[ccp4bb] sfall in "MODE ATMMAP RESMOD" mode misreads PDB coordinates
Dear everyone, I am using sfall in "MODE ATMMAP RESMOD" to compute a tagged map with which to score a model against a map. The input PDB is OK and regularly read by all programs - but sfall seems to interpret the coordinates wrongly (see the list of the first 10 atoms coordinates that the program outputs, both in orthogonal and fractional): First 10 atoms of atsort - orthog coordinates 1NCYS 140283.0180251.2380115.7960 1.00 500.003 fractional coordinates 1.57127 1.39484 0.64288 First 10 atoms of atsort - orthog coordinates 2CA CYS 140281.9190250.4390115.2740 1.00 500.002 fractional coordinates 1.56517 1.39040 0.63998 3CCYS 140280.7020251.2870114.8740 1.00 500.002 fractional coordinates 1.55842 1.39511 0.63776 4OCYS 140279.7890250.8050114.1860 1.00 500.004 fractional coordinates 1.55335 1.39243 0.63394 5CB CYS 140281.5250249.4400116.3580 1.00 500.002 fractional coordinates 1.56299 1.38485 0.64600 6SG CYS 140280.3360248.2280115.8470 1.00 500.005 fractional coordinates 1.55638 1.37813 0.64317 7NSER 141280.6900252.5440115.3270 1.00 500.003 fractional coordinates 1.55835 1.40209 0.64028 8CA SER 141279.5620253.4720115.1380 1.00 500.002 fractional coordinates 1.55209 1.40724 0.63923 9CB SER 141279.7800254.7110116.0260 1.00 500.002 fractional coordinates 1.55330 1.41412 0.64416 10OG SER 141280.9710255.4130115.6440 1.00 500.004 fractional coordinates 1.55991 1.41802 0.64204 11CSER 141279.3470253.9090113.6680 1.00 500.002 fractional coordinates 1.55089 1.40967 0.63107 First 10 atoms of sorted file in asym unit - 10.55639 0.64317 0.37813699.89 1.00 5 102SG CYS 10.56299 0.64600 0.38485599.89 1.00 2 102CB CYS 10.56517 0.63998 0.39040299.89 1.00 2 102CA CYS 10.55335 0.63394 0.39243499.89 1.00 4 102O CYS 10.57127 0.64288 0.39484199.89 1.00 3 102N CYS 10.55842 0.63776 0.39511399.89 1.00 2 102C CYS 10.55835 0.64028 0.40209799.89 1.00 3 103N SER 10.55209 0.63923 0.40724899.89 1.00 2 103CA SER 10.55330 0.64416 0.41412999.89 1.00 2 103CB SER 10.55991 0.64204 0.41802 1099.89 1.00 4 103OG SER As a result of course the tagged map is completely screwed. Has anyone encountered this problem and if so how did they solve it? I would rather avoid digging into the code if I can! Best regards Pietro -- Pietro Roversi Sir William Dunn School of Pathology, Oxford University South Parks Road, Oxford OX1 3ER, England UK Tel. 0044-1865-275385
[ccp4bb] Dashed lines in coot
Hi All, These days I found the model and the density map changed to dashed lines after typed something wrong in the keyboard, I donnot know what have been typed and couldnot change it back to the normal appearance until rerun coot. The version of my coot is 0.3.3 in fedora. Does anybody know about it? Or maybe there is some functions behind those dashed lines? Thanks!
Re: [ccp4bb] sfall in "MODE ATMMAP RESMOD" mode misreads PDB coordinates
Hi Pietro, It looks as though SFALL thinks you have fractional coords and has coverted them into orthog ones. Does the PDB have a CRYST1 and SCALEi cards? Adam On Thu, 24 Apr 2008, Pietro Roversi wrote: > Dear everyone, > > I am using sfall in "MODE ATMMAP RESMOD" to compute a > tagged map with > which to score a model against a map. The input PDB is OK and regularly > read by all programs - but sfall seems to interpret the coordinates > wrongly (see the list of the first 10 atoms coordinates that the program > outputs, both in orthogonal and fractional): > > First 10 atoms of atsort - orthog coordinates > 1NCYS 140283.0180251.2380115.7960 1.00 500.003 >fractional coordinates 1.57127 1.39484 0.64288 > > > First 10 atoms of atsort - orthog coordinates > 2CA CYS 140281.9190250.4390115.2740 1.00 500.002 >fractional coordinates 1.56517 1.39040 0.63998 > 3CCYS 140280.7020251.2870114.8740 1.00 500.002 >fractional coordinates 1.55842 1.39511 0.63776 > 4OCYS 140279.7890250.8050114.1860 1.00 500.004 >fractional coordinates 1.55335 1.39243 0.63394 > 5CB CYS 140281.5250249.4400116.3580 1.00 500.002 >fractional coordinates 1.56299 1.38485 0.64600 > 6SG CYS 140280.3360248.2280115.8470 1.00 500.005 >fractional coordinates 1.55638 1.37813 0.64317 > 7NSER 141280.6900252.5440115.3270 1.00 500.003 >fractional coordinates 1.55835 1.40209 0.64028 > 8CA SER 141279.5620253.4720115.1380 1.00 500.002 >fractional coordinates 1.55209 1.40724 0.63923 > 9CB SER 141279.7800254.7110116.0260 1.00 500.002 >fractional coordinates 1.55330 1.41412 0.64416 > 10OG SER 141280.9710255.4130115.6440 1.00 500.004 >fractional coordinates 1.55991 1.41802 0.64204 > 11CSER 141279.3470253.9090113.6680 1.00 500.002 >fractional coordinates 1.55089 1.40967 0.63107 > First 10 atoms of sorted file in asym unit - >10.55639 0.64317 0.37813699.89 1.00 5 102SG > CYS >10.56299 0.64600 0.38485599.89 1.00 2 102CB > CYS >10.56517 0.63998 0.39040299.89 1.00 2 102CA > CYS >10.55335 0.63394 0.39243499.89 1.00 4 102O > CYS >10.57127 0.64288 0.39484199.89 1.00 3 102N > CYS >10.55842 0.63776 0.39511399.89 1.00 2 102C > CYS >10.55835 0.64028 0.40209799.89 1.00 3 103N > SER >10.55209 0.63923 0.40724899.89 1.00 2 103CA > SER >10.55330 0.64416 0.41412999.89 1.00 2 103CB > SER >10.55991 0.64204 0.41802 1099.89 1.00 4 103OG > SER > > As a result of course the tagged map is completely screwed. > > Has anyone encountered this problem and if so how did they solve it? I > would rather avoid digging into the code if I can! > > Best regards > > Pietro > -- > Pietro Roversi > Sir William Dunn School of Pathology, Oxford University > South Parks Road, Oxford OX1 3ER, England UK > Tel. 0044-1865-275385 >
