[ccp4bb] AW: [ccp4bb] Rfree problem
Dear Nazia, you give very little details, so it is difficult to give a specific advice. Here are some comments: -How high is your Rfree? Depending on the resolution of your data, an Rfree in the 25-30% range while not good, may still be acceptable. -Are there problems like ice-rings in your data? -Did you check the complete protein chain with a graphics program (coot) for residues/loops which may need rebuilding? -Did you try other refinement programs? (Buster, Refmac, Phenix). -Try different weights (in Refmac: weight MATRIX keyword). I recently had a case where only with a very specific weight the R and Rfree would go down. Just run the same refinement job several times with different weights. Good luck! Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Nazia Nasir Phd2009,ProteinCrystall.Lab Gesendet: Dienstag, 10. Dezember 2013 07:52 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Rfree problem Hi, I am facing a problem in refining one of my structures. With every refinement the Rwork is going down. However, the Rfree is stuck and is not going down with refinement. I have already solved the structure without the co-factor bound. This structure is with the co-factor bound. Can anyone suggest the reason for the Rfree getting stuck? Thanks -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi
Re: [ccp4bb] scala discrepancy between versions
Dear Jens, did you try aimless instead of scala? I understood it is the successor of scala and I would not have thought scala is still being developed. Phil should correct me if I am wrong. Best, Tim P.S.: I hope it will be a long time before the last release of ccp4. ;-) On 12/10/2013 05:05 AM, Jens Kaiser wrote: Dear developers, We have been doing the local scaling with scala of multiple wavelengths for some time. We scaled one dataset collected remotely from the absorption edge, and input this to scala as a reference as one batch without any problems. With the latest release of CCP4, though, we get the following error (reference dataset is assigned to batch 9): Missing phi limits for batch 9 No valid orientation data for batch 9: this is not allowed with TAILS, SECONDARY, ABSORPTION or BEAMS options Scala: Failed in SETSCL Times: User: 0.0s System:0.0s Elapsed: 0:00 Our current workaround is to use the last, not the latest, release of CCP4. Any help is appreciated, Jens -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] scala discrepancy between versions
Dear Jens I'm surprised by this as Scala hasn't changed for quite a long time, as it is now now superseded by Aimless. However, I haven't implemented a reference dataset in Aimless since in the end I decided that it wasn't very useful, so if you find it useful then you will indeed need to use Scala. I will investigate, but in the meantime you could just copy an old version of Scala into the updated ccp4 directory (i.e. into $CBIN == CCP4/bin/) Phil On 10 Dec 2013, at 04:05, Jens Kaiser kai...@caltech.edu wrote: Dear developers, We have been doing the local scaling with scala of multiple wavelengths for some time. We scaled one dataset collected remotely from the absorption edge, and input this to scala as a reference as one batch without any problems. With the latest release of CCP4, though, we get the following error (reference dataset is assigned to batch 9): Missing phi limits for batch 9 No valid orientation data for batch 9: this is not allowed with TAILS, SECONDARY, ABSORPTION or BEAMS options Scala: Failed in SETSCL Times: User: 0.0s System:0.0s Elapsed: 0:00 Our current workaround is to use the last, not the latest, release of CCP4. Any help is appreciated, Jens
Re: [ccp4bb] small crystals
Dear Careina, you can also apply for beamtime at PETRA-III. You get away with the same size crystals but only require a nano liter drop rather than a few ml of your sample. And you probably get beamtime much quicker because all the equipment is installed and collecting a data set takes very short time. This was demonstrated at the ECM in Warwick this year, so no need for FEL (at least for structure determination). Best, Tim On 12/10/2013 04:36 AM, Jens Kaiser wrote: Careina, If your target is interesting enough, try to reproduce the small crystals in batch and apply for FELS time. Small crystals are actually an advantage there. Cheers, Jens On Wed, 2013-12-04 at 21:41 -0800, Careina Edgooms wrote: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] small crystals
