Re: [ccp4bb] precipitates
Dear All, Many thanks for the suggestions. Cheers, Amit 2008/10/1 Chavas Leo <[EMAIL PROTECTED]> > Dear Amit -- > On 30 Sep 2008, at 14:16, amit sharma wrote: > > I am trying to crystallize a protein with a peptide attached to it via a > 9aa linker. I am mostly getting precipitates in conditions carrying salts. I > have tried optimization screens by varying the pH and salt concentrations, > but in vain. Can anyone please suggest ways by which I could optimize the > conditions further? > > > Among all the possibilities, you can: > - try to crystallize without salt ; > - change the temperature ; > - dilute your protein ; > - add some isopropanol (or other) to make the solution "smoother" ; > - change your linker size ; > - ... > > HTH > Kind regards. > > -- Leo -- > > Chavas Leonard, Ph.D. @ home > Research Associate > Marie Curie Actions Fellow > > Faculty of Life Sciences > The University of Manchester > The Michael Smith Building > Oxford Road > Manchester Lancashire > M13 9PT > > Tel: +44(0)161-275-1586 > e-mail: [EMAIL PROTECTED] > http://personalpages.manchester.ac.uk/staff/leonard.chavas/ > > > -- Amit Sharma, Ph.D. Research Associate, Department of Biology, University of York, YO10 5DD UK
Re: [ccp4bb] precipitates
Dear Amit -- On 30 Sep 2008, at 14:16, amit sharma wrote: I am trying to crystallize a protein with a peptide attached to it via a 9aa linker. I am mostly getting precipitates in conditions carrying salts. I have tried optimization screens by varying the pH and salt concentrations, but in vain. Can anyone please suggest ways by which I could optimize the conditions further? Among all the possibilities, you can: - try to crystallize without salt ; - change the temperature ; - dilute your protein ; - add some isopropanol (or other) to make the solution "smoother" ; - change your linker size ; - ... HTH Kind regards. -- Leo -- Chavas Leonard, Ph.D. @ home Research Associate Marie Curie Actions Fellow Faculty of Life Sciences The University of Manchester The Michael Smith Building Oxford Road Manchester Lancashire M13 9PT Tel: +44(0)161-275-1586 e-mail: [EMAIL PROTECTED] http://personalpages.manchester.ac.uk/staff/leonard.chavas/
[ccp4bb] mtz library in windows
Dear all, I am using CCP4 mtz library and other libraries to develop a program. Because I am using a PC at home, I am actually using the CCP4 in windows. However, when I link the mtz library(hence libccp4c.lib), it happens that the compiler complains error LNK2001: unresolved external symbol [EMAIL PROTECTED] and some other functions that should be in libccp4c not found. I checked the project setting(I am using compaq visual fortran 6.6), I have included libccp4c, libccp4f in the library, and library path in /link/input/Additional Library Path, so I don't understand why the compiler still cannot resolve the functions. Can anybody help, please? Your suggestion is greatly appreciated. Alex
Re: [ccp4bb] CCP4i can NOT run
Hi Boaz,
Thanks for your suggestion. I have tried as you suggested. In my case,
CCP4_MASTER
can NOT set to be /home/prog.
The ccp4i did not launched because it can not locate the ccp4i.tcl file.
On Tue, Sep 30, 2008 at 8:53 PM, Boaz Shaanan <[EMAIL PROTECTED]> wrote:
> Your setup in ccp4.setup seems to be incorrect.
> You should have:
> setenv CCP4_MASTER/home/prog
>
> and then you would have:
>
> echo $CCP4 /home/prog/ccp4-6.0.2
>
> I don´t think your problem has anything to do with tcl setup.
>
>Boaz
>
> - Original Message -
> From: Yingjie Peng <[EMAIL PROTECTED]>
> Date: Tuesday, September 30, 2008 15:45
> Subject: Re: [ccp4bb] CCP4i can NOT run
> To: CCP4BB@JISCMAIL.AC.UK
>
> > Hi Iain,
> >
> > Thanks for your reply. The path for tclsh is
> > "/usr/local/tcltkblt/bin/".There are six files: bltsh, bltwish,
> > tclsh, tclsh8.4, wish and wish8.4. The
> > tclsh
> > and wish are symbolic links for tclsh8.4 and wish8.4,
> > respectively. For the
> > permission reason you mentioned, I have also tried this change:
> > sudo chown yjpeng:yjpeng -R /usr/local/tcltkblt
> > It does NOT work!
