[ccp4bb] software/web server to determine ligand volume
Dear all, Is there any software or web server available to calculate the volume of a ligand if the ligand coordinates are provided? Google seems to come up only with options to calculate protein cavity volume. Thanks in advance, Sreetama Das, phd student, Physics, IISc
[ccp4bb] RMSD between structures of homologous proteins
Dear all, When calculating the RMSD between structures of homologous proteins, where there are large changes in the loop region(s), which RMSD should be reported - an overall value which may be inflated due to the deviations in the loops, or separate values for the core and loop regions? What is the best way to calculate the RMSD for superposition of the cores - should I prepare a separate PDB file by removing the coordinates of the loop residues and then superpose? Thanks in advance, Sreetama das, PhD student, Physics, IISc
Re: [ccp4bb] RMSD between structures of homologous proteins
Sreetama, See the following URL whether it is useful to solve your issue http://cluster.physics.iisc.ernet.in/3dss/ best, Sekar Dear all, When calculating the RMSD between structures of homologous proteins, where there are large changes in the loop region(s), which RMSD should be reported - an overall value which may be inflated due to the deviations in the loops, or separate values for the core and loop regions? What is the best way to calculate the RMSD for superposition of the cores - should I prepare a separate PDB file by removing the coordinates of the loop residues and then superpose? Thanks in advance, Sreetama das, PhD student, Physics, IISc -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. Could you kindly confirm the safe receipt of the mail please. All best wishes and regards, Yours sincerely, Dr. K. Sekar, Ph.D. Associate Professor Supercomputer Education and Research Centre Room No. 341, old Ecological Sciences Building (Second Floor) Indian Institute of Science Bangalore 560 012 INDIA E-mail:se...@physics.iisc.ernet.in se...@serc.iisc.ernet.in Tel: 91-(0)80-22933059/22933060/23600551 Fax: 91-(0)80-23600085 Home page: http://www.physics.iisc.ernet.in/~dichome/sekhome/index.html Google Scholar: http://scholar.google.co.in/citations?hl=enuser=CT0Z5kIJ -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
[ccp4bb] AW: [ccp4bb] RMSD between structures of homologous proteins
Dear Sreetama, I use an ancient superposition program which rejects all atom pairs deviating more than 3 sigma and repeats this procedure until convergence. It then reports the RMSD for all atoms and for the atoms deviating less than 3 sigma. I find this an excellent method to separate the core from variable loop regions. It also ensures a robust superposition of the cores, ignoring variable loops. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von sreetama das Gesendet: Mittwoch, 13. August 2014 08:12 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] RMSD between structures of homologous proteins Dear all, When calculating the RMSD between structures of homologous proteins, where there are large changes in the loop region(s), which RMSD should be reported - an overall value which may be inflated due to the deviations in the loops, or separate values for the core and loop regions? What is the best way to calculate the RMSD for superposition of the cores - should I prepare a separate PDB file by removing the coordinates of the loop residues and then superpose? Thanks in advance, Sreetama das, PhD student, Physics, IISc
[ccp4bb] AW: [ccp4bb] Protein_Ligand Suggestion
Dear Monica, here are some comments from my side: -one can ask the bulletin board, or just decrease the occupancy during refinement and see what happens. If the statistics get better: keep it, if they get worse: reject the result. -I think it is not a question of correct or incorrect structures. You have two structures: one with a short soak and low ligand concentration, and one with a longer soak and higher ligand concentration, but with 88% complete data. I would refine both structures independently and in both cases refine a group occupancy for the ligand. If this is done correctly, I would expect that both structures will look very similar and provide independent proof for the binding mode of your ligand. One question is: what to do with ambiguities in the low-occupancy structure? I would look in the high-occupancy structure for hints how to resolve these, but more puritan crystallographers may have different opinions. Also 88% completeness is not great, but also not that bad. If the electron density looks good, I would trust it. However, if both independently refined structures show a very different binding mode, I would get suspicious. In this case I would not just ignore the low-occupancy structure but would try to find out what happened: was the ligand incorrectly fitted? Where the soaking conditions (precipitant, buffer, pH) different, could it be that the ligand was degraded/turned over during the long soak? Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Monica Mittal Gesendet: Mittwoch, 13. August 2014 07:48 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Protein_Ligand Suggestion Dear all I have solved a structure of protein with ligand at 2.7 and 2.8 A. Both have ligand bound in it as i soaked it. For the first structure i soaked for lesser time and lesser concentration of ligand and solved it at 2.7 A resolution. Although the ligand has good density (2Fo-Fc at 0.9 sigma) but it is little unconnected from its middle portion. Its RSCC value is 0.76 and B-factor of 70.0. Overall completeness of structure is 91%. So If i decrease the occupancy of ligand here, will it improve the ligand statistics esp. the Real space R-value?? However for the second structure, i soaked for longer time and higher ligand concentration, so the ligand is perfect with clear 2Fo-Fc map at 1.2 sigma and Fo-Fc map at 2.8 sigma along with well defined simulated annealing and composite omit maps. The RSCC value is 0.81 with B-factor of 40.1.The only issue is the overall completeness of the structure is 88%. But if i get evrything OK at this completeness, den can i consider the second structure with ligand as the correct one?? Thanks in advance. Monica
Re: [ccp4bb] software/web server to determine ligand volume
Hi A rough calculation is 18Å^3 per non-hydrogen atom in the ligand - I use a pencil and a bit of paper for the mechanics of this! On 13 Aug 2014, at Wed13 Aug 07:06, sreetama das wrote: Dear all, Is there any software or web server available to calculate the volume of a ligand if the ligand coordinates are provided? Google seems to come up only with options to calculate protein cavity volume. Thanks in advance, Sreetama Das, phd student, Physics, IISc Harry -- ** note change of address ** Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing)
