Re: [ccp4bb] isopropanol as a precipitant
Hi, if you look at papers on crystallization of the E.coli protein ROP, you might find interesting info, in particular e.g.: Papanikolau Y, Kotsifaki D, Fadouloglou VE, Gazi AD, Glykos NM, Cesareni G, Kokkinidis M. Ionic strength reducers: an efficient approach to protein purification and crystallization. Application to two Rop variants. Acta Crystallogr D Biol Crystallogr. 2004 Jul;60(Pt 7):1334-7 (sounds like a lot of work, but you asked for it ... ;-) ) volatility is obviously a big problem, but as for cryo use paratone-N (or related dry highly viscous, see previous discussions on its behaviour on ccp4bb) oil, thats probably the easiest thing to do. (only thing ever work for me with methanol and ethanol) obvioulsy you can try butanols etc and MDP as the less volatile relatives. HTH, Tommi ps sounds suspiciously close to ROP in size... not a fourhelix bundle is it??? (none of my business obviously though) (on another note i think it would be nice if people would subscribe with their institute email addresses so that we are all on the same level in public discussions. not that it should be mandatory but it would be sort of nice given its a professional forum) Quoting shivesh kumar [EMAIL PROTECTED]: Dear all Sorry for non-CCP4 query. I have crystallized a 7kDa protein in 50-60% of isopropanol,pH 4.0-4.6.Theinteresting thing is that the xtal appears within 5 hrs at 16 degree. The protein conc is 5mg/ml and drop size is 3+1 with mother liquor 300 micro lt.Numerous xtals appears and but small is size.Do anyone can share their experience with Isopropanol as a precipitant and to improve the xtal quality with other precipitants.All suggestions are welcome. Thanx in advance. Shivesh -- Tommi Kajander, Ph.D. Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] multiple sequence alignment from multiple pairwise structural alignments
Hi Stephen, please check the links in CCP4 wiki at http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Structure_based_sequence_alignment best, Kay -- Kay Diederichs http://strucbio.biologie.uni-konstanz.de email: [EMAIL PROTECTED] Tel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz smime.p7s Description: S/MIME Cryptographic Signature
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
Hi Evette, Have you tried to mutate one glycosylation site at the time and check for expression? Often glycosylation is necessary for secretion -that's the case with my protein. Also these sites tend to have a defined glycosylation pattern which eliminates the need of deglycosylation. You could try a mammalian expression system as an alternative. I have been using Invitrogen's Flp-In system to generate stable cell lines in HEK293 Flp-In cells and has worked very well in my hands (I'm not affiliated with Invitrogen). Hope this helps. Vangelis On Mar 4, 2008, at 22:25, Radisky, Evette S., Ph.D. wrote: Dear all, Our lab is new to working with Pichia pastoris, also new to working with glycosylated proteins. We have a construct for a secreted protein that expresses pretty well in Picha, but upon mutation of the 2 N-linked glycosylation sites to Ala, we get no expression at all, nada. The nucleic acid sequence appears to be correct, i.e. we have not introduced any unintentional frame shifts, stop codons, or anything like that. Is this a common phenomenon? Are there any tricks to get the Pichia to do its thing? Any chance that alternative substitutions will work when Ala does not? Or are we better off (a) trying to deglycosylate enzymatically, or (b) trying a different expression host? All opinions and anecdotes welcome. Thanks! Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
Dear Evette, it is quite common that mutation of glycosylation sites completely kills expression. One rationale is that the glycans cover hydrophobic patches on the surface of the protein, and when exposed, the protein won't fold properly anymore. Pichia is a pain because the glycans it produces are enormous and heterogenous. That means that when you deglycosylate, you end up with a protein sample that has glycans of varying length which certainly does not help in crystallization. There are some commercial pichia strains out there with a modified glycosylation machinery. But I would certainly commend Artem's suggestion to switch to baculo. Cheers, Rob Meijers Synchrotron Soleil Radisky, Evette S., Ph.D. [EMAIL PROTECTED] wrote: Removal of glycosylation sites in Picha expression construct Dear all, Our lab is new to working with Pichia pastoris, also new to working with glycosylated proteins. We have a construct for a secreted protein that expresses pretty well in Picha, but upon mutation of the 2 N-linked glycosylation sites to Ala, we get no expression at all, nada. The nucleic acid sequence appears to be correct, i.e. we have not introduced any unintentional frame shifts, stop codons, or anything like that. Is this a common phenomenon? Are there any tricks to get the Pichia to do its thing? Any chance that alternative substitutions will work when Ala does not? Or are we better off (a) trying to deglycosylate enzymatically, or (b) trying a different expression host? All opinions and anecdotes welcome. Thanks! Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) - Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.
