Re: [ccp4bb] Resolution and data/parameter ratio, which one is more important?
Dear Guangyu, 80% solvent is an awful lot. The first thing I would do is to check that there is not another protein molecule hiding somewhere in the asymmetric unit. What I usually do in these cases is to set a very large map radius (say 40-60 Å) and look at the complete solvent region to see if there are regions which are significantly more noisy. I would also check that the crystal packing makes sense, e.g. continuous contacts in all three dimensions and no layers without any protein contacts. Of course that best results are obtained by building and refining both crystal forms and cross-checking the results from one crystal form in the other crystal form. Having said that, I have had some amazingly clear and unbiased electron density maps from low-resolution, high solvent crystals. It were molecular replacement structures, but due to the very strong solvent flattening effect, the phases looked like experimental ones. If your low-resolution structure genuinly has 80% solvent, I would be tempted to start building in that map. Best, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Guangyu Zhu Sent: Friday, March 15, 2013 1:28 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Resolution and data/parameter ratio, which one is more important? I have this question. For exmaple, a protein could be crystallized in two crystal forms. Two crystal form have same space group, and 1 molecule/asymm. One crystal form diffracts to 3A with 50% solvent; and the other diffracts to 3.6A with 80% solvent. The cell volume of 3.6A crystal must be 5/2=2.5 times larger because of higher solvent content. If both data collecte to same completeness (say 100%), 3.6A data actually have higher data/parameter ratio, 5/2/(3.6/3)**3= 1.45 times to 3A data. For refinement, better data/parameter should give more accurate structure, ie. 3.6A data is better. But higher resolution should give a better resolved electron density map. So which crystal form really give a better (more reliable and accurate) protein structure?
[ccp4bb] How to use SHELXE to perform phasing and density modification
Dear all, I have an original sca file with anomalous signal and a heavy atoms sites file in PDB format. PDB FILE : CRYST1 77.780 77.780 187.640 90.00 90.00 120.00 P 61 2 2 SCALE1 0.012857 0.007423 -0.00 -0.0 SCALE2 -0.00 0.014846 -0.000.0 SCALE3 0.00 -0.00 0.005329 -0.0 ATOM 1 S HAT 1 -62.495 123.694 12.804 0.36 20.00 S ATOM 9 S HAT 9 -49.984 90.531 2.130 0.32 20.00 S ATOM 10 S HAT10 -59.282 106.437 9.760 0.74 20.00 S ATOM 84 S HAT84 -60.153 114.024 15.399 0.52 20.00 S ... ... Can I use SHELXE to perform phasing and density modification? How to do it? Thank you for your help! Wei
Re: [ccp4bb] How to use SHELXE to perform phasing and density modification
Dear Wei, There is a new SHELX homepage with extensive documentation and downloading instructions at: http://shelx.uni-ac.gwdg.de/SHELX/ SHELXE requires reflection files name.hkl (native) and name_fa.hkl (data for phasing) and and the heavy atoms in SHELX format in name_fa.res. I recommend running SHELXC to prepare the files and SHELXD to find the heavy atoms, then everything will be in the right format. You can either run the programs from the command line or use a GUI such as hkl2map. If (as it seems) your heavy atoms come from an isomorphous structure, then you can run SHELXC to read XDS_ASCII.HKL or .sca files to make the .hkl, files followed by AnoDe (also part of SHELX) to read name_fa.hkl and name.ent (your original full PDB file, no just the heavy atoms) to make name_fa.res containing the heavy atom sites from the anomalous map. Then you have the files you need to run SHELXE, e.g. shelxe name name_fa -a5 -s0.5 -q but see the documentation for more information about the command line switches, e.g. -n for NCS. The advantage of this is that your final structure will be relative to the same origin as your original PDB file. Best wishes, George On 03/15/2013 10:13 AM, Wei Feng wrote: Dear all, I have an original sca file with anomalous signal and a heavy atoms sites file in PDB format. PDB FILE : CRYST1 77.780 77.780 187.640 90.00 90.00 120.00 P 61 2 2 SCALE1 0.012857 0.007423 -0.00 -0.0 SCALE2 -0.00 0.014846 -0.00 0.0 SCALE3 0.00 -0.00 0.005329 -0.0 ATOM 1 S HAT 1 -62.495 123.694 12.804 0.36 20.00 S ATOM 9 S HAT 9 -49.984 90.531 2.130 0.32 20.00 S ATOM 10 S HAT 10 -59.282 106.437 9.760 0.74 20.00 S ATOM 84 S HAT 84 -60.153 114.024 15.399 0.52 20.00 S ... ... Can I use SHELXE to perform phasing and density modification? How to do it? Thank you for your help! Wei -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582
Re: [ccp4bb] How to use SHELXE to perform phasing and density modification
