Re: [ccp4bb] coot: Show Symmetry
Your pdb-file should contain a line starting with CRYST1 containing cell dimensions and space group. Maybe this is not the case for your pdb-file? What is the output of grep CRYST1 pdffile where you replace 'pdffile' with the name of the file you are loading into coot? Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Mon, 3 Dec 2007, Vineet Gaur wrote: Hi All i want to display symmetry molecules in COOT, but regularly getting the following warning: There are no model molecules that can display symmetry (Cryst1 problem). How to troubleshoot this Cryst1 problem thanks in advance Vineet gaur
Re: [ccp4bb] coot: Show Symmetry
This happens if you try to read a .pdb file from SHELXL into Coot directly. SHELXL doesn't know the name of the space group (!), only the symmetry operators (with the advantage that it has no problems with non-standard settings) so it doesn't put the name of the space group onto the CRYST1 line. An elegant way around this problem is to read the .res file from SHELXL into Coot instead, or you can edit the CRYST1 line in the .pdb file. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-2582 On Tue, 4 Dec 2007, Tim Gruene wrote: Your pdb-file should contain a line starting with CRYST1 containing cell dimensions and space group. Maybe this is not the case for your pdb-file? What is the output of grep CRYST1 pdffile where you replace 'pdffile' with the name of the file you are loading into coot? Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Mon, 3 Dec 2007, Vineet Gaur wrote: Hi All i want to display symmetry molecules in COOT, but regularly getting the following warning: There are no model molecules that can display symmetry (Cryst1 problem). How to troubleshoot this Cryst1 problem thanks in advance Vineet gaur
Re: [ccp4bb] how to change a membrane protein into a water soluble protein?
Another option is refolding which can increase soluble protein content and is used routinely to achieve soluble protein such as the TIMPs http://peds.oxfordjournals.org/cgi/content/abstract/7/8/1035 http://www.proteinscience.org/cgi/reprint/11/10/2493.pdf?ck=nck that said, this is not true of all membrane proteins. Addition of a fusion partner, MBP, to the normally membrane associated FMO3 has been shown to generate stable, soluble protein and the addition of a fusion protein allows purification downstream more easily. Here is a paper where they did as the original poster suggested and tried mutagenesis of hydrophobic regions, including a truncation of a membrane anchor. They achieved increased solubility with this in combination with use of detergents. Krueger SK, Siddens LK, Henderson MC, VanDyke JE, Karplus PA, Pereira CB, Williams DE. Abstract C-Terminal truncation of rabbit flavin-containing monooxygenase isoform 2 enhances solubility. Arch Biochem Biophys. 2006 Jun 15;450(2):149-56. Epub 2006 Mar 29. cheers Quoting Bil Clemons [EMAIL PROTECTED]: There is also the soluble KcsA. Computational design of water-soluble analogues of the potassium channel KcsA. A. M. Slovic, H. Kono, J. D. Lear, J. G. Saven, and W. F. DeGrado (2004) PNAS 101, 1828-1833 Bil Bil Clemons, PhD Assistant Professor of Biochemistry Caltech 157 Broad Center MC 114-96 Pasadena, CA 91125 (626) 395-1796 [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] From: Thomas J Magliery PhD [EMAIL PROTECTED] Reply-To: Thomas J Magliery PhD [EMAIL PROTECTED] Date: Mon, 3 Dec 2007 16:50:03 -0500 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to change a membrane protein into a water solub= le protein? =20 It's hard. See: =20 J Mol Biol. 2005 May 6;348(3):777-87. X-ray structure of a water-soluble analog of the membrane protein phospholamban:=20 sequence determinants defining the topology of tetrameric and pentameric coiled coils. Slovic AM, Stayrook SE, North B, Degrado WF. =20 Slovic, A. M., Summa, C. M., Lear, J. D. DeGrado, W. F. (2002). Computational design of a water-soluble analog of phospholamban. Protein Sci. 12, 337=AD348. =20 Li, H., Cocco, M. J., Steitz, T. A. Engelman, D. E. (2001). Conversion