Re: [ccp4bb] Help with pseudosymmetry problem
Hi Peter, Can you try to run xtriage and see what it tells you in terms of possible twin laws and merging statistics in higher symmetry space groups? The log file is attached. xtriage does not find any clear signs of pseudosymmetry or higher metric symmetry, but it does detect the pseudo-translation peak at (0.129, 0.475, 0.218) with 10% of the origin height, which it doesn't consider as significant (I get 20% when I do it using FFT) If for some reason no twin laws are found, you can manually change the unit cell on the command line to have beta exactly equal to 90... I didn't do this, as possible twin law was found. Or have I misunderstood the logic? Let me know what the logfile tells you. If the translation is 'special' xtraige will tell you what the approximate pseudo symmetry would be. It doesn't seem to think so. Derek P.S. Great program by the way! xtriage.log Description: Binary data 2008/4/23, Derek Logan <[EMAIL PROTECTED]>: Hi everyone, Can anyone help me with interpretation of a self rotation function and native Patterson from a dataset with pseudosymmetry? I've always been a bit poor on spherical polars. The space group is P21 with beta = 92.2°. The kappa=180° section of the SRF, calculated using Molrep, is at http://mole.mbfys.lu.se/~derek/selfRF_180.png and contains two big peaks around 7 sigma. I'm having trouble identifying these in the list of peaks from Molrep: thetaphi chialphabeta gamma Isym_i Isym_j Sol_RF 1 0.000.000.00 0.000.000.00 1 1 Sol_RF 1 90.00 -90.00 180.00 0.00 180.000.00 1 2 Sol_RF 1 90.00 90.00 180.00 0.00 180.000.00 2 1 Sol_RF 1 0.000.000.00 0.000.000.00 2 2 Sol_RF 2158.56 180.00 180.00 0.00 42.89 -180.00 1 1 Sol_RF 2111.440.00 180.00 -180.00 137.110.00 1 2 Sol_RF 2111.440.00 180.00180.00 137.110.00 2 1 Sol_RF 2 21.440.00 180.00180.00 -42.890.00 2 2 Sol_RF 3165.650.00 180.00 -180.00 28.700.00 1 1 Sol_RF 3104.35 -180.00 180.00 0.00 151.30 180.00 1 2 Sol_RF 3104.35 180.00 180.00 0.00 151.30 -180.00 2 1 Sol_RF 3 14.35 -180.00 180.00 0.00 -28.70 180.00 2 2 It seems to me to be two copies of peak 2. I believe theta starts in the middle, perpendicular to the page and phi starts on the x axis, thus the peak just below the centre would be (21.44, 0, 180). I presume that the second peak is the symmetry-related (158.56, 180, 0)? However where is (111.44 0 180)? I would expect to see this near the bottom of the plot, but it's not there. I'm sure I'm missing something fundamental about the symmetry of the SRF projection, but unfortunately I don't have a supervisor to bug about this (I *am* the supervisor...) In the native Patterson http://mole.mbfys.lu.se/~derek/nativePatterson.png there are two peaks of almost equal height. How can this be reconciled with having only one strong peak in the SRF? There are most likely two dimers in the asymmetric unit, but there may only be one, with very high resulting solvent content. What's more the molecules are leucine-rich repeat proteins and have weak internal symmetry. I believe this was an issue with the ribonuclease inhibitor, but looking briefly at the crystallisation article and structure article I wasn't able to find a rationalisation of this problem. The 2-fold is perpendicular to b*. How could this cause the two peaks? Thanks Derek -- - P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -
Re: [ccp4bb] Help with pseudosymmetry problem
Frankly when faced with these problems of generating symmetry equivalents i revert to almn, where a) I can guarantee the orthogonalisation is as I expect, and b) it generates an exhaustive set of symmetry equivalent peaks. But that is old technology.. If you have two dimers in the asymmetric unit, and only one self rotation peak, it is reasonable to assume the 2 fold NCS axes are in the same orientation. That doesnt necessarily mean the dimers have to be in exactly the same orientation about that 2 fold, s0 I guess you might expect a split peak for a pseudo translation. another trick that sometimes helps - is there a dimer model with exptl data available? again back to almn but sometimes you can fit the two sets of hklin together. Although theoretically you should get the same information from search with a dimer model matching Fcalc to Fobs. Eleanor Derek Logan wrote: Thanks to everyone who helped with the self RF problem: Eleanor, Ian, Claudine, Pietro & Alexei. Eleanor wrote: 1) It is a bit hard to find out how MOLREP defines its orthogonal axes - many programs use X0 || a, Yo || b* and in P21 hence Zortho is || to c* If that is what Molrep does then your 2 fold is in the a c* plane, 21 degrees or 111 degrees from c*. The 2 peaks you see are symmetry