Re: [ccp4bb] Problem with DM
Dear Yuhua I think that you may have failed to specify a solvent content fraction before attempting to run DM - please try rerunning the job and checking the Required Parameters folder about halfway down the window. If the box next to the text Fraction solvent content is empty and coloured yellow then this is likely to be the problem. The solvent fraction should be between 0.0 (all protein, no solvent) and 1.0 (all solvent, no protein). Please check the DM documentation for more information. Hope that this helps, best wishes Peter yuhua wrote: Dear Crystallographers, I failed to run DM in ccp4 package recently and got the errors as below: a href=http://www.yorvic.york.ac.uk/~cowtan/dm/refs.html;dm reference:/a blockquote K. Cowtan (1994), dm: An automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography, 31, p34-38. /blockquotep a name=tocdmh2Contents/h2/a ul lia href=#commanddmCommand input/a lia href=#commentsdmComments/a lia href=#mtzindmMTZ input/a lia href=#datachkdmData Checking/a lia href=#datascldmData Scaling/a lia href=#solmskdmSolvent Mask/a lia href=#cyc0001dmFirst Cycle/a lia href=#dataoutdmOutput/a /ul a name=commanddmh2Command Input/h2/a pre Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#modeMODE /a SOLV Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#combineCOMBINE /a PERT Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#schemeSCHEME /a ALL Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#ncycleNCYCLE /a AUTO Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#solcSOLCONT /a *** Warning Real sub-argument expected Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#ncsmaskNCSMASK /a Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#labinLABIN /a FP=F_p2 SIGFP=SIGF_p2 PHIO=PHIC FOMO=FOM Data line--- a href=/usr/local/ccp4-6.0.2/docs/dm.html#laboutLABOUT /a FDM=FDM PHIDM=PHIDM FOMDM=FOMDM dm: Input error (see above) Times: User: 0.0s System:0.0s Elapsed: 0:00 /pre /html *** * Information from CCP4Interface script *** The program run with command: dm HKLIN /Users/Research/CNS/newtls_refmac4.mtz HKLOUT /Users/Research/CNS/newtls_dm1.mtz SOLOUT /Users/Research/CNS/newtls_dm1.msk has failed with error message dm: Input error (see above) *** #CCP4I TERMINATION STATUS 0 dm: Input error (see above) #CCP4I TERMINATION TIME 04 Mar 2008 22:15:09 #CCP4I MESSAGE Task failed I can not fix it. Can anyone give me some help? Thanks very much! -- ___ Peter J Briggs, [EMAIL PROTECTED] Tel: +44 1925 603826 CCP4, [EMAIL PROTECTED] Fax: +44 1925 603825 http://www.ccp4.ac.uk/ Daresbury Laboratory, Daresbury, Warrington WA4 4AD
[ccp4bb] Missing fonts...
Hi folks I've had a couple of reports recently of people trying to run ipmosflm on Fedora Core 8 machines, and they get the following error - ** xdl_view error in routine xdl_open_view ** ** Unable to load *adobe-courier-medium-r*--8* font ** I'm assuming that the install for FC8 is not including all the Courier fonts; since I don't have an FC8 machine handy, could anyone here offer advice about the best way to install these? Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 2QH
Re: [ccp4bb] Copper Staining
One other staining method deserves to be mentioned with the others already in this thread: We use standard issue Coomassie Blue staining, but do the staining and destaining with heated solutions (place the gel in a tray on a hotplate set to ~90drg in a fumehood). We routinely have the result after 10-15 minutes (5 min stain, 5-10 min destain). -bjørn -- Bjørn Pañella Pedersen Ph.D Student, MSc Department of Molecular Biology Office: +45 89425021 University of Aarhus Lab: +45 89425010 Gustav Wieds Vej 10c Email: [EMAIL PROTECTED] DK-8000 Aarhus C Lab WWW: http://www.bioxray.dk Denmark Jacob Keller wrote: Dear Crystallographers: just to share something I found out recently, that is really helpful to know: 5 min Copper Stain protocol Normal SDS-PAGE gels can be stained in 5 min with 300mM CuCl2. Simply remove the gel, rinse ~10s in water, place 5 min in CuCl2, and the gel is negatively stained, with protein bands appearing clear, and background opaque white. To visualize, I put it on a flatbed scanner in a darkened room. It works in my hands every bit as well as coomassie (at least as sensitive, if not more), and if you want, you can stain afterwards in coomassie with the usual effect. To destain, place gel in EDTA. The original paper is Lee et al, Anal. Biochem. 1987. Instead of waiting hours, you can see the results in 5 min with copper! Also, there is no smell, and it can be re-used, to a point. Gels are stable in H2O for months, say Lee et al. Jacob p.s. I am not in copper comodities... p.p.s. if anybody knows of down-sides to this, please let me know.