of phospholamban into a soluble pentameric helical bundle. Biochemistry, 40, 6636=AD6645. =20 Frank, S., Kammerer, R. A., Hellstern, S., Pegoraro, S., Stetefeld, J., Lustig, A. et al. (2000). Toward a high resolution structure of phospholamban: design of soluble transmembrane domain mutants. Biochemistry, 39, 6825=AD6831. =20 Tom =20 =20 Daniel Jin wrote: Hi, I am wondering whether there is a way to turn a membrane protein with known crystal structure into a water soluble protein by systematic mutagenesis. I guess it should be doable if we introduce enough hydrophilic residues on the surface. Has anyone tested this crazy idea before? Thank you for your help. Best, Chen =20 Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.=20 http://us.rd.yahoo.com/evt=3D51733/*http://mobile.yahoo.com/;_ylt=3DAhu06i6= 2sR8H DtDypao8Wcj9tAcJ%20 =20 =20 --=20 Thomas J. Magliery, Ph.D. Assistant Professor Department of Chemistry Department of Biochemistry The Ohio State University 1043 Evans Laboratory 100 West 18th Ave. Columbus, OH 43210-1185 =20 (614) 247-8425 office (614) 292-1685 fax [EMAIL PROTECTED] http://www.chemistry.ohio-state.edu/~magliery =20
[ccp4bb] I have my bound ligand (I think), now what?
Hello all, right, I think I have found a ligand bound to my protein. I used the COOT utility to find the ligands after reading in a model and library file generated by sketcher of my ligand. Now I am a bit unsure as to how to proceed? How can I 'accept' a state and/or refine it? Maybe there is a better way to model my ligand into the electron density not accounted for by my protein? Any general suggestions or comments would be appreciated as I am very inexperienced. cheers Brenda
Re: [ccp4bb] how to change a membrane protein into a water soluble protein?
Don't know if anyone has mentioned this paper but its an exact example how to make a K channel soluble. Roosild TP, Choe S. Redesigning an integral membrane K+ channel into a soluble protein. Protein Eng Des Sel. 2005 Feb;18(2):79-84. Epub 2005 Mar 23. PMID: 15788421 [PubMed - indexed for MEDLINE] Scott On Tue, December 4, 2007 4:04 am, Brenda Patterson wrote: Another option is refolding which can increase soluble protein content and is used routinely to achieve soluble protein such as the TIMPs http://peds.oxfordjournals.org/cgi/content/abstract/7/8/1035 http://www.proteinscience.org/cgi/reprint/11/10/2493.pdf?ck=nck that said, this is not true of all membrane proteins. Addition of a fusion partner, MBP, to the normally membrane associated FMO3 has been shown to generate stable, soluble protein and the addition of a fusion protein allows purification downstream more easily. Here is a paper where they did as the original poster suggested and tried mutagenesis of hydrophobic regions, including a truncation of a membrane anchor. They achieved increased solubility with this in combination with use of detergents. Krueger SK, Siddens LK, Henderson MC, VanDyke JE, Karplus PA, Pereira CB, Williams DE. Abstract C-Terminal truncation of rabbit flavin-containing monooxygenase isoform 2 enhances solubility. Arch Biochem Biophys. 2006 Jun 15;450(2):149-56. Epub 2006 Mar 29. cheers Quoting Bil Clemons [EMAIL PROTECTED]: There is also the soluble KcsA. Computational design of water-soluble analogues of the potassium channel KcsA. A. M. Slovic, H. Kono, J. D. Lear, J. G. Saven, and W. F. DeGrado (2004) PNAS 101, 1828-1833 Bil Bil Clemons, PhD Assistant Professor of Biochemistry Caltech 157 Broad Center MC 114-96 Pasadena, CA 91125 (626) 395-1796 [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] From: Thomas J Magliery PhD [EMAIL PROTECTED] Reply-To: Thomas J Magliery PhD [EMAIL PROTECTED] Date: Mon, 3 Dec 2007 16:50:03 -0500 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to change a membrane protein into a water solub= le protein? =20 It's hard. See: =20 J Mol Biol. 2005 May 6;348(3):777-87. X-ray structure of a water-soluble analog of the membrane protein phospholamban:=20 sequence determinants defining the topology of