[ccp4bb] determining the domain for overexpression and crystallization
Dear all, Before we try to study the crystal structure of an unknown protein, we need to determine the sequence that can fold into a compact and stable 3D domain. What kinds of methods can we choose? -- Best regards, XH Wu
Re: [ccp4bb] determining the domain for overexpression and crystallization
Hi, There's a whole bunch of programs that can help you out there. The 2 methods I think of right now are DISPROT (there's a server I believe, http://www.ist.temple.edu/disprot/ ) - Must admit I haven't been to that one for quite a while; DISPROT provides areas of your sequence with high probability of disorder hydrophobic cluster analysis. There are many others as well, can't think of them right now. Also, common sense (like trying to crystallise with and without tags) can be helpful. You may have to try to crystallise several constructs. And there's more than just compact and stable to crystallisation. Monodispersity is quite important too. Fred. Xianhui Wu wrote: Dear all, Before we try to study the crystal structure of an unknown protein, we need to determine the sequence that can fold into a compact and stable 3D domain. What kinds of methods can we choose? -- Best regards, XH Wu
[ccp4bb] sealing tapes
Dear all, Sorry for this slightly off-topic email, but I am looking for a transparent sealing tape for 96-well crystallization plates, with the following properties: - high sealing performances - compatible with UV screening - optionally, available as rolls Thanks for your help JL -- Jean-Luc Ferrer |---| |Institut de Biologie Structurale J.P. Ebel CEA/CNRS/UJF| |41 rue Jules Horowitz - 38027 Grenoble cedex 1 - FRANCE| |tel.: +33 (0)4-38-78-59-10 - fax : +33 (0)4-38-78-51-22| |---|
Re: [ccp4bb] sealing tapes
Hi Jean-Luc, We have complied a list of UV compatible cover media and plates. The list is by all means not complete yet, but the best seals we found are all sheets (that does not mean all sheets are UV compatible!). If you want I can send you a PDF file outside of the BB. Flip On 3/7/2011 10:31, ferrer wrote: Dear all, Sorry for this slightly off-topic email, but I am looking for a transparent sealing tape for 96-well crystallization plates, with the following properties: - high sealing performances - compatible with UV screening - optionally, available as rolls Thanks for your help JL
Re: [ccp4bb] disulfide bridge in red density
Hello everyone, Thank you very much for giving me such a valuable information. I will try one by one each suggestion and then ask again if i feel more problem. thanks regards Sadaf Iqbal PhD Scholar ICCBS, University of Karachi, Pakistan. Visiting Scientist University of Hamburg, Germany. From: Sanishvili, Ruslan rsanishv...@anl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, March 7, 2011 1:22:14 Subject: Re: [ccp4bb] disulfide bridge in red density Hi Sadaf, Most likely you are dealing with radiation damage. If that's the case, you would need to refine occupancies as Mario has already suggested. However, you may not find a double conformation. Instead, you may find that some of your sulfurs are gone. There is a ton of publications on this topic, take a loo. If you have high enough redundancy, you may be able to use only the part of the data, collected at the beginning. This would have suffered less from radiation. You can also use zero dose extrapolations - look up Diederichs' and Borek's publications. Good luck! Nukri -Original Message- From: CCP4 bulletin board on behalf of sadaf iqbal Sent: Sun 3/6/2011 12:57 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] disulfide bridge in red density Hello everyone, Recently, i have solved a protein structure at 1.3 Å resolution. I have encountered one problem in this structure while refining in COOT. There are three disulfide bridges in my structure and if i used 2.0 Å resolution range in refmac then the density around disulfide bridges is ok but if i used 1.3 Å resolution range in refmac then red density is appeared along with blue one. Can anybody help me in this regard? I already played by increasing temperature factor of sulfur atoms but red density is still there. Thanks in advance Sadaf Iqbal PhD Scholar ICCBS, University of Karachi, Pakistan. Visiting Scientist University of Hamburg, Germany.
Re: [ccp4bb] mosflm gain
I have to say that I don't fully agree with James' recommendation to
adjust the GAIN in MOSFLM until the calculated SDFAC parameter in
SCALA is 1.0.