Re: [ccp4bb] Copper Staining
You can also do this more quickly by microwaving your gels in stain/destain solutions just for the lazy or impatient Regards Mike On Wed, 2008-03-05 at 12:09 +0100, Bjørn Pañella Pedersen wrote: One other staining method deserves to be mentioned with the others already in this thread: We use standard issue Coomassie Blue staining, but do the staining and destaining with heated solutions (place the gel in a tray on a hotplate set to ~90drg in a fumehood). We routinely have the result after 10-15 minutes (5 min stain, 5-10 min destain). -bjørn DIVFONT size=1 color=grayThis e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom /FONT/DIV
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
Evette, As the glycosylation seems to be critical for production of your protein in P. pastoris I would imagine it would be the same in both insect and mammalian cells. As I'm sure you've invested plenty of time getting this expression system to work it would seem a waste not to try enzymatic treatment with either Endo H or PNGaseF (although you may need to denature the protein to get complete removal with the latter enzyme) I'm curious about your choice of mutations though. Although I know it's standard to perform Alanine substitution, switching from a known surface Asn to an Ala seems pretty drastic to me. If I perform a google survey of the literature it seems mutating the Asn to an Asp is an alternative (and better ?) choice, I also have seen publications where mutation of the Ser/Thr residue of the N-glycosylation recognition site to an Alanine appears to work. I also agree with others on the CCP4 bb of trying the mutations individually. Stephen -- Stephen Weeks, Ph. D. Drexel University College of Medicine Department of Biochemistry and Molecular Biology Room 10102 New College Building 245 N. 15th St. Philadelphia, PA 19102 Phone: (+) 215-762-7316 Fax: (+) 215-762-4452 Radisky, Evette S., Ph.D. wrote: Dear all, Our lab is new to working with Pichia pastoris, also new to working with glycosylated proteins. We have a construct for a secreted protein that expresses pretty well in Picha, but upon mutation of the 2 N-linked glycosylation sites to Ala, we get no expression at all, nada. The nucleic acid sequence appears to be correct, i.e. we have not introduced any unintentional frame shifts, stop codons, or anything like that. Is this a common phenomenon? Are there any tricks to get the Pichia to do its thing? Any chance that alternative substitutions will work when Ala does not? Or are we better off (a) trying to deglycosylate enzymatically, or (b) trying a different expression host? All opinions and anecdotes welcome. Thanks! Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
Thanks to everyone for the great suggestions so far. To clarify and answer a few questions, with the mutated construct we get no protein either secreted or in the pellet. The protein in question is about 20 Kda; with glycosylation at both sites it is around 29 kDa (both in mammalian cells and in Pichia). The protein has been previously produced and crystallized by another lab; in that case the double Ala mutant was expressed in CHO cells. It has been shown that glycosylation is not required for function. There are several reports of related (but natively nonglycosylated) proteins being produced in Pichia. We decided to try Pichia because we were already using it for another project, and we decided to introduce the Ala mutations because that mutant protein had apparently been crystallographically well behaved previously. Thanks again, Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
Re: [ccp4bb] Missing fonts...