Yes, Tim is right… for now… in few years, with the E-XFEL, we'll get to much less sample, and much more time available. But that's in few years. Leo On Dec 10, 2013, at 10:01 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Careina, you can also apply for beamtime at PETRA-III. You get away with the same size crystals but only require a nano liter drop rather than a few ml of your sample. And you probably get beamtime much quicker because all the equipment is installed and collecting a data set takes very short time. This was demonstrated at the ECM in Warwick this year, so no need for FEL (at least for structure determination). Best, Tim On 12/10/2013 04:36 AM, Jens Kaiser wrote: Careina, If your target is interesting enough, try to reproduce the small crystals in batch and apply for FELS time. Small crystals are actually an advantage there. Cheers, Jens On Wed, 2013-12-04 at 21:41 -0800, Careina Edgooms wrote: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] Rfree problem
Dear Nazia Nasir, it is important to know what you do BETWEEN the refinement cycles. Rfree not moving and R1 dropping is often a sign of overfitting, i.e. you might be adding water molecules into the noise or refine the occupancy of single atoms, e.g. in a ligand or of the water molecules. As a general rule, carry out as much model building as possible and run as few refinement runs (not cycles within a run!!!) as possible. Best, Tim On 12/10/2013 07:51 AM, Nazia Nasir Phd2009,ProteinCrystall.Lab wrote: Hi, I am facing a problem in refining one of my structures. With every refinement the Rwork is going down. However, the Rfree is stuck and is not going down with refinement. I have already solved the structure without the co-factor bound. This structure is with the co-factor bound. Can anyone suggest the reason for the Rfree getting stuck? Thanks -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] small crystals
'Will' is a fashionable word - but since even a Nobel prize was given based on promises never kept, who would be surprised... Best wishes from now, Tim On 12/10/2013 10:11 AM, CHAVAS Leonard wrote: Yes, Tim is right… for now… in few years, with the E-XFEL, we'll get to much less sample, and much more time available. But that's in few years. Leo On Dec 10, 2013, at 10:01 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Careina, you can also apply for beamtime at PETRA-III. You get away with the same size crystals but only require a nano liter drop rather than a few ml of your sample. And you probably get beamtime much quicker because all the equipment is installed and collecting a data set takes very short time. This was demonstrated at the ECM in Warwick this year, so no need for FEL (at least for structure determination). Best, Tim On 12/10/2013 04:36 AM, Jens Kaiser wrote: Careina, If your target is interesting enough, try to reproduce the small crystals in batch and apply for FELS time. Small crystals are actually an advantage there. Cheers, Jens On Wed, 2013-12-04 at 21:41 -0800, Careina Edgooms wrote: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] small crystals
Indeed nothing is predictable. We can only assume things on what is *planed* and *promised*, and you can take my words on the fact that I will do my best to have it done properly and as close to the expectations as possible. But again, nobody can really predict the future. Finger crossed. Cheers, Leo On Dec 10, 2013, at 10:13 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: 'Will' is a fashionable word - but since even a Nobel prize was given based on promises never kept, who would be surprised... Best wishes from now, Tim On 12/10/2013 10:11 AM, CHAVAS Leonard wrote: Yes, Tim is right… for now… in few years, with the E-XFEL, we'll get to much less sample, and much more time available. But that's in few years. Leo On Dec 10, 2013, at 10:01 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Careina, you can also apply for beamtime at PETRA-III. You get away with the same size crystals but only require a nano liter drop rather than a few ml of your sample. And you probably get beamtime much quicker because all the equipment is installed and collecting a data set takes very short time. This was demonstrated at the ECM in Warwick this year, so no need for FEL (at least for structure determination). Best, Tim On 12/10/2013 04:36 AM, Jens Kaiser wrote: Careina, If your target is interesting enough, try to reproduce the small crystals in batch and apply for FELS time. Small crystals are actually an advantage there. Cheers, Jens On Wed, 2013-12-04 at 21:41 -0800, Careina Edgooms wrote: Hi all Any advice on how to get bigger crystals from conditions that give showers of tiny crystals? I am getting small pretty looking individual crystals but they are too small and they don't seem to grow. In fact, in some instances if left for a couple of days they actually dissolve. I have fiddled around with mother liquor volume, protein concentration as well as drop volume (I am using hanging drop method) but none seem to make any difference and I always get the same tiny crystals. I think I might try microseeding but I haven't tried that yet. Any suggestions or tricks would be welcome Careina. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