equivalents. This was my interpretation. Glad we agree ;-) The documentation says "A parallel to X , Cstar parallel to Z" As for the Patterson - what height are those peaks relative to the origin? The peaks are u = 0.129, v = 0.473, w = 0.220 (20% of origin peak height) and u = 0.180, v = 0.500, w = 0.248 (19%). What I don't get is why there are two and only one strong 2-fold. 2 dimers in the AU gives 50% solvent, 1 dimer 75%. The crystals diffract to 2.3Å, which would tip the balance in favour of 50% solvent in my opinion. With 2 dimers in the asymm unit and with the non-cryst 2-fold perpendicular to b* you could have such translations between one monomer and another. Would the 2-folds of both dimers have to be very similarly oriented? Maybe one peak masks the other at this resolution? is there a model - easiest to solve it then analyse this sort of stuff later! Believe me, we've been trying for a very long time! The problem is that it's a leucine rich repeat protein with under 30% sequence identity to any of the other LRR models out there. I think the failure of MR is down to a combination of a) the low homology, b) the pseudosymmetry, c) the nature of the LRR, which means you can get MR solutions that are out by one or more repeats. Maybe even the internal symmetry of the whole LRR structure can add to this pathology? We've had some solutions that looked almost right, but we can never see much more than what's already in the MR solution. Ian wrote: The symmetry of the self-RF is explained in detail in the documentation for POLARRFN, in fact I would advise you to use this because you can then plot monoclinic space groups with the unique b axis along the orthogonal Z axis (NCODE = 3) and then the symmetry is *much* easier to interpret. The reason I started using Molrep was that POLARRFN always used to choke on these data. However that problem seems to have disappeared. Using ORTH 3 indeed gives a more interpretable plot, as you say. According to polarrfn.doc the symmetry generated by a 2-fold along b parallel to Z is (180-theta, 180-phi, kappa) so the peak in the list (159,180,180) is the same as (21,0,180) which is a NCS 2-fold that you can see just below centre. The peak (111,0,180) is thus the same as (69,180,180) near the top which is another NCS 2-fold perp to the first generated by the crystallographic 2-fold. Indeed, I see the peak (69, 180, 180) but I don't find it in the list in the log file from Molrep. I thought that list was supposed to be exhaustive. Also the plot is not well documented for Molrep. I wrote to the BB a while ago to ask what the contour levels were but no-one answered. By Googling I found a crystallisation paper where it was described as "from 0.5 sigma in steps of 0.5 sigma" but that information appears to have come by word of mouth. Also, is it just the "north hemisphere", as Claudine put it, that is plotted? Anyway, I feel somewhat wiser now... Derek
Re: [ccp4bb] sfall in "MODE ATMMAP RESMOD" mode misreads PDB coordinates
As far as I know SFALL reads PDB files - I dont think there is an option to input fractional coordinates.. I suspect it thinks everything is displaced one space to the left or right .. Can you run pdbset xyzin now.pdb xyzout now+.pdb end and see if there is any difference? When I run the map correlation task which does this the answers look sort of sensible.. Data line--- MODE ATMMAP RESMOD Data line--- grid 130 140 170 Data line--- symmetry 'P 1' fractional coordinates 0.49431 1.19518 0.40395 9NGLN34-11.0320 40.7750 20.4450 0.50 12.613 fractional coordinates 0.38022 1.19991 0.40616 10CA GLN34-12.3450 40.1470 20.3370 0.50 12.122 fractional coordinates 0.34151 1.18372 0.40401 11CGLN34-12.3150 38.6620 20.7240 0.50 12.772 fractional coordinates 0.32789 1.15251 0.41170 First 10 atoms of sorted file in asym unit - 10.48580 0.43335 0.16420713.64 0.50 2 33CD1 LEU Eleanor Adam Ralph wrote: Hi Pietro, It looks as though SFALL thinks you have fractional coords and has coverted them into orthog ones. Does the PDB have a CRYST1 and SCALEi cards? Adam On Thu, 24 Apr 2008, Pietro Roversi wrote: Dear everyone, I am using sfall in "MODE ATMMAP RESMOD" to compute a tagged map with which to score a model against a map. The input PDB is OK and regularly read by all programs - but sfall seems to interpret the coordinates wrongly (see the list of the first 10 atoms coordinates that the program outputs, both in orthogonal and fractional): First 10 atoms of atsort - orthog coordinates 1NCYS 140283.0180251.2380115.7960 1.00 500.003 fractional coordinates 1.57127 1.39484 0.64288 First 10 atoms of atsort - orthog coordinates 2CA CYS 140281.9190250.4390115.2740 1.00 500.002 fractional coordinates 1.56517 1.39040 0.63998 3CCYS 140280.7020251.2870114.8740 1.00 500.002 fractional coordinates 1.55842 1.39511 0.63776 4OCYS 140279.7890250.8050114.1860 1.00 500.004 fractional coordinates 1.55335 1.39243 0.63394 5CB CYS 140281.5250249.4400116.3580 1.00 500.002 fractional coordinates 1.56299 1.38485 0.64600 6SG CYS 140280.3360248.2280115.8470 1.00 500.005 fractional coordinates 1.55638 1.37813 0.64317 7NSER 141280.6900252.5440115.3270 