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Wei, if you are not too familiar with shelx c/d/e, I suggest the following procedure, provided shelx c/d/e are installed: - - get hkl2map from http://webapps.embl-hamburg.de/hkl2map/ - - run hkl2map from a terminal and from the directory where your .sca file is - - provide a project name (I call it 'foo' for now) and run the first two steps, i.e. shelxc and shelxd - - close hkl2map now inside the directory there will be several files, the important ones being foo.hkl, foo_fa.hkl, and foo_fa.res - - use the program coordconv from CCP4 in order to get the fractional coordinates of your substructure and copy the coordinates into the foo_fa.res - - type 'shelxe foo foo_fa -a -h -q' and 'shelxe foo foo_fa -a -h -q - -i' and check if one of the resulting PDB files foo.pdb and foo_i.pdb (together with the corresponding map files foo.phs and foo_i.phs) make sense. Cheers, Tim On 03/15/2013 10:13 AM, Wei Feng wrote: Dear all, I have an original sca file with anomalous signal and a heavy atoms sites file in PDB format. PDB FILE : CRYST1 77.780 77.780 187.640 90.00 90.00 120.00 P 61 2 2 SCALE1 0.012857 0.007423 -0.00 -0.0 SCALE2 -0.00 0.014846 -0.000.0 SCALE3 0.00 -0.00 0.005329 -0.0 ATOM 1 S HAT 1 -62.495 123.694 12.804 0.36 20.00 S ATOM 9 S HAT 9 -49.984 90.531 2.130 0.32 20.00 S ATOM 10 S HAT10 -59.282 106.437 9.760 0.74 20.00 S ATOM 84 S HAT84 -60.153 114.024 15.399 0.52 20.00 S ... ... Can I use SHELXE to perform phasing and density modification? How to do it? Thank you for your help! Wei - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRQvEhUxlJ7aRr7hoRApLYAJ99A/i6FFsWJqlAOOx4zdwhKOTzHACgopdu OBNV+xUMee8l8fo2jwnac+U= =vAB5 -END PGP SIGNATURE-
Re: [ccp4bb] How to use SHELXE to perform phasing and density modification (correction)
Sorry, a better command line for running SHELXE in this case would have been: shelxe name name_fa -a5 -s0.5 -q -h -z this ensures that the heavy atoms are refined before calculating the initial phases, this often gives much better maps. If you are not sure whether the space group is P6122 or P6522, then you should run shelxe twice, once with and once without -i (which would invert the space group and atom coordinates). It is usually worth trying different solvent contents (-s). George On 03/15/2013 11:09 AM, George Sheldrick wrote: Dear Wei, There is a new SHELX homepage with extensive documentation and downloading instructions at: http://shelx.uni-ac.gwdg.de/SHELX/ SHELXE requires reflection files name.hkl (native) and name_fa.hkl (data for phasing) and and the heavy atoms in SHELX format in name_fa.res. I recommend running SHELXC to prepare the files and SHELXD to find the heavy atoms, then everything will be in the right format. You can either run the programs from the command line or use a GUI such as hkl2map. If (as it seems) your heavy atoms come from an isomorphous structure, then you can run SHELXC to read XDS_ASCII.HKL or .sca files to make the .hkl, files followed by AnoDe (also part of SHELX) to read name_fa.hkl and name.ent (your original full PDB file, no just the heavy atoms) to make name_fa.res containing the heavy atom sites from the anomalous map. Then you have the files you need to run SHELXE, e.g. shelxe name name_fa -a5 -s0.5 -q but see the documentation for more information about the command line switches, e.g. -n for NCS. The advantage of this is that your final structure will be relative to the same origin as your original PDB file. Best wishes, George On 03/15/2013 10:13 AM, Wei Feng wrote: Dear all, I have an original sca file with anomalous signal and a heavy atoms sites file in PDB format. PDB FILE : CRYST1 77.780 77.780 187.640 90.00 90.00 120.00 P 61 2 2 SCALE1 0.012857 0.007423 -0.00 -0.0 SCALE2 -0.00 0.014846 -0.00 0.0 SCALE3 0.00 -0.00 0.005329 -0.0 ATOM 1 S HAT 1 -62.495 123.694 12.804 0.36 20.00 S ATOM 9 S HAT 9 -49.984 90.531 2.130 0.32 20.00 S ATOM 10 S HAT 10 -59.282 106.437 9.760 0.74 20.00 S ATOM 84 S HAT 84 -60.153 114.024 15.399 0.52 20.00 S ... ... Can I use SHELXE to perform phasing and density modification? How to do it? Thank you for your help! Wei -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582
Re: [ccp4bb] Resolution and data/parameter ratio, which one is more important?
Hi Guangyu, I think it's not as straightforward as comparing d/p ratios, that is only one of several factors that influences precision. Another important factor would be the overall level of thermal motion disorder which will most likely be significantly higher in the 3.6A 80% case; after all that's probably the reason that it only diffracts to 3.6A! All things considered I would go for the 3A form. Cheers -- Ian On 15 March 2013 00:27, Guangyu Zhu g...@hwi.buffalo.edu wrote: I have this question. For exmaple, a protein could be crystallized in two crystal forms. Two crystal form have same space group, and 1 molecule/asymm. One crystal form diffracts to 3A with 50% solvent; and the other diffracts to 3.6A with 80% solvent. The cell volume of 3.6A crystal must be 5/2=2.5 times larger because of higher solvent content. If both data collecte to same completeness (say 100%), 3.6A data actually have higher data/parameter ratio, 5/2/(3.6/3)**3= 1.45 times to 3A data. For refinement, better data/parameter should give more accurate structure, ie. 3.6A data is better. But higher resolution should give a better resolved electron density map. So which crystal form really give a better (more reliable and accurate) protein structure?
Re: [ccp4bb] RNA 3D structure alignment
superpose and gesamt in ccp4 On 14 Mar 2013, at 20:53, Chen Zhao wrote: Dear all, I am now struggling to align two 3D RNA structures. I know there are a bunch of web servers, but they either just generated a pdb file with a single aligned structure, or they left the ligand out. Does any of you have some recommendations? Alternatively, is there some software that can calculate the transformation matrix between the coordinates in 2 pdb files? Then I could add the ligand back by myself. I suspect that Matlab is able to do this, but I would save it as the last resort. Thank you so much! Best, Chen -- Scanned by iCritical.
Re: [ccp4bb] Resolution and data/parameter ratio, which one is more important?