tetrameric and pentameric coiled coils. Slovic AM, Stayrook SE, North B, Degrado WF. =20 Slovic, A. M., Summa, C. M., Lear, J. D. DeGrado, W. F. (2002). Computational design of a water-soluble analog of phospholamban. Protein Sci. 12, 337=AD348. =20 Li, H., Cocco, M. J., Steitz, T. A. Engelman, D. E. (2001). Conversion of phospholamban into a soluble pentameric helical bundle. Biochemistry, 40, 6636=AD6645. =20 Frank, S., Kammerer, R. A., Hellstern, S., Pegoraro, S., Stetefeld, J., Lustig, A. et al. (2000). Toward a high resolution structure of phospholamban: design of soluble transmembrane domain mutants. Biochemistry, 39, 6825=AD6831. =20 Tom =20 =20 Daniel Jin wrote: Hi, I am wondering whether there is a way to turn a membrane protein with known crystal structure into a water soluble protein by systematic mutagenesis. I guess it should be doable if we introduce enough hydrophilic residues on the surface. Has anyone tested this crazy idea before? Thank you for your help. Best, Chen =20 Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.=20 http://us.rd.yahoo.com/evt=3D51733/*http://mobile.yahoo.com/;_ylt=3DAhu06i6= 2sR8H DtDypao8Wcj9tAcJ%20 =20 =20 --=20 Thomas J. Magliery, Ph.D. Assistant Professor Department of Chemistry Department of Biochemistry The Ohio State University 1043 Evans Laboratory 100 West 18th Ave. Columbus, OH 43210-1185 =20 (614) 247-8425 office (614) 292-1685 fax [EMAIL PROTECTED] http://www.chemistry.ohio-state.edu/~magliery =20 -- Scott D. Pegan, Ph.D. Visiting Senior Research Specialist Center for Pharmaceutical Biotechnology University of Illinois at Chicago
[ccp4bb] Postdoctoral Research Associate at Brookhaven National Laboratory
POSTDOCTORAL RESEARCH ASSOCIATE - Project Title: Novel Methods for Microcrystal Structure Determination at NSLS and NSLS-II Location: Brookhaven National Laboratory, Upton, NY, U.S.A. This new project is under the direction of A. M. Orville, and is also associated with the PXRR group (www.px.nsls.bnl.gov) at Brookhaven National Laboratory (BNL). The PXRR group conducts research, technology development, operations, and training at six beamlines (X8, X12b, X12c, X25, X26c, X29) at the National Synchrotron Light Source (NSLS). A long-term goal for the PXRR group is to create a resource for macromolecular crystallography at NSLS-II, the new synchrotron light source that is being created at BNL during the next five to seven years. Microcrystals only a few microns along an edge are often easy to obtain; but, are typically of no use for traditional diffraction studies. In contrast, the brightness and focused beam anticipated at NSLS-II provides an ideal opportunity to use microcrystals for diffraction studies. A major barrier that remains to be overcome is how to manipulate microcrystals for structure determination. Part of the research strategy will be to use a large number of randomly oriented microcrystals, from each of which only a small fraction of the whole dataset is collected, in order to build-up the complete dataset. Full _expression_ of these methods may approach the physical limits of crystal diffraction and will clearly help motivate the optimum performance of NSLS-II. Consequently, we will develop novel microcrystallography methods at the NSLS and look forward to utilizing the outstanding characteristics of NSLS-II facility. The position requires a Ph.D. in biochemistry, structural biology, chemistry, or physics. Experience in macromolecular crystallography and/or synchrotron x-ray sources is desired. The prospective candidate must be able to interact with a diverse group of scientists and engineers, as well as work independently. Send CV and contact information for four mentors or referees to [EMAIL PROTECTED] referring to Position No. FH4835. AMO ** Allen M. Orville, Ph.D. Biology Department Brookhaven National Laboratory Upton, NY 11973-5000 e-mail: [EMAIL PROTECTED] phone 631-344-4739 fax 631-344-2741 www.biology.bnl.gov/structure/orville.html
Re: [ccp4bb] how to change a membrane protein into a water soluble protein?