[ccp4bb] Twinning in space group Pc
Dear, I’m struggling with the following (small molecule) problem: We are trying to solve the structure of a metal-organic framework containing a chiral compound. The space group is most probably Pc, but when refining, SHELX gives the error “Possible racemic twin or wrong absolute structure - try TWIN refinement”. As we know our compound is enantiopure, a racemic twin is very unlikely. In this regard, also a centro-symmetric space group is not possible (although CrysAlisPro always gives P2/c as the proper space group). As a matter of fact, trying different space groups is not solving the problem. The second problem is that half of the structure is visible, but the other half is completely not clear. Refinement is not possible at all (R-value of 33%). When running TwinRotMat (Platon), I get the following possible 2-fold twin axes: 2-axis ( 0 1 -1 ) [ -2 5 -4 ], Angle () [] = 2.31 Deg, Freq =14 * (-0.992 -0.0190.015) (h1) (h2) Nr Overlap =84 (-0.4300.075 -0.860) * (k1) = (k2) BASF = 0.96 ( 0.459 -1.146 -0.083) (l1) (l2)DEL-R =-0.064 2-axis ( 0 1 1 ) [ 2 5 4 ], Angle () [] = 2.31 Deg, Freq =15 * (-0.9920.0190.015) (h1) (h2) Nr Overlap = 229 ( 0.4300.0750.860) * (k1) = (k2) BASF = 0.94 ( 0.4591.146 -0.083) (l1) (l2)DEL-R =-0.050 2-axis ( 1 0 -2 ) [ 3 0 -2 ], Angle () [] = 0.54 Deg, Freq =19 * (-0.1360.000 -0.576) (h1) (h2) Nr Overlap = 992 ( 0.000 -1.0000.000) * (k1) = (k2) BASF = 0.86 (-1.7030.0000.136) (l1) (l2)DEL-R =-0.030 2-axis ( 1 2 -1 ) [ 5 5 -1 ], Angle () [] = 0.39 Deg, Freq =13 * (-0.3800.620 -0.124) (h1) (h2) Nr Overlap = 854 ( 1.2560.256 -0.251) * (k1) = (k2) BASF = 0.88 (-0.617 -0.617 -0.877) (l1) (l2)DEL-R =-0.019 However, none of these do actually improve the refinement. Has anyone encountered possible twinning/twin laws in Pc please? Or any other suggestions are most welcome? Thank you very much Kristof
Re: [ccp4bb] Twinning in space group Pc
Hi, Pc may not be the space group for your crystal, if the molecule is chiral. Seems like the data were forced to be reduced to a mirror_related SG. Lijun On Aug 13, 2014 2:00 AM, Kristof Van Hecke kristofrg.vanhe...@gmail.com wrote: Dear, I’m struggling with the following (small molecule) problem: We are trying to solve the structure of a metal-organic framework containing a chiral compound. The space group is most probably Pc, but when refining, SHELX gives the error “Possible racemic twin or wrong absolute structure - try TWIN refinement”. As we know our compound is enantiopure, a racemic twin is very unlikely. In this regard, also a centro-symmetric space group is not possible (although CrysAlisPro always gives P2/c as the proper space group). As a matter of fact, trying different space groups is not solving the problem. The second problem is that half of the structure is visible, but the other half is completely not clear. Refinement is not possible at all (R-value of 33%). When running TwinRotMat (Platon), I get the following possible 2-fold twin axes: 2-axis ( 0 1 -1 ) [ -2 5 -4 ], Angle () [] = 2.31 Deg, Freq = 14 * (-0.992 -0.0190.015) (h1) (h2) Nr Overlap = 84 (-0.4300.075 -0.860) * (k1) = (k2) BASF = 0.96 ( 0.459 -1.146 -0.083) (l1) (l2)DEL-R =-0.064 2-axis ( 0 1 1 ) [ 2 5 4 ], Angle () [] = 2.31 Deg, Freq = 15 * (-0.9920.0190.015) (h1) (h2) Nr Overlap = 229 ( 0.4300.0750.860) * (k1) = (k2) BASF = 0.94 ( 0.4591.146 -0.083) (l1) (l2)DEL-R =-0.050 2-axis ( 1 0 -2 ) [ 3 0 -2 ], Angle () [] = 0.54 Deg, Freq = 19 * (-0.1360.000 -0.576) (h1) (h2) Nr Overlap = 992 ( 0.000 -1.0000.000) * (k1) = (k2) BASF = 0.86 (-1.7030.0000.136) (l1) (l2)DEL-R =-0.030 2-axis ( 1 2 -1 ) [ 5 5 -1 ], Angle () [] = 0.39 Deg, Freq = 13 * (-0.3800.620 -0.124) (h1) (h2) Nr Overlap = 854 ( 1.2560.256 -0.251) * (k1) = (k2) BASF = 0.88 (-0.617 -0.617 -0.877) (l1) (l2)DEL-R =-0.019 However, none of these do actually improve the refinement. Has anyone encountered possible twinning/twin laws in Pc please? Or any other suggestions are most welcome? Thank you very much Kristof
Re: [ccp4bb] AW: [ccp4bb] RMSD between structures of homologous proteins
I think PISA does this for you - it overlaps structural features and gives an RMSD on those parts that fit and a useful list of matching residues. You can run it from CCP4I or at PDBe. If you ant more than CA RMSD then you will need to select out the spans to fit and use the LSQKAB overlap procedure FIT MAIN RESi n n+m CHAIN A MATCH RESI b b+m CHAIN C FIT …. etc for example - you can do that in the GUI Eleanor On 13 August 2014 02:48, herman.schreu...@sanofi.com wrote: Dear Sreetama, I use an ancient superposition program which rejects all atom pairs deviating more than 3 sigma and repeats this procedure until convergence. It then reports the RMSD for all atoms and for the atoms deviating less than 3 sigma. I find this an excellent method to separate the core from variable loop regions. It also ensures a robust superposition of the cores, ignoring variable loops. Best, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *sreetama das *Gesendet:* Mittwoch, 13. August 2014 08:12 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] RMSD between structures of homologous proteins Dear all, When calculating the RMSD between structures of homologous proteins, where there are large changes in the loop region(s), which RMSD should be reported - an overall value which may be inflated due to the deviations in the loops, or separate values for the core and loop regions? What is the best way to calculate the RMSD for superposition of the cores - should I prepare a separate PDB file by removing the coordinates of the loop residues and then superpose? Thanks in advance, Sreetama das, PhD student, Physics, IISc
Re: [ccp4bb] Twinning in space group Pc