Ian, Because it is same protein, the high thermal motion is likely caused by crystal packing, and should be corrected by TLS refinement. The B left over should be similar. Anyway, this is just a hypothetical question. I tried to make other things same and just compare resolution and d/p. But you can still find difference. So if 80% crystal diffract to 3.0A too, then d/p ratio is much higher than 3.0A 50% crystal, it must be a more accurate refinement. What if 80% crystal diffract to 3.1, 3.2A, or 3.3A? Or I change the question to: could d/p ratio compensate some resolution? Thanks! Guangyu From: Ian Tickle ianj...@gmail.commailto:ianj...@gmail.com Date: Friday, March 15, 2013 6:33 AM To: System Administrator g...@hwi.buffalo.edumailto:g...@hwi.buffalo.edu Cc: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@jiscmail.ac.ukmailto:CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Resolution and data/parameter ratio, which one is more important? Hi Guangyu, I think it's not as straightforward as comparing d/p ratios, that is only one of several factors that influences precision. Another important factor would be the overall level of thermal motion disorder which will most likely be significantly higher in the 3.6A 80% case; after all that's probably the reason that it only diffracts to 3.6A! All things considered I would go for the 3A form. Cheers -- Ian On 15 March 2013 00:27, Guangyu Zhu g...@hwi.buffalo.edumailto:g...@hwi.buffalo.edu wrote: I have this question. For exmaple, a protein could be crystallized in two crystal forms. Two crystal form have same space group, and 1 molecule/asymm. One crystal form diffracts to 3A with 50% solvent; and the other diffracts to 3.6A with 80% solvent. The cell volume of 3.6A crystal must be 5/2=2.5 times larger because of higher solvent content. If both data collecte to same completeness (say 100%), 3.6A data actually have higher data/parameter ratio, 5/2/(3.6/3)**3= 1.45 times to 3A data. For refinement, better data/parameter should give more accurate structure, ie. 3.6A data is better. But higher resolution should give a better resolved electron density map. So which crystal form really give a better (more reliable and accurate) protein structure?
Re: [ccp4bb] Resolution and data/parameter ratio, which one is more important?
Well, wouldn't NCS be a parallel situation? I have heard, for example, that the maps of viruses are considerably better at a given resolution than monomeric proteins. So I would guess that someone has looked at this topic in the case of NCS. Maybe high solvent content would be equivalent to filling the solvent holes with equivalent proteins (assuming (unrealistically) that the crystal diffract to the same resolution, since the parameter ratio would be the same? JPK On Fri, Mar 15, 2013 at 9:27 AM, Guangyu Zhu g...@hwi.buffalo.edu wrote: Ian, Because it is same protein, the high thermal motion is likely caused by crystal packing, and should be corrected by TLS refinement. The B left over should be similar. Anyway, this is just a hypothetical question. I tried to make other things same and just compare resolution and d/p. But you can still find difference. So if 80% crystal diffract to 3.0A too, then d/p ratio is much higher than 3.0A 50% crystal, it must be a more accurate refinement. What if 80% crystal diffract to 3.1, 3.2A, or 3.3A? Or I change the question to: could d/p ratio compensate some resolution? Thanks! Guangyu From: Ian Tickle ianj...@gmail.com Date: Friday, March 15, 2013 6:33 AM To: System Administrator g...@hwi.buffalo.edu Cc: CCP4BB@JISCMAIL.AC.UK CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Resolution and data/parameter ratio, which one is more important? Hi Guangyu, I think it's not as straightforward as comparing d/p ratios, that is only one of several factors that influences precision. Another important factor would be the overall level of thermal motion disorder which will most likely be significantly higher in the 3.6A 80% case; after all that's probably the reason that it only diffracts to 3.6A! All things considered I would go for the 3A form. Cheers -- Ian On 15 March 2013 00:27, Guangyu Zhu g...@hwi.buffalo.edu wrote: I have this question. For exmaple, a protein could be crystallized in two crystal forms. Two crystal form have same space group, and 1 molecule/asymm. One crystal form diffracts to 3A with 50% solvent; and the other diffracts to 3.6A with 80% solvent. The cell volume of 3.6A crystal must be 5/2=2.5 times larger because of higher solvent content. If both data collecte to same completeness (say 100%), 3.6A data actually have higher data/parameter ratio, 5/2/(3.6/3)**3= 1.45 times to 3A data. For refinement, better data/parameter should give more accurate structure, ie. 3.6A data is better. But higher resolution should give a better resolved electron density map. So which crystal form really give a better (more reliable and accurate) protein structure? -- *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Farms Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org ***
Re: [ccp4bb] How to use SHELXE to perform phasing and density modification