Dear Harry, The simplest solution may well be to do what we are starting to do with RasMol to deal with the divergence in font sets on Linux systems. We are gathering font kits, and then setting up install and run scripts that put the fonts into convenient directories in the user account and then do an xset +fp on each run. Regards, Herbert At 10:34 AM + 3/5/08, Harry Powell wrote: Hi folks I've had a couple of reports recently of people trying to run ipmosflm on Fedora Core 8 machines, and they get the following error - ** xdl_view error in routine xdl_open_view ** ** Unable to load *adobe-courier-medium-r*--8* font ** I'm assuming that the install for FC8 is not including all the Courier fonts; since I don't have an FC8 machine handy, could anyone here offer advice about the best way to install these? Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 2QH -- = Herbert J. Bernstein, Professor of Computer Science Dowling College, Kramer Science Center, KSC 121 Idle Hour Blvd, Oakdale, NY, 11769 +1-631-244-3035 [EMAIL PROTECTED] =
[ccp4bb] PhD studentship available
Dear Colleague, A BBSRC PhD studentship (for UK students only - EU students may be eligible for funding) is available for three years from October 2008 to work in my laboratory at Queen Mary, University of London. A provisional project title is Conformational changes during the photocycle of a bacterial phytochrome. Further information is available from http://photosynthesis.sbcs.qmul.ac.uk/Krauss_PhDProject.pdf The main requirement is for a motivated and qualified scientist with a commitment to solving important biological problems by means of structural analysis, including X-ray crystallography. Please bring this opportunity to the attention of any interested graduate or final-year undergraduate in your research group or department. Applicants are invited to apply at the earliest opportunity. The position is open until filled. Best wishes, Norbert Krauß ___ Dr Norbert Krauss Senior Lecturer in Structural Biology School of Biological and Chemical Sciences Queen Mary, University of London Walter Besant Building Mile End Road London E1 4NS UK Tel.:+44 20 7882 8445 e-mail: [EMAIL PROTECTED] http://www.sbcs.qmul.ac.uk/people/norbert_krauss.shtml http://photosynthesis.sbcs.qmul.ac.uk/
[ccp4bb] New service from IUCr Journals
Dear All You may be interested to hear of a new service we have just launched for IUCr Journals that allows authors to create enhanced interactive illustrations of structures to include in their articles. The first illustration prepared in this way appears in the article of Mueller-Dieckmann et al. [Acta Cryst. (2008), F64, 156-162] on the crystal structure of mouse ADP-ribosylhydrolase 3. The illustration is a view rendered within an interactive Jmol applet so readers may change the orientation and size of the view and, by right-clicking within the illustration, they have access to the full repertoire of menu options provided by Jmol. The toolkit used to create this illustration is now available and forms an integral part of the IUCr submission and review systems. It can be accessed from the Acta D or Acta F author services pages (http://journals.iucr.org/). More details have been published in an editorial [Acta Cryst. (2008), F64, 154-155] and any comments or feedback on the service are very welcome. Best wishes Louise Louise Jones Technical Editor Acta Crystallographica Sections D F IUCr 5 Abbey Square Chester CH1 2HU England E-mail [EMAIL PROTECTED] Tel +44 1244 342878 FAX +44 1244 314888
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
Evette, This is quite intriguing. At the outset I want to say that just because glycoslyation is not essential for function it still may be absolutely necessary for correct trafficking. Obviously in your case the mutant has been made successfully in CHO cells, assuming the mutant transcript is present in Pichia I wonder if this is an effect of fusing it to the alpha mating factor secretion signal. Have you - or anyone else for that matter - tried alternative secretion signals ? Sorry but I have two more questions for you. How do you lyse you cells to see production of the protein in the pellet ? I found boiling Pichia in SDS buffer pretty inefficient, do you use glass beads ? Secondly Im curious as to what residues are present when you align you protein to the related non-natively glycosylated proteins (that where successfully expressed in Pichia) specifically at the site of glycosylation. Stephen -- Stephen Weeks, Ph. D. Drexel University College of Medicine Department of Biochemistry and Molecular Biology Room 10102 New College Building 245 N. 15th St. Philadelphia, PA 19102 Phone: (+) 215-762-7316 Fax: (+) 215-762-4452
[ccp4bb] Beamtime Available for Macrocrystalography at NE-CAT
BEAM TIME AVAILABLE FOR MACROMOLECULAR CRYSTALLOGRAPHY AT THE TWO ID BEAMLINES OF NE-CAT AT APS. Beamline 24ID-C: Variable energy between 6-18keV, ADSC Q315 Detector, MAD Capable. Beamline 24ID-E: Equipped with MD2 microdiffractometer, delivering beam as small as 5 microns. Single energy (12662eV), ADSC Q315 Detector. Contact [EMAIL PROTECTED] for further details. See http://necat.chem.cornell.edu