Twin laws are possible if there are 2 ways to index your cell, and non-merefedral twinning is possible in any system depending on the cell. I am not sure of the small molecule tools to check twinning though. Eleanor Dodson On 13 August 2014 05:58, Lijun Liu lijunli...@gmail.com wrote: Hi, Pc may not be the space group for your crystal, if the molecule is chiral. Seems like the data were forced to be reduced to a mirror_related SG. Lijun On Aug 13, 2014 2:00 AM, Kristof Van Hecke kristofrg.vanhe...@gmail.com wrote: Dear, I’m struggling with the following (small molecule) problem: We are trying to solve the structure of a metal-organic framework containing a chiral compound. The space group is most probably Pc, but when refining, SHELX gives the error “Possible racemic twin or wrong absolute structure - try TWIN refinement”. As we know our compound is enantiopure, a racemic twin is very unlikely. In this regard, also a centro-symmetric space group is not possible (although CrysAlisPro always gives P2/c as the proper space group). As a matter of fact, trying different space groups is not solving the problem. The second problem is that half of the structure is visible, but the other half is completely not clear. Refinement is not possible at all (R-value of 33%). When running TwinRotMat (Platon), I get the following possible 2-fold twin axes: 2-axis ( 0 1 -1 ) [ -2 5 -4 ], Angle () [] = 2.31 Deg, Freq = 14 * (-0.992 -0.0190.015) (h1) (h2) Nr Overlap = 84 (-0.4300.075 -0.860) * (k1) = (k2) BASF = 0.96 ( 0.459 -1.146 -0.083) (l1) (l2)DEL-R =-0.064 2-axis ( 0 1 1 ) [ 2 5 4 ], Angle () [] = 2.31 Deg, Freq = 15 * (-0.9920.0190.015) (h1) (h2) Nr Overlap = 229 ( 0.4300.0750.860) * (k1) = (k2) BASF = 0.94 ( 0.4591.146 -0.083) (l1) (l2)DEL-R =-0.050 2-axis ( 1 0 -2 ) [ 3 0 -2 ], Angle () [] = 0.54 Deg, Freq = 19 * (-0.1360.000 -0.576) (h1) (h2) Nr Overlap = 992 ( 0.000 -1.0000.000) * (k1) = (k2) BASF = 0.86 (-1.7030.0000.136) (l1) (l2)DEL-R =-0.030 2-axis ( 1 2 -1 ) [ 5 5 -1 ], Angle () [] = 0.39 Deg, Freq = 13 * (-0.3800.620 -0.124) (h1) (h2) Nr Overlap = 854 ( 1.2560.256 -0.251) * (k1) = (k2) BASF = 0.88 (-0.617 -0.617 -0.877) (l1) (l2)DEL-R =-0.019 However, none of these do actually improve the refinement. Has anyone encountered possible twinning/twin laws in Pc please? Or any other suggestions are most welcome? Thank you very much Kristof
[ccp4bb] off topic
Sorry for off topic question, just wondering if anyone has come across a study that shows the residue pka of certain amino acids is different in vitro compared to in vivo? Best Careina
Re: [ccp4bb] Twinning in space group Pc
Hi folks Pc *must* have both enantiomers, since it's got a glide plane ( = mirror + translation parallel to mirror). So the sample *cannot* be enantiopure if the space group is Pc (or P2/ c)... BTW, Pc isn't a centrosymmetric space group. Unless I'm wrong... On 13 Aug 2014, at Wed13 Aug 12:37, Eleanor Dodson wrote: Twin laws are possible if there are 2 ways to index your cell, and non-merefedral twinning is possible in any system depending on the cell. I am not sure of the small molecule tools to check twinning though. Eleanor Dodson On 13 August 2014 05:58, Lijun Liu lijunli...@gmail.com wrote: Hi, Pc may not be the space group for your crystal, if the molecule is chiral. Seems like the data were forced to be reduced to a mirror_related SG. Lijun On Aug 13, 2014 2:00 AM, Kristof Van Hecke kristofrg.vanhe...@gmail.com wrote: Dear, I’m struggling with the following (small molecule) problem: We are trying to solve the structure of a metal-organic framework containing a chiral compound. The space group is most probably Pc, but when refining, SHELX gives the error “Possible racemic twin or wrong absolute structure - try TWIN refinement”. As we know our compound is enantiopure, a racemic twin is very unlikely. In this regard, also a centro-symmetric space group is not possible (although CrysAlisPro always gives P2/c as the proper space group). As a matter of fact, trying different space groups is not solving the problem. The second problem is that half of the structure is visible, but the other half is completely not clear. Refinement is not possible at all (R-value of 33%). When running TwinRotMat (Platon), I get the following possible 2- fold twin axes: 2-axis ( 0 1 -1 ) [ -2 5 -4 ], Angle () [] = 2.31 Deg, Freq =14 * (-0.992 -0.0190.015) (h1) (h2) Nr Overlap =84 (-0.4300.075 -0.860) * (k1) = (k2) BASF = 0.96 ( 0.459 -1.146 -0.083) (l1) (l2)DEL- R =-0.064 2-axis ( 0 1 1 ) [ 2 5 4 ], Angle () [] = 2.31 Deg, Freq =15 * (-0.9920.0190.015) (h1) (h2) Nr Overlap = 229 ( 0.4300.0750.860) * (k1) = (k2) BASF = 0.94 ( 0.4591.146 -0.083) (l1) (l2)DEL- R =-0.050 2-axis ( 1 0 -2 ) [ 3 0 -2 ], Angle () [] = 0.54 Deg, Freq =19 * (-0.1360.000 -0.576) (h1) (h2) Nr Overlap = 992 ( 0.000 -1.0000.000) * (k1) = (k2) BASF = 0.86 (-1.7030.0000.136) (l1) (l2)DEL- R =-0.030 2-axis ( 1 2 -1 ) [ 5 5 -1 ], Angle () [] = 0.39 Deg, Freq =13 * (-0.3800.620 -0.124) (h1) (h2) Nr Overlap = 854 ( 1.2560.256 -0.251) * (k1) = (k2) BASF = 0.88 (-0.617 -0.617 -0.877) (l1) (l2)DEL- R =-0.019 However, none of these do actually improve the refinement. Has anyone encountered possible twinning/twin laws in Pc please? Or any other suggestions are most welcome? Thank you very much Kristof Harry -- ** note change of address ** Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing)
[ccp4bb] AW: [ccp4bb] Twinning in space group Pc
Dear Kristof, Lijun is right, space group Pc is not compatible with chiral molecules. Maybe diffraction of your non-chiral metal structure overwhelmed the chiral contribution of your organic framework. Why not use a trick from protein crystallography: process and solve your structure in P1? According to the international tables there are two asymmetric units in Pc, so the crystallographic problem should remain manageable. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Lijun Liu Gesendet: Mittwoch, 13. August 2014 11:59 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Twinning in space group Pc Hi, Pc may not be the space group for your crystal, if the molecule is chiral. Seems like the data were forced to be reduced to a mirror_related SG. Lijun On Aug 13, 2014 2:00 AM, Kristof Van Hecke kristofrg.vanhe...@gmail.commailto:kristofrg.vanhe...@gmail.com wrote: Dear, I’m struggling with the following (small molecule) problem: We are trying to solve the structure of a metal-organic framework containing a chiral compound. The space group is most probably Pc, but when refining, SHELX gives the error “Possible racemic twin or wrong absolute structure - try TWIN refinement”. As we know our compound is enantiopure, a racemic twin is very unlikely. In this regard, also a centro-symmetric space group is not possible (although CrysAlisPro always gives P2/c as the proper space group). As a matter of fact, trying different space groups is not solving the problem. The second problem is that half of the structure is visible, but the other half is completely not clear. Refinement is not possible at all (R-value of 33%). When running TwinRotMat (Platon), I get the following possible 2-fold twin axes: 2-axis ( 0 1 -1 ) [ -2 5 -4 ], Angle () [] = 2.31 Deg, Freq =14 * (-0.992 -0.0190.015) (h1) (h2) Nr Overlap =84 (-0.4300.075 -0.860) * (k1) = (k2) BASF = 0.96 ( 0.459 -1.146 -0.083) (l1) (l2)DEL-R =-0.064 2-axis ( 0 1 1 ) [ 2 5 4 ], Angle () [] = 2.31 Deg, Freq =15 * (-0.9920.0190.015) (h1) (h2) Nr Overlap = 229 ( 0.4300.0750.860) * (k1) = (k2) BASF = 0.94 ( 0.4591.146 -0.083) (l1) (l2)DEL-R =-0.050 2-axis ( 1 0 -2 ) [ 3 0 -2 ], Angle () [] = 0.54 Deg, Freq =19 * (-0.1360.000 -0.576) (h1) (h2) Nr Overlap = 992 ( 0.000 -1.0000.000) * (k1) = (k2) BASF = 0.86 (-1.7030.0000.136) (l1) (l2)DEL-R =-0.030 2-axis ( 1 2 -1 ) [ 5 5 -1 ], Angle () [] = 0.39 Deg, Freq =13 * (-0.3800.620 -0.124) (h1) (h2) Nr Overlap = 854 ( 1.2560.256 -0.251) * (k1) = (k2) BASF = 0.88 (-0.617 -0.617 -0.877) (l1) (l2)DEL-R =-0.019 However, none of these do actually improve the refinement. Has anyone encountered possible twinning/twin laws in Pc please? Or any other suggestions are most welcome? Thank you very much Kristof
