[ccp4bb] Glycosylation in Gram-positive bacteria such as bacillus and paenibacillus
Colleagues, Many bacilli are known to have S-layer proteins outlining their surface and such proteins are known to be glycosylated. I have a few questions below: 1) What's the signature motif of such glycosylation and what pathway is involved? 2) Do you know any experts in this area, esp. in Paenibacillus? Thanks, Yong-Fu Li Elanco Animal Health
Re: [ccp4bb] Local service for AKTA explorer in Maryland or east coast
Thanks for the inputs from Markus, Kim, and a local friend. Per Jacqueline's request, here is a short list of capable services. 1) James Nathlar, Hopeful, Inc, j...@nathlar.com. Jim is happy to help out, and he is in DC area. 2) ATG Service, Analytical Technologies Group, Ph: 860-449-0886. Jodi Dwyer jdw...@atgservice.com, Jodi is very responsive. It seems they have expertise. 3) Tritech Inc, www.tritechinc.com, O - 410.798.7610. They are also responsive. I hope this list can grow longer over time. Regards, Yong-Fu On Fri, Jul 20, 2012 at 2:58 PM, Jacqueline Vitali jackie.vit...@gmail.comwrote: if you find a service please post -- Thanks On Thu, Jul 19, 2012 at 4:03 PM, Yong-Fu Li yongf...@gmail.com wrote: Colleagues, Does anyone use a local service on an AKTA explorer (or other models) machine ideally in the east coast or in Maryland? GE Healthcare will stop supporting this model in a few years, and I am not sure if they are willing to cover an old machine. I remember someone recommended a local service company in UK some time ago, so I am wondering if such a reliable service exists in the US or better near Maryland. Your suggestions are much appreciated. Yong-Fu Li
[ccp4bb] Local service for AKTA explorer in Maryland or east coast
Colleagues, Does anyone use a local service on an AKTA explorer (or other models) machine ideally in the east coast or in Maryland? GE Healthcare will stop supporting this model in a few years, and I am not sure if they are willing to cover an old machine. I remember someone recommended a local service company in UK some time ago, so I am wondering if such a reliable service exists in the US or better near Maryland. Your suggestions are much appreciated. Yong-Fu Li
[ccp4bb] CCP4i doesn't run
Hi, I am using CCP4 6.1.13 in Windows 7. It worked fine after installation, but now stopped running after I moved CCP4-DATABASE to a subdirectory for consolidation. So I uninstalled the whole program and removed folders (CCP4-Packages and Ccp4Temp) from C: drive, and then I downloaded it again and reinstalled the program, however the problem doesn't go away. It did ask me to override a lock once. It stops with the following message: ERROR no project database directory: C:/Ccp4Temp/CCP4_DATABASE Please set up a project directory using the DirectoriesProjectDir window from the main CCP4i interface, and then rerun this job. In the assigned project directory, CCP4_DATABASE folder exists and contains 2 files: database (def file), and database (Lock file). C:/Ccp4Temp is empty. I shouldn't have messed up with the directories, but can anyone help? Thanks, Yong-Fu
[ccp4bb] CCP4 installation error 1911
Hi, I am trying to install the latest version and downloaded the .exe file. But it failed with error message 1911. CCP4Wiki doesn't have any entry for it. CCP4 6.1.2 (it appears to be 6.1.3) Windows XP professional system Here is the error message: Error 1911. Could not register type library for file C:\CCP4-Packages\GraphViz\bin\comdlg32.ocx. Contact your support personnel. Do I need to have administrator's privilege to install in C: drive? I installed previous versions without any problem. Thanks for any help. Yong-Fu Li
[ccp4bb] DS Visualizer ActiveX Control in PowerPoint
Hi, This is non-CCP4 related question, I apologize to those who are not interested. But I hope there might be someone over there with a good answer. Program: Accelry DS Visualizer 2.0 (Free ware). It has some interesting features. My intention: Create an image from pdb files and save as .msv file. Then in PowerPoint this file can be inserted via an ActiveX Control window. In 2003 version it works this way: (1) Open ppt, then bring in “Visual Basic” and “Control Toolbox” panel. (2) Click “More Controls” in “Control Toolbox” penal, then select “Accelrys DSVisualizer ControlR1” (3) The cursor becomes cross then makes a window in ppt slide. (4) Switch ppt to “Full Screen” then right-click on black window to import molecule file. (5) Now you can manipulate the molecule as in DS Visualizer when ppt is in “Full Screen” However, in 2007 version the layout is different and the Visual Basic and Control ToolBox exist in the Developer menu. I could not locate the Accelrys DSVisualizer ControlR1. Instead there are entries such as DSDisplayPanel Class and DSStatusBar Class. Choosing either one and trying to draw a window will give the same error: Could not access system registry. I also posted a similar question to the Accelrys forum but no reply yet. Thanks in advance. Yong-Fu Li IAVI, Brooklyn, NY