> >
> > For better analysis, I paste my ccp4.setup file as following:
> > ### THIS SECTION MUST BE EDITED #
> >
> > # CCP4_MASTER is the location of the top-level directory containing
> > `ccp4-'
> > # This is usually the directory in which you ran the tar command
> > to unpack
> > the
> > # code, and is assumed to be shared between machines at a multi-
> > machinesite.
> >
> > setenv CCP4_MASTER/home/prog/ccp4-6.0.2
> > setenv CCP4
> > $CCP4_MASTER/ccp4-6.0.2
> >
> > # Check for existence of CCP4_MASTER
> > if (! -e $CCP4_MASTER) then
> > echo "* WARNING
> > **"echo "The directory
> > $CCP4_MASTER"echo "(assigned to CCP4_MASTER)
> > does not exist."
> > echo "The CCP4 programs will not run
> > correctly, and any"
> > echo "installation attempt will have errors
> > or will fail."
> > echo "* WARNING
> > **"endif
> >
> > # CCP4_SCR: a per-user directory for run-time-generated scratch
> > # files. A dedicated scratch filesystem is probably better than
> > (/usr)/tmp# BINSORT_SCR: a scratch directory for binsort's use;
> > normally same as
> > CCP4_SCR
> >
> > setenv CCP4_SCR /data/tmp
> > # check to see if this exists and if not try to make it
> > if (! -e $CCP4_SCR) mkdir $CCP4_SCR
> > if (! -e $CCP4_SCR) \
> > echo "Unable to make CCP4_SCR. CCP4 progs
> > will not run correctly."
> >
> > setenv BINSORT_SCR $CCP4_SCR
> >
> > ### CCP4i setup - you may need to edit CCP4I_TCLTK ###
> > # CCP4I_TOP - the top directory of the interface
> > setenv CCP4I_TOP ${CCP4}/ccp4i
> > # CCP4I_TCLTK - directory containing tclsh,wish and bltwish
> > executables# as used in
> $CCP4I_TOP/bin/ccp4i,ccp4ish,loggraph
> > # For 'standard' installations this is /usr/local/bin
> > # but note the SGI distributed version of Tcl/Tk is not
> > # appropriate version
> > setenv CCP4I_TCLTK /usr/local/tcltkblt/bin
> > # CCP4I_HELP - directory contain ccp4i help - default is
> > $CCP4I_TOP/helpsetenv CCP4I_HELP ${CCP4I_TOP}/help
>
> >
> > ### Optional - setting http_proxy environment
> > # The commented out 'setenv' line below may have to be declared
> > to download
> > # and edit protein sequences using the new "Import/Edit Protein
> > Sequences"# (This may also be required for remote job submission
> > in arp/warp.)
> > # task. If so, uncomment this line and replace the example proxy
> > URL with
> > your
> > # relevant URL
> >
> > #setenv HTTP_PROXY wwwblah.blah.ac.uk:/blah.blah
> >
> > ### NOVICE USERS STOP HERE #
> >
> > ### OPTIONS TO CUSTOMISE CCP4 #
> >
> > # By default, CCP4 directories are appended to the end of paths (PATH,
> > # LD_LIBRARY_PATH, and DYLD_LIBRARY_PATH). If ccp4_first_in_path
> > is set
> > # to 1, then they will be prepended to the beginning of paths.
> > # When deciding local policy, bear in mind the possible
> > existence of
> > # other CCP4 installations, and the possibility of non-CCP4 programs
> > # with conflicting names.
> > set ccp4_first_in_path = 1
> >
> > # The commented-out switch statement below provides an example
> > of how
> > # to use this file for several machines/architectures sharing
> > $CCP4_MASTER.# If necessary, uncomment and edit this.
> > Otherwise, if you're only
> > supporting
> > # a single system alter the uncommented part as necessary.
