best for calculation of non covalent interactions present in several pdb structures? Thanks for any insights provided by the community members... Cheers GM
[ccp4bb] 2,3-butanediol
Dear all, I would like to know which is the more suitable method of using 2,3-Butanediol as cryoprotectant? Addition in to reservoir buffer or crystal soaking or both can be tried? Cheers Gauri
