[ccp4bb] Protein-DNA complex crystallisation
Hello all I am novice to this field and trying to crystallize a protein-protein complex verses DNA i.e a ternary complex of protein complex and DNA, which stochiometry is 2:!. I am wondering 1-what should be the DNA and protein ratio for the ternary complex like this typically for binary complex it is taken as 1.2 :1. 2- what should be the minimum concentation of DNA in the crystallization drop while protein-DNA complex (binary complex of protein-DNA) crystallization. similary what care should be taken for the ternary complex crystallisation. 3- What are the screen would be better to start the screening of crystallisation of the complex. 4- Whether crystallisation under oil could be done for the complexes or hanging drop would be better than the crystallisation under oil in this concern. I would really apprecitae the suggestions. Thanks in advance thomas
[ccp4bb] Precipitation of protein-protein complex under most of the crystallization condition
Hello BB I am working with small protein-protein complex of Molecular weight 40kDa. This protein expresses well and soluble in 20mM Tris-Cl pH-8.0 and 50mM of KCl, could be concentrated upto 20mg per ml in this buffer. I have purifed this protein with Ni-NTA resin and they are free from any contaminants. Therefore, this protein have been directly used for the screening. I started crystallisation screening with INDEX screen of hampton research and some other lab made random screen. It precipitates immediately in the most of the condition (85%) except those conditions where PEG and Jeffamine are as precipitant with HEPES or Tris-Cl buffers above the pH value 7. Another conditions where it doesnot precipiatate are 0.1M Tris-Bis pH-5.5 + 3M NaCl , and 0.1M Na-Acetate pH-4.5 + 3M NaCl. This is a strange behaviour of the protein complex. I would appreciate the suggestions in this concern. What are the other possibilties, which can be explored through this observation? Although I can proceed for the screening with the other screens like PEG Screen or crystal screen or some other random screens, it would be better to find out the root problem of the immediate precipiation of the proteins in the crystallisation conditions. Thanks in advance Jhon
[ccp4bb] degradation of protein durring freez thaw
Hello BB I apolozize an off topic query. I am working with small proetin-protein complex of 24kDa. I purify this N-terminal His-tagged complex through tylon resin in 20mM of Tris pH-8.0, 0.3M NaCl . After purification this protein complex are dialysed in 20mM tris pH=8.0.I am able to purify enough amount of protein for crystallisation, which can be concentrated upto 10mg per ml. Then i check the dgradation on the polyacrylamide gel after concentration of the protein. I donot see any degdation protein band on the gel. I store the protein at -80 in aliquotes of 100ul immedaitely after concentration in same buffer. protein concentartion are done at 4 degree by centrifugation. Next day before setting up the trays for crystallisation screening, protein solution concentration check is being done. it turns out that this complex has degraded and concentration is only 1-2 mg per ml. i would appreciate the suggestions to prevent the degradation of complex or How should i make it more stable? so, that i can proceed for the crystallisation. I would really appreciate the suggestions. Thanks in advance Thomas
[ccp4bb] surface area calcualtion
Hello all can any one suggest any server or tool which can calculate the burried surface area between the domains of the same monomer? PISA calculates the interfacial surace area between the monomer or oligomer. Can areamol calculate it? Thanks in advance Thomas
[ccp4bb] very low factor of coordiantes
Hi all I am refining an dataset of 2.9 A resolution. I am using TLS+restained refinemnet+ isotropic B factor and simple scaling. After reinement 10 cycle of refinemnt after geometry optimisation i found that b factor of the coordinates drastically goes down to 2.0 of att the atoms of the PDB. If i use this PDB file for further refinment of model optimisation, its R factor suddenly shoots up upto 60.0 Current (Rfree 32.0 and Rwork -25.0). what could be the problem ? can any one suggest the solution? I will appreciate that. Thanks in advance Thomas
[ccp4bb] refinemnet of the ligand in CNS
HEllo all i would like to refine a ligand along with the proetin molecule in CNS. When i google about this query i get all these informations to refine the ligands in cns. The critical box to fill in is the protein coordinate file (your pdb filename). Then save this file to your working directory, and run it using the cns.com file which you will need to change to: #!/bin/tcsh cns_solve generate.inp generate.log If you have ligand in your structure you can also use generate.inp to incorporate the ligand library into the mtf file. To do this you need to generate and then save a CNS topology (.top) and a parameters (.par or .param) file using the PRODRG2http://davapc1.bioch.dundee.ac.uk/programs/prodrg/server. You then need to give CNS the pdb file for your ligand as a separate pdb file AFTER you have build it into your density, and also tell generate.inp where to find your .top and .par files. When i tried this website, it is not working or i donot know how to use. can any one suggest me how to create this dictionary file. i.e topology and parameter file for the CNS of the ligands etc. Thanks in advance Thomas
[ccp4bb] program to create cartoon image from a PDB file which have only C-alpha coordinates
Hi all I am interested to make a cartoon image file from a old PDB. This PDB contains the coordinates of the only C-alpha atoms. Is there is any program to create cartoon image from this PDB? please, can any one suggest me? Thanks in advance With Best Regards Jhon Thomas
Re: [ccp4bb] ccp4i unable to read mtz files
Hi Harry Yes, i have solved the problem. There was permission problem in the /tmp/thomas directory,that's causes this problem to me. Since,my account (login) in my computer had the root (administrative) permission and i was trying to run the program from my login after setenv as well as defining path in my .bashrc, i always use to bump with this problem.But, whenever i ran the program as root(administrator), program runs and complete the any job successfully.As, i looked for the permission of my home directory as well as the tmp directory both were had permission root (administrative) as i changed the permission of my account(login) instead of root(administrative) to my myself i.e thomas, solved the problem and now, i am able to run it very successfully. I guess yours problem is also similar and somewhere there will be problem with the permission of the directories only.So, look carefully about this. With regards Jhon1 thomas
[ccp4bb] ccp4i unable to extract data from project Directory
Hi all I have successfully installed CCP-6.0.2 on Fedora core 6 by automated download page of the CCP4.But, as i tried to run the CCP4i, after defining the project directory , it is not able to extract thye file from the project directory and i get a massage of this kind CCP4 encounterd an error when trying to extract the data from home/thomas/thomas1/test.mtz. Please, can anyone suggest that, what could be the problem ? Is it inatallation problem, or something else? Thanks in advance With regards JT