> > #
> > # The significance of the environment variables is as follows:
> > # CBIN: location of the executables -- must be on
> > your path (see below);
> > # CLIB: location of (binary) library files such as
> > libccp4.a and
> > libccp4.so;
> > # MANPATH: set this if your system's `man' looks at
> > such an environment
> > # variable to determine
Re: [ccp4bb] CCP4i can NOT run
I have checked /etc/hosts, it is similar to what you say. I also tried to set it exactly the same as yours. But it did NOT work. Thanks! On Tue, Sep 30, 2008 at 10:24 PM, Ben Eisenbraun <[EMAIL PROTECTED]>wrote: > On Tue, Sep 30, 2008 at 10:48:27AM +0800, Yingjie Peng wrote: > > I have installed the latest version of CCP4i under Fedora 9 on my laptop > > HP Compaq 6515b (two processors). The system setup was done and the CCP4i > > interface can run normally. But if I submit a job, it displayes > > "STARTING" the job in the job window and will NOT actually run the job. > > Also I can NOT kill the job with its status as STARTING. > > I have seen similar behavior before. Is your /etc/hosts correct? The > localhost line should look like this: > > 127.0.0.1 localhost > > Some of the CCP4 utilities act as miniature servers and bind to localhost > to communicate between the GUI and the process. If this is actually the > problem, you should see output in the shell that started ccp4i similar > to this: > > ERROR running script can not connect to server port (RunNotification) > SERVER_HOST localhost SERVER_PORT 4441 > > -ben > > -- > Ben Eisenbraun > Structural Biology Grid Harvard Medical School > http://sbgrid.org http://hms.harvard.edu >
Re: [ccp4bb] CCP4i can NOT run
On Tue, Sep 30, 2008 at 10:48:27AM +0800, Yingjie Peng wrote: > I have installed the latest version of CCP4i under Fedora 9 on my laptop > HP Compaq 6515b (two processors). The system setup was done and the CCP4i > interface can run normally. But if I submit a job, it displayes > "STARTING" the job in the job window and will NOT actually run the job. > Also I can NOT kill the job with its status as STARTING. I have seen similar behavior before. Is your /etc/hosts correct? The localhost line should look like this: 127.0.0.1 localhost Some of the CCP4 utilities act as miniature servers and bind to localhost to communicate between the GUI and the process. If this is actually the problem, you should see output in the shell that started ccp4i similar to this: ERROR running script can not connect to server port (RunNotification) SERVER_HOST localhost SERVER_PORT 4441 -ben -- Ben Eisenbraun Structural Biology Grid Harvard Medical School http://sbgrid.org http://hms.harvard.edu
[ccp4bb] precipitates
Dear CCP4bbers, I am trying to crystallize a protein with a peptide attached to it via a 9aa linker. I am mostly getting precipitates in conditions carrying salts. I have tried optimization screens by varying the pH and salt concentrations, but in vain. Can anyone please suggest ways by which I could optimize the conditions further? thanks in advance, -- Amit Sharma
Re: [ccp4bb] cryo-protection for crystals grown in ethanol
Dear Rongjin, I would: -prepare different cryosolutions, adding glycerol, replacing water with glycerol, replacing ethanol with other more cryogenic alcohols: if you have enough crystals, see how these behave when transferred to these solutions, if they crack, decrease precipitant, if they dissolve, increase. once you have measurement time: -first measure a couple of crystals in a capillary or mitegen "loop and sleeve" at room temperature to get a "best case" diffraction. On a home source you might even get a dataset, but surely you'll get good estimates of mosaic spread, spacegroup, and other crystallographic parameters, like for instance an idea about if the crystals may be twinned. -then measure a couple of crystals directly frozen from the drop (i.e. "worst-case" scenario). -then go for complete cryoprotected datasets, using the combined knowledge of previous experiments. Even if the upcoming "beamtime" is not enough for all of this and you have to book more time in the coming weeks, I would go through all these steps and resist the temptation to go for "quick and dirty". If you only ever measure at 100K you never know how good the crystals were before freezing. Mark Quoting Rongjin Guan <[EMAIL PROTECTED]> Dear All I got crystals from 20% Ethanol with 0.1M Tris pH 8.5. This is my first time to have crystals in Ethanol and want to get some suggestions of cryo-protection from those who have done this before. I am waiting for my time on home X-ray facility, and hope I can get some suggestions before that. Thanks, Rongjin Guan
Re: [ccp4bb] CCP4i can NOT run
Hi Iain,
Thanks for your reply. The path for tclsh is "/usr/local/tcltkblt/bin/".
There are six files: bltsh, bltwish, tclsh, tclsh8.4, wish and wish8.4. The
tclsh
and wish are symbolic links for tclsh8.4 and wish8.4, respectively. For the
permission reason you mentioned, I have also tried this change:
sudo chown yjpeng:yjpeng -R /usr/local/tcltkblt
It does NOT work!