1.00 500.003 fractional coordinates 1.55835 1.40209 0.64028 8CA SER 141279.5620253.4720115.1380 1.00 500.002 fractional coordinates 1.55209 1.40724 0.63923 9CB SER 141279.7800254.7110116.0260 1.00 500.002 fractional coordinates 1.55330 1.41412 0.64416 10OG SER 141280.9710255.4130115.6440 1.00 500.004 fractional coordinates 1.55991 1.41802 0.64204 11CSER 141279.3470253.9090113.6680 1.00 500.002 fractional coordinates 1.55089 1.40967 0.63107 First 10 atoms of sorted file in asym unit - 10.55639 0.64317 0.37813699.89 1.00 5 102SG CYS 10.56299 0.64600 0.38485599.89 1.00 2 102CB CYS 10.56517 0.63998 0.39040299.89 1.00 2 102CA CYS 10.55335 0.63394 0.39243499.89 1.00 4 102O CYS 10.57127 0.64288 0.39484199.89 1.00 3 102N CYS 10.55842 0.63776 0.39511399.89 1.00 2 102C CYS 10.55835 0.64028 0.40209799.89 1.00 3 103N SER 10.55209 0.63923 0.40724899.89 1.00 2 103CA SER 10.55330 0.64416 0.41412999.89 1.00 2 103CB SER 10.55991 0.64204 0.41802 1099.89 1.00 4 103OG SER As a result of course the tagged map is completely screwed. Has anyone encountered this problem and if so how did they solve it? I would rather avoid digging into the code if I can! Best regards Pietro -- Pietro Roversi Sir William Dunn School of Pathology, Oxford University South Parks Road, Oxford OX1 3ER, England UK Tel. 0044-1865-275385
[ccp4bb] Setting drops with proteins that are hard to concentrate
Dear All, I have a 50kDa protein that is soluble and monodisperse at up to approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). However, it aggregates (probably both via disulphides and via 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ml, even in the presence of several detergents. I don't want to add DTT since my protein should have several intramolecular disulphidesalthough I do have 2 free Cysteines, partially exposed. I have already tried mutating the Cysteines, with little improvement. Any suggestions for obtaining 5-10mg/ml? Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, 2004, pages 8933...) Thanks for any input! Yours, Matt Matthew J. Bottomley, Ph.D. Senior Research Biochemist IRBM / MRL-Rome Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Setting drops with proteins that are hard to concentrate
I have a similar problem Matthew. Just got some NVoy from Novexin in which some claim can help if hydrophobic patches are the main problem. Will see. Regards, Roberto On 24 Apr 2008, at 12:35, Bottomley, Matthew wrote: Dear All, I have a 50kDa protein that is soluble and monodisperse at up to approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). However, it aggregates (probably both via disulphides and via 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ ml, even in the presence of several detergents. I don't want to add DTT since my protein should have several intramolecular disulphidesalthough I do have 2 free Cysteines, partially exposed. I have already tried mutating the Cysteines, with little improvement. Any suggestions for obtaining 5-10mg/ml? Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, 2004, pages 8933...) Thanks for any input! Yours, Matt Matthew J. Bottomley, Ph.D. Senior Research Biochemist IRBM / MRL-Rome Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/ contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. --- Dr. Roberto Steiner Randall Division of Cell and Molecular Biophysics New Hunt's House King's College London Guy's Campus London, SE1 1UL Phone +44 (0)20-7848-8216 Fax +44 (0)20-7848-6435 e-mail [EMAIL PROTECTED]
Re: [ccp4bb] Setting drops with proteins that are hard to concentrate
can't you try different volumes: i.e. 360 nl protein solution in very little buffer and salt + 40 nl precipitant. Should give roughly 10 mg/ml after equilibration. Best wishes Kornelius On Thu, 24 Apr 2008 12:42:42 +0100 Roberto Steiner <[EMAIL PROTECTED]> wrote: > I have a similar problem Matthew. > Just got some NVoy from Novexin in which some claim can help if hydrophobic > patches are the main problem. Will see. > > Regards, > Roberto > > On 24 Apr 2008, at 12:35, Bottomley, Matthew wrote: > > > Dear All, > > > > I have a 50kDa protein that is soluble and monodisperse at up to > > approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). > > > > However, it aggregates (probably both via disulphides and via > > 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ > > ml, even in the presence of several detergents. I don't want to add > > DTT since my protein should have several intramolecular > > disulphidesalthough I do have 2 free Cysteines, partially > > exposed. I have already tried mutating the Cysteines, with little > > improvement. > > > > Any suggestions for obtaining 5-10mg/ml? > > > > Does anybody have good experiences with usin L-Arg and L-Glu (e.g. > > At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, > > 2004, pages 8933...) > > > > Thanks for any input! > > > > Yours, > > > > Matt > > > > > > Matthew J. Bottomley, Ph.D. > > Senior Research Biochemist > > IRBM / MRL-Rome > > > > > > Notice: This e-mail message, together with any attachments, > > contains information of Merck & Co., Inc. (One Merck