Re: [ccp4bb] AW: [ccp4bb] RMSD between structures of homologous proteins
It is probably Gesamt/SSM (Superpose in CCP4), or PDBeFold at EBI rather than PISA -- Eugene On 13 Aug 2014, at 12:33, Eleanor Dodson wrote: I think PISA does this for you - it overlaps structural features and gives an RMSD on those parts that fit and a useful list of matching residues. You can run it from CCP4I or at PDBe. If you ant more than CA RMSD then you will need to select out the spans to fit and use the LSQKAB overlap procedure FIT MAIN RESi n n+m CHAIN A MATCH RESI b b+m CHAIN C FIT …. etc for example - you can do that in the GUI Eleanor On 13 August 2014 02:48, herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com wrote: Dear Sreetama, I use an ancient superposition program which rejects all atom pairs deviating more than 3 sigma and repeats this procedure until convergence. It then reports the RMSD for all atoms and for the atoms deviating less than 3 sigma. I find this an excellent method to separate the core from variable loop regions. It also ensures a robust superposition of the cores, ignoring variable loops. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von sreetama das Gesendet: Mittwoch, 13. August 2014 08:12 An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] RMSD between structures of homologous proteins Dear all, When calculating the RMSD between structures of homologous proteins, where there are large changes in the loop region(s), which RMSD should be reported - an overall value which may be inflated due to the deviations in the loops, or separate values for the core and loop regions? What is the best way to calculate the RMSD for superposition of the cores - should I prepare a separate PDB file by removing the coordinates of the loop residues and then superpose? Thanks in advance, Sreetama das, PhD student, Physics, IISc -- Scanned by iCritical.
Re: [ccp4bb] [ccp4bb] Twinning in space group Pc
Dear, Indeed,. Pc is not compatible with chiral molecules,. my mistake. I’m trying to process/solve the structure in P1 now and see how far I get. Thank you very much for pointing things out. Regards Kristof On 13 Aug 2014, at 13:58, herman.schreu...@sanofi.com wrote: Dear Kristof, Lijun is right, space group Pc is not compatible with chiral molecules. Maybe diffraction of your non-chiral metal structure overwhelmed the chiral contribution of your organic framework. Why not use a trick from protein crystallography: process and solve your structure in P1? According to the international tables there are two asymmetric units in Pc, so the crystallographic problem should remain manageable. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Lijun Liu Gesendet: Mittwoch, 13. August 2014 11:59 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Twinning in space group Pc Hi, Pc may not be the space group for your crystal, if the molecule is chiral. Seems like the data were forced to be reduced to a mirror_related SG. Lijun On Aug 13, 2014 2:00 AM, Kristof Van Hecke kristofrg.vanhe...@gmail.com wrote: Dear, I’m struggling with the following (small molecule) problem: We are trying to solve the structure of a metal-organic framework containing a chiral compound. The space group is most probably Pc, but when refining, SHELX gives the error “Possible racemic twin or wrong absolute structure - try TWIN refinement”. As we know our compound is enantiopure, a racemic twin is very unlikely. In this regard, also a centro-symmetric space group is not possible (although CrysAlisPro always gives P2/c as the proper space group). As a matter of fact, trying different space groups is not solving the problem. The second problem is that half of the structure is visible, but the other half is completely not clear. Refinement is not possible at all (R-value of 33%). When running TwinRotMat (Platon), I get the following possible 2-fold twin axes: 2-axis ( 0 1 -1 ) [ -2 5 -4 ], Angle () [] = 2.31 Deg, Freq =14 * (-0.992 -0.0190.015) (h1) (h2) Nr Overlap =84 (-0.4300.075 -0.860) * (k1) = (k2) BASF = 0.96 ( 0.459 -1.146 -0.083) (l1) (l2)DEL-R =-0.064 2-axis ( 0 1 1 ) [ 2 5 4 ], Angle () [] = 2.31 Deg, Freq =15 * (-0.9920.0190.015) (h1) (h2) Nr Overlap = 229 ( 0.4300.0750.860) * (k1) = (k2) BASF = 0.94 ( 0.4591.146 -0.083) (l1) (l2)DEL-R =-0.050 2-axis ( 1 0 -2 ) [ 3 0 -2 ], Angle () [] = 0.54 Deg, Freq =19 * (-0.1360.000 -0.576) (h1) (h2) Nr Overlap = 992 ( 0.000 -1.0000.000) * (k1) = (k2) BASF = 0.86 (-1.7030.0000.136) (l1) (l2)DEL-R =-0.030 2-axis ( 1 2 -1 ) [ 5 5 -1 ], Angle () [] = 0.39 Deg, Freq =13 * (-0.3800.620 -0.124) (h1) (h2) Nr Overlap = 854 ( 1.2560.256 -0.251) * (k1) = (k2) BASF = 0.88 (-0.617 -0.617 -0.877) (l1) (l2)DEL-R =-0.019 However, none of these do actually improve the refinement. Has anyone encountered possible twinning/twin laws in Pc please? Or any other suggestions are most welcome? Thank you very much Kristof
Re: [ccp4bb] Twinning in space group Pc