Dear George, Thank you very much for your help! Wei At 2013-03-15 18:09:09,George Sheldrick gshe...@shelx.uni-ac.gwdg.de wrote: Dear Wei, There is a new SHELX homepage with extensive documentation and downloading instructions at: http://shelx.uni-ac.gwdg.de/SHELX/ SHELXE requires reflection files name.hkl (native) and name_fa.hkl (data for phasing) and and the heavy atoms in SHELX format in name_fa.res. I recommend running SHELXC to prepare the files and SHELXD to find the heavy atoms, then everything will be in the right format. You can either run the programs from the command line or use a GUI such as hkl2map. If (as it seems) your heavy atoms come from an isomorphous structure, then you can run SHELXC to read XDS_ASCII.HKL or .sca files to make the .hkl, files followed by AnoDe (also part of SHELX) to read name_fa.hkl and name.ent (your original full PDB file, no just the heavy atoms) to make name_fa.res containing the heavy atom sites from the anomalous map. Then you have the files you need to run SHELXE, e.g. shelxe name name_fa -a5 -s0.5 -q but see the documentation for more information about the command line switches, e.g. -n for NCS. The advantage of this is that your final structure will be relative to the same origin as your original PDB file. Best wishes, George On 03/15/2013 10:13 AM, Wei Feng wrote: Dear all, I have an original sca file with anomalous signal and a heavy atoms sites file in PDB format. PDB FILE : CRYST1 77.780 77.780 187.640 90.00 90.00 120.00 P 61 2 2 SCALE1 0.012857 0.007423 -0.00 -0.0 SCALE2 -0.00 0.014846 -0.000.0 SCALE3 0.00 -0.00 0.005329 -0.0 ATOM 1 S HAT 1 -62.495 123.694 12.804 0.36 20.00 S ATOM 9 S HAT 9 -49.984 90.531 2.130 0.32 20.00 S ATOM 10 S HAT10 -59.282 106.437 9.760 0.74 20.00 S ATOM 84 S HAT84 -60.153 114.024 15.399 0.52 20.00 S ... ... Can I use SHELXE to perform phasing and density modification? How to do it? Thank you for your help! Wei -- Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582
[ccp4bb]
*Postgraduate Studentship available in the Ciulli Laboratory* *Chemical Structural Biology of Protein-Protein Interactions* A fully-funded PhD studentship is available immediately in our new laboratory within the College of Life Sciences at the University of Dundee ( http://www.lifesci.dundee.ac.uk/people/alessio-ciulli) to investigate the Chemical Structural Biology of protein-protein interfaces (PPIs) that recognise post-translational modifications of protein amino acids. This multi-disciplinary project will comprise structure-function studies of PPIs and protein-ligand interactions using a combination of molecular/structural biology and biophysical techniques with small molecule drug design and organic synthesis. The goals of the project are two-fold: 1) to elucidate structure-function relationships within multidomain proteins containing paired chromatin reader domains that act as epigenetics effectors; 2) to exploit this information towards designing and developing novel small molecule chemical probes that modulate these PPIs. For a recent example of our research on PPIs see Van Molle et al. *Chemistry Biology* *2012*, 19, 1300-12 (doihttp://dx.doi.org/10.1016/j.chembiol.2012.08.015 ) Applicants should have (or expect to obtain) at least the equivalent of a UK II.1 honours degree (and preferably a Masters) in chemistry, biochemistry, chemical biology, structural biology or other relevant discipline. Applications from students with either a strong chemical or biological science background are encouraged, where the applicant is interested in learning the other discipline. To apply please complete the online application form at: http://www.lifesci.dundee.ac.uk/phdprog/division-biological-chemistry-and-drug-discovery/dr-alessio-ciulli Please do not hesitate to contact me by email for informal enquiries or if you want to discuss the project further. -- Alessio Ciulli, PhD Reader in Chemical and Structural Biology BBSRC David Phillips Fellow Division: Biological Chemistry and Drug Discovery College of Life Sciences, University of Dundee, Dundee Email: a.ciu...@dundee.ac.uk Email: ac...@cam.ac.uk http://www.lifesci.dundee.ac.uk/people/alessio-ciulli http://www-ciulli.ch.cam.ac.uk/ -- The University of Dundee is a registered Scottish Charity, No: SC015096
Re: [ccp4bb] Resolution and data/parameter ratio, which one is more important?
Guangyu, If I'm understanding your question correctly; you're asking if all other things are equal (resolution, degree of disorder, etc), does improving the data/parameter ratio result in an improved model? The short answer is: (at least sometimes) yes. Pete Guangyu Zhu wrote: Ian, Because it is same protein, the high thermal motion is likely caused by crystal packing, and should be corrected by TLS refinement. The B left over should be similar. Anyway, this is just a hypothetical question. I tried to make other things same and just compare resolution and d/p. But you can still find difference. So if 80% crystal diffract to 3.0A too, then d/p ratio is much higher than 3.0A 50% crystal, it must be a more accurate refinement. What if 80% crystal diffract to 3.1, 3.2A, or 3.3A? Or I change the question to: could d/p ratio compensate some resolution? Thanks! Guangyu From: Ian Tickle ianj...@gmail.commailto:ianj...@gmail.com Date: Friday, March 15, 2013 6:33 AM To: System Administrator g...@hwi.buffalo.edumailto:g...@hwi.buffalo.edu Cc: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK CCP4BB@jiscmail.ac.ukmailto:CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] Resolution and data/parameter ratio, which one is more important? Hi Guangyu, I think it's not as straightforward as comparing d/p ratios, that is only one of several factors that influences precision. Another important factor would be the overall level of thermal motion disorder which will most likely be significantly higher in the 3.6A 80% case; after all that's probably the reason that it only diffracts to 3.6A! All things considered I would go for the 3A form. Cheers -- Ian On 15 March 2013 00:27, Guangyu Zhu g...@hwi.buffalo.edumailto:g...@hwi.buffalo.edu wrote: I have this question. For exmaple, a protein could be crystallized in two crystal forms. Two crystal form have same space group, and 1 molecule/asymm. One crystal form diffracts to 3A with 50% solvent; and the other diffracts to 3.6A with 80% solvent. The cell volume of 3.6A crystal must be 5/2=2.5 times larger because of higher solvent content. If both data collecte to same completeness (say 100%), 3.6A data actually have higher data/parameter ratio, 5/2/(3.6/3)**3= 1.45 times to 3A data. For refinement, better data/parameter should give more accurate structure, ie. 3.6A data is better. But higher resolution should give a better resolved electron density map. So which crystal form really give a better (more reliable and accurate) protein structure?