Drive, > > Whitehouse Station, New Jersey, USA 08889), and/or its affiliates > > (which may be known outside the United States as Merck Frosst, > > Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact > > information for affiliates is available at http://www.merck.com/ > > contact/contacts.html) that may be confidential, proprietary > > copyrighted and/or legally privileged. It is intended solely for > > the use of the individual or entity named on this message. If you > > are not the intended recipient, and have received this message in > > error, please notify us immediately by reply e-mail and then delete > > it from your system. > > --- > Dr. Roberto Steiner > Randall Division of Cell and Molecular Biophysics > New Hunt's House > King's College London > Guy's Campus > London, SE1 1UL > Phone +44 (0)20-7848-8216 > Fax +44 (0)20-7848-6435 > e-mail [EMAIL PROTECTED] > > > > -- Kornelius Zeth Max Planck Institute for Developmental Biology Dept. Protein Evolution Spemannstr. 35 72076 Tuebingen, Germany [EMAIL PROTECTED] Tel -49 7071 601 323 Fax -49 7071 601 349
Re: [ccp4bb] Help with pseudosymmetry problem
we have NCS rotation (158.56, 180, 0) - rotation matrix [R] and we have two CS operators (P21) - rotation matrix (0 0 0) [1] and (90 90 180) [2]. So, all symmetry related (for [R]) rotations are [1][R][1] = [R] - (158.56, 180, 0) [1][R][2] = [R][2] - ( 111.44 0.0 180.0) [2][R][1] = [2][R] - ( 111.44 0.0 180.0) [2][R][2] - (21.44, 0, 180) Really we have two rotations, because (158.56, 180, 0) and (21.44, 0, 180) are idendical. There is nothing special. If there is pure dimer one rotation is pure 2-fold axis another is 2-fold axis with translation. Alexei On 23 Apr 2008, at 22:29, Derek Logan wrote: Thanks to everyone who helped with the self RF problem: Eleanor, Ian, Claudine, Pietro & Alexei. Eleanor wrote: 1) It is a bit hard to find out how MOLREP defines its orthogonal axes - many programs use X0 || a, Yo || b* and in P21 hence Zortho is || to c* If that is what Molrep does then your 2 fold is in the a c* plane, 21 degrees or 111 degrees from c*. The 2 peaks you see are symmetry equivalents. This was my interpretation. Glad we agree ;-) The documentation says "A parallel to X , Cstar parallel to Z" As for the Patterson - what height are those peaks relative to the origin? The peaks are u = 0.129, v = 0.473, w = 0.220 (20% of origin peak height) and u = 0.180, v = 0.500, w = 0.248 (19%). What I don't get is why there are two and only one strong 2-fold. 2 dimers in the AU gives 50% solvent, 1 dimer 75%. The crystals diffract to 2.3Å, which would tip the balance in favour of 50% solvent in my opinion. With 2 dimers in the asymm unit and with the non-cryst 2-fold perpendicular to b* you could have such translations between one monomer and another. Would the 2-folds of both dimers have to be very similarly oriented? Maybe one peak masks the other at this resolution? is there a model - easiest to solve it then analyse this sort of stuff later! Believe me, we've been trying for a very long time! The problem is that it's a leucine rich repeat protein with under 30% sequence identity to any of the other LRR models out there. I think the failure of MR is down to a combination of a) the low homology, b) the pseudosymmetry, c) the nature of the LRR, which means you can get MR solutions that are out by one or more repeats. Maybe even the internal symmetry of the whole LRR structure can add to this pathology? We've had some solutions that looked almost right, but we can never see much more than what's already in the MR solution. Ian wrote: The symmetry of the self-RF is explained in detail in the documentation for POLARRFN, in fact I would advise you to use this because you can then plot monoclinic space groups with the unique b axis along the orthogonal Z axis (NCODE = 3) and then the symmetry is *much* easier to interpret. The reason I started using Molrep was that POLARRFN always used to choke on these data. However that problem seems to have disappeared. Using ORTH 3 indeed gives a more interpretable plot, as you say. According to polarrfn.doc the symmetry generated by a 2-fold along b parallel to Z is (180-theta, 180-phi, kappa) so the peak in the list (159,180,180) is the same as (21,0,180) which is a NCS 2-fold that you can see just below centre. The peak (111,0,180) is thus the same as (69,180,180) near the top which is another NCS 2-fold perp to the first generated by the crystallographic 2-fold. Indeed, I see the peak (69, 180, 180) but I don't find it in the list in the log file from Molrep. I thought that list was supposed to be exhaustive. Also the plot is not well documented for Molrep. I wrote to the BB a while ago to ask what the contour levels were but no-one answered. By Googling I found a crystallisation paper where it was described as "from 0.5 sigma in steps of 0.5 sigma" but that information appears to have come by word of mouth. Also, is it just the "north hemisphere", as Claudine put it, that is plotted? Anyway, I feel somewhat wiser now... Derek
Re: [ccp4bb] Setting drops with proteins that are hard to concentrate