how many (quasi) equivalent positions do you think the chiral compound can adopt in the organo-metal framework? Or the organo-metal framework in the crystal? If it's two, you can probably do alternate conformations - if there are more, it could become impossible, even in P1. Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 13 Aug 2014, at 11:00, Kristof Van Hecke wrote: Dear, I’m struggling with the following (small molecule) problem: We are trying to solve the structure of a metal-organic framework containing a chiral compound. The space group is most probably Pc, but when refining, SHELX gives the error “Possible racemic twin or wrong absolute structure - try TWIN refinement”. As we know our compound is enantiopure, a racemic twin is very unlikely. In this regard, also a centro-symmetric space group is not possible (although CrysAlisPro always gives P2/c as the proper space group). As a matter of fact, trying different space groups is not solving the problem. The second problem is that half of the structure is visible, but the other half is completely not clear. Refinement is not possible at all (R-value of 33%). When running TwinRotMat (Platon), I get the following possible 2-fold twin axes: 2-axis ( 0 1 -1 ) [ -2 5 -4 ], Angle () [] = 2.31 Deg, Freq =14 * (-0.992 -0.0190.015) (h1) (h2) Nr Overlap =84 (-0.4300.075 -0.860) * (k1) = (k2) BASF = 0.96 ( 0.459 -1.146 -0.083) (l1) (l2)DEL-R =-0.064 2-axis ( 0 1 1 ) [ 2 5 4 ], Angle () [] = 2.31 Deg, Freq =15 * (-0.9920.0190.015) (h1) (h2) Nr Overlap = 229 ( 0.4300.0750.860) * (k1) = (k2) BASF = 0.94 ( 0.4591.146 -0.083) (l1) (l2)DEL-R =-0.050 2-axis ( 1 0 -2 ) [ 3 0 -2 ], Angle () [] = 0.54 Deg, Freq =19 * (-0.1360.000 -0.576) (h1) (h2) Nr Overlap = 992 ( 0.000 -1.0000.000) * (k1) = (k2) BASF = 0.86 (-1.7030.0000.136) (l1) (l2)DEL-R =-0.030 2-axis ( 1 2 -1 ) [ 5 5 -1 ], Angle () [] = 0.39 Deg, Freq =13 * (-0.3800.620 -0.124) (h1) (h2) Nr Overlap = 854 ( 1.2560.256 -0.251) * (k1) = (k2) BASF = 0.88 (-0.617 -0.617 -0.877) (l1) (l2)DEL-R =-0.019 However, none of these do actually improve the refinement. Has anyone encountered possible twinning/twin laws in Pc please? Or any other suggestions are most welcome? Thank you very much Kristof
Re: [ccp4bb] software/web server to determine ligand volume
VOIDOO will do this On 13 Aug 2014, at 2:06 AM, sreetama das wrote: Dear all, Is there any software or web server available to calculate the volume of a ligand if the ligand coordinates are provided? Google seems to come up only with options to calculate protein cavity volume. Thanks in advance, Sreetama Das, phd student, Physics, IISc --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pjl...@gmail.com pl...@drexelmed.edu
[ccp4bb] AW: [ccp4bb] off topic
Dear Careina, The pka of amino acids is not dependent on in vivo/in vitro, but on the local environment, e.g. free in solution vs. part of a folded protein. As part of a folded protein the shifts will be specific for the particular protein and I am not aware of any general rules. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Careina Edgooms Gesendet: Mittwoch, 13. August 2014 13:41 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] off topic Sorry for off topic question, just wondering if anyone has come across a study that shows the residue pka of certain amino acids is different in vitro compared to in vivo? Best Careina
Re: [ccp4bb] off topic
Dear Careina, Following on from what Herman said, if you have a structure you can use the propKa server http://propka.ki.ku.dk/ to predict pKas for amino acids in the local environment as found in your structure. Perhaps some of the propKa literature might also be helpful? HTH, Dave [image: David Briggs on about.me] David Briggs about.me/david_briggs http://about.me/david_briggs On 13 August 2014 12:40, Careina Edgooms 02531c126adf-dmarc-requ...@jiscmail.ac.uk wrote: Sorry for off topic question, just wondering if anyone has come across a study that shows the residue pka of certain amino acids is different in vitro compared to in vivo? Best Careina
Re: [ccp4bb] off topic
thank you, yes I am aware of propka and I know very well that the local environment of the amino acid is what contributes to its pka based on things like whether it is buried or exposed or involved in H-bonding. I am in the process of responding to a reviewer who suggests but does not give references that in vivo conditions can alter the ionisation of some amino acids differently to in vitro. I am struggling to respond to this without some sort of proof or study on the matter C On Wednesday, August 13, 2014 2:38 PM, David Briggs drdavidcbri...@gmail.com wrote: Dear Careina, Following on from what Herman said, if you have a structure you can use the propKa server http://propka.ki.ku.dk/ to predict pKas for amino acids in the local environment as found in your structure. Perhaps some of the propKa literature might also be helpful? HTH, Dave David Briggs about.me/david_briggs On 13 August 2014 12:40, Careina Edgooms 02531c126adf-dmarc-requ...@jiscmail.ac.uk wrote: Sorry for off topic question, just wondering if anyone has come across a study that shows the residue pka of certain amino acids is different in vitro compared to in vivo? Best Careina
Re: [ccp4bb] AW: [ccp4bb] RMSD between structures of homologous proteins
Thanks to the mailing list I came across this: http://www.ncbi.nlm.nih.gov/pubmed/24167157 The C code to compute is available on the link mentioned in the paper. Best wishes, George On Wed, Aug 13, 2014 at 8:01 AM, Eugene Krissinel eugene.krissi...@stfc.ac.uk wrote: It is probably Gesamt/SSM (Superpose in CCP4), or PDBeFold at EBI rather than PISA -- Eugene On 13 Aug 2014, at 12:33, Eleanor Dodson wrote: I think PISA does this for you - it overlaps structural features and gives an RMSD on those parts that fit and a useful list of matching residues. You can run it from CCP4I or at PDBe. If you ant more than CA RMSD then you will need to select out the spans to fit and use the LSQKAB overlap procedure FIT MAIN RESi n n+m CHAIN A MATCH RESI b b+m CHAIN C FIT …. etc for example - you can do that in the GUI Eleanor On 13 August 2014 02:48, herman.schreu...@sanofi.commailto: herman.schreu...@sanofi.com wrote: Dear Sreetama, I use an ancient superposition program which rejects all atom pairs deviating more than 3 sigma and repeats this procedure until convergence. It then reports the RMSD for all atoms and for the atoms deviating less than 3 sigma. I find this an excellent method to separate the core from variable loop regions. It also ensures a robust superposition of the cores, ignoring variable loops. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto: CCP4BB@JISCMAIL.AC.UK] Im Auftrag von sreetama das Gesendet: Mittwoch, 13. August 2014 08:12 An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] RMSD between structures of homologous proteins Dear all, When calculating the RMSD between structures of homologous proteins, where there are large changes in the loop region(s), which RMSD should be reported - an overall value which may be inflated due to the deviations in the loops, or separate values for the core and loop regions? What is the best way to calculate the RMSD for superposition of the cores - should I prepare a separate PDB file by removing the coordinates of the loop residues and then superpose? Thanks in advance, Sreetama das, PhD student, Physics, IISc -- Scanned by iCritical.