Re: [ccp4bb] space group determination problem
what happened to all the even l h reflections? Phil On 15 Mar 2013, at 15:09, gengxiang zhao gzh...@gmail.com wrote: Dear CCP4s, I am looking for more experienced concerns to determine which space group my crystal is. At present, we take it as P42212 (#94). HKL is below: Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 9 -58.6 40.8 -1.4 0 0 11-204.4 53.9 -3.8 0 0 13 -57.1 62.8 -0.9 0 0 15-470.6 92.7 -5.1 0 0 17-626.1 105.1 -6.0 0 0 19 -64.7 62.4 -1.0 0 0 21 266.6 75.9 3.5 0 0 231372.4 116.4 11.8 0 0 25-543.9 84.8 -6.4 0 0 27-396.8 93.1 -4.3 0 0 29-598.8 102.1 -5.9 0 0 31 617.4 116.2 5.3 0 0 33 445.4 93.8 4.7 0 0 35 -64.5 89.5 -0.7 7 0 0-241.4 134.7 -1.8 9 0 0-375.8 55.5 -6.8 11 0 0 -39.1 61.8 -0.6 13 0 0-356.1 78.1 -4.6 15 0 0-262.6 65.6 -4.0 17 0 0-324.7 89.3 -3.6 19 0 0-178.7 88.5 -2.0 21 0 0-726.3 115.3 -6.3 23 0 0-189.4 131.0 -1.4 25 0 0 157.7 157.5 1.0 27 0 0-591.5 213.4 -2.8 29 0 0-111.7 198.4 -0.6 31 0 0 -94.2 247.0 -0.4 33 0 0-169.8 306.5 -0.6 35 0 0 -71.2 347.8 -0.2 39 0 0 -82.8 417.9 -0.2 Thanks a lot. Best Wishes, Gengxiang
Re: [ccp4bb] space group determination problem
Hi Gengxiang, Personally I find it impossible to reliably assign a space group from integrated reflections because you just don't know if the apparent systematic absence violations are due to a TDS streak or overlapping neighbouring strong spots. In the old days (i.e. when we had precession cameras) we would never do this: we would look at the images and see if there was actually a spot at the Bragg position. Now technology has advanced and with rotation images it's much harder to do this. Maybe it's possible to make pseudo-precession images? What I would do is assume the worst and assign it temporarily as P422; then let the HA or MR program sort out the space group by trying all the possibilities; it's only CPU time after all! My 2p's worth. -- Ian On 15 March 2013 15:09, gengxiang zhao gzh...@gmail.com wrote: Dear CCP4s, I am looking for more experienced concerns to determine which space group my crystal is. At present, we take it as P42212 (#94). HKL is below: Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 9 -58.6 40.8 -1.4 0 0 11-204.4 53.9 -3.8 0 0 13 -57.1 62.8 -0.9 0 0 15-470.6 92.7 -5.1 0 0 17-626.1 105.1 -6.0 0 0 19 -64.7 62.4 -1.0 0 0 21 266.6 75.9 3.5 0 0 231372.4 116.4 11.8 0 0 25-543.9 84.8 -6.4 0 0 27-396.8 93.1 -4.3 0 0 29-598.8 102.1 -5.9 0 0 31 617.4 116.2 5.3 0 0 33 445.4 93.8 4.7 0 0 35 -64.5 89.5 -0.7 7 0 0-241.4 134.7 -1.8 9 0 0-375.8 55.5 -6.8 11 0 0 -39.1 61.8 -0.6 13 0 0-356.1 78.1 -4.6 15 0 0-262.6 65.6 -4.0 17 0 0-324.7 89.3 -3.6 19 0 0-178.7 88.5 -2.0 21 0 0-726.3 115.3 -6.3 23 0 0-189.4 131.0 -1.4 25 0 0 157.7 157.5 1.0 27 0 0-591.5 213.4 -2.8 29 0 0-111.7 198.4 -0.6 31 0 0 -94.2 247.0 -0.4 33 0 0-169.8 306.5 -0.6 35 0 0 -71.2 347.8 -0.2 39 0 0 -82.8 417.9 -0.2 Thanks a lot. Best Wishes, Gengxiang
Re: [ccp4bb] space group determination problem
Well - you seem to have absences for virtually all the (0 0 4n+1 4n+3) and (2n+1 0 0) reflections which is consistent with P42 212 but you can be misled. Is there any pseudo translation with either X or Z 0.5 - that could give you similar absences. As Ian says, be wary.. Eleanor On 15 March 2013 15:51, Ian Tickle ianj...@gmail.com wrote: Hi Gengxiang, Personally I find it impossible to reliably assign a space group from integrated reflections because you just don't know if the apparent systematic absence violations are due to a TDS streak or overlapping neighbouring strong spots. In the old days (i.e. when we had precession cameras) we would never do this: we would look at the images and see if there was actually a spot at the Bragg position. Now technology has advanced and with rotation images it's much harder to do this. Maybe it's possible to make pseudo-precession images? What I would do is assume the worst and assign it temporarily as P422; then let the HA or MR program sort out the space group by trying all the possibilities; it's only CPU time after all! My 2p's worth. -- Ian On 15 March 2013 15:09, gengxiang zhao gzh...@gmail.com wrote: Dear CCP4s, I am looking for more experienced concerns to determine which space group my crystal is. At present, we take it as P42212 (#94). HKL is below: Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 9 -58.6 40.8 -1.4 0 0 11-204.4 53.9 -3.8 0 0 13 -57.1 62.8 -0.9 0 0 15-470.6 92.7 -5.1 0 0 17-626.1 105.1 -6.0 0 0 19 -64.7 62.4 -1.0 0 0 21 266.6 75.9 3.5 0 0 231372.4 116.4 11.8 0 0 25-543.9 84.8 -6.4 0 0 27-396.8 93.1 -4.3 0 0 29-598.8 102.1 -5.9 0 0 31 617.4 116.2 5.3 0 0 33 445.4 93.8 4.7 0 0 35 -64.5 89.5 -0.7 7 0 0-241.4 134.7 -1.8 9 0 0-375.8 55.5 -6.8 11 0 0 -39.1 61.8 -0.6 13 0 0-356.1 78.1 -4.6 15 0 0-262.6 65.6 -4.0 17 0 0-324.7 89.3 -3.6 19 0 0-178.7 88.5 -2.0 21 0 0-726.3 115.3 -6.3 23 0 0-189.4 131.0 -1.4 25 0 0 157.7 157.5 1.0 27 0 0-591.5 213.4 -2.8 29 0 0-111.7 198.4 -0.6 31 0 0 -94.2 247.0 -0.4 33 0 0-169.8 306.5 -0.6 35 0 0 -71.2 347.8 -0.2 39 0 0 -82.8 417.9 -0.2 Thanks a lot. Best Wishes, Gengxiang
[ccp4bb] Please recommend linux workstation
Dear All I am planning to buy a linux workstation for crystallography. It seems that Dell does not offer workstations with linux right now. Any good experience to recommend? Thank you! Jie Liu
Re: [ccp4bb] Please recommend linux workstation
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Jie Liu, most Linux distributions are not too difficult to install, and with a decent internet connection you only need to download a few MB for a bootable CD for network installation - do you have a reason why you do not want this yourself? Kind regards, Tim On 03/15/2013 05:34 PM, jie liu wrote: Dear All I am planning to buy a linux workstation for crystallography. It seems that Dell does not offer workstations with linux right now. Any good experience to recommend? Thank you! Jie Liu - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRQ09xUxlJ7aRr7hoRAvJ+AJ9OM3AiV7RixUFaNqPCMbKhkKzqlwCgomP/ 7o166epVERXwzoBe0/xp2s0= =SbA8 -END PGP SIGNATURE-