Dear Matt, make sure that you don't have slow or even fast formation of unspecific intermolecular disulfides by simple checks at several time points on SDS-PAGE using sample buffer without DTT. Check the content of free thiols and disulfides of your protein over a period of time. If you need easy and solid protocols, just send me a mail. Guenter Bottomley, Matthew wrote: Dear All, I have a 50kDa protein that is soluble and monodisperse at up to approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). However, it aggregates (probably both via disulphides and via 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ml, even in the presence of several detergents. I don't want to add DTT since my protein should have several intramolecular disulphidesalthough I do have 2 free Cysteines, partially exposed. I have already tried mutating the Cysteines, with little improvement. Any suggestions for obtaining 5-10mg/ml? Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, 2004, pages 8933...) Thanks for any input! Yours, Matt Matthew J. Bottomley, Ph.D. Senior Research Biochemist IRBM / MRL-Rome Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates (which may be known outside the United States as Merck Frosst, Merck Sharp & Dohme or MSD and in Japan, as Banyu - direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system. -- *** Priv.Doz.Dr. Guenter Fritz Fachbereich Biologie Sektion Naturwissenschaften Universitaet Konstanz http://www.biologie.uni-konstanz.de/fritz Universitaetsstrasse 10 Postfach M665 D-78457 Konstanz e-mail: [EMAIL PROTECTED] Tel. Office: +49-(0)7531 88 3205 Tel. Lab : +49-(0)7531 88 3687 Fax: +49-(0)7531 88 2966
Re: [ccp4bb] Setting drops with proteins that are hard to concentrate
Dear Matt, In one of our project, the protein of interest used to elute from Ni-column with ~20 other proteins. I also guessed it due to surface Cysteines. Addition of 2mM free Cys in the protein sample helped, which competed with free Cys on the protein surface and, ultimately, gave happy single band on sds-page. All the best Manish -- Center for Advanced Research in Biotechnology University of Maryland Biotechnology Institute 9600 Gudelsky Drive, Rockville MD 20850 USA Tel: +1-240-314-6130; fax: +1-240-314-6255 - Original Message From: "Bottomley, Matthew" <[EMAIL PROTECTED]> To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, April 24, 2008 7:35:46 AM Subject: [ccp4bb] Setting drops with proteins that are hard to concentrate Setting drops with proteins that are hard to concentrate Dear All, I have a 50kDa protein that is soluble and monodisperse at up to approx 1mg/ml (after Ni-affinity and size-exclusion chromatography). However, it aggregates (probably both via disulphides and via 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ml, even in the presence of several detergents. I don't want to add DTT since my protein should have several intramolecular disulphidesalthough I do have 2 free Cysteines, partially exposed. I have already tried mutating the Cysteines, with little improvement. Any suggestions for obtaining 5-10mg/ml? Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, 2004, pages 8933...) Thanks for any input! Yours, Matt Matthew J. Bottomley, Ph.D. Senior Research Biochemist IRBM / MRL-Rome Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now. http://mobile.yahoo.com/;_ylt=Ahu06i62sR8HDtDypao8Wcj9tAcJ
[ccp4bb] arp/warp and refmac problem on os x
Hi, I've got a rather weird problem when trying to run arp/warp. When trying to run arp/warp (latest version 7.0.1) it complains that I should install a newer version of Refmac (5.4.0045). The version of Refmac I have installed is 5.4.0066, and thats what is being used if I run Refmac both from terminal or via CCP4i. However, when arp/warp starts to run it magically finds a refmac 5.2.something and uses that. Reinstalling arp/warp did not help, even though the install script showed that I had refmac 5.4.0066 installed... I'm running OS X 10.5.2 and have installed ccp4 and refmac via fink. All other CCP4 programs work fine and Refmac runs normally as well, it's only when it's called from arp/warp that something goes wrong.. All ideas to fix this problem are most welcome. Cheers, Ronnie Berntsson
Re: [ccp4bb] arp/warp and refmac problem on os x
On 24 Apr 2008, at 3:38 PM, Ronnie Berntsson wrote: I've got a rather weird problem when trying to run arp/warp. When trying to run arp/warp (latest version 7.0.1) it complains that I should install a newer version of Refmac (5.4.0045). The version of Refmac I have installed is 5.4.0066, and thats what is being used if I run Refmac both from terminal or via CCP4i. However, when arp/warp starts to run it magically finds a refmac 5.2.something and uses that. Reinstalling arp/warp did not help, even though the install script showed that I had refmac 5.4.0066 installed... I'm running OS X 10.5.2 and have installed ccp4 and refmac via fink. All other CCP4 programs work fine and Refmac runs normally as well, it's only when it's called from arp/warp that something goes wrong.. All ideas to fix this problem are most welcome. This is an