[ccp4bb] PSDI 2014, Abstract and Registration Info
[image: Inline image 1] *PROTEIN STRUCTURE DETERMINATION IN INDUSTRY 2014* The 22nd PSDI will take place at the Hotel Miragem in Cascais, Portugal, a coastal town located 30 kilometers west of Lisbon, Portugal. The meeting will start with registration and an opening reception on Sunday, 2nd November. This will be followed by two and a half days of scientific program starting Monday morning and finishing at noon on Wednesday, 5th November. A half day excursion to Lisbon is scheduled as a part of the meeting. The scientific program will include the following sessions: ·Drug Discovery Stories ·Challenging Targets ·Biophysics Supporting Structural Biology ·Binding Kinetics in Drug Discovery ·Structural Biology of Biopharmaceuticals Registration for participants and exhibitors is open at the website: www.psdi2014.org *Please note the deadline for registration and payment: 31 Aug 2014* If you are interested in speaking in any of the scientific sessions, please contact the organizers at psdi2...@merckgroup.com with an abstract. Abstracts for “Binding Kinetics in Drug Discovery” are particularly encouraged. Additional information concerning the conference and further updates can be found at the PSDI2014 website http://www.psdi2014.org/. Vendor/Sponsor opportunities are available. If you have any questions, please contact the organizers at psdi2...@merckgroup.com. Please feel free to forward this announcement to anyone who may be interested in participating. We look forward to a great conference this fall. With kind regards, The PSDI 2014 organizing committee: ·Tiago Bandeiras, IBET, Portugal ·Anna Gardberg, EMD Serono, USA ·Theresa Johnson, EMD Serono, USA ·Beate Matlok, Merck KGaA, Germany ·Djordje Musil, Merck KGaA, Germany psdi2...@merckgroup.com http://www.psdi2014.org
Re: [ccp4bb] off topic
Hi Careina, Regardless of what I think of this reviewer's comment, the only technique of which I'm aware that can measure in vivo (and in vitro of course) the pKa's of amino-acids is NMR. Perhaps you can find some NMR literature where in-vivo and in-vitro pKa's have been compared? Just a thought. Good luck with the reviewer. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Careina Edgooms [02531c126adf-dmarc-requ...@jiscmail.ac.uk] Sent: Wednesday, August 13, 2014 3:45 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic thank you, yes I am aware of propka and I know very well that the local environment of the amino acid is what contributes to its pka based on things like whether it is buried or exposed or involved in H-bonding. I am in the process of responding to a reviewer who suggests but does not give references that in vivo conditions can alter the ionisation of some amino acids differently to in vitro. I am struggling to respond to this without some sort of proof or study on the matter C On Wednesday, August 13, 2014 2:38 PM, David Briggs drdavidcbri...@gmail.com wrote: Dear Careina, Following on from what Herman said, if you have a structure you can use the propKa server http://propka.ki.ku.dk/ to predict pKas for amino acids in the local environment as found in your structure. Perhaps some of the propKa literature might also be helpful? HTH, Dave David Briggs about.me/david_briggs On 13 August 2014 12:40, Careina Edgooms 02531c126adf-dmarc-requ...@jiscmail.ac.uk wrote: Sorry for off topic question, just wondering if anyone has come across a study that shows the residue pka of certain amino acids is different in vitro compared to in vivo? Best Careina
Re: [ccp4bb] thin shell Rfree set selection
By all means, try it both ways and see whether the R-Rfree gap narrows with random vs thin shell selection. Depending on resolution and data quality, you may also consider imposing NCS restraints. Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Xianchi Dong dongxian...@gmail.com /divdivDate:08/12/2014 4:45 PM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] thin shell Rfree set selection /divdiv /divDear all, I have a dataset of C2 symmetry and 2 molecules in the ASU. I am wondering if I have to use thin shell Rfree set selection to avoid Rfree bias by NCS. And which Rfree selection method is better? Thanks in advance. Xianchi
[ccp4bb] AW: [ccp4bb] off topic
Dear Careina, even in vivo, the pka depends on the local environment, so the question boils down to: is the local environment in vivo different from in vitro? E.g. is your protein embedded or in contact with a membrane, is it part of a large multiprotein complex etc? Does your crystallization solution or the in vivo compartment of your protein contain molecules that could influence the pka? Since I assume that you did not measure pka's experimentally, I assume that the remarks of the referee concern the discussion of your paper. In this case, I would use some arguments like above as response to the referee, depending on the context of course, which I do not know. Good luck! Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Careina Edgooms Gesendet: Mittwoch, 13. August 2014 14:45 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] off topic thank you, yes I am aware of propka and I know very well that the local environment of the amino acid is what contributes to its pka based on things like whether it is buried or exposed or involved in H-bonding. I am in the process of responding to a reviewer who suggests but does not give references that in vivo conditions can alter the ionisation of some amino acids differently to in vitro. I am struggling to respond to this without some sort of proof or study on the matter C On Wednesday, August 13, 2014 2:38 PM, David Briggs drdavidcbri...@gmail.commailto:drdavidcbri...@gmail.com wrote: Dear Careina, Following on from what Herman said, if you have a structure you can use the propKa server http://propka.ki.ku.dk/ to predict pKas for amino acids in the local environment as found in your structure. Perhaps some of the propKa literature might also be helpful? HTH, Dave David Briggs about.me/david_briggs On 13 August 2014 12:40, Careina Edgooms 02531c126adf-dmarc-requ...@jiscmail.ac.ukmailto:02531c126adf-dmarc-requ...@jiscmail.ac.uk wrote: Sorry for off topic question, just wondering if anyone has come across a study that shows the residue pka of certain amino acids is different in vitro compared to in vivo? Best Careina