Re: [ccp4bb] multiple sequence alignment from multiple pairwise structural alignments
Not so long ago someone posted this link in this bord: http://www.charite.de/bioinf/strap/ It convinced me. Maybe that should also be included in the CCP4wiki a collection of what program do I use when I want to make X Y or Z Juergen Ashley Buckle wrote: I can recommend MUSTANG: http://p2p.cs.mu.oz.au/mustang/ Web server version is http://p2p.cs.mu.oz.au/mustang/php/ paper is here: http://www.ncbi.nlm.nih.gov/pubmed/16736488?ordinalpos=5itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum Ashley On 05/03/2008, at 6:13 AM, Stephen Graham wrote: Hi all, I would like to generate a structure-based multiple sequence alignment using 4 structures. I have already generated pairwise alignments for each 'pair' of structures (6 alignments in all). Is there a program out there that can take a number of aligned structures (or even just a number of pairwise sequence alignments) and calculate the 'best' multiple sequence alignment? Please note that there is absolutely no sequence conservation between these structures, making standard sequence-based alignment tools pretty useless. Thanks, Stephen -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549 Ashley Buckle Ph.D NHMRC Senior Research Fellow The Department of Biochemistry and Molecular Biology School of Biomedical Sciences, Faculty of Medicine Victorian Bioinformatics Consortium (VBC) Monash University, Clayton, Vic 3800 Australia http://www.med.monash.edu.au/biochem/staff/abuckle.html iChat/AIM: blindcaptaincat skype: ashley.buckle Tel: (613) 9902 0269 (office) Tel: (613) 9905 1653 (lab) Fax : (613) 9905 4699 -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
Re: [ccp4bb] finicky protein
Something else you could try is adding know ligands to your purification step - depending on your proteein (or even during expression, e.g. metals come to mind). Juergen [EMAIL PROTECTED] wrote: Just beware that changing how you break the cells open can change the average size of chromosome chunks, which can change how DNA binding proteins behave in the lysate. Original message Date: Mon, 3 Mar 2008 15:21:15 + From: Mads Gabrielsen [EMAIL PROTECTED] Subject: [ccp4bb] finicky protein To: CCP4BB@JISCMAIL.AC.UK I am not a big fan of sonication. Try changing your way of disrupting the cells. I have compared sonication vs mechanical stress on several unrelated proteins, and for me a good old french press wins every time. If you want to get all modern and fancy, a cell disruptor gives similar results. Cheers, Mads Gabrielsen [Hide Quoted Text] On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote: Hi all sorry, for offtopic query... I am trying to purify my protein by Ni-NTA affinity chromatography. After sonication as i centrifuge bacterial lysate, soon after 10 min whole lysates get precipitated during loading on the column and some time it remain soluble too. if i get purified through the column without precipitation, it gets precipitated during dialysis. I have tried lot, by chnaging buffers, increasing salt or deacreasing salt or no salt at are helpless. I do purifiaction in cold room. can any one suggest some solution? Thanks in advance. NSH -- Dr. Mads Gabrielsen GBRC, B217 Division of Biochemistry and Molecular Biology IBLS University of GlasgowPhone Office: 01413308119 G12 8QQ Phone Lab: 01413306449 UK E-mail: [EMAIL PROTECTED] This message was sent using IMP, the Internet Messaging Program. -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
[ccp4bb] BIOXHIT - Sulphur SAD Workshop - June, 18th-21st - REMINDER
Dear all, the Oeiras TID center at ITQB is organizing the second edition of the BIOXHIT workshop entitled S-SAD diffraction data phasing of macromolecule single crystals from home and Synchrotron X-ray sources that will take place June 18-21. The course will be lectured by Gordon Leonard, George Sheldrick, Clemens Vonrhein and Manfred Weiss. The aim of the course is to teach the state of the art methods and protocols in the collection, processing, scaling and phasing of S-SAD diffraction data from crystals of macromolecules to obtain the best possible interpretable map prior to model building. The number of participants will be limited to 16. If you are interested to participate, send by e-mail a short CV and a letter describing your experience in crystallography as one-PDF file to the organization ([EMAIL PROTECTED] and [EMAIL PROTECTED]) before the 31st of March. More information and the programme (http://tid.itqb.unl.pt/images/BioxHit%20SSAD%20preliminary.pdf) of the course can be found on the TID center web page (http://tid.itqb.unl.pt/ssad.html) Best regards Daniele de Sanctis and Pedro Matias -- Daniele de Sanctis, PhD Homo sum humani nil alienum a me puto __ phone ++ 351 21 4469 662fax ++ 351 21 4433 664 e-mail [EMAIL PROTECTED], [EMAIL PROTECTED] Mailing address: ITQB, Av. República, Apartado 127 2781-901 Oeiras Portugal
Re: [ccp4bb] Off topic: General rule for maximum flow rate for affinity column?
