Re: [ccp4bb] why I can't reproduce R based on the same program?
Hi Hailiang, I want to reproduce the R factor provided by PDB file. The structure was refined by REFMAC, and so I think if I try a REFMAC refinement based on the pdb file and reflection data, the initial R factor given by REFMAC should be it. The pdb file provides residual B factors with TLS given by the header. I therefore generated the PDB with the total anisotropic B, based on which I tried REFMAC. However, the initial R_free was higher than provide by PDB (0.226 vs 0.216). Not sure why I can't reproduce R based on the same program. Thanks for any advice. 1. Unless something isn't right (with the data, model or your scripts or all of them), (normally) you don't have to do the refinement to reproduce the R-factor reported in PDB file header of a deposited structure. 2. For particular structure you mentioned in this thread (1ss8) the R-factor is easily reproducible with phenix.model_vs_data (*): phenix.model_vs_data pdb1ss8.ent 1ss8.mtz gives me: (...) Model_vs_Data: r_work(re-computed): 0.214 r_free(re-computed): 0.243 (...) Information extracted from PDB file header: program_name: REFMAC year: 4 r_work : 0.215 r_free : 0.249 (...) which is close given the diffeernces in how the bulk-solvent and anisotropic scaling is done and loss of accuracy due to back-and-forth conversions of ADPs (from total to partial+TLS matrices and from partial+TLS matrices to total). Pavel. (*) Afonine PV, Grosse-Kunstleve RW, Chen VB, Headd JJ, Moriarty NW, Richardson JS, Richardson DC, Urzhumtsev A, Zwart PH, Adams PD: phenix.model_vs_data: a high-level tool for the calculation of crystallographic model and data statistics. J. Appl. Cryst. 2010, 43:677-685.
Re: [ccp4bb] why I can't reproduce R based on the same program?
Thanks for all the advices. The REFMAC PDB didn't provide ksol and bsol in the author's refinement, otherwise I would fix them in my refinement. Best Regards, Hailiang Hi Hailiang, I want to reproduce the R factor provided by PDB file. The structure was refined by REFMAC, and so I think if I try a REFMAC refinement based on the pdb file and reflection data, the initial R factor given by REFMAC should be it. The pdb file provides residual B factors with TLS given by the header. I therefore generated the PDB with the total anisotropic B, based on which I tried REFMAC. However, the initial R_free was higher than provide by PDB (0.226 vs 0.216). Not sure why I can't reproduce R based on the same program. Thanks for any advice. 1. Unless something isn't right (with the data, model or your scripts or all of them), (normally) you don't have to do the refinement to reproduce the R-factor reported in PDB file header of a deposited structure. 2. For particular structure you mentioned in this thread (1ss8) the R-factor is easily reproducible with phenix.model_vs_data (*): phenix.model_vs_data pdb1ss8.ent 1ss8.mtz gives me: (...) Model_vs_Data: r_work(re-computed): 0.214 r_free(re-computed): 0.243 (...) Information extracted from PDB file header: program_name: REFMAC year: 4 r_work : 0.215 r_free : 0.249 (...) which is close given the diffeernces in how the bulk-solvent and anisotropic scaling is done and loss of accuracy due to back-and-forth conversions of ADPs (from total to partial+TLS matrices and from partial+TLS matrices to total). Pavel. (*) Afonine PV, Grosse-Kunstleve RW, Chen VB, Headd JJ, Moriarty NW, Richardson JS, Richardson DC, Urzhumtsev A, Zwart PH, Adams PD: phenix.model_vs_data: a high-level tool for the calculation of crystallographic model and data statistics. J. Appl. Cryst. 2010, 43:677-685.