[ccp4bb] PhD position in membrane protein structural biology
Dear colleagues, A PhD position is available in (membrane) protein biochemistry and X-ray crystallography in the newly established research group of Dr. Henning Tidow at the Institute for Biochemistry and Molecular Biology, University of Hamburg, Germany. Our group studies the structure-function relationship of calcium pumps and channels from various organisms and their regulation. The PhD project will involve expression, purification and structural characterization by X-ray crystallography of membrane proteins involved in calcium transport as well as functional studies to elucidate their regulation. The ideal candidate is a highly motivated student with a strong interest in structural biology. Previous experience in molecular biology, protein biochemistry and/or crystallization would be an advantage. Good English language skills are essential; knowledge of German is not required. The PhD position is funded by the Emmy Noether Program of the German Research Foundation (DFG) and initially available for three years. Starting date would be March 2014 or later. Salary will be according to the collective agreement (German TVL scale). In case of equivalent qualifications, disabled applicants will be preferentially considered. Applications and informal inquiries about the position should be sent to Henning Tidow by email (ti...@chemie.uni-hamburg.de). Applications should include a CV, a letter of motivation detailing research interests and expertise, and contact details for at least two referees. Closing date is 10 January 2014 but applications will be considered after that period until a suitable candidate is found. --- Henning Tidow Department of Chemistry Institute of Biochemistry and Molecular Biology University of Hamburg Martin-Luther-King-Platz 6 20146 Hamburg Germany
Re: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain
Well the density strongly suggests a Tryptophan side chain. Sometimes discrepancies (errors) appear in sequences. How certain are you of the accuracy of your sequence? HTH, Fred. On 10/12/13 13:44, PriyankMaindola wrote: dear members i am trying to solve this crystal structure but I am puzzled with an undefined blob that appeared at a glutamine residue after refinement. I have attached pics of that below. Is it a covalent modification of acid-amide side chain.. ... as there is no charged environment around and density seems continuous. please suggest following reagents were encountered by protein during purification, crystallization and soaking : phenyl methyl sulfonyl fluoride benzamidine tris dtt (could it be cyclized dtt?) k[au(cn)2] acidic pH isopropanol citrate sulfate phosfate K+, Na+, Cl- map contour: 2fo-fc: 1rmsd fo-fc (green): 3 rmsd -- *Priyank* -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS2 / ELMA Campus EPN 6 rue Jules Horowitz F-38042 Grenoble Tel: +33 457428605 (Fax: +33 438785494)
Re: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain
check an anomalous map! The obvious thing to do to rule out gold binding - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 12/10/2013 12:44 PM, PriyankMaindola wrote: dear members i am trying to solve this crystal structure but I am puzzled with an undefined blob that appeared at a glutamine residue after refinement. I have attached pics of that below. Is it a covalent modification of acid-amide side chain.. ... as there is no charged environment around and density seems continuous. please suggest following reagents were encountered by protein during purification, crystallization and soaking : phenyl methyl sulfonyl fluoride benzamidine tris dtt (could it be cyclized dtt?) k[au(cn)2] acidic pH isopropanol citrate sulfate phosfate K+, Na+, Cl- map contour: 2fo-fc: 1rmsd fo-fc (green): 3 rmsd -- *Priyank*
[ccp4bb] Post-doctoral fellowship in fragment-based drug discovery