dollins, I do have an excellent reference for you to read J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Mar 25;769(1):133-44. Strategies for the purification and on-column cleavage of glutathione-S-transferase fusion target proteins.Dian C, Eshaghi S, Urbig T, McSweeney S, Heijbel A, Salbert G, Every bit of the method in the paper is easily customizable and in many cases proven worked in our hands PSP On Thu, Feb 28, 2008 at 5:26 PM, Eric Dollins [EMAIL PROTECTED] wrote: Dear protein purifiers, Off topic question: Is there a general rule for how fast you can load, wash and elute from affinity columns, e.g. glutathione agarose? The product insert from Sigma says load under gravity flow. For the volume of cell lysate I have, gravity loading would take an excruciatingly long time. I want to hook up a peristaltic pump to speed things along, but don't really have a feel for just how fast one can load a column in general (I realize this is also dependent on the construct, the buffer, etc). What about the subsequently wash or elution? Thanks for help Eric -- D. Eric Dollins, Ph.D. C266 LSRC, Research Dr. Duke University Medical Center Durham, NC 27710 (919) 681-1668, [EMAIL PROTECTED] -- Pius S Padayatti Department of Biochemistry, Structural Biology Division, School of Medicine, RT-500 Case Western Reserve University, Cleveland, Ohio-44106, 216-368-6833
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
I don't know much about Pichia expression system. You can consult glycobiologists if you are 100% sure that no mistake was made during the experiments. Our lab has been using Drosophila S2 insect cell expression system. With this system, you can maintain cell culture using roller bottles or flasks (so better than the baculovirus expression system). The N-linked glycan is about 1000 dalton per site which can be removed easily by enzymes. The yield is typically above several mg per liter for even large proteins (80 kDa). Holly Date: Wed, 5 Mar 2008 08:38:46 -0600 From: [EMAIL PROTECTED] Subject: Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct To: CCP4BB@JISCMAIL.AC.UK Thanks to everyone for the great suggestions so far. To clarify and answer a few questions, with the mutated construct we get no protein either secreted or in the pellet. The protein in question is about 20 Kda; with glycosylation at both sites it is around 29 kDa (both in mammalian cells and in Pichia). The protein has been previously produced and crystallized by another lab; in that case the double Ala mutant was expressed in CHO cells. It has been shown that glycosylation is not required for function. There are several reports of related (but natively nonglycosylated) proteins being produced in Pichia. We decided to try Pichia because we were already using it for another project, and we decided to introduce the Ala mutations because that mutant protein had apparently been crystallographically well behaved previously. Thanks again, Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) _ Helping your favorite cause is as easy as instant messaging. You IM, we give. http://im.live.com/Messenger/IM/Home/?source=text_hotmail_join
[ccp4bb] Neutron Protein Crystallography Call for Proposals New Deadline
Dear CCP4, The deadline for proposals for all beam lines at LANSCE has been extended by 2 weeks. Below is the reposted call for the PCS. PROTEIN CRYSTALLOGRAPHY STATION AT LANSCE -- CALL FOR PROPOSALS for Run Cycle Beginning the Week of June 5 2008 You are invited to apply for beam time on the neutron Protein Crystallography Station (PCS) at the Los Alamos Neutron Science Center. The deadline for submitting proposals is 6:00 p.m. (1800) Mountain Standard Time on Sunday, March 17th, 2008. Successful proposals will be scheduled during the period June 5, 2008 through mid-December 2008. Please note that there will only be one proposal cycle in calendar year 2008. Because of the long duration of this run cycle, fast access proposals will be accepted throughout, but require compelling evidence of sufficient scientific urgency to justify scheduling. The PCS is a high performance neutron protein crystallography beam line funded by the Office of Biological and Environmental Research of the U.S. Department of Energy. Users of the PCS have access to free neutron beam-time, free perdeuration services and also support for data reduction and structure analysis (http://mnc.lanl.gov). For more technical information about the PCS and experimental requirements, contact Paul Langan (505) 665 8125, [EMAIL PROTECTED] PROPOSAL GUIDANCE AND SUBMISSION Proposals must be submitted using the process on the LANSCE website; only proposals submitted in this manner will be accepted. To access the proposal submission sites, log onto the LANSCE home page (http://lansce.lanl.gov). On this page you will find the link Submit a Proposal. Click on this link and submit your PCS proposal at the site for the Lujan Neutron Scattering Center. Detailed instructions for preparing the proposals can be found on the proposal submission sites under Step-by-Step Guide to Submitting an Online Proposal. STUDENT TRAVEL SUPPORT Travel support is available for students to participate in an experiment via the STONE (Student Travel Opportunities for Neutron Experiments) Program. For information about this program go to the relevant proposal submission site as described previously and click on the STONE Program link. Instructions about how to apply for support from the STONE program are found here. Should you have any questions or require assistance, please contact the LANSCE User Office by e-mail at mailto:[EMAIL PROTECTED][EMAIL PROTECTED] or by telephone at (505) 665-1010.
