[ccp4bb] Off Topic: How to delete loops from a protein
Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
[ccp4bb] Off Topic: How to delete loops from a protein
Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
[ccp4bb] Off Topic: How to delete loops from a protein
Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
[ccp4bb] Off Topic: How to delete loops from a protein
Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
Re: [ccp4bb] Off Topic: How to delete loops from a protein
You could run your sequence through this web server first: http://ffas.burnham.org/XtalPred-cgi/xtal.pl Then regarding your loops you could use MUSTANG (or any other 3D alignment tool which generates a superimposed ensemble of structures) http://www.ncbi.nlm.nih.gov/pubmed/16736488 to identify with your other homologs which regions should be trimmed off. Then you turn to your favorite site directed mutagenesis kit and chop off the loops. How big is your protein ? Have you tried different expression systems ? Good luck ! Jürgen On Jul 18, 2011, at 9:01 PM, Obayed Ullah wrote: Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah .. 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/
Re: [ccp4bb] Off Topic: How to delete loops from a protein
Hi Obayed, you could give in situ protolysis a try. This is where you add a bit of protease along with you target protein to the crystallization drop. It has been quite successful for the folks at the SGC. Here are the relevant references: Dong A, et al. In situ proteolysis for protein crystallization and structure determination. Nat Methods. 2007 Dec;4(12):1019-21.PMID: 17982461. (http://www.ncbi.nlm.nih.gov/pubmed/17982461) Wernimont A, Edwards A. In situ proteolysis to generate crystals for structure determination: an update. PLoS One. 2009;4(4):e5094. PMID: 19352432. (http://www.ncbi.nlm.nih.gov/pubmed/19352432) good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Mon, 18 Jul 2011, Obayed Ullah wrote: Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
Re: [ccp4bb] Off Topic: How to delete loops from a protein
Hi Obayed, If I understood your question well, you are looking for something called "secondary structure prediction". I googled these keywords and found this server: http://bioinf.cs.ucl.ac.uk/psipred/ You may find other interesting servers on the web and some literature comparing them. I think such methods need only the sequence of your protein to predict its secondary structures. Hope this helps, Francois. On 07/19/2011 02:14 PM, Eric Larson wrote: Hi Obayed, you could give in situ protolysis a try. This is where you add a bit of protease along with you target protein to the crystallization drop. It has been quite successful for the folks at the SGC. Here are the relevant references: Dong A, et al. In situ proteolysis for protein crystallization and structure determination. Nat Methods. 2007 Dec;4(12):1019-21.PMID: 17982461. (http://www.ncbi.nlm.nih.gov/pubmed/17982461) Wernimont A, Edwards A. In situ proteolysis to generate crystals for structure determination: an update. PLoS One. 2009;4(4):e5094. PMID: 19352432. (http://www.ncbi.nlm.nih.gov/pubmed/19352432) good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Mon, 18 Jul 2011, Obayed Ullah wrote: Hi all I wrote last time but got only one feedback. I know some of you guys must have this experience that how to delete loops from the protein. Please help me with suggestions. I am working with a human protein which have around 20% sequence identity with the other proteins of the same family. Structure of some of the proteins from this family have been solved. All the solved structures have around 20% identity with my protein. I am trying to crystallize the protein but it looks like very hard to get crystal. I have tried different N and C terminally truncated constructs for crystallization but no crystal. My feeling is that probably there is some flexible loops with in the protein which limiting the crystallization. So I want to delete the loops with in the protein (not to truncate in the terminal, I already have done this). I am not asking suggestion about how to delete the loop rather how to decide where the loop is. I am not sure how much it will be helpful to get a homology model of such a protein having low sequence identity. Is there any strategy to decide where the loop could be? Does anybody know any established/ rational method to do that. Waiting for your suggestions Obayed Ullah
Re: [ccp4bb] Off Topic: How to delete loops from a protein
I've found that predator is one of the best services of this sort: Ref: PROTEINS: Structure, Function, and Genetics 27:329–335 (1997) Server: http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::predator The server is slow but the service is good. James On Jul 18, 2011, at 10:47 PM, Francois Berenger wrote: > Hi Obayed, > > If I understood your question well, > you are looking for something called "secondary structure prediction". > > I googled these keywords and found this server: > http://bioinf.cs.ucl.ac.uk/psipred/ > > You may find other interesting servers on the web and > some literature comparing them. > > I think such methods need only the sequence of your > protein to predict its secondary structures. > > Hope this helps, > Francois. > > On 07/19/2011 02:14 PM, Eric Larson wrote: >> Hi Obayed, >> >> you could give in situ protolysis a try. This is where you add a bit of >> protease along with you target protein to the crystallization drop. It >> has been quite successful for the folks at the SGC. Here are the >> relevant references: >> >> Dong A, et al. In situ proteolysis for protein crystallization and >> structure determination. Nat Methods. 