Another reference: N. Sukumar, Y.Xu, D.L. Gatti, B.Mitra and F.S. Mathews Structure of an Active Soluble Mutant of the Membrane-Associated (S)-Mandelate Dehydrogenase Biochemistry 40,9870-9878 (2001). In this paper, the membrane protein is converted into the soluble protein by replacing an intra-chain integral membrane-binding portion (39 residues) by a segment from its closely related soluble homologue (20 residues) and crystallized without any detergents. Sukumar -- N. Sukumar NE-CAT, Building 436E Argonne National Laboratory 9700 South Cass Avenue Argonne, IL 60439 e-mail: [EMAIL PROTECTED] Tel: 630-252-0681 Fax: 630-252-0687 On Tue, 2007-12-04 at 08:06, Scott Pegan wrote: Don't know if anyone has mentioned this paper but its an exact example how to make a K channel soluble. Roosild TP, Choe S. Redesigning an integral membrane K+ channel into a soluble protein. Protein Eng Des Sel. 2005 Feb;18(2):79-84. Epub 2005 Mar 23. PMID: 15788421 [PubMed - indexed for MEDLINE] Scott On Tue, December 4, 2007 4:04 am, Brenda Patterson wrote: Another option is refolding which can increase soluble protein content and is used routinely to achieve soluble protein such as the TIMPs http://peds.oxfordjournals.org/cgi/content/abstract/7/8/1035 http://www.proteinscience.org/cgi/reprint/11/10/2493.pdf?ck=nck that said, this is not true of all membrane proteins. Addition of a fusion partner, MBP, to the normally membrane associated FMO3 has been shown to generate stable, soluble protein and the addition of a fusion protein allows purification downstream more easily. Here is a paper where they did as the original poster suggested and tried mutagenesis of hydrophobic regions, including a truncation of a membrane anchor. They achieved increased solubility with this in combination with use of detergents. Krueger SK, Siddens LK, Henderson MC, VanDyke JE, Karplus PA, Pereira CB, Williams DE. Abstract C-Terminal truncation of rabbit flavin-containing monooxygenase isoform 2 enhances solubility. Arch Biochem Biophys. 2006 Jun 15;450(2):149-56. Epub 2006 Mar 29. cheers Quoting Bil Clemons [EMAIL PROTECTED]: There is also the soluble KcsA. Computational design of water-soluble analogues of the potassium channel KcsA. A. M. Slovic, H. Kono, J. D. Lear, J. G. Saven, and W. F. DeGrado (2004) PNAS 101, 1828-1833 Bil Bil Clemons, PhD Assistant Professor of Biochemistry Caltech 157 Broad Center MC 114-96 Pasadena, CA 91125 (626) 395-1796 [EMAIL PROTECTED] mailto:[EMAIL PROTECTED] From: Thomas J Magliery PhD [EMAIL PROTECTED] Reply-To: Thomas J Magliery PhD [EMAIL PROTECTED] Date: Mon, 3 Dec 2007 16:50:03 -0500 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to change a membrane protein into a water solub= le protein? =20 It's hard. See: =20 J Mol Biol. 2005 May 6;348(3):777-87. X-ray structure of a water-soluble analog of the membrane protein phospholamban:=20 sequence determinants defining the topology of tetrameric and pentameric coiled coils. Slovic AM, Stayrook SE, North B, Degrado WF. =20 Slovic, A. M., Summa, C. M., Lear, J. D. DeGrado, W. F. (2002). Computational design of a water-soluble analog of phospholamban. Protein Sci. 12, 337=AD348. =20 Li, H., Cocco, M. J., Steitz, T. A. Engelman, D. E. (2001). Conversion of phospholamban into a soluble pentameric helical bundle. Biochemistry, 40, 6636=AD6645. =20 Frank, S., Kammerer, R. A., Hellstern, S., Pegoraro, S., Stetefeld, J., Lustig, A. et al. (2000). Toward a high resolution structure of phospholamban: design of soluble transmembrane domain mutants. Biochemistry, 39, 6825=AD6831. =20 Tom =20 =20 Daniel Jin wrote: Hi, I am wondering whether there is a way to turn a membrane protein with known crystal structure into a water soluble protein by systematic mutagenesis. I guess it should be doable if we introduce enough hydrophilic residues on the surface. Has anyone tested this crazy idea before? Thank you for your help. Best, Chen =20 Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.=20 http://us.rd.yahoo.com/evt=3D51733/*http://mobile.yahoo.com/;_ylt=3DAhu06i6= 2sR8H DtDypao8Wcj9tAcJ%20 =20 =20 --=20 Thomas J. Magliery, Ph.D. Assistant Professor Department of Chemistry Department of Biochemistry The Ohio State University 1043 Evans Laboratory 100 West 18th Ave. Columbus, OH 43210-1185 =20 (614) 247-8425 office (614) 292-1685 fax [EMAIL PROTECTED] http://www.chemistry.ohio-state.edu/~magliery =20
Re: [ccp4bb] I have my bound ligand (I think), now what?