[ccp4bb] coot question for any/all?
Hello, So sorry to post this here but I'm at wits end and am just hoping that somebody here has had a similar problem (and subsequent fix). Here goes. I have 7 systems, close to identical, all running FC7. For all, I used the autobuild script on the ccp4 website to generate the entire ccp4 package (including coot0.3.1). As I don't want to deal with network problems, I install the programs on every computer following the same build methods. I know I could do this differently, but for now I don't want to change methods. Now, everything appears to be running normally with no issues. However, I just recently tried building a model from scratch, using the baton-mode and then CAatoms - mainchain button. One one computer, this works great, and I can build, combine fragments, renumber, blah blah blah and get a structure. For the other 6 machines, I can do the baton mode (and it seems to work), but when I hit the CA - mainchain button, nothing happens (it just spins for a few seconds then appears to work but there are no new atoms on the screen). I tried downloading the newest ccp4 with no luck, and also the newest coot (from the coot website). They seem to run fine, but again get stalled when I try to do what I state above. It just doesn't work (though coot appears to be working normally - I can display molecules/maps and move things around and such). The problem is, I don't even know where to start here. I would gladly replace/uninstall/reinstall, but what is it that is broken and needs fixing? Like I said, I tried several different reinstalls today with no luck at all (even the new coot gave the same problem). I went to the coot faq and did the soft-link of the reference-libraries (which is an old fix but you never know) and it had no effect. I will say the one machine that is working is my one at home, and so I can't simply share that directory via nfs and see if it works on the other machines (not trivial). All were built the same way, but obviously I missed a file/link or something that is not allowing this important part of the program to work. Any helpful tips? Thanks so much. Dave Roberts [EMAIL PROTECTED]
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
Stephen, We confirmed by PCR that our gene was chromosomally integrated in the many Pichia colonies that we screened for expression, but we did not directly assess for mRNA transcripts. We are indeed using the alpha mating factor secretion signal and have not tested any alternative secretion signals, nor am I aware of any other groups that have tried other signal sequences in Pichia with this family of proteins. We have been extracting proteins from the pellets by a quick alkaline extraction method, variations of which have been published for S. cerevisiae and S. pombe; to judge from our Coomassie stained gels, it is pretty efficient for the Pichia as well. I'd never before thought of checking the homologous residues in the other family members that have been produced in Pichia. The residues at these positions by sequence alignment are D, P, G, and S. However, when I do a structure-based alignment with the one other family member that has been crystallized, I see that structural conservation at these sites is poor; they are on loops that look completely dissimilar between the proteins, so I don't think I can read much into the substitutions. Thanks for your help. Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab) -Original Message- From: Stephen Weeks [mailto:[EMAIL PROTECTED] Sent: Wednesday, March 05, 2008 10:43 AM To: CCP4BB@JISCMAIL.AC.UK; Radisky, Evette S., Ph.D. Subject: Re: Removal of glycosylation sites in Picha expression construct Evette, This is quite intriguing. At the outset I want to say that just because glycoslyation is not essential for function it still may be absolutely necessary for correct trafficking. Obviously in your case the mutant has been made successfully in CHO cells, assuming the mutant transcript is present in Pichia I wonder if this is an effect of fusing it to the alpha mating factor secretion signal. Have you - or anyone else for that matter - tried alternative secretion signals ? Sorry but I have two more questions for you. How do you lyse you cells to see production of the protein in the pellet ? I found boiling Pichia in SDS buffer pretty inefficient, do you use glass beads ? Secondly I'm curious as to what residues are present when you align you protein to the related non-natively glycosylated proteins (that where successfully expressed in Pichia) specifically at the site of glycosylation. Stephen -- Stephen Weeks, Ph. D. Drexel University College of Medicine Department of Biochemistry and Molecular Biology Room 10102 New College Building 245 N. 15th St. Philadelphia, PA 19102 Phone: (+) 215-762-7316 Fax: (+) 215-762-4452
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
Radisky, Evette S., Ph.D. wrote: The residues at these positions by sequence alignment are D, P, G, and S. Don't Perturb Glycosylation Sites. Sorry, I couldn't resist. -- Paul Paukstelis, Ph.D. Research Associate Institute for Cellular and Molecular Biology The University of Texas at Austin P: 512-471-4778, F: 512-232-3420 [EMAIL PROTECTED]
Re: [ccp4bb] coot question for any/all?