2007 Dec;4(12):1019-21.PMID: >> 17982461. (http://www.ncbi.nlm.nih.gov/pubmed/17982461) >> >> Wernimont A, Edwards A. In situ proteolysis to generate crystals for >> structure determination: an update. PLoS One. 2009;4(4):e5094. PMID: >> 19352432. (http://www.ncbi.nlm.nih.gov/pubmed/19352432) >> >> good luck, >> >> Eric >> >> >> Eric T. Larson, PhD >> Biomolecular Structure Center >> Department of Biochemistry >> Box 357742 >> University of Washington >> Seattle, WA 98195 >> >> email: larso...@u.washington.edu >> >> >> On Mon, 18 Jul 2011, Obayed Ullah wrote: >> >>> >>> Hi all >>> >>> I wrote last time but got only one feedback. I know some of you guys >>> must have this experience that how to delete loops from the >>> protein. Please help me with suggestions. >>> >>> I am working with a human protein which have around 20% sequence >>> identity with the other proteins of the same family. Structure >>> of some of the proteins from this family have been solved. All the >>> solved structures have around 20% identity with my protein. I >>> am trying to crystallize the protein but it looks like very hard to >>> get crystal. I have tried different N and C terminally >>> truncated constructs for crystallization but no crystal. My feeling is >>> that probably there is some flexible loops with in the >>> protein which limiting the crystallization. >>> >>> So I want to delete the loops with in the protein (not to truncate in >>> the terminal, I already have done this). I am not asking >>> suggestion about how to delete the loop rather how to decide where the >>> loop is. I am not sure how much it will be helpful to get a >>> homology model of such a protein having low sequence identity. Is >>> there any strategy to decide where the loop could be? Does >>> anybody know any established/ rational method to do that. >>> >>> Waiting for your suggestions >>> >>> Obayed Ullah >>> >>> >>> >>> >>>
Re: [ccp4bb] Off Topic: How to delete loops from a protein/homology modeling
Hi Obayed, even though there is 20% sequence identity you may be able to get a very good homology model, especially if there is more than one protein structure in the PDB with 20% homology. Then you can overlap the pdbs and find out what structurally needs to be preserved as opposed to what is in total homology preserved. Typically it is the position of turns residues G, D, S, P, N etc. You won't know until you thread your protein through both pdbs and compare them all. Swiss Pro's Expasy has an easy program that will take an alignment with a pdb and generate a homology model with loops spliced in and energy minimized. There are many other more or less complicated programs, but it's a good one to start with. Paul Dr. Paul Kraft Structural Biologist cell 586-596-2770 email: haresea...@yahoo.com email: kraft_proteome_resea...@yahoo.com This communication and any attachments contain information which is confidential and may also be privileged. It is for the exclusive use of the intended recipient(s). If you are not the intended recipient(s) please note that any form of disclosure, distribution, copying or use of this communication or the information in it or in any attachments is strictly prohibited and may be unlawful. If you have received this communication in error, please notify the sender and delete the email and destroy any copies of it. E-mail communications cannot be guaranteed to be secure or error free, as information could be intercepted, corrupted, amended, lost, destroyed, arrive late or incomplete, or contain viruses. We do not accept liability for any such matters or their consequences. Anyone who communicates with us by e-mail is taken to accept the risks in doing so. --- On Tue, 7/19/11, James Stroud wrote: From: James Stroud Subject: Re: [ccp4bb] Off Topic: How to delete loops from a protein To: CCP4BB@JISCMAIL.AC.UK Date: Tuesday, July 19, 2011, 2:37 AM I've found that predator is one of the best services of this sort: Ref: PROTEINS: Structure, Function, and Genetics 27:329–335 (1997) Server: http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::predator The server is slow but the service is good. James On Jul 18, 2011, at 10:47 PM, Francois Berenger wrote: > Hi Obayed, > > If I understood your question well, > you are looking for something called "secondary structure prediction". > > I googled these keywords and found this server: > http://bioinf.cs.ucl.ac.uk/psipred/ > > You may find other interesting servers on the web and > some literature comparing them. > > I think such methods need only the sequence of your > protein to predict its secondary structures. > > Hope this helps, > Francois. > > On 07/19/2011 02:14 PM, Eric Larson wrote: >> Hi Obayed, >> >> you could give in situ protolysis a try. This is where you add a bit of >> protease along with you target protein to the crystallization drop. It >> has been quite successful for the folks at the SGC. Here are the >> relevant references: >> >> Dong A, et al. In situ proteolysis for protein crystallization and >> structure determination. Nat Methods. 