Dear colleagues, below is an advert for an postdoc position in my group. best, Marko Applications are invited from post-doctoral scientists with expertise in protein crystallography to join the group of Dr Marko Hyvonen (http://www-cryst.bioc.cam.ac.uk/groups/hyvonen/) at the Department of Biochemistry as part of a multidisciplinary drug discovery programme between the Departments of Chemistry (Prof Chris Abell and Dr David Spring) and Biochemistry (Dr Marko Hyvonen and Prof Tom Blundell) and the MRC Cancer Unit (Prof Ashok Venkitaraman and Dr Grahame McKenzie) at the University of Cambridge. Candidates must have experience in all aspects from protein crystallography from protein expression and purification through crystallisation and structure determination. Previous experience in high-throughput crystallography, in scripting and automation of crystallographic work-flow is highly desirable and background in drug discovery would also be advantageous. Candidates must have a PhD in structural biology and relevant post-doctoral experience is desirable, as is a track record of achievement exemplified by academic publications and/or industrial experience. Applicants will be expected to work closely and interactively with colleagues in different disciplines, to have good communications skills and the ability to work under pressure to meet deadlines. Informal enquiries can be sent to Dr Marko Hyvonen (mh...@cam.ac.uk). Fixed-term: The funds for this post are available for 15 months in the first instance. More details of the application process can be found at http://www.jobs.cam.ac.uk/job/2689/ _ Marko Hyvonen Department of Biochemistry, University of Cambridge ma...@cryst.bioc.cam.ac.uk http://www-cryst.bioc.cam.ac.uk/groups/hyvonen tel:+44-(0)1223-766 044 mobile: +44-(0)7796-174 877 fax:+44-(0)1223-766 002 --
[ccp4bb] AW: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain
My first guess was also a metal ion. However, a tryptophan as Fred suggested cannot be ruled out. A simple preliminary test is to scroll up the contouring level and look when the contours of the blob disappear. If the contours quickly disappear, you have something disordered or light. If the contours of the blob disappear at the same moment or later as e.g. sulfur atoms, you have something heavy like a metal ion. You still have to fit all possibilities and see what refines best. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Matthias Zebisch Gesendet: Dienstag, 10. Dezember 2013 14:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain check an anomalous map! The obvious thing to do to rule out gold binding - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.ukmailto:matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 12/10/2013 12:44 PM, PriyankMaindola wrote: dear members i am trying to solve this crystal structure but I am puzzled with an undefined blob that appeared at a glutamine residue after refinement. I have attached pics of that below. Is it a covalent modification of acid-amide side chain.. ... as there is no charged environment around and density seems continuous. please suggest following reagents were encountered by protein during purification, crystallization and soaking : phenyl methyl sulfonyl fluoride benzamidine tris dtt (could it be cyclized dtt?) k[au(cn)2] acidic pH isopropanol citrate sulfate phosfate K+, Na+, Cl- map contour: 2fo-fc: 1rmsd fo-fc (green): 3 rmsd -- Priyank
[ccp4bb] coot error on a Scientific Linux box (32 bit)
Hi all, I was wondering if anyone had seen this behaviour with Coot. Whatever version I was using, or the latest that comes with ccp4 now gives me the following error message. Rather cryptic message for me. Thanks, Fred. The program 'coot-real' received an X Window System error. This probably reflects a bug in the program. The error was 'BadWindow (invalid Window parameter)'. (Details: serial 323 error_code 3 request_code 153 minor_code 4) (Note to programmers: normally, X errors are reported asynchronously; that is, you will receive the error a while after causing it. To debug your program, run it with the --sync command line option to change this behavior. You can then get a meaningful backtrace from your debugger if you break on the gdk_x_error() function.) Gtk-Message: Failed to load module pk-gtk-module Gtk-Message: Failed to load module canberra-gtk-module coot-exe: /home/prog/ccp4-6.4.0/bin/coot-real coot-version: /home/prog/ccp4-6.4.0/bin/coot-real platform: /bin/uname core: #f No core file found. No debugging This is not helpful. Please turn on core dumps before sending a crash report -- Fred. Vellieux (B.Sc., Ph.D., hdr) IBS2 / ELMA Campus EPN 6 rue Jules Horowitz F-38042 Grenoble Tel: +33 457428605 (Fax: +33 438785494)
Re: [ccp4bb] coot error on a Scientific Linux box (32 bit)