Re: [ccp4bb] Please recommend linux workstation
Dell Precision workstations are sold with RHEL, Ubuntu, or no OS (FreeDOS), but you can't see those options from the public websites. You need to try to get a Dell Premier account, talk to the Dell Rep for your university about that, here's a screen shot from our uni's premier site : http://i.imgur.com/NlekuSq.png We usually end up purchasing one with a Windows license though and just install CentOS over that. On Fri, Mar 15, 2013 at 11:34 AM, jie liu li...@umdnj.edu wrote: Dear All I am planning to buy a linux workstation for crystallography. It seems that Dell does not offer workstations with linux right now. Any good experience to recommend? Thank you! Jie Liu
Re: [ccp4bb] Please recommend linux workstation
I've been pretty happy with the machines from System76https://www.system76.com/. They come with the most recent version of Ubuntu pre-installed. However, as Tim says - installing Linux on any machine you can buy is a pretty simple process now. Cheers, Jim On Fri, Mar 15, 2013 at 9:42 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Jie Liu, most Linux distributions are not too difficult to install, and with a decent internet connection you only need to download a few MB for a bootable CD for network installation - do you have a reason why you do not want this yourself? Kind regards, Tim On 03/15/2013 05:34 PM, jie liu wrote: Dear All I am planning to buy a linux workstation for crystallography. It seems that Dell does not offer workstations with linux right now. Any good experience to recommend? Thank you! Jie Liu - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRQ09xUxlJ7aRr7hoRAvJ+AJ9OM3AiV7RixUFaNqPCMbKhkKzqlwCgomP/ 7o166epVERXwzoBe0/xp2s0= =SbA8 -END PGP SIGNATURE- -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructures http://www.emeraldbiostructures.com/ Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.com jfair...@embios.com
Re: [ccp4bb] space group determination problem
As a follow up to Ian's suggestion, the LABELIT software (sorry for a non-CCP4 suggestion) will create artificial precession images from your raw oscillation images. Documentation can be found here: http://adder.lbl.gov/labelit/ And an article describing the functionality can be found here: http://cci.lbl.gov/publications/download/CCN_2011_p15.pdf Hope it helps, Mike - Original Message - From: Ian Tickle ianj...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, March 15, 2013 8:51:47 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] space group determination problem Hi Gengxiang, Personally I find it impossible to reliably assign a space group from integrated reflections because you just don't know if the apparent systematic absence violations are due to a TDS streak or overlapping neighbouring strong spots. In the old days (i.e. when we had precession cameras) we would never do this: we would look at the images and see if there was actually a spot at the Bragg position. Now technology has advanced and with rotation images it's much harder to do this. Maybe it's possible to make pseudo-precession images? What I would do is assume the worst and assign it temporarily as P422; then let the HA or MR program sort out the space group by trying all the possibilities; it's only CPU time after all! My 2p's worth. -- Ian On 15 March 2013 15:09, gengxiang zhao gzh...@gmail.com wrote: Dear CCP4s, I am looking for more experienced concerns to determine which space group my crystal is. At present, we take it as P42212 (#94). HKL is below: Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 9 -58.6 40.8 -1.4 0 0 11 -204.4 53.9 -3.8 0 0 13 -57.1 62.8 -0.9 0 0 15 -470.6 92.7 -5.1 0 0 17 -626.1 105.1 -6.0 0 0 19 -64.7 62.4 -1.0 0 0 21 266.6 75.9 3.5 0 0 23 1372.4 116.4 11.8 0 0 25 -543.9 84.8 -6.4 0 0 27 -396.8 93.1 -4.3 0 0 29 -598.8 102.1 -5.9 0 0 31 617.4 116.2 5.3 0 0 33 445.4 93.8 4.7 0 0 35 -64.5 89.5 -0.7 7 0 0 -241.4 134.7 -1.8 9 0 0 -375.8 55.5 -6.8 11 0 0 -39.1 61.8 -0.6 13 0 0 -356.1 78.1 -4.6 15 0 0 -262.6 65.6 -4.0 17 0 0 -324.7 89.3 -3.6 19 0 0 -178.7 88.5 -2.0 21 0 0 -726.3 115.3 -6.3 23 0 0 -189.4 131.0 -1.4 25 0 0 157.7 157.5 1.0 27 0 0 -591.5 213.4 -2.8 29 0 0 -111.7 198.4 -0.6 31 0 0 -94.2 247.0 -0.4 33 0 0 -169.8 306.5 -0.6 35 0 0 -71.2 347.8 -0.2 39 0 0 -82.8 417.9 -0.2 Thanks a lot. Best Wishes, Gengxiang -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] Please recommend linux workstation
Some of those system76 desktops look overclocked. I've always wanted to try one to speed up processing but have been warned away from it for fear of errors and stability. Does anyone have any experience of overclocked desktops for PX software? Alan On 15/03/2013 17:55, Jim Fairman wrote: I've been pretty happy with the machines from System76 https://www.system76.com/. They come with the most recent version of Ubuntu pre-installed. However, as Tim says - installing Linux on any machine you can buy is a pretty simple process now. Cheers, Jim On Fri, Mar 15, 2013 at 9:42 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de mailto:t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Jie Liu, most Linux distributions are not too difficult to install, and with a decent internet connection you only need to download a few MB for a bootable CD for network installation - do you have a reason why you do not want this yourself? Kind regards, Tim On 03/15/2013 05:34 PM, jie liu wrote: Dear All I am planning to buy a linux workstation for crystallography. It seems that Dell does not offer workstations with linux right now. Any good experience to recommend? Thank you! Jie Liu - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRQ09xUxlJ7aRr7hoRAvJ+AJ9OM3AiV7RixUFaNqPCMbKhkKzqlwCgomP/ 7o166epVERXwzoBe0/xp2s0= =SbA8 -END PGP SIGNATURE- -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructures http://www.emeraldbiostructures.com/ Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.com mailto:fairman@gmail.com jfair...@embios.com mailto:jfair...@embios.com -- Alan Cheung Gene Center Ludwig-Maximilians-University Feodor-Lynen-Str. 25 81377 Munich Germany Phone: +49-89-2180-76845 Fax: +49-89-2180-76999 E-mail: che...@lmb.uni-muenchen.de