[ccp4bb] Fab purification and crystallization
Again you should read the spectacular paper by Obmolova and co. where they solved the structures of three Fab-antigen complexes using MMS microseeding (seeding into random screens), starting with one hit containing clusters of needles - which could not themselves be optimized The paper is available on open access (free) http://journals.iucr.org/d/issues/2010/08/00/issconts.html http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf Acta Cryst. (2010). D66, 927-933 [ doi:10.1107/S0907444910026041 http://dx.doi.org/10.1107/S0907444910026041 ] Promoting crystallization of antibody-antigen complexes via microseed matrix screening G. Obmolova http://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Obmo lova%2C%20G%2E , T. J. Malia http://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Mali a%2C%20T%2EJ%2E , A. Teplyakov http://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Tepl yakov%2C%20A%2E , R. Sweet http://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Swee t%2C%20R%2E and G. L. Gilliland http://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Gill iland%2C%20G%2EL%2E Synopsis: The application of microseed matrix screening to the crystallization of related antibodies in complex with IL-13 is described. Both self-seeding or cross-seeding helped promote nucleation and increase the hit rate. Online 14 July 2010 The application of microseed matrix screening to the crystallization of antibody-antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate. -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of xaravich ivan Sent: 10 September 2010 04:00 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fab purification and crystallization Hi CCP4bb, I have two questions regarding Fab purification and Fab-antigen complex crystallization and would really appreciate any input from the experienced board. 1) I have got some hits for Fab-antigen complex (150 kD) but they are all needle clusters. Whatever fine screen I formulate, it always gives me these needle clusters. Are there some better common ways to change needles to single crystals? 2) I have certain IgGs from which I purify the Fab by papain digestion (resin from ThermoSci). One of the first steps is to dialyze the IgG with the digestion buffer,( 20mM Na-phosphate and 10mM EDTA pH 7.0) Over night. I always get 30-60% of the IgG precipitated during this overnight dialysis. I tried to increase the salt by adding 200mM NaCl but of no effect. Have anyone experienced such problem? Is there any thing that could be tried to stop this precipitation. thanks in advance. ivan
[ccp4bb] How do I set up a Refmac5 dictionary to constrain octahedrally coordinated Ca++
Dear all, How do I set up a Refmac dictionary to constrain octahedrally coordinated Ca++? Refmac5 seems to detect the potential bond between the Ca++ and the ligands: INFO: link is found (not be used) dist= 2.229 ideal_dist= 2.320 ch:AA res: 263 GLU at:O .-ch:Ag res: 600 CA at:CA . but it does not enforce them, and I cannot figure out what the CCP4 convention for a O-Ca++ bond is. Thanks for any suggestions! Pietro
Re: [ccp4bb] How do I set up a Refmac5 dictionary to constrain octahedrally coordinated Ca++
Pietro, You will need to set "Make links between: All others if...residues are close only" in the Refmac GUI and write out a .cif file for the Ca-ligand distance restraints. See this link: http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Model+Refinement#Restraining_metal_ligand_distances_during_refinement_in_REFMAC Cheers. Pietro Roversi wrote: Dear all, How do I set up a Refmac dictionary to constrain octahedrally coordinated Ca++? Refmac5 seems to detect the potential bond between the Ca++ and the ligands: INFO: link is found (not be used) dist= 2.229 ideal_dist= 2.320 ch:AA res: 263 GLU at:O .-ch:Ag res: 600 CA at:CA . but it does not enforce them, and I cannot figure out what the CCP4 convention for a O-Ca++ bond is. Thanks for any suggestions! Pietro -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@mail.colgate.edu
Re: [ccp4bb] How do I set up a Refmac5 dictionary to constrain octahedrally coordinated Ca++
Hi Pietro, There are monomer library entries for Ca in various states of coordination by water, entries, OC1, OC2,OC3 etc but unfortunately these are incomplete (no distances or angles with sd's), at least in our CCP4 installation. The entry for octahedral MG-O6, file MO6, is complete so you could use that as a model if you want to. The advantage of these entries is that they restrain angles as well as distances (eg O-MG-O, or O-CA-O in your case), ie it will keep it octahedral. Andrew On 10 Sep 2010, at 14:56, Pietro Roversi wrote: Dear all, How do I set up a Refmac dictionary to constrain octahedrally coordinated Ca++? Refmac5 seems to detect the potential bond between the Ca++ and the ligands: INFO: link is found (not be used) dist= 2.229 ideal_dist= 2.320 ch:AA res: 263 GLU at:O .-ch:Ag res: 600 CA at:CA . but it does not enforce them, and I cannot figure out what the CCP4 convention for a O-Ca++ bond is. Thanks for any suggestions! Pietro
Re: [ccp4bb] Fab purification and crystallization