David, check if your personal environment (login scripts, paths, etc...) is the same on your home machine and the other machines. Looks as if there might be a difference. What does the coot console say, any error messages or hints? HTH Carsten -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of David Roberts Sent: Wednesday, March 05, 2008 2:39 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] coot question for any/all? Hello, So sorry to post this here but I'm at wits end and am just hoping that somebody here has had a similar problem (and subsequent fix). Here goes. I have 7 systems, close to identical, all running FC7. For all, I used the autobuild script on the ccp4 website to generate the entire ccp4 package (including coot0.3.1). As I don't want to deal with network problems, I install the programs on every computer following the same build methods. I know I could do this differently, but for now I don't want to change methods. Now, everything appears to be running normally with no issues. However, I just recently tried building a model from scratch, using the baton-mode and then CAatoms - mainchain button. One one computer, this works great, and I can build, combine fragments, renumber, blah blah blah and get a structure. For the other 6 machines, I can do the baton mode (and it seems to work), but when I hit the CA - mainchain button, nothing happens (it just spins for a few seconds then appears to work but there are no new atoms on the screen). I tried downloading the newest ccp4 with no luck, and also the newest coot (from the coot website). They seem to run fine, but again get stalled when I try to do what I state above. It just doesn't work (though coot appears to be working normally - I can display molecules/maps and move things around and such). The problem is, I don't even know where to start here. I would gladly replace/uninstall/reinstall, but what is it that is broken and needs fixing? Like I said, I tried several different reinstalls today with no luck at all (even the new coot gave the same problem). I went to the coot faq and did the soft-link of the reference-libraries (which is an old fix but you never know) and it had no effect. I will say the one machine that is working is my one at home, and so I can't simply share that directory via nfs and see if it works on the other machines (not trivial). All were built the same way, but obviously I missed a file/link or something that is not allowing this important part of the program to work. Any helpful tips? Thanks so much. Dave Roberts [EMAIL PROTECTED]
Re: [ccp4bb] Missing fonts...
Harry Powell wrote: Hi folks I've had a couple of reports recently of people trying to run ipmosflm on Fedora Core 8 machines, and they get the following error - ** xdl_view error in routine xdl_open_view ** ** Unable to load *adobe-courier-medium-r*--8* font ** I'm assuming that the install for FC8 is not including all the Courier fonts; since I don't have an FC8 machine handy, could anyone here offer advice about the best way to install these? Fedora 8 no longer installs 75dpi fonts with the default installation. 'yum install xorg-x11-fonts-75dpi' will get you the needed fonts. Thomas
Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct
If you have poor conservation in this region and the region itself is a loop, I would perhaps attempt to replace the entire loop with another one - from a homologue that does not have the glycosylation site(s). Not that this is guarranteed to work, but it might. Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Radisky, Evette S., Ph.D. Sent: Wednesday, March 05, 2008 9:39 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Removal of glycosylation sites in Picha expression construct Thanks to everyone for the great suggestions so far. To clarify and answer a few questions, with the mutated construct we get no protein either secreted or in the pellet. The protein in question is about 20 Kda; with glycosylation at both sites it is around 29 kDa (both in mammalian cells and in Pichia). The protein has been previously produced and crystallized by another lab; in that case the double Ala mutant was expressed in CHO cells. It has been shown that glycosylation is not required for function. There are several reports of related (but natively nonglycosylated) proteins being produced in Pichia. We decided to try Pichia because we were already using it for another project, and we decided to introduce the Ala mutations because that mutant protein had apparently been crystallographically well behaved previously. Thanks again, Evette Evette S. Radisky, Ph.D. Assistant Professor and Associate Consultant II Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 (office) (904) 953-0046 (lab)