Re: [ccp4bb] Twinning in space group Pc
Dear Kristof, Have you tried to solve it with the new SHELXT? You can force it to consider only chiral (Sohnke) space groups by putting -c on the command line. Best wishes, George On 13.08.2014 11:00, Kristof Van Hecke wrote: Dear, I’m struggling with the following (small molecule) problem: We are trying to solve the structure of a metal-organic framework containing a chiral compound. The space group is most probably Pc, but when refining, SHELX gives the error “Possible racemic twin or wrong absolute structure - try TWIN refinement”. As we know our compound is enantiopure, a racemic twin is very unlikely. In this regard, also a centro-symmetric space group is not possible (although CrysAlisPro always gives P2/c as the proper space group). As a matter of fact, trying different space groups is not solving the problem. The second problem is that half of the structure is visible, but the other half is completely not clear. Refinement is not possible at all (R-value of 33%). When running TwinRotMat (Platon), I get the following possible 2-fold twin axes: 2-axis ( 0 1 -1 ) [ -2 5 -4 ], Angle () [] = 2.31 Deg, Freq =14 * (-0.992 -0.0190.015) (h1) (h2) Nr Overlap =84 (-0.4300.075 -0.860) * (k1) = (k2) BASF = 0.96 ( 0.459 -1.146 -0.083) (l1) (l2)DEL-R =-0.064 2-axis ( 0 1 1 ) [ 2 5 4 ], Angle () [] = 2.31 Deg, Freq =15 * (-0.9920.0190.015) (h1) (h2) Nr Overlap = 229 ( 0.4300.0750.860) * (k1) = (k2) BASF = 0.94 ( 0.4591.146 -0.083) (l1) (l2)DEL-R =-0.050 2-axis ( 1 0 -2 ) [ 3 0 -2 ], Angle () [] = 0.54 Deg, Freq =19 * (-0.1360.000 -0.576) (h1) (h2) Nr Overlap = 992 ( 0.000 -1.0000.000) * (k1) = (k2) BASF = 0.86 (-1.7030.0000.136) (l1) (l2)DEL-R =-0.030 2-axis ( 1 2 -1 ) [ 5 5 -1 ], Angle () [] = 0.39 Deg, Freq =13 * (-0.3800.620 -0.124) (h1) (h2) Nr Overlap = 854 ( 1.2560.256 -0.251) * (k1) = (k2) BASF = 0.88 (-0.617 -0.617 -0.877) (l1) (l2)DEL-R =-0.019 However, none of these do actually improve the refinement. Has anyone encountered possible twinning/twin laws in Pc please? Or any other suggestions are most welcome? Thank you very much Kristof -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-33021 or -33068 Fax. +49-551-39-22582
Re: [ccp4bb] random half data sets
On Tue, Aug 12, 2014 at 10:28 PM, Keller, Jacob kell...@janelia.hhmi.org wrote: A somewhat similar question, with a quick answer I hope: when programs output CC's of 1/2 datasets, are several random halvings compared/averaged, and if not, does this make a difference, or are the scores so similar there's no point? The latter, I think. It probably only matters for data where you have a lot of erroneous observations (like ice rings) or at the fringes where there's almost no signal anyway. In my hands, a dataset with mulitplicity of 3.9 has an outer shell with a CC1/2 between 0.497 and 0.503 depending on random seed, which isn't worth worrying about. -Nat
Re: [ccp4bb] thin shell Rfree set selection
It depends to a large extent on the orientation of the Ncs operator. If that aligns with a crystal axis then there can be bias in the Rfree selection but if it doesn't NCS does not affect the Free R much ( in my opinion..) Eleanor On 13 August 2014 09:14, Ed Pozharski pozharsk...@gmail.com wrote: By all means, try it both ways and see whether the R-Rfree gap narrows with random vs thin shell selection. Depending on resolution and data quality, you may also consider imposing NCS restraints. Sent on a Sprint Samsung Galaxy S® III Original message From: Xianchi Dong Date:08/12/2014 4:45 PM (GMT-05:00) To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] thin shell Rfree set selection Dear all, I have a dataset of C2 symmetry and 2 molecules in the ASU. I am wondering if I have to use thin shell Rfree set selection to avoid Rfree bias by NCS. And which Rfree selection method is better? Thanks in advance. Xianchi
[ccp4bb] desiring untouched ligand
Hi everyone, Does anyone know of a way to refine with CCP4 - Refmac5 (restrained refinement is what I do) fixing a part of the the ligand? Thank you very much for your input, Remie
Re: [ccp4bb] desiring untouched ligand
Hi Remie, You can add harmonic restraints for parts you do not want to move too much. Instructions could be found here: http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/refmac/refmac_keywords.html#Harmonic Regards Garib On 13 Aug 2014, at 15:44, Remie Fawaz-Touma remiefa...@gmail.com wrote: Hi everyone, Does anyone know of a way to refine with CCP4 - Refmac5 (restrained refinement is what I do) fixing a part of the the ligand? Thank you very much for your input, Remie Dr Garib N Murshudov MRC-LMB Francis Crick Avenue Cambridge CB2 0QH UK Web http://www.mrc-lmb.cam.ac.uk, http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/
[ccp4bb] Computational Crystallography Newsletter - Volume 5, Number 2
I am pleased to announce the publication of the latest issue of the Computational Crystallography Newsletter: http://www.phenix-online.org/newsletter/ A listing of the articles and short communications is given below. Please note that the newsletter accepts articles of a general nature of interest to all crystallographers. Please send any articles to me at nwmoria...@lbl.gov noting that there is a Word Template on the website to streamline production. Articles Details of the Conformation-Dependent Library http://phenix-online.org/newsletter/CCN_2014_07.pdf#page=12 Short Communications Connectivity analysis tools in CCTBX http://phenix-online.org/newsletter/CCN_2014_07.pdf#page=4 phenix.fab_elbow_angle: Fragment Antigen-Binding elbow angle calculation tool http://phenix-online.org/newsletter/CCN_2014_07.pdf#page=8 Fitting tips #8 – Acetyl Groups are like Peptides: Planar and trans http://phenix-online.org/newsletter/CCN_2014_07.pdf#page=2 Cheers Nigel -- Nigel W. Moriarty Building 64R0246B, Physical Biosciences Division Lawrence Berkeley National Laboratory Berkeley, CA 94720-8235 Phone : 510-486-5709 Email : nwmoria...@lbl.gov Fax : 510-486-5909 Web : CCI.LBL.gov
[ccp4bb] Off topic: Cloning multi genes/multi cistronic in yeast