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Fred, without further info my guess is that you try to run coot through X11-forwarding of an ssh-connection which sometimes causes this error message, or you need to recompile your graphics driver, e.g. because your kernel was updated and the driver was not. Best, Tim On 12/10/2013 03:53 PM, vellieux wrote: Hi all, I was wondering if anyone had seen this behaviour with Coot. Whatever version I was using, or the latest that comes with ccp4 now gives me the following error message. Rather cryptic message for me. Thanks, Fred. The program 'coot-real' received an X Window System error. This probably reflects a bug in the program. The error was 'BadWindow (invalid Window parameter)'. (Details: serial 323 error_code 3 request_code 153 minor_code 4) (Note to programmers: normally, X errors are reported asynchronously; that is, you will receive the error a while after causing it. To debug your program, run it with the --sync command line option to change this behavior. You can then get a meaningful backtrace from your debugger if you break on the gdk_x_error() function.) Gtk-Message: Failed to load module pk-gtk-module Gtk-Message: Failed to load module canberra-gtk-module coot-exe: /home/prog/ccp4-6.4.0/bin/coot-real coot-version: /home/prog/ccp4-6.4.0/bin/coot-real platform: /bin/uname core: #f No core file found. No debugging This is not helpful. Please turn on core dumps before sending a crash report - -- - -- 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 Icedove - http://www.enigmail.net/ iD8DBQFSpzDQUxlJ7aRr7hoRAgO/AJ9d2O1prlFBUEIOyFReQo3X2hscYQCeOTZ3 6QeY2C8XGNlNuwHXWgVA0lY= =Tqjo -END PGP SIGNATURE-
Re: [ccp4bb] Rfree problem
Hi, The current rwork is 0.31 and Rfree is 0.4, after 10 sets of refinement. I build quite a large portion before sending it for refinement. Thanks a lot for all the answers. I have started working as per suggestions. -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi
Re: [ccp4bb] Rfree problem
Hi Nazia, you did not say what Rwork and Rfree values are... If, for instance, Rwork=20% and Rfree=21% then a situation when Rfree stays the same or even increases and Rwork goes down is totally valid as long as a reasonable Rfree-Rwork gap is maintained. The point is that you need to look at three numbers Rwork, Rfree and Rfree-Rwork, not either one of them. Pavel On Mon, Dec 9, 2013 at 10:51 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab nazia.nasi...@nii.ac.in wrote: Hi, I am facing a problem in refining one of my structures. With every refinement the Rwork is going down. However, the Rfree is stuck and is not going down with refinement. I have already solved the structure without the co-factor bound. This structure is with the co-factor bound. Can anyone suggest the reason for the Rfree getting stuck? Thanks -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi
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Re: [ccp4bb] AW: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain
It doesn't look like you left a CN on your gold atom. These things are pretty much covalently bound. Dale Tronrud On 12/10/2013 08:13 AM, PriyankMaindola wrote: dear all: I. i am not able to fit trp, since 1. trp doesnt fit well 2. positive density comes in fo-fc map after refinement 3. this is a soaked crystal str with heavy atom soln, the native one has perfect density for gln, so mutation to trp is unlikely II. on increasing contour level, 2fo-fc map fades above 4.5 rmsd if I do not put anything in the blob and see the refined map III. placing Au+ and refining (occ 1 ; B-fac 63 A2) gives figure 4.png (attached below) however anomalous diff map does give positive density but not a clear round, spherical one. On 10 December 2013 19:29, herman.schreu...@sanofi.com mailto:herman.schreu...@sanofi.com wrote: My first guess was also a metal ion. However, a tryptophan as Fred suggested cannot be ruled out. A simple preliminary test is to scroll up the contouring level and look when the contours of the blob disappear. If the contours quickly disappear, you have something disordered or light. If the contours of the blob disappear at the same moment or later as e.g. sulfur atoms, you have something heavy like a metal ion. You still have to fit all possibilities and see what refines best. __ __ Best, Herman __ __ *Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Matthias Zebisch *Gesendet:* Dienstag, 10. Dezember 2013 14:21 *An:* CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK *Betreff:* Re: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain __ __ check an anomalous map! The obvious thing to do to rule out gold binding - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK __ __ Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk mailto:matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 12/10/2013 12:44 PM, PriyankMaindola wrote: dear members __ __ i am trying to solve this crystal structure but I am puzzled with an undefined blob that appeared at a glutamine residue after refinement. I have attached pics of that below. Is it a covalent modification of acid-amide side chain.. ... as there is no charged environment around and density seems continuous. __ __ please suggest __ __ following reagents were encountered by protein during purification, crystallization and soaking : phenyl methyl sulfonyl fluoride benzamidine tris dtt (could it be cyclized dtt?) __ __ k[au(cn)2] acidic pH isopropanol citrate sulfate phosfate K+, Na+, Cl- map contour: 2fo-fc: 1rmsd fo-fc (green): 3 rmsd __ __ __ __ -- *Priyank* __ __ -- *Priyank Maindola*
