Re: [ccp4bb] determining the domain for overexpression and crystallization
Dear all, Thank you very much for all of information about domain determination! I think the limited proteolysis is a good choice for the domain determination as we have no information about a protein. However, if we do have a domain information by the bioinformatics, how can we truncate the domain? Are we need to keep three or five more amino acids in two ends? Thanks. Best regards, Xianhui On Tue, Mar 8, 2011 at 2:22 AM, Mark J van Raaij mjvanra...@cnb.csic.eswrote: for an experimental way to determine soluble domains see the following paper: ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets. Yumerefendi H, Tarendeau F, Mas PJ, Hart DJ. J Struct Biol. 2010 Oct;172(1):66-74. Epub 2010 Mar 4. Review. PMID: 20206698 [PubMed - indexed for MEDLINE] Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij/index.php?l=1 On 7 Mar 2011, at 19:18, gauri misra wrote: 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 -- Best regards, Xianhui
Re: [ccp4bb] determining the domain for overexpression and crystallization
yes limited proteolysis is the best choice for the domain determination of unknown protein from our lab some people did this. after doing limited proteolysis just sequence digested part N terminally or the C terminally and find out the region from where its getting digested. side by side u can do fold prediction using phyre or u can use fold index to know the compact part of the protein. go through J Biol Chem. http://www.ncbi.nlm.nih.gov/pubmed/18974091 2008 Dec 26;283(52):36532-41. Epub 2008 Oct 30. Characterization of Rv3868, an essential hypothetical protein of the ESX-1 secretion system in Mycobacterium tuberculosis. Luthra Ahttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Luthra%20A%22%5BAuthor%5D , Mahmood Ahttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Mahmood%20A%22%5BAuthor%5D , Arora Ahttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Arora%20A%22%5BAuthor%5D , Ramachandran Rhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Ramachandran%20R%22%5BAuthor%5D . did similar work . On Fri, Mar 18, 2011 at 4:19 AM, Xianhui Wu wuxian...@gmail.com wrote: Dear all, Thank you very much for all of information about domain determination! I think the limited proteolysis is a good choice for the domain determination as we have no information about a protein. However, if we do have a domain information by the bioinformatics, how can we truncate the domain? Are we need to keep three or five more amino acids in two ends? Thanks. Best regards, Xianhui On Tue, Mar 8, 2011 at 2:22 AM, Mark J van Raaij mjvanra...@cnb.csic.eswrote: for an experimental way to determine soluble domains see the following paper: ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets. Yumerefendi H, Tarendeau F, Mas PJ, Hart DJ. J Struct Biol. 2010 Oct;172(1):66-74. Epub 2010 Mar 4. Review. PMID: 20206698 [PubMed - indexed for MEDLINE] Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij/index.php?l=1 On 7 Mar 2011, at 19:18, gauri misra wrote: 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 -- Best regards, Xianhui -- Vandana Kukshal Postdoc Fellow structure biology group international Center For Genetic Engineering and Biotechnolgy Aruna Asaf Ali Marg 110 067 New Delhi INDIA
Re: [ccp4bb] determining the domain for overexpression and crystallization
Hi, There's a whole bunch of programs that can help you out there. The 2 methods I think of right now are DISPROT (there's a server I believe, http://www.ist.temple.edu/disprot/ ) - Must admit I haven't been to that one for quite a while; DISPROT provides areas of your sequence with high probability of disorder hydrophobic cluster analysis. There are many others as well, can't think of them right now. Also, common sense (like trying to crystallise with and without tags) can be helpful. You may have to try to crystallise several constructs. And there's more than just compact and stable to crystallisation. Monodispersity is quite important too. Fred. Xianhui Wu 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] 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] determining the domain for overexpression and crystallization
for an experimental way to determine soluble domains see the following paper: ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets. Yumerefendi H, Tarendeau F, Mas PJ, Hart DJ. J Struct Biol. 2010 Oct;172(1):66-74. Epub 2010 Mar 4. Review. PMID: 20206698 [PubMed - indexed for MEDLINE] Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij/index.php?l=1 On 7 Mar 2011, at 19:18, gauri misra wrote: 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