Dear all One of my human membrane proteins have been described to interact with additional subunits for its activity. To obtain functional form in yeast (Saccharomyces), I can think of two approach of either cloning all the subunits under one promoter or reconstitute in vitro. For the first option, what is the length of base pairs between the stop codon of one gene and the start of Kozak sequence for the next one? Is there any preference for the order so that only one subunit is His tagged? Second option will need multiple purification steps and some trials with protein ratios. Is this better? Thank you. Theresa
[ccp4bb] monomer/dimer protein
Hello All- I am interested in monomer/dimer contamination when building a crystal lattice, ie. if you are building a crystal lattice with a monomeric species of protein, incorporation of dimers may yield lattice or surface defects. This species may be considered a macromolecular contaminant. I have read a few papers on this subject, a couple are listed here: I. Yoshizaki et al. / Journal of Crystal Growth 290 (2006) 185–191 Caylor CL1, Dobrianov I, Lemay SG, Kimmer C, Kriminski S, Finkelstein KD, Zipfel W, Webb WW, Thomas BR, Chernov AA, Thorne RE. Proteins. 1999 Aug 15;36(3):270-81. In each of the studies that I have read, lysozyme is the model protein for these studies. I have not seen studies thus far that have been done with other proteins. Can anyone point me toward other studies, specifically non-lysozyme studies, where incorporation of two different oligomerization states has been shown to yield crystals with higher level of defects? Microscopy studies of such would be great too. Thanks in advance- Todd
Re: [ccp4bb] Off topic: Cloning multi genes/multi cistronic in yeast
On 8/13/14 2:29 PM, Theresa Hsu wrote: Dear all One of my human membrane proteins have been described to interact with additional subunits for its activity. To obtain functional form in yeast (Saccharomyces), I can think of two approach of either cloning all the subunits under one promoter or reconstitute in vitro. For the first option, what is the length of base pairs between the stop codon of one gene and the start of Kozak sequence for the next one? Is there any preference for the order so that only one subunit is His tagged? Second option will need multiple purification steps and some trials with protein ratios. Is this better? Natively, Saccharomyces does not have operons. And I am unaware of operon-like constructs (single promoter with multiple genes oriented in the same direction) working in Saccharomyces. (The GAL1-10 divergent promoter is probably the closest analog, but this involves transcription of the GAL1 and GAL10 genes that are encoded on opposite strands in opposite orientations.) For multiprotein complexes, one strategy for in vivo complex assembly is to encode each protein (with its own promoter) on separate plasmid (with distinct selectable markers) and transform them all into the same strain. This avoids the problems of in vitro reconstitution of the complex as well as the problem of subunits that cannot be stably expressed alone. Chris.
Re: [ccp4bb] Off topic: Cloning multi genes/multi cistronic in yeast
Hey, in my old lab we developed a system to express two proteins from one plasmid using the before mentioned GAL1-10 Promoter. Giving the use of different plasmids with different selection markers, coexpression of multiple proteins is possible in yeast. The system was used in a study published in Structure last year and you could contact my old lab to request plasmids. We used a TRP1 and a LEU2 plasmid and coexpressed three proteins in this study, but it is easily extendable to at least 4 proteins. You can find the paper here: http://www.ncbi.nlm.nih.gov/pubmed/23954503 To receive plasmids from the study, please contact Ed Hurt, the corresponding author at the BZH in Heidelberg/Germany. I stumbled across a similar system recently, which is also based on the GAL promoter, but don't find the reference now... Good luck, Karsten Am 13.08.2014 15:31, schrieb Chris Putnam: On 8/13/14 2:29 PM, Theresa Hsu wrote: Dear all One of my human membrane proteins have been described to interact with additional subunits for its activity. To obtain functional form in yeast (Saccharomyces), I can think of two approach of either cloning all the subunits under one promoter or reconstitute in vitro. For the first option, what is the length of base pairs between the stop codon of one gene and the start of Kozak sequence for the next one? Is there any preference for the order so that only one subunit is His tagged? Second option will need multiple purification steps and some trials with protein ratios. Is this better? Natively, Saccharomyces does not have operons. And I am unaware of operon-like constructs (single promoter with multiple genes oriented in the same direction) working in Saccharomyces. (The GAL1-10 divergent promoter is probably the closest analog, but this involves transcription of the GAL1 and GAL10 genes that are encoded on opposite strands in opposite orientations.) For multiprotein complexes, one strategy for in vivo complex assembly is to encode each protein (with its own promoter) on separate plasmid (with distinct selectable markers) and transform them all into the same strain. This avoids the problems of in vitro reconstitution of the complex as well as the problem of subunits that cannot be stably expressed alone. Chris. -- Karsten Thierbach, Dr. rer. nat. California Institute of Technology Division of Chemistry Chemical Engineering Hoelz laboratory 1200 E. California Blvd., M/C 147-75 Pasadena, CA 91125, U.S.A.
Re: [ccp4bb] software/web server to determine ligand volume
You can calculate volumes and much more, here: http://www.molinspiration.com/cgi-bin/properties Javier On Wed, Aug 13, 2014 at 12:06 AM, sreetama das somon_...@yahoo.co.in wrote: Dear all, Is there any software or web server available to calculate the volume of a ligand if the ligand coordinates are provided? Google seems to come up only with options to calculate protein cavity volume. Thanks in advance, Sreetama Das, phd student, Physics, IISc -- Javier M. Gonzalez, PhD. Protein Crystallography Station Bioscience Division Bioenergy and Biome Sciences Group (B-11) Los Alamos National Laboratory TA-3, Building 4200, Room 202B Mailstop T007 Los Alamos, NM 87545 Phone: +1 (505) 667-9376 LinkedIn http://www.linkedin.com/pub/javier-gonz%C3%A1lez/22/7b/83a Email bio...@gmail.com
