Re: [ccp4bb] update-MBP fusion protein
On Nov 13, 2010, at 11:04 PM, Jerry McCully wrote: Dear ALL; A few weeks ago, I posted my problem with MBP fusion protein. Thank folks very much for the help. Here is my latest result. 1) Shortening the linker region would signifcantly reduce the cleavage of target proteins from MBP; 2) Adding a C-terminal tag would minimize the degradation of target proteins and facilitate the purification; 3) Low temperature induction(18 degree) would significantly reduce the soluble aggregates; However, all the optimization procedures resulted in a ~150KD-200KD oligomer of MBP fusion protein after gel-filtration. Only a very small portion of MBP fusion protein exsited as monomers even in the expression condition with low temperature and 0.1mM IPTG. Does anyone experience the oligomerization of MBP fusion protein? Thanks again and have a nice weekend! Jerry McCully Dear Jerry, As Artem already pointed out, this is indeed quite common. For an example and a possible way to recover whatever amount of monomer you are getting, see Supplementary Figure 1 of: http://www.ncbi.nlm.nih.gov/pubmed/19052627?dopt=Abstract having said that, in this case we managed since our our monomeric species was still ~20% of the total... if it's significantly less, I'd also suggest that you consider insect/mammalian cell expression. Good luck, Luca Luca Jovine, Ph.D. Group Leader EMBO Young Investigator Karolinska Institutet Department of Biosciences and Nutrition Center for Biosciences Hälsovägen 7, SE-141 57 Huddinge, Sweden Voice: +46.(0)8.6083-301 FAX: +46.(0)8.6081-501 E-mail: luca.jov...@ki.se W3: http://jovinelab.org
Re: [ccp4bb] Graphics for notebook
Dear all, Thanks for your suggestions. From what I learned new GPUs from NVIDIA are using the Optimus technology which does not support Linux, meaning that only the dedicated graphics on the system will be used in Linux. Does it still make sense to go for NVIDIA instead of ATI? Eric
[ccp4bb] Question on calculation of RMSD
Dear All I have two structures of homo-dimeric protein complex with different DNA. I want to calculate RMS deviation between second monomer from these two complexes by fixing superposed first monomer. This I require to know what is the effect of DNA on relative orientation of two monomers in the dimer. Previously I was using MOLEMAN2 to do this calculation. Please can you suggest me any other program to do this calculation. Thanking you Raj E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile)
Re: [ccp4bb] Question on calculation of RMSD
Coot And I would use LSQKAB instead of the SSM method. First look at what stays rigid in one subunit and define those residues for the superposition, then look at the text window where it will report you the rmsd for each position and chain. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/http://web.me.com/bosch_lab/ On Nov 14, 2010, at 4:52 PM, E rajakumar wrote: Dear All I have two structures of homo-dimeric protein complex with different DNA. I want to calculate RMS deviation between second monomer from these two complexes by fixing superposed first monomer. This I require to know what is the effect of DNA on relative orientation of two monomers in the dimer. Previously I was using MOLEMAN2 to do this calculation. Please can you suggest me any other program to do this calculation. Thanking you Raj E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile)
Re: [ccp4bb] Question on calculation of RMSD
DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/). Choose the pairwise alignment function. It is all automatic and will give you the lowest rmsd. Note, however, that it will omit parts that are very different for calculation. Within CCP4, SUPERPOSE or COOT can also do that. You need to specify the residues you want to use for calculation for the lsq fit function. Regards, Clement Angkawidjaja, PhD Specially Appointed CMP Assistant Professor Graduate School of Engineering Osaka University 2-1 Yamadaoka GSE Commoon East 8F Suita-shi, Osaka 565-0871, Japan Tel/Fax +81-6-6879-4580 http://www.mls.eng.osaka-u.ac.jp/~bio_ext/mlsbe123/clement.html /// G30 Chemistry/Biology Combined Major Program http://cmp.sci.osaka-u.ac.jp/CMP/ -Original Message- From: E rajakumar Sent: Monday, November 15, 2010 6:52 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Question on calculation of RMSD Dear All I have two structures of homo-dimeric protein complex with different DNA. I want to calculate RMS deviation between second monomer from these two complexes by fixing superposed first monomer. This I require to know what is the effect of DNA on relative orientation of two monomers in the dimer. Previously I was using MOLEMAN2 to do this calculation. Please can you suggest me any other program to do this calculation. Thanking you Raj E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile)
Re: [ccp4bb] Question on calculation of RMSD
The lowest rmsd might not be the biological relevant one As confucius would say, don't trust the output of a program if you have not programmed it yourself or know what it's doing. Last words of wisdom for tonight. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/http://web.me.com/bosch_lab/ On Nov 14, 2010, at 8:32 PM, Clement Angkawidjaja wrote: DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/). Choose the pairwise alignment function. It is all automatic and will give you the lowest rmsd. Note, however, that it will omit parts that are very different for calculation. Within CCP4, SUPERPOSE or COOT can also do that. You need to specify the residues you want to use for calculation for the lsq fit function. Regards, Clement Angkawidjaja, PhD Specially Appointed CMP Assistant Professor Graduate School of Engineering Osaka University 2-1 Yamadaoka GSE Commoon East 8F Suita-shi, Osaka 565-0871, Japan Tel/Fax +81-6-6879-4580 http://www.mls.eng.osaka-u.ac.jp/~bio_ext/mlsbe123/clement.html /// G30 Chemistry/Biology Combined Major Program http://cmp.sci.osaka-u.ac.jp/CMP/ -Original Message- From: E rajakumar Sent: Monday, November 15, 2010 6:52 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Question on calculation of RMSD Dear All I have two structures of homo-dimeric protein complex with different DNA. I want to calculate RMS deviation between second monomer from these two complexes by fixing superposed first monomer. This I require to know what is the effect of DNA on relative orientation of two monomers in the dimer. Previously I was using MOLEMAN2 to do this calculation. Please can you suggest me any other program to do this calculation. Thanking you Raj E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile)
Re: [ccp4bb] Question on calculation of RMSD
Andrew Martin's ProFit program is another option: http://www.bioinf.org.uk/software/profit/doc/node11.html Having fitted the structures using the ZONE and ATOMS commands to specify which residues and atoms should be included in the fitting, the RMS deviation may then be calculated over a different region of the structure and/or a different atom set. This is achieved using the RZONE and RATOMS commands. The syntax of these commands is identical to that of the ZONE and ATOMS commands described in Sections 8 and 9. -Eric -Original Message- From: E rajakumar Sent: Monday, November 15, 2010 6:52 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Question on calculation of RMSD Dear All I have two structures of homo-dimeric protein complex with different DNA. I want to calculate RMS deviation between second monomer from these two complexes by fixing superposed first monomer. This I require to know what is the effect of DNA on relative orientation of two monomers in the dimer. Previously I was using MOLEMAN2 to do this calculation. Please can you suggest me any other program to do this calculation. Thanking you Raj E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) -- -- Eric Bennett, er...@pobox.com Drawing on my fine command of the language, I said nothing. -Robert Benchley