For better analysis, I paste my ccp4.setup file as following:
### THIS SECTION MUST BE EDITED #
# CCP4_MASTER is the location of the top-level directory containing
`ccp4-'
# This is usually the directory in which you ran the tar command to unpack
the
# code, and is assumed to be shared between machines at a multi-machine
site.
setenv CCP4_MASTER/home/prog/ccp4-6.0.2
setenv CCP4$CCP4_MASTER/ccp4-6.0.2
# Check for existence of CCP4_MASTER
if (! -e $CCP4_MASTER) then
echo "* WARNING **"
echo "The directory $CCP4_MASTER"
echo "(assigned to CCP4_MASTER) does not exist."
echo "The CCP4 programs will not run correctly, and any"
echo "installation attempt will have errors or will fail."
echo "* WARNING **"
endif
# CCP4_SCR: a per-user directory for run-time-generated scratch
# files. A dedicated scratch filesystem is probably better than (/usr)/tmp
# BINSORT_SCR: a scratch directory for binsort's use; normally same as
CCP4_SCR
setenv CCP4_SCR /data/tmp
# check to see if this exists and if not try to make it
if (! -e $CCP4_SCR) mkdir $CCP4_SCR
if (! -e $CCP4_SCR) \
echo "Unable to make CCP4_SCR. CCP4 progs will not run correctly."
setenv BINSORT_SCR $CCP4_SCR
### CCP4i setup - you may need to edit CCP4I_TCLTK ###
# CCP4I_TOP - the top directory of the interface
setenv CCP4I_TOP ${CCP4}/ccp4i
# CCP4I_TCLTK - directory containing tclsh,wish and bltwish executables
# as used in $CCP4I_TOP/bin/ccp4i,ccp4ish,loggraph
# For 'standard' installations this is /usr/local/bin
# but note the SGI distributed version of Tcl/Tk is not
# appropriate version
setenv CCP4I_TCLTK /usr/local/tcltkblt/bin
# CCP4I_HELP - directory contain ccp4i help - default is $CCP4I_TOP/help
setenv CCP4I_HELP ${CCP4I_TOP}/help
### Optional - setting http_proxy environment
# The commented out 'setenv' line below may have to be declared to download
# and edit protein sequences using the new "Import/Edit Protein Sequences"
# (This may also be required for remote job submission in arp/warp.)
# task. If so, uncomment this line and replace the example proxy URL with
your
# relevant URL
#setenv HTTP_PROXY wwwblah.blah.ac.uk:/blah.blah
### NOVICE USERS STOP HERE #
### OPTIONS TO CUSTOMISE CCP4 #
# By default, CCP4 directories are appended to the end of paths (PATH,
# LD_LIBRARY_PATH, and DYLD_LIBRARY_PATH). If ccp4_first_in_path is set
# to 1, then they will be prepended to the beginning of paths.
# When deciding local policy, bear in mind the possible existence of
# other CCP4 installations, and the possibility of non-CCP4 programs
# with conflicting names.
set ccp4_first_in_path = 1
# The commented-out switch statement below provides an example of how
# to use this file for several machines/architectures sharing $CCP4_MASTER.
# If necessary, uncomment and edit this. Otherwise, if you're only
supporting
# a single system alter the uncommented part as necessary.
#
# The significance of the environment variables is as follows:
# CBIN: location of the executables -- must be on your path (see below);
# CLIB: location of (binary) library files such as libccp4.a and
libccp4.so;