2007 Dec;4(12):1019-21.PMID: >> 17982461. (http://www.ncbi.nlm.nih.gov/pubmed/17982461) >> >> Wernimont A, Edwards A. In situ proteolysis to generate crystals for >> structure determination: an update. PLoS One. 2009;4(4):e5094. PMID: >> 19352432. (http://www.ncbi.nlm.nih.gov/pubmed/19352432) >> >> good luck, >> >> Eric >> >> >> Eric T. Larson, PhD >> Biomolecular Structure Center >> Department of Biochemistry >> Box 357742 >> University of Washington >> Seattle, WA 98195 >> >> email: larso...@u.washington.edu >> >> >> On Mon, 18 Jul 2011, Obayed Ullah wrote: >> >>> >>> Hi all >>> >>> I wrote last time but got only one feedback. I know some of you guys >>> must have this experience that how to delete loops from the >>> protein. Please help me with suggestions. >>> >>> I am working with a human protein which have around 20% sequence >>> identity with the other proteins of the same family. Structure >>> of some of the proteins from this family have been solved. All the >>> solved structures have around 20% identity with my protein. I >>> am trying to crystallize the protein but it looks like very hard to >>> get crystal. I have tried different N and C terminally >>> truncated constructs for crystallization but no crystal. My feeling is >>> that probably there is some flexible loops with in the >>> protein which limiting the crystallization. >>>
Re: [ccp4bb] Off Topic: How to delete loops from a protein/homology modeling
Homer is one of the best services for homology modeling: http://protein.cribi.unipd.it/Homer/ James On Jul 19, 2011, at 2:06 PM, Paul Kraft wrote: > Hi Obayed, > even though there is 20% sequence identity you may be able to get a very good > homology model, especially if there is more than one protein structure in the > PDB with 20% homology. Then you can overlap the pdbs and find out what > structurally needs to be preserved as opposed to what is in total homology > preserved. Typically it is the position of turns residues G, D, S, P, N etc. > You won't know until you thread your protein through both pdbs and compare > them all. Swiss Pro's Expasy has an easy program that will take an alignment > with a pdb and generate a homology model with loops spliced in and energy > minimized. There are many other more or less complicated programs, but it's a > good one to start with. > Paul > > Dr. Paul Kraft > Structural Biologist > cell 586-596-2770 > email: haresea...@yahoo.com > email: kraft_proteome_resea...@yahoo.com > > > This communication and any attachments contain information which is > confidential and may also be privileged. It is for the exclusive use of the > intended recipient(s). If you are not the intended recipient(s) please note > that any form of disclosure, distribution, copying or use of this > communication or the information in it or in any attachments is strictly > prohibited and may be unlawful. If you have received this communication in > error, please notify the sender and delete the email and destroy any copies > of it. > > E-mail communications cannot be guaranteed to be secure or error free, as > information could be intercepted, corrupted, amended, lost, destroyed, arrive > late or incomplete, or contain viruses. We do not accept liability for any > such matters or their consequences. Anyone who communicates with us by e-mail > is taken to accept the risks in doing so. > > --- On Tue, 7/19/11, James Stroud wrote: > > From: James Stroud > Subject: Re: [ccp4bb] Off Topic: How to delete loops from a protein > To: CCP4BB@JISCMAIL.AC.UK > Date: Tuesday, July 19, 2011, 2:37 AM > > I've found that predator is one of the best services of this sort: > > Ref: PROTEINS: Structure, Function, and Genetics 27:329–335 (1997) > > Server: http://mobyle.pasteur.fr/cgi-bin/portal.py?#forms::predator > > The server is slow but the service is good. > > James > > > > On Jul 18, 2011, at 10:47 PM, Francois Berenger wrote: > > > Hi Obayed, > > > > If I understood your question well, > > you are looking for something called "secondary structure prediction". > > > > I googled these keywords and found this server: > > http://bioinf.cs.ucl.ac.uk/psipred/ > > > > You may find other interesting servers on the web and > > some literature comparing them. > > > > I think such methods need only the sequence of your > > protein to predict its secondary structures. > > > > Hope this helps, > > Francois. > > > > On 07/19/2011 02:14 PM, Eric Larson wrote: > >> Hi Obayed, > >> > >> you could give in situ protolysis a try. This is where you add a bit of > >> protease along with you target protein to the crystallization drop. It > >> has been quite successful for the folks at the SGC. Here are the > >> relevant references: > >> > >> Dong A, et al. In situ proteolysis for protein crystallization and > >> structure determination. Nat Methods. 2007 Dec;4(12):1019-21.PMID: > >> 17982461. (http://www.ncbi.nlm.nih.gov/pubmed/17982461) > >> > >> Wernimont A, Edwards A. In situ proteolysis to generate crystals for > >> structure determination: an update. PLoS One. 2009;4(4):e5094. PMID: > >> 19352432. (http://www.ncbi.nlm.nih.gov/pubmed/19352432) > >> > >> good luck, > >> > >> Eric > >> > >> > >> Eric T. Larson, PhD > >> Biomolecular Structure Center > >> Department of Biochemistry > >> Box 357742 > >> University of Washington > >> Seattle, WA 98195 > >> > >> email: larso...@u.washington.edu > >> > >> > >> On Mon, 18 Jul 2011, Obayed Ullah wrote: > >> > >>> > >>> Hi all > >>> > >>> I wrote last time but got only one feedback. I know some of you guys > >>> must have this experience that how to delete loops from the > >>> protein. Please help me w