Thanks everyone, I have really a lot to do now. Jurgen, yes I do separate the complex over size-exclusion column before setting trays. Patrick, Thanks for the wonderful reference. Sivaram, Thank you.It was really nice of you to send the link of your thesis.It is a wonderful gesture and I appreciate it. Could you remember how dilute your IgG starting sample was and the time of dialysis prior to papain digestion? Also could you elaborate a bit on how you specifically did the microseeding steps. Having a glance on your thesis, I could not find these of the top. Thanks again, Ivan On Fri, Sep 10, 2010 at 4:35 AM, Patrick Shaw Stewart patr...@douglas.co.uk wrote: Again you should read the spectacular paper by Obmolova and co. where they solved the structures of three Fab-antigen complexes using “MMS” microseeding (seeding into random screens), starting with one hit containing clusters of needles - which could not themselves be optimized The paper is available on open access (free) http://journals.iucr.org/d/issues/2010/08/00/issconts.html http://journals.iucr.org/d/issues/2010/08/00/bw5361/bw5361.pdf *Acta Cryst.* (2010). D*66*, 927-933 [ doi:10.1107/S0907444910026041http://dx.doi.org/10.1107/S0907444910026041 ] Promoting crystallization of antibody-antigen complexes *via* microseed matrix screening G. Obmolovahttp://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Obmolova%2C%20G%2E , T. J. Maliahttp://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Malia%2C%20T%2EJ%2E , A. Teplyakovhttp://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Teplyakov%2C%20A%2E , R. Sweethttp://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Sweet%2C%20R%2E and G. L. Gillilandhttp://scripts.iucr.org/cgi-bin/citedin?search_on=nameauthor_name=Gilliland%2C%20G%2EL%2E *Synopsis:** *The application of microseed matrix screening to the crystallization of related antibodies in complex with IL-13 is described. Both self-seeding or cross-seeding helped promote nucleation and increase the hit rate. Online 14 July 2010 The application of microseed matrix screening to the crystallization of antibody–antigen complexes is described for a set of antibodies that include mouse anti-IL-13 antibody C836, its humanized version H2L6 and an affinity-matured variant of H2L6, M1295. The Fab fragments of these antibodies were crystallized in complex with the antigen human IL-13. The initial crystallization screening for each of the three complexes included 192 conditions. Only one hit was observed for H2L6 and none were observed for the other two complexes. Matrix self-microseeding using these microcrystals yielded multiple hits under various conditions that were further optimized to grow diffraction-quality H2L6 crystals. The same H2L6 seeds were also successfully used to promote crystallization of the other two complexes. The M1295 crystals appeared to be isomorphous to those of H2L6, whereas the C836 crystals were in a different crystal form. These results are consistent with the concept that the conditions that are best for crystal growth may be different from those that favor nucleation. Microseed matrix screening using either a self-seeding or cross-seeding approach proved to be a fast, robust and reliable method not only for the refinement of crystallization conditions but also to promote crystal nucleation and increase the hit rate. -- For information and discussion about protein crystallization and automation, please join our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en patr...@douglas.co.ukDouglas Instruments Ltd. DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *xaravich ivan *Sent:* 10 September 2010 04:00 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Fab purification and crystallization Hi CCP4bb, I have two questions regarding Fab purification and Fab-antigen complex crystallization and would really appreciate any input from the experienced board. 1) I have got some hits for Fab-antigen complex (150 kD) but they are all needle clusters. Whatever fine screen I formulate, it always gives me these needle clusters. Are there some better common ways to change needles to single crystals? 2) I have certain IgGs from which I purify the Fab by papain digestion (resin from ThermoSci). One of the first steps is to dialyze the IgG with the digestion buffer,( 20mM Na-phosphate and 10mM EDTA pH 7.0) Over night. I always get 30-60% of the IgG precipitated during this overnight dialysis. I tried to increase the salt by adding 200mM NaCl but of no effect. Have anyone
Re: [ccp4bb] Fab purification and crystallization
Hi Ivan, Did you try using a buffer other than phosphate? Also maybe a different pH can help keeping the IgG in solution. Although papain prefers pH 6-7, it is a fairly robust enzyme and will cleave with 20% efficiency in the range of pH4-9 (Hoover S Kokes E ,1946, http://www.jbc.org/content/167/1/199.full.pdf, and Lowe G Yuthavong Y 1971, http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1177120/pdf/biochemj00648-0125.pdf). Also the un-beaded form of papain is super cheap. So there is no reason to constrain your cleavage in a condition that your IgG doesn't like. Why not cleave the IgG in the original purification buffer supplemented with EDTA and cysteine? Zhijie From: xaravich ivan Sent: Thursday, September 09, 2010 10:59 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Fab purification and crystallization Hi CCP4bb, I have two questions regarding Fab purification and Fab-antigen complex crystallization and would really appreciate any input from the experienced board. 1) I have got some hits for Fab-antigen complex (150 kD) but they are all needle clusters. Whatever fine screen I formulate, it always gives me these needle clusters. Are there some better common ways to change needles to single crystals? 2) I have certain IgGs from which I purify the Fab by papain digestion (resin from ThermoSci). One of the first steps is to dialyze the IgG with the digestion buffer,( 20mM Na-phosphate and 10mM EDTA pH 7.0) Over night. I always get 30-60% of the IgG precipitated during this overnight dialysis. I tried to increase the salt by adding 200mM NaCl but of no effect. Have anyone experienced such problem? Is there any thing that could be tried to stop this precipitation. thanks in advance. ivan