# MANPATH: set this if your system's `man' looks at such an environment
# variable to determine where to look for man pages. By adding
# $CCP4/man to the normal path you get the CCP4 man pages
# with keyword searching as system ones.
# MCTYPE: used for LAUE make -- see LAUE documentation
# CCP4_BROWSER: set this if you intend to use the html documentation
# (recommended). It should have the path and name of a html browser eg
# /usr/bin/X11/netscape or /usr/local/bin/lynx. The browser will be
# started with the alias ccp4help and will open the file
# $CHTML/INDEX.html.
# switch (`hostname`)# edit this switch statement if used
# # for multiple systems
# case 'foo':
setenv CBIN $CCP4/bin
setenv CLIB $CCP4/lib
setenv CCP4_BROWSER firefox
if (${?MANPATH}) then
if ($ccp4_first_in_path) then
setenv MANPATH ${CCP4}/man:${MANPATH}
else
setenv MANPATH ${MANPATH}:${CCP4}/man
endif
else
if ($ccp4_first_in_path) then
setenv MANPATH ${CCP4}/man:/usr/share/man
else
setenv MANPATH /usr/share/man:${CCP4}/man
endif
endif
setenv MCTYP
Re: [ccp4bb] Putting ligand in the protein structure
Yes: There are details in the coot manual 1) Get a dictionary for your ligand from PRODRG or somewhere. - you have obviously done this.. 2) CHECK IT IS CORRECT!!! Right chirality, planar groups, etc.. 3) Import dictionary into coot, and a set of coordinates. 4) Import 2mFo-Dfc and mFo-Dfc maps ( auto mtz from REFMAC will do this) 5) Hit find ligand. 6) Real space refine it - you might need to do some brutal gradding and shoving of bits.. Eleanor George Kontopidis wrote: You need protein structure, a different e. density map (1Fo-1Fc) and your ligand structure. Then from COOT menu select Other Modelling Tools and click Find Ligand. You can also move manually the ligand into the density map. George -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Anshul Awasthi Sent: Tuesday, September 30, 2008 10:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Putting ligand in the protein structure Hi all the crystallographers, I am trying to solve a structure of a protein with some inhibitor. I want to know how I can put in my inhibitor in the density map of the data i got. I can see some density in the active site where the inhibitor should be. I generated the topoly file of the inhibitor (in both pdba nd refmac5 top formats) from the Dundee PRODRG server. Now do i need to incorporate the structure of the inhibitor in ccp4 or can i do in coot?? I am not sure of how to do it. ANy sugegstion will be very valuable for me. No virus found in this incoming message. Checked by AVG - http://www.avg.com Version: 8.0.173 / Virus Database: 270.7.5/1697 - Release Date: 29/9/2008 7:25 ìì
Re: [ccp4bb] Putting ligand in the protein structure
You need protein structure, a different e. density map (1Fo-1Fc) and your ligand structure. Then from COOT menu select Other Modelling Tools and click Find Ligand. You can also move manually the ligand into the density map. George -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Anshul Awasthi Sent: Tuesday, September 30, 2008 10:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Putting ligand in the protein structure Hi all the crystallographers, I am trying to solve a structure of a protein with some inhibitor. I want to know how I can put in my inhibitor in the density map of the data i got. I can see some density in the active site where the inhibitor should be. I generated the topoly file of the inhibitor (in both pdba nd refmac5 top formats) from the Dundee PRODRG server. Now do i need to incorporate the structure of the inhibitor in ccp4 or can i do in coot?? I am not sure of how to do it. ANy sugegstion will be very valuable for me. No virus found in this incoming message. Checked by AVG - http://www.avg.com Version: 8.0.173 / Virus Database: 270.7.5/1697 - Release Date: 29/9/2008 7:25 ìì
[ccp4bb] PhD Position at Structural Biology Brussels
A PhD Position in Structural Biology is available at Structural Biology Brussels, Vrije Universiteit Brussel, Belgium. We are looking for a highly motivated PhD student interested in biochemical and structural studies on tRNA modifying enzymes and enzyme complexes. The Structural Biology Brussels laboratory (for more info: http://www.structuralbiology.be/) is located at the Free University of Brussels and is affiliated with the Flanders Institute for Biotechnology (VIB). Project description: Transfer RNA (tRNA) molecules are pivotal in the translation of the genetic code into protein sequences. In this process, the recognition of the mRNA codon by the correct tRNA does not solely dependent on the primary sequence of the tRNA molecule. Rather, around 100 different posttranscriptional nucleoside modifications have been described, particularly in the anticodon loop, that can modify cognate codon recognition, stabilize the codon- anticodon wobble base pairing and/or affect aminoacylation properties. The current project will deal with the study of the structure/function relationship of tRNA modifying enzymes from both prokaryotic and eukaryotic origin. To study these often multi-subunit complexes and the interaction with their tRNA substrates, an integrated approach of X-ray crystallography, enzyme kinetics and different biophysical methods to study the protein-protein and protein- nucleic acid interactions, will be used. The position is available immediately Candidates should have a recent Masters degree in Biochemistry, Biology, Chemistry or related. Skills in either molecular biology or protein chemistry & purification or protein crystallography would be an asset. Please send applications (including CV, research experience, and at least two names and contact information for references) to: Wim Versées, Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel - [EMAIL PROTECTED]
[ccp4bb] Putting ligand in the protein structure
Hi all the crystallographers, I am trying to solve a structure of a protein with some inhibitor. I want to know how I can put in my inhibitor in the density map of the data i got. I can see some density in the active site where the inhibitor should be. I generated the topoly file of the inhibitor (in both pdba nd refmac5 top formats) from the Dundee PRODRG server. Now do i need to incorporate the structure of the inhibitor in ccp4 or can i do in coot?? I am not sure of how to do it. ANy sugegstion will be very valuable for me.