Re: [ccp4bb] OT: VectorNTI alternatives
I like BioEdit too. It is PC based, downloadable, and very easy to use. It allows copy-paste of a word or text file, and does alignment, translation, back translation, etc, and more. Fabulous program. I also use Lasergene which has the long standing DNA Star, Megalign, but you have to buy a license. It also requires changing format of files to text and saving with a specific suffix such as .seq which is inconvenient. You cannot copy and paste, and when you see a good alignment, you cannot copy and paste out either. Yong-Fu Li On Wed, Jan 28, 2009 at 7:43 AM, Mark Brooks mark.x.bro...@gmail.comwrote: Hi Darren, My favourite program for editing sequences (apart from Vector NTI, which I suppose I'm going to have to delete soon), is BioEdit: http://www.mbio.ncsu.edu/BioEdit/bioedit.html It has an old fashioned cluttered interface, but does do sequence editing, translation into proteins, ClustalW alignments and contig assemblies (a bit like ContigExpress in Vector NTI). It opens ABI files for sequencing data, to view the chromatograms. It uses the external programs such as clustalw alignments or cap3 to do the contig assemblies, and its licence doesn't expire! BioEdit is quite impressive, and sometimes I use it instead of Vector NTI, even (honestly!). For storing everything, I put my primers, plasmid sequences, insert sequences in a MySQL database, with an HTML front end I wrote: http://plasmidb.sourceforge.net/ Plasmi::db also has a homespun feel to it, and only works with Firefox, for example (not other browsers). There is a primer designer page, for traditional cloning by restriction digestion etc.. I can't pretend it's in the same league as Vector NTI, though. The data is stored in a non-proprietary format; database tables which can be viewed with either the HTML pages, or MS Excel, for example. I never really believed that Vector NTI was going to stay free (even to universities etc.) for a long time, and I do think that they deserve some money for their (excellent) product. I hope that they can decide on a reasonable pricing scheme though, instead of vacillating between huge sums and nothing. They seem to be heading towards a moderate price nowadays, at least. Mark 2009/1/28 Darren Hart h...@embl.fr: Hello, After several years of offering the molecular biology software VectorNTI free to the academic community (their open access program) and building up a huge user base, Invitrogen have suddenly announced that they will no longer renew these free licences and the existing ones will be left to expire within the year. There are heavy renewal fees for anyone wishing to continue use of this software. Can anyone recommend decent alternative PC compatible alternatives? Main uses are construct and primer design, plus simple quick alignments, sequence data analysis etc. The database structure for storing sequences was pretty useful also. Ideally free, otherwise reasonably priced. I've seen CLCbio and Geneious have products, both free and paid. Any experience? Thanks, Darren EMBL Grenoble ps anyone using VNTI might consider a backup of their work by exporting files to .gb format. I don't know if a locked up (expired) version permits this and you will have no notice that it is about to expire. -- Mark Brooks, IBBMC, UMR8619 - Bâtiment 430, Université de Paris-Sud, 91405 Orsay CEDEX. Tel: 0169157968 Fax: 0169853715 Skype: markabrooks
[ccp4bb] ccp4 6.1.0 does not run