Re: [ccp4bb] Follow-up: Phaser output in QtRView under Windows XP
Hi, David Waterman and Andrey Lebedev sorted out my problem with Phaser log file output in QtRView on Windows. It turns out that Phaser on Windows fails to write a User: line just under the program banner in the log. This line is necessary for proper processing of the log file. You can correct the broken log by adding a line like this: # # # ### CCP4 PROGRAM SUITE: Phaser 2.5.5 ### # User: fcx32934 Run time: Thu Dec 05 13:28:16 2013 David has promised that the developers will release a fix for this in an update in the near future. It could be through a change to Phaser or to the log file parsing, I don't know for sure. /Derek On 4 Dec 2013, at 11:42, Derek Logan derek.lo...@biochemistry.lu.se wrote: Hi again, Following up on my mail earlier this morning, I installed CCP4 in a virtual Windows XP machine on my Mac. When I run Phaser in Windows the QtRView output is lacking the graphical section, as described for the course computers, but I can open the log file generated in the Mac OS X version of Phaser using the Windows CCP4I and the graphs are visible. This suggests it's not a problem with the GUI but with the content of the log file generated by the Windows Phaser. Any ideas? Thanks Derek
Re: [ccp4bb] Fwd: undefined edensity blob at glutamine sidechain
Priyank, I think it's too far out from the Calpha to be Trp, and also not quite flat enough. The map contains more information than just the shape as to identity. Compare the max peak height of the 2Fo-Fc blob near the Gln to neighboring element heights. Much higher than C/N/O ? Probably not citrate, propanol etc Much higher than S ? Probably not Cl, Mg, S, SO4, PO4 K is only a little heavier than S. So you might cut down the range of possibilities considerably. Partially occupied elements can be an issue, but you could narrow your range of options. You can also do the expedient thing and drop an Au in there with e.g. 0.5 occupancy (or a range of occupancies) and see what the refinement does. Phil Jeffrey Princeton On 12/10/13 7:44 AM, PriyankMaindola wrote: dear members i am trying to solve this crystal structure but I am puzzled with an undefined blob that appeared at a glutamine residue after refinement. I have attached pics of that below. Is it a covalent modification of acid-amide side chain.. ... as there is no charged environment around and density seems continuous. please suggest following reagents were encountered by protein during purification, crystallization and soaking : phenyl methyl sulfonyl fluoride benzamidine tris dtt (could it be cyclized dtt?) k[au(cn)2] acidic pH isopropanol citrate sulfate phosfate K+, Na+, Cl- map contour: 2fo-fc: 1rmsd fo-fc (green): 3 rmsd -- *Priyank*
Re: [ccp4bb] Follow-up: Phaser output in QtRView under Windows XP
Yes, a bugfix from the phaser code side has been committed to the phaser svn. Airlie On Dec 10 2013, Derek Logan wrote: Hi, David Waterman and Andrey Lebedev sorted out my problem with Phaser log file output in QtRView on Windows. It turns out that Phaser on Windows fails to write a User: line just under the program banner in the log. This line is necessary for proper processing of the log file. You can correct the broken log by adding a line like this: # # # ### CCP4 PROGRAM SUITE: Phaser 2.5.5 ### # User: fcx32934 Run time: Thu Dec 05 13:28:16 2013 David has promised that the developers will release a fix for this in an update in the near future. It could be through a change to Phaser or to the log file parsing, I don't know for sure. /Derek On 4 Dec 2013, at 11:42, Derek Logan derek.lo...@biochemistry.lu.se wrote: Hi again, Following up on my mail earlier this morning, I installed CCP4 in a virtual Windows XP machine on my Mac. When I run Phaser in Windows the QtRView output is lacking the graphical section, as described for the course computers, but I can open the log file generated in the Mac OS X version of Phaser using the Windows CCP4I and the graphs are visible. This suggests it's not a problem with the GUI but with the content of the log file generated by the Windows Phaser. Any ideas? Thanks Derek
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