[ccp4bb] Deglycosylation enzymes
Hi all, I am looking to obtain plasmids encoding the deglycosylating enzymes peptide-N-glycosidase F and endoglycosidase F1 for expression as fusion proteins with GST. They were described in the following paper: Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases. F. Grueninger-Leitch, A. D'Arcy, B. D'Arcy, and C. Chène Department of Gene Technologies, Pharma Preclinical Research, F. Hoffmann-La Roche AG, Basel, Switzerland. Protein Sci. 1996 December; 5(12): 2617–2622. Has anyone here had any luck obtaining them? I understand that a materials transfer agreement is needed. I have tried contacting the authors and Hoffmann-La Roche via four difference channels and have met with a wall of silence! Any help here would be appreciated! With thanks, a++
[ccp4bb] model refinement
Dear all I am trying to solve structure of data set at 1.9 A,r merge 9.3,it belongs to space group P3221 unit cell 160.78 157.32 135.62 90.0 90.0 90.0,i has no NCS ,i did tls refinement as well but after water addition the r factor and r free is stucked at 22 and 25 respectively.suggestions are requested for the same. regards Atul Kumar
Re: [ccp4bb] model refinement (corrections, apologies!)
Dear all Thanks for your replies. And thanks to all those who noticed that I had posted wrong unit cell dimensions for P3221 (which I picked from the header of the template pdb). My space group is P3221 (unit cell 54.8 54.8 100.1 90 90 120). Data quality appears fine. I/sigI=4.9; Rmerge=0.088; Completeness=99.9 (outershell values are: 2.3; 0.333; 99.9 respectively) As mentioned above, I solved the structure by molecular replacement and there appears to be no twinning. There are no major unmodelled densities. A couple of small blobs are present, which I think may be glycerol or some other molecule from my crystallization buffer but I still need to model these. Other than that, there are no major issues. I was just wondering if the present R-factor values seem alright for a 1.9 ang data, which many of you have suggested it does? Thanks again. Any other suggestions/comments are still welcome. Atul On Fri, Jun 18, 2010 at 7:39 AM, atul kumar atulsingh21...@gmail.comwrote: Dear all I am trying to solve structure of data set at 1.9 A,r merge 9.3,it belongs to space group P3221 unit cell 160.78 157.32 135.62 90.0 90.0 90.0,i has no NCS ,i did tls refinement as well but after water addition the r factor and r free is stucked at 22 and 25 respectively.suggestions are requested for the same. regards Atul Kumar
Re: [ccp4bb] model refinement (corrections, apologies!)
Hi Atul, With an I/sig of 2.3 and 99.9% completeness in the highest resolution shell you could have collected to an even higher resolution it would seem That might help in modeling the solvent molecules. Do you have more crystals? Flip atul kumar wrote: Dear all Thanks for your replies. And thanks to all those who noticed that I had posted wrong unit cell dimensions for P3221 (which I picked from the header of the template pdb). My space group is P3221 (unit cell 54.8 54.8 100.1 90 90 120). Data quality appears fine. I/sigI=4.9; Rmerge=0.088; Completeness=99.9 (outershell values are: 2.3; 0.333; 99.9 respectively) As mentioned above, I solved the structure by molecular replacement and there appears to be no twinning. There are no major unmodelled densities. A couple of small blobs are present, which I think may be glycerol or some other molecule from my crystallization buffer but I still need to model these. Other than that, there are no major issues. I was just wondering if the present R-factor values seem alright for a 1.9 ang data, which many of you have suggested it does? Thanks again. Any other suggestions/comments are still welcome. Atul On Fri, Jun 18, 2010 at 7:39 AM, atul kumar atulsingh21...@gmail.com mailto:atulsingh21...@gmail.com wrote: Dear all I am trying to solve structure of data set at 1.9 A,r merge 9.3,it belongs to space group P3221 unit cell 160.78 157.32 135.62 90.0 90.0 90.0,i has no NCS ,i did tls refinement as well but after water addition the r factor and r free is stucked at 22 and 25 respectively.suggestions are requested for the same. regards Atul Kumar
Re: [ccp4bb] model refinement (corrections, apologies!)