Happy Holidays! I kept having the following error when running 6.1.0 on Fedora 9 in a csh shell. CCP4 is in a directory: /usr/local/progs/CCP4/. The setup file is called from this directory: /usr/local/progs/CCP4/ccp4-6.1.0/include/ccp4.setup (This file was copied from ccp4.setup-dist) [...@localhost ccp4logs]$ ccp4i /usr/local/progs/CCP4/tcltk++/bin/bltwish: error while loading shared libraries: libtk8.4.so: cannot open shared object file: No such file or directory I also tested as root in the ccp4-6.1.0/ccp4i directory: [r...@localhost bin]# ./ccp4i 20:29:45 Creating a home directory for CCP4 at /root/.CCP4 20:29:45 Creating CCP4i shadow area at /root/.CCP4/CCP4I_TOP 20:29:45 Creating shadow subdirectory /root/.CCP4/CCP4I_TOP/bin 20:29:45 Creating shadow subdirectory /root/.CCP4/CCP4I_TOP/src 20:29:45 Creating shadow subdirectory /root/.CCP4/CCP4I_TOP/etc 20:29:45 Creating shadow subdirectory /root/.CCP4/CCP4I_TOP/tasks 20:29:45 Creating shadow subdirectory /root/.CCP4/CCP4I_TOP/scripts 20:29:45 Creating shadow subdirectory /root/.CCP4/CCP4I_TOP/templates 20:29:45 Creating shadow subdirectory /root/.CCP4/CCP4I_TOP/utils 20:29:45 Creating shadow subdirectory /root/.CCP4/CCP4I_TOP/loggraph 20:29:45 Creating shadow subdirectory /root/.CCP4/CCP4I_TOP/mapslicer 20:29:45 Creating shadow subdirectory /root/.CCP4/CCP4I_TOP/sketch 20:29:45 Creating local monomer directory /root/.CCP4/monomer 20:29:45 Running auto-configure for configure Creating new configure parameters file /usr/local/progs/CCP4/ccp4-6.1.0/ccp4i/etc/unix/configure.def python: error while loading shared libraries: libstdc++.so.5: cannot open shared object file: No such file or directory CCP4i failed to connect to database server after 1 attempt(s) Accessing the project database directly for this session Top level CCP4 directory is /usr/local/progs/CCP4/ccp4-6.1.0 Using CCP4 programs from /usr/local/progs/CCP4/ccp4-6.1.0/bin ccp4i did start, but with error message saying the server database cannot be accessed. 6.0.2 worked fine with the above setting. I am not sure where was wrong. Any help is appreciated. Yong-Fu Li IAVI Design Lab @ BAT, NY
Re: [ccp4bb] insect expression system
Thanks to all that replied. You might find this useful. The question was: How to select an insect expression system for mammalian/viral glycoproteins? The following is a summary of the replies. *1. Baculovirus system* 1) For most proteins, the level of expression is far greater with bacuovirus. (Dima Klenchin) 2) An interesting system: BacMam. BacMam recombinant baculovirus in transporter expression: a study of BCRP and OATP1B1. Protein Expr Purif. 2006 Jun;47(2):591-8. Epub 2006 Jan 30. (Pius Stephen Padayatti) *2. Drosophila system* 1) With S2 you can get away without figuring out if the virus works, then again you probably need a stable cell line... (so which ever works...?) (OBS! cant do SeMet labelling with S2!! or if someone can please tell me) (Tommi Kajander) 2) We use S2 (drosophila cells) for generating our protein. Baculovirus is much more complicated to get running whereas S2 may generate slightly lower amounts of protein, but you will be able to get a system up and running quite quickly. In my experience I've had cases where I was only able to get very low virus titres for use, whereas the drosophila cells were able to go rapidly to expression for the same protein. The S2 cells are also a non-lytic system, so your protein will not get degraded so easily as they might in a lytic virus system. In my case I've tried them side-by-side and found S2 cells to be best (but that's obviously just in one case). (Paul A. McEwan) *3. Just use mammalian system!* 1) Why would you worry about insect expression systems if you already can secrete your constructs in mammalian cells? For such proteins, transient expression in HEK cells for example gives higher yields than baculo, is faster, cheaper, you can nicely control glycosylation, easily do Se-Met labelling and so on. Here are some references (PMID): 17355862, 17001101, 16823037, 11788735, 16082028. (Radu Aricescu) 2) I couldn't agree more with Radu. We had great success in expressing mg amounts of a secreted protein in HEK293 cells (with Radu's help :-) . The same protein was initially expressed in Sf9 cells but with much lower yields. Furthermore, we could very easily generate a stable HEK293 cell line expressing the same protein (at similar levels with transient transfections) with the Flp-In system in just a couple of weeks. We also have a LIC vector which is compatible with the Flp-In system. (Vangelis Christodoulou)
[ccp4bb] insect expression system
Hi, I searched my mail box for possible answers you have given but couldn't find any. The question is about selecting insect expression systems for mammalian or viral glycosylated proteins. There is confirmed expression of the target proteins in mammalian cells. They are secreted into the media. 1) Baculovirus system 2) Drosophila system Which system would you recommend for high expression and convenience? Has anyone ever compared those systems side by side? Any suggestions or references are greatly appreciated. Yongfu Li