interesting feature of the Fink CCP4/Refmac installation. When you install the new refmac in Fink, you get an executable called refmac-5.4, which lives in /sw/share/xtal/ccp4-6.0.2/bin. The refmac5 that you get from the terminal lives in /sw/bin and is a symbolic link to this executable. However, in /sw/share/xtal/ccp4-6.0.2/bin there still lurks an executable refmac5, and this is version 5.2.0019. When you run ARP/wARP from the GUI, it seems that this is the executable that it uses - hence the version warnings. In our experience, this also breaks flex-wARP when run through the GUI. Whether this is just a symptom of the way I use Fink to install things, or whether it's a problem for everyone, I don't know. I have a solution to this, although it may not be the most elegant one. I move the old refmac5 out of the way, and create a new symbolic link mv /sw/share/xtal/ccp4-6.0.2/bin/refmac5 /sw/share/xtal/ ccp4-6.0.2/bin/refmac5.old ln -s /sw/share/xtal/ccp4-6.0.2/bin/refmac-5.4 /sw/share/xtal/ ccp4-6.0.2/bin/refmac5 (This is from memory, so the details may not be correct.) Hope this helps, Chris The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
Re: [ccp4bb] arp/warp and refmac problem on os x
in arp/warp classic the script does not call refmac5 (the first thing in your path) but $CBIN/refmac5 (refmac from the CBIN place) I suspect that you still kept refmac 5.2 in the official CBIN directory and put refmac5 in another location but earlier in your path, so its picked up first ... try refmac5 -i and $CBIN/refmac5 -i I suspect they will give different results. My best guess at least ... A. On Apr 24, 2008, at 16:38, Ronnie Berntsson wrote: Hi, I've got a rather weird problem when trying to run arp/warp. When trying to run arp/warp (latest version 7.0.1) it complains that I should install a newer version of Refmac (5.4.0045). The version of Refmac I have installed is 5.4.0066, and thats what is being used if I run Refmac both from terminal or via CCP4i. However, when arp/warp starts to run it magically finds a refmac 5.2.something and uses that. Reinstalling arp/warp did not help, even though the install script showed that I had refmac 5.4.0066 installed... I'm running OS X 10.5.2 and have installed ccp4 and refmac via fink. All other CCP4 programs work fine and Refmac runs normally as well, it's only when it's called from arp/warp that something goes wrong.. All ideas to fix this problem are most welcome. Cheers, Ronnie Berntsson
Re: [ccp4bb] arp/warp and refmac problem on os x
Thanks for a very quick answer, it indeed solved the problem. Cheers, Ronnie On Apr 24, 2008, at 4:55 PM, Chris Richardson wrote: On 24 Apr 2008, at 3:38 PM, Ronnie Berntsson wrote: I've got a rather weird problem when trying to run arp/warp. When trying to run arp/warp (latest version 7.0.1) it complains that I should install a newer version of Refmac (5.4.0045). The version of Refmac I have installed is 5.4.0066, and thats what is being used if I run Refmac both from terminal or via CCP4i. However, when arp/ warp starts to run it magically finds a refmac 5.2.something and uses that. Reinstalling arp/warp did not help, even though the install script showed that I had refmac 5.4.0066 installed... I'm running OS X 10.5.2 and have installed ccp4 and refmac via fink. All other CCP4 programs work fine and Refmac runs normally as well, it's only when it's called from arp/warp that something goes wrong.. All ideas to fix this problem are most welcome. This is an interesting feature of the Fink CCP4/Refmac installation. When you install the new refmac in Fink, you get an executable called refmac-5.4, which lives in /sw/share/xtal/ ccp4-6.0.2/bin. The refmac5 that you get from the terminal lives in /sw/bin and is a symbolic link to this executable. However, in / sw/share/xtal/ccp4-6.0.2/bin there still lurks an executable refmac5, and this is version 5.2.0019. When you run ARP/wARP from the GUI, it seems that this is the executable that it uses - hence the version warnings. In our experience, this also breaks flex-wARP when run through the GUI. Whether this is just a symptom of the way I use Fink to install things, or whether it's a problem for everyone, I don't know. I have a solution to this, although it may not be the most elegant one. I move the old refmac5 out of the way, and create a new symbolic link mv /sw/share/xtal/ccp4-6.0.2/bin/refmac5 /sw/share/xtal/ ccp4-6.0.2/bin/refmac5.old ln -s /sw/share/xtal/ccp4-6.0.2/bin/refmac-5.4 /sw/share/xtal/ ccp4-6.0.2/bin/refmac5 (This is from memory, so the details may not be correct.) Hope this helps, Chris The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network. On Apr 24, 2008, at 4:56 PM, Anastassis Perrakis wrote: in arp/warp classic the script does not call refmac5 (the first thing in your path) but $CBIN/refmac5 (refmac from the CBIN place) I suspect that you still kept refmac 5.2 in the official CBIN directory and put refmac5 in another location but earlier in your path, so its picked up first ... try refmac5 -i and $CBIN/refmac5 -i I suspect they will give different results. My best guess at least ... A.