Re: [ccp4bb] The number of protein-protein complex in recent years
On the subject: Are there any protein-protein complexes commercially available (for cheap)? The only one I could think of was hemoglobin. Morten On 14 March 2013 01:56, Wei Feng ccp4...@hotmail.com wrote: Dear Lucas, Thank you for your help! Wei At 2013-03-13 23:05:54,Lucas lucasbleic...@gmail.com wrote: That probably won't be very precise. A better option would be doing an advanced search, then select Structure Features - Number of Entities as a protein query, select protein as entity type, and then a very wide interval starting with 2 (say, between 2 and 100). That returns 14453 hits if I click result count. Then you can add search criteria to refine your search - in your case, Deposition-Deposit Date and then select the desired timespan will return what you're looking for. Lucas 2013/3/13 Wei Feng ccp4...@hotmail.com: Dear all, I want to do a statistics about the number of protein-protein complexes deposited in PDB in recent years.(1972~1992,1993...2012) I tried keywords protein-protein complex, protein complex etc. in the search of PDB but all of them are fail. Can everyone tell me how to do? Thank you very much! Wei -- Morten K Grøftehauge, PhD Pohl Group Durham University
Re: [ccp4bb] space group determination problem
Michael, yes sorry I had (temporarily) forgotten about LABELIT. In the pseudo-precession image in the article (Fig. 3a) one can clearly see the TDS streaks along the axes which could easily fool you into misassigning the space group if all you have are the integrated intensities. Very nice! Cheers -- Ian On 15 March 2013 17:10, Michael Thompson mi...@chem.ucla.edu wrote: As a follow up to Ian's suggestion, the LABELIT software (sorry for a non-CCP4 suggestion) will create artificial precession images from your raw oscillation images. Documentation can be found here: http://adder.lbl.gov/labelit/ And an article describing the functionality can be found here: http://cci.lbl.gov/publications/download/CCN_2011_p15.pdf Hope it helps, Mike - Original Message - From: Ian Tickle ianj...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, March 15, 2013 8:51:47 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] space group determination problem Hi Gengxiang, Personally I find it impossible to reliably assign a space group from integrated reflections because you just don't know if the apparent systematic absence violations are due to a TDS streak or overlapping neighbouring strong spots. In the old days (i.e. when we had precession cameras) we would never do this: we would look at the images and see if there was actually a spot at the Bragg position. Now technology has advanced and with rotation images it's much harder to do this. Maybe it's possible to make pseudo-precession images? What I would do is assume the worst and assign it temporarily as P422; then let the HA or MR program sort out the space group by trying all the possibilities; it's only CPU time after all! My 2p's worth. -- Ian On 15 March 2013 15:09, gengxiang zhao gzh...@gmail.com wrote: Dear CCP4s, I am looking for more experienced concerns to determine which space group my crystal is. At present, we take it as P42212 (#94). HKL is below: Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 9 -58.6 40.8 -1.4 0 0 11 -204.4 53.9 -3.8 0 0 13 -57.1 62.8 -0.9 0 0 15 -470.6 92.7 -5.1 0 0 17 -626.1 105.1 -6.0 0 0 19 -64.7 62.4 -1.0 0 0 21 266.6 75.9 3.5 0 0 23 1372.4 116.4 11.8 0 0 25 -543.9 84.8 -6.4 0 0 27 -396.8 93.1 -4.3 0 0 29 -598.8 102.1 -5.9 0 0 31 617.4 116.2 5.3 0 0 33 445.4 93.8 4.7 0 0 35 -64.5 89.5 -0.7 7 0 0 -241.4 134.7 -1.8 9 0 0 -375.8 55.5 -6.8 11 0 0 -39.1 61.8 -0.6 13 0 0 -356.1 78.1 -4.6 15 0 0 -262.6 65.6 -4.0 17 0 0 -324.7 89.3 -3.6 19 0 0 -178.7 88.5 -2.0 21 0 0 -726.3 115.3 -6.3 23 0 0 -189.4 131.0 -1.4 25 0 0 157.7 157.5 1.0 27 0 0 -591.5 213.4 -2.8 29 0 0 -111.7 198.4 -0.6 31 0 0 -94.2 247.0 -0.4 33 0 0 -169.8 306.5 -0.6 35 0 0 -71.2 347.8 -0.2 39 0 0 -82.8 417.9 -0.2 Thanks a lot. Best Wishes, Gengxiang -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
[ccp4bb] anti-His6 Ab
Dear All, would you be able to recommend a primary anti-His6 Ab for western blotting? Thus far, the ones we've used are not very specific to the N-terminal hexahistidines and we pick up lots of background. Also, there's one (others?) from Sigma that is conjugated to HRP, but costs ~$650. Thanks! Elias
Re: [ccp4bb] anti-His6 Ab
Hi Elias, Not sure if you've looked at this option, but Pierce sells a polyHis tag detection kit that is not based on an antibody, but instead is a nickel-activated derivative of HRP. Has worked quite well for us in the past. Brian Mark On 2013-03-15, at 12:36 PM, Elias Fernandez efern...@utk.edumailto:efern...@utk.edu wrote: Dear All, would you be able to recommend a primary anti-His6 Ab for western blotting? Thus far, the ones we’ve used are not very specific to the N-terminal hexahistidines and we pick up lots of background. Also, there’s one (others?) from Sigma that is conjugated to HRP, but costs ~$650. Thanks! Elias
Re: [ccp4bb] anti-His6 Ab
We haven't found a great primary (or secondary) antibody, but we have used the NTA Atto 488 dye (Sigma 39625) with some success at primary detection of His-tagged proteins directly in our gels, without the need for a transfer step to a membrane. We run BSA alongside our samples to determine the amount of non-specific binding and it is usually very low. Our collaborators use the InVision kit from Invitrogen (similar dye) after a crude NTA purification, and it only picks up the protein of interest. It might be worth a try depending on your needs, but I'm not sure of the detection limits of this dye. Lauren Boucher Bosch Lab|Johns Hopkins School of Public Health 615 N Wolfe St, W8710|Baltimore, MD 21205 lbouc...@jhu.edu
[ccp4bb] Strange density in solvent channel and high Rfree
*Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure.* *A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel???* *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* * * *Do someone have an explanation or solution for this?* * * *Cheers,* *Andrey*
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Check for translational NCS And you are way too conservative with resolution. Even those holding onto the Rmerge-dictated past would probably acquiesce to lower I/sig cutoff. If you are using aimless, follow its recommendations based on CC1/2, it's good for you. Cheers, Ed. On Fri, 2013-03-15 at 15:39 -0300, Andrey Nascimento wrote: Dear all, I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure. A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel??? I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf Do someone have an explanation or solution for this? Cheers, Andrey -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Wow, Usually on the default settings of adxv, I can see spots at this I/sig! I suspect your data goes higher than 1.77. Include more of the high resolution data. You very likely don't have the correct space group. F On Mar 15, 2013, at 11:39 AM, Andrey Nascimento andreynascime...@gmail.com wrote: I/Isig.=4.4 - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Strange density in solvent channel and high Rfree
What happens if you solvent-flatten/flip/massage that map, but tell the software the solvent content much lower than what you think it is now? Maybe you'll find another copy of the molecule? From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Andrey Nascimento [andreynascime...@gmail.com] Sent: Friday, March 15, 2013 1:39 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Strange density in solvent channel and high Rfree Dear all, I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure. A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel??? I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf Do someone have an explanation or solution for this? Cheers, Andrey
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Hi Andrey, I am taking a risky guess: From your slide #2, it looks like a termini (unless this correspond to the beginning or end of disordered a loop) of the protein chain(s) is (are) extending toward(s) the solvent channel. Are the density features shown in slides #3,4, and 5 extend from this terminal residue?!! If this is true, did you missed to model any uncleaved-tag residues?!! I would also look at 2Fo-Fc at 1.0 sigma. Hope this helps, Partha On Fri, Mar 15, 2013 at 2:39 PM, Andrey Nascimento andreynascime...@gmail.com wrote: *Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure.* *A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel???* *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* * * *Do someone have an explanation or solution for this?* * * *Cheers,* *Andrey*
Re: [ccp4bb] Strange density in solvent channel and high Rfree
Or give it to arp/warp, either with the current model to improve or with phases from the current model to build new model? Phoebe A. Rice wrote: What happens if you solvent-flatten/flip/massage that map, but tell the software the solvent content much lower than what you think it is now? Maybe you'll find another copy of the molecule? *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Andrey Nascimento [andreynascime...@gmail.com] *Sent:* Friday, March 15, 2013 1:39 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Strange density in solvent channel and high Rfree *Dear all,* *I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure.* *A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel???* *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf* * * *Do someone have an explanation or solution for this?* ** *Cheers,* *Andrey*
Re: [ccp4bb] Strange density in solvent channel and high Rfree
I second Phoebe's suggestion. Looks like another molecule to me. If you are doing molecular replacement you may need to get creative about trimming. When we had a case like that it wasn't until the third person worked on it that we go a solution because he trimmed the model differently than the first two who tried. Kendall On Mar 15, 2013, at 3:29 PM, Phoebe A. Rice pr...@uchicago.edumailto:pr...@uchicago.edu wrote: What happens if you solvent-flatten/flip/massage that map, but tell the software the solvent content much lower than what you think it is now? Maybe you'll find another copy of the molecule? From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] on behalf of Andrey Nascimento [andreynascime...@gmail.commailto:andreynascime...@gmail.com] Sent: Friday, March 15, 2013 1:39 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Strange density in solvent channel and high Rfree Dear all, I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure. A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel??? I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf Do someone have an explanation or solution for this? Cheers, Andrey
Re: [ccp4bb] CCP4 Update 019
Dear CCP4 Users A CCP4 update has just been released, consisting of the following changes. * Aimless: Task interface fixed in part of input customisation section * Molrep: Improvements to scoring system and bug fixes * Logview: Command-prompt routine for viewing individual log files with QtRView Simply call it with path to the log file as the only parameter * QtRView: Cosmetic improvements and changes for Logview * Gesamt: Now prints superposition matrices also in fractional coordinates * Superpose: Now prints superposition matrices also in fractional coordinates If you do not currently receive updates, consider re-installing your CCP4 setup using the latest binary packages, which now have the CCP4 Update Manager (ccp4um) integrated. Note that auto-updates will work correctly only with CCP4 release 6.3.0, therefore upgrade if necessary. Please report any bugs to c...@stfc.ac.ukmailto:c...@stfc.ac.uk Many thanks for using CCP4 Andrey Lebedev -- Scanned by iCritical.