On Fri, Jun 18, 2010 at 09:14:28AM +, atul kumar wrote: Dear all [...] these. Other than that, there are no major issues. I was just wondering if the present R-factor values seem alright for a 1.9 ang data, which many of you have suggested it does? Atul Yes, it does. The R-values are not the numbers you should look at to decide whether or not model building and refinement have finished. It's the map and e.g. the molprobity output you should look at. Tim On Fri, Jun 18, 2010 at 7:39 AM, atul kumar atulsingh21...@gmail.comwrote: Dear all I am trying to solve structure of data set at 1.9 A,r merge 9.3,it belongs to space group P3221 unit cell 160.78 157.32 135.62 90.0 90.0 90.0,i has no NCS ,i did tls refinement as well but after water addition the r factor and r free is stucked at 22 and 25 respectively.suggestions are requested for the same. regards Atul Kumar -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Deglycosylation enzymes
Hi Aaron, There is a very simple way around (or through :-)) such walls of silence. Both enzymes are rather small (see UniProt Q9XBM8 and P36911) and in a week or so you can have synthetic cDNAs built as you wish. The cost should be equivalent to the purchase of a few enzyme aliquots, so not a bad deal in long term. Best wishes, radu -- A. Radu Aricescu, PhD University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive, Oxford OX3 7BN United Kingdom Phone: +44-1865-287564 Fax: +44-1865-287547 Original message Date: Fri, 18 Jun 2010 17:10:55 +1000 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Aaron Oakley aar...@uow.edu.au) Subject: [ccp4bb] Deglycosylation enzymes To: CCP4BB@JISCMAIL.AC.UK Hi all, I am looking to obtain plasmids encoding the deglycosylating enzymes peptide-N-glycosidase F and endoglycosidase F1 for expression as fusion proteins with GST. They were described in the following paper: Deglycosylation of proteins for crystallization using recombinant fusion protein glycosidases. F. Grueninger-Leitch, A. D'Arcy, B. D'Arcy, and C. Chène Department of Gene Technologies, Pharma Preclinical Research, F. Hoffmann-La Roche AG, Basel, Switzerland. Protein Sci. 1996 December; 5(12): 2617–2622. Has anyone here had any luck obtaining them? I understand that a materials transfer agreement is needed. I have tried contacting the authors and Hoffmann-La Roche via four difference channels and have met with a wall of silence! Any help here would be appreciated! With thanks, a++
Re: [ccp4bb] about the Hampton Research FoldIt kit
I can make a short summary. It seems that Hampton research does not offer the kit continually. Similar kit from other companies might be a substitution. 1.Pierce http://www.piercenet.com/products/browse.cfm?fldID=F5674C5F-898D-4BA7-8514-CE57D20F7018 2. Molecular Dimensions http://www.moleculardimensions.com/shopdisplayproducts.asp?id=94cat=Protein+Refolding+ http://www.moleculardimensions.com/shopdisplayproducts.asp?id=94cat=Protein+Refolding+ 3.Athenaes http://www.athenaes.com/QuickFoldProteinRefoldingKit.phphttp://www.athenaes.com/QuickFoldProteinRefoldingKit.php Thanks and best wishes. http://www.athenaes.com/QuickFoldProteinRefoldingKit.php On Fri, Jun 18, 2010 at 11:41 AM, francois@cbs.cnrs.fr wrote: Hi Jiamu You can find a similar kit here http://www.athenaes.com/QuickFoldProteinRefoldingKit.php François = Francois Hoh, PhD CENTRE de BIOCHIMIE STRUCTURALE 29 rue de Navacelles 34090 MONTPELLIER France Tel : (33) 4 67 41 77 06 Fax : (33) 4 67 41 79 13 e-mail francois@cbs.cnrs.fr (http://www.cbs.cnrs.fr) -- Jiamu Du, Ph.D. Postdoctoral Research Fellow Laboratory of Structural Biology Memorial Sloan-Kettering Cancer Center RRL 269, 430 E 67th Street New York, NY, 10021 E-mail: d...@mskcc.org Tel: (917) - 292 - 4616
[ccp4bb] Cryo tongs/manipulator
Hello all, Does anyone know where one could purchase cryo-vial tongs/manipulatos with the plastic coated handles (See attached image)? We have checked with Blake Industries (where I think we originally got them eons ago), however their new catalog does not list them any more. Thanks in advance. -Rajintha attachment: Cryo_Vial_Manipulator.jpg
Re: [ccp4bb] Cryo tongs/manipulator
These just look like ordinary hemostats that have been dipped in rubber goo stuff. You can get rubber goo stuff from McMaster-Carr: http://www.mcmaster.com/#plastic-coating-dip/=7l6uk4 -James Holton MAD Scientist On 6/18/2010 10:12 AM, Bandaranayake, Rajintha wrote: Hello all, Does anyone know where one could purchase cryo-vial tongs/manipulatos with the plastic coated handles (See attached image)? We have checked with Blake Industries (where I think we originally got them eons ago), however their new catalog does not list them any more. Thanks in advance. -Rajintha
Re: [ccp4bb] Cryo tongs/manipulator
When searching, avoid the almost overwhelming temptation to spell it as vile manipulator or you will just wind up with a listing of university administrators. Bandaranayake, Rajintha wrote: Hello all, Does anyone know where one could purchase cryo-vial tongs/manipulatos with the plastic coated handles (See attached image)? We have checked with Blake Industries (where I think we originally got them eons ago), however their new catalog does not list them any more. Thanks in advance. -Rajintha
Re: [ccp4bb] model refinement
Not sure if anyone has mentioned this but for example what sort of maps are you using for your refinement. Have you tried density modification, or simulated annealing and or omit maps. These can really be useful for finding parts of your model that are not consistent with the data. Good luck! Gina From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of atul kumar Sent: Friday, June 18, 2010 5:14 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] model refinement (corrections, apologies!) Dear all Thanks for your replies. And thanks to all those who noticed that I had posted wrong unit cell dimensions for P3221 (which I picked from the header of the template pdb). My space group is P3221 (unit cell 54.8 54.8 100.1 90 90 120). Data quality appears fine. I/sigI=4.9; Rmerge=0.088; Completeness=99.9 (outershell values are: 2.3; 0.333; 99.9 respectively) As mentioned above, I solved the structure by molecular replacement and there appears to be no twinning. There are no major unmodelled densities. A couple of small blobs are present, which I think may be glycerol or some other molecule from my crystallization buffer but I still need to model these. Other than that, there are no major issues. I was just wondering if the present R-factor values seem alright for a 1.9 ang data, which many of you have suggested it does? Thanks again. Any other suggestions/comments are still welcome. Atul On Fri, Jun 18, 2010 at 7:39 AM, atul kumar atulsingh21...@gmail.com wrote: Dear all I am trying to solve structure of data set at 1.9 A,r merge 9.3,it belongs to space group P3221 unit cell 160.78 157.32 135.62 90.0 90.0 90.0,i has no NCS ,i did tls refinement as well but after water addition the r factor and r free is stucked at 22 and 25 respectively.suggestions are requested for the same. regards Atul Kumar 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 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] model refinement (corrections, apologies!)