[ccp4bb] Question about Phoenix crystallization robot
BACKGROUND: Recently we acquired an Art Robbins Phoenix crystallization robot. This instrument is in a shared environment, accessible to labs with projects that range from small, well-behaved soluble cytosolic proteins to large complexes and integral membrane proteins. Many of our users obtain only small quantities (a few hundred microliters) of purified proteins, and they are always looking for ways to maximize the number of crystallization screens they can set up with their samples. QUESTION: Several users have recently asked if they could use a protocol that allows them to aspirate enough protein into the Nanoneedle (the needle used for dispensing protein) to set up 3-5 or more different 96-condition screens. They feel this would minimize any sample waste and maximize their time. Our concern is that this might result in clogging of the Nanoneedle due to evaporation and subsequent precipitation of their protein, as the amount of time required for such a protocol would be greater than 10 minutes. Our local Art Robbins representative has agreed with us that this is not a recommended protocol. We are in a bit of a dilemma as we do not have enough experience with this robot to definitively say that the users should not follow this kind of protocol, but perhaps there is a better way to achieve their desired goal. Does anyone out there have any practical suggestions and experience that could help us accommodate such user requests? Thank you in advance, I'll post a summary of responses, Diana * * * * * * * * * * * * * * * * * * * * * * * * * * * * Diana R. Tomchick Associate Professor University of Texas Southwestern Medical Center Department of Biochemistry 5323 Harry Hines Blvd. Rm. ND10.214B Dallas, TX 75390-8816, U.S.A. Email: [EMAIL PROTECTED] 214-645-6383 (phone) 214-645-6353 (fax)
Re: [ccp4bb] Scala summary and log file- sometimes shortened
Hello everyone, It seems that the reason I get "short summaries" in scala is because the "Show Summary button" is affected by a possible intermitently appearing change in scala log file formatting. When I looked for the text "Summary" in the log file I did find the entire table -intact. SO just for completeness - here is what the "Show summary" gave . And below this I have the intact summary in the log file. It seems like ccp4i "Show summary" button for scala log file viewing does not include the entire table in the window everytime. Summary data for Project: cy Crystal: P10_2 Dataset: P10_2 Overall InnerShell OuterShell Low resolution limit 102.06102.06 2.11 High resolution limit 2.00 6.34 2.00 Rmerge 0.252 0.112 5.247 Rmeas (within I+/I-) 0.269 0.118 6.982 Scala: ** Normal termination ** Times: User: 403.8s System:7.8s Elapsed: 6:52 ### INTACT summary: When I do Full log file view : I get the entire summary Summary data for Project: cy Crystal: P10_2 Dataset: P10_2 Overall InnerShell OuterShell Low resolution limit 102.06102.06 2.11 High resolution limit 2.00 6.34 2.00 Rmerge 0.252 0.112 5.247 Rmeas (within I+/I-) 0.269 0.118 6.982 Scala: ** Normal termination ** Times: User: 403.8s System:7.8s Elapsed: 6:52 Rmeas (all I+ & I-)0.269 0.118 6.982 Rpim (within I+/I-)0.091 0.036 4.552 Rpim (all I+ & I-) 0.091 0.036 4.552 Fractional partial bias -0.113-0.122-1.163 Total number of observations 1458009111004 8307 Total number unique 212719 9864 4327 Mean((I)/sd(I)) 6.4 16.6 0.3 Completeness67.9 99.6 9.4 Multiplicity 6.9 11.3 1.9 ### User: hari Run date: 22/ 4/2008 Run time: 16:39:47 On Tue, Apr 22, 2008 at 4:14 PM, hari jayaram <[EMAIL PROTECTED]> wrote: > For some reason scala does not output the same log file for me everytime. I > got used to looking at the summary for my scala run, using the Show summary > button in ccp4i after picking view log file. > In most cases I get a detailed summary. For this particular dataset , > despite not changing any parameters in the GUI I get a very shortened > summary. > Is it that I clicked some button by mistake, that shortens the log-summary. > > I am using the latest version of ccp4 6.2.2 and fink installed ccp4 on OSX > Leopard. > I am reproducing the short and long summary here. > > Thanks for your help > Hari Jayaram > > > BAD Case: > > Summary data for Project: cy Crystal: P10_2 Dataset: P10_2 > >Overall InnerShell OuterShell > > Low resolution limit 102.06102.06 2.11 > High resolution limit 2.00 6.34 2.00 > > Rmerge 0.252 0.112 5.247 > Rmeas (within I+/I-) 0.269 0.118 6.982 > Scala: ** Normal termination ** > Times: User: 404.5s System:8.0s Elapsed: 6:53 > > > > GOOD case: > > Summary data for Project: cy Crystal: p10_1 Dataset: p10_1 > >Overall InnerShell OuterShell > > Low resolution limit 102.60102.60 2.12 > High resolution limit 2.01 6.35 2.01 > > Rmerge 0.164 0.053 3.283 > Rmeas (within I+/I-) 0.183 0.058 4.363 > Rmeas (all I+ & I-)0.183 0.058 4.363 > Rpim (within I+/I-)0.080 0.021 2.835 > Rpim (all I+ & I-) 0.080 0.021 2.835 > Fractional partial bias -0.022-0.018-0.072 > Total number of observations 835420 73666 4479 > Total number unique 196397 9701 3022 > Mean((I)/sd(I)) 2.2 4.2 0.2 > Completeness64.3 99.8
[ccp4bb] Daily Digest Version of CCP4BB
In case someone do not know this feature or how to set it up, you can choose to get the daily digest version of CCP4BB so that your mail box will not be filled with mails that you can't find time to read individually. I have just changed my settings at JISCMAIL web site. You would have to click 'CCP4BB' link to change the settings (the table version didn't work for me, on Mac at least). Anyhow, I just thought it would be good to tell others about this feature, because I believe it would help saving time for a lot of busy people. You can still read/reply messages whenever you want, but you would be able to do these in a more 'consolidated' or efficient way. HTH. Holly Jing, PhD TSRI, La Jolla, USA _ Spell a grand slam in this game where word skill meets World Series. Get in the game. http://club.live.com/word_slugger.aspx?icid=word_slugger_wlhm_admod_april08