Hi Brenda, you'll need a cif description file for your ligand which you will read in either Coot or Refmac so that it will also refine correctly. I usually get my cif's from the Dundee PRODRG server, you can either paste your current coordinates as ATOMS in the provided field or sketch your molecule as indicated on the website. Here's the link: http://davapc1.bioch.dundee.ac.uk/programs/prodrg/ Then once the cif file is generated you will need to include this in your refmac script with the following command: #!/bin/tcsh -f refmac5 \ HKLIN $hklin HKLOUT $hklot \ LIBIN YOUR_SMALL_MOLECULE.cif \ XYZIN my_current.pdb XYZOUT $xyzot \ EOF $log YOUR REFINEMENT PROTOCOL EOF Brenda Patterson wrote: Hello all, right, I think I have found a ligand bound to my protein. I used the COOT utility to find the ligands after reading in a model and library file generated by sketcher of my ligand. Now I am a bit unsure as to how to proceed? How can I 'accept' a state and/or refine it? Maybe there is a better way to model my ligand into the electron density not accounted for by my protein? Any general suggestions or comments would be appreciated as I am very inexperienced. cheers Brenda -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
[ccp4bb] PEG MW vs. cryoprotectivity
We've been having a discussion in the lab about whether or not middle-sized PEGs such as 4000 can be expected to serve as cryoprotectants (and if not, why certain commercial kits are formulated the way they are). Can anybody shed some light / references on the question of the size of PEGs vs. their ability to help in freezing? thanks, Phoebe --- Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 fax 773 702 0439 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
[ccp4bb] skin on a drop
Hi All, As we know, there is a skin on a crystallization drop, especially when the drop contains PEG. Does anyone know what the skin is, a degraded protein ? How to prevent it ? Thanks Simon
Re: [ccp4bb] skin on a drop
Hi Try setting up at 4C can help J Hi All, As we know, there is a skin on a crystallization drop, especially when the drop contains PEG. Does anyone know what the skin is, a degraded protein ? How to prevent it ? Thanks Simon
[ccp4bb] How to make a structure-based multiple sequence alignment on DALI server?
Hi all， I want to produce structure-based multiple sequence alignment of my protein with five of its homologs on DALI server. However, when I tried the Database Search Form, only one homolog was picked up from PDB. If I align my protein with each homolog by the DaliLite Pairwise comparison, how can I combine them together? Thanks in advance. Best regards, Sincerely, Dalei Wu Drug Discovery and Design Center Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences 555 Zu Chongzhi Road, Shanghai, 201203, China [EMAIL PROTECTED] 2007-12-05
Re: [ccp4bb] How to make a structure-based multiple sequence alignment on DALI server?
You can perform a multiple structural alignment using MUSTANG ( http://www.bx.psu.edu/arun/research/mustang/), which draws upon some of the underpinnings of the DALI approach. James