Hi Atul, On Fri, Jun 18, 2010 at 09:14:28AM +, atul kumar wrote: Dear all [...] these. Other than that, there are no major issues. I was just wondering if the present R-factor values seem alright for a 1.9 ang data, which many of you have suggested it does? Atul the typical R-factors for all structures in PDB refined at around 1.9A are: Histogram of Rwork for models in PDB at resolution 1.80-2.00 A: 0.093 - 0.112 : 1 0.112 - 0.132 : 15 0.132 - 0.151 : 186 0.151 - 0.171 : 773 0.171 - 0.190 : 1721 0.190 - 0.209 : 1924 0.209 - 0.229 : 991 your structure 0.229 - 0.248 : 342 0.248 - 0.268 : 54 0.268 - 0.287 : 17 Histogram of Rfree for models in PDB at resolution 1.80-2.00 A: 0.138 - 0.160 : 6 0.160 - 0.182 : 117 0.182 - 0.204 : 739 0.204 - 0.226 : 1565 0.226 - 0.248 : 1974 0.248 - 0.270 : 1133 your structure 0.270 - 0.292 : 389 0.292 - 0.314 : 85 0.314 - 0.336 : 13 0.336 - 0.358 : 3 Histogram of Rfree-Rwork for all model in PDB at resolution 1.80-2.00 A: 0.001 - 0.011 : 64 0.011 - 0.021 : 409 0.021 - 0.031 : 1214 your structure 0.031 - 0.041 : 1797 0.041 - 0.050 : 1388 0.050 - 0.060 : 697 0.060 - 0.070 : 282 0.070 - 0.080 : 111 0.080 - 0.090 : 47 0.090 - 0.100 : 15 Number of structures considered: 6024 It looks like you are more or less good (overall), but it is also likely that some more improvement can be done to the model which you can be easily pin-pointed by Comprehensive Validation option available from PHENIX GUI. Pavel. PS The command to get the above statistics is (in PHENIX): phenix.r_factor_statistics 1.9
[ccp4bb] f2mtz error: problems reading reflection
Dear Experts, After a new installation on Mac OS 10.6, i am getting this error (related to f2mtz) during the Find SITES step by SHELX in Autosharp. Data line--- LABO H K L FA SIGFA ALPHA Number of columns to be read in: 6 Data line--- CTYP H H H F Q P Data line--- FORM '(3F4.0,2F8.2,F4.0)' $TEXT:Warning: $$ comment $$ WARNING: Format I replaced with F $$ FORMATTED OLD file opened on unit 1 Logical name: HKLIN, Filename: SHELX/1_fa.hkl $TEXT:Warning: $$ comment $$ WARNING: PROJECTNAME not assigned $$ $TEXT:Warning: $$ comment $$ WARNING: CRYSTALNAME not assigned $$ $TEXT:Warning: $$ comment $$ WARNING: DATASETNAME not assigned $$ *** Read error Check FORMAT -- especially the need for REALs f2mtz: problems reading reflection 9 f2mtz: problems reading reflection 9 I am running ccp4-6.1.13. It looks like a FORTRAN error but do not know how to fix this or which file to edit particularly. SHARP otherwise running fine if i exclude SHELX step by feeding known sites. Thanks for your help. Yogesh -- Yogesh K. Gupta, Ph.D. Dept of Structural Chemical Biology Mount Sinai School of Medicine Icahn Medical Institute 1425, Madison Av. (Rm 16-26) New York, NY 10029 Tel:+1 212-659-8639 -