(Background information, the sigmas from Mosflm sd(I) are corrected in
SCALA according to
sd(I) corrected = SdFac * sqrt{sd(I)**2 + SdB*Ihl +
(SdAdd*Ihl)**2}
in order to get the best agreement between corrected sigmas and the
observed differences between symmtery/Friedel related intensities)
While I fully agree with his argument that systematic errors such as
absorption, etc give an error proportional to the intensity, and
therefore should be corrected by the SDADD term rather than SDFAC, in
any real world data set that I have come across the situation is not
so simple. Indeed, according to the usual treatment of errors there
should be no need for the SDB term in SCALA, but in practice it is
essential to have this term to be able to match corrected sigmas with
the observed differences between symmetry related reflections. It also
turns out that the three variable parameters SDFAC, SDB and SDADD are
highly correlated, so one can get rather different values for any
individual parameter from very similar datasets. Radiation damage is
certainly one source of error which would not be expected to follow a
simple error model, or non-isomorphism if multiple crystals have been
used.
Phil Evans is not entirely happy with the behaviour of the refinement
of these parameters and is in fact currently looking at this, but
there is a basic problem here that one is trying to use a simple
error model for a situation where (for whatever reason) it does not
really apply.
The sigma estimates from MOSFLM are only intended to give an estimate
of the random error in the intensities. In my opinion, trying to
account for systematic errors is best done at the point of merging the
data where much more information is available (ie symmetry related
measurements).
I would be most interested to hear of any examples where the default
value of the GAIN in MOSFLM is clearly wrong, but to the best of my
current knowledge the default GAIN is perfectly adequate.
Best wishes
Andrew
On 4 Mar 2011, at 19:47, James Holton wrote:
I have found that the best way to get the GAIN right in MOSFLM is
to have a look at the optimum Sdfac parameter at the end of SCALA
(the first of the three SDCORRection values). Specifically, if
SDFac is 1, then you need to increase the GAIN. This is because
SDFac1 means that the spots were noisier than MOSFLM thought they
should be, and if a given number of ADU is noisier than expected,
then there must have been fewer photons involved in generating the
signal. This means that the true gain was higher. Yes, there are
other sources of error, like shutter jitter, beam flicker,
calibration errors, absorption effects, scale factor errors, etc.
But these are all directly proportional to the intensity, and
therefore accounted for by adjusting SDadd (the last of the three
SDCORR values). SDfac accounts for noise proportional to the square
root of intensity, and only shot noise (like photon counting)
behaves like that.
David Waterman makes an excellent point that the point-spread
function (PSF) acts like a smoothing filter and makes the background
look less noisy than photon-counting error permits. This makes the
BGRATIO-estimated GAIN lower than the true GAIN. However, one can
argue that this is not always a bad thing, since the error in
measuring the intensity of a given area of flat background really is
better than photon counting. This is because you have the
smoothing effect of the PSF working for you: bringing in signal
from areas outside the region you are measuring (prior knowledge of
flatness if you will). However, this smoothing effect of the PSF
does not apply to spots because spot photons all arrive in
essentially the same place, and no smoothing will change the
intrinsic noise of the total number of photons that actually
arrived. The upshot of this is that we really need two different
values for GAIN, one for the background and one for the background-
subtracted spot intensity. The influence on sigma(I) would depend
on the relative contributions from the spot vs the background under
it. I am pretty sure this is not implemented.
It is perhaps interesting that there is also a third type of noise
which is independent of the spot intensity: read-out noise. This
used to be called fog on film detectors. Despite all the money we
spend on detectors that minimize it, there is no specific accounting
for read-out noise in MOSFLM or any other integration package I am
aware of. However, a trick to account for it is to simply lower
the ADCOFFSET. For example, using 1 A X-rays on an ADSC Q315r
detector in hwbin mode, the true GAIN is 1.8 ADU/photon, the
ADCOFFSET is 40 ADU, and the read-out noise is equivalent
Re: [ccp4bb] disulfide bridge in red density
Hi Sadaf It is also possible that the features you observe are also due to anisotropy of the ADPs. At 1.3A resolution you can perform anisotropic refinement. Give it a go and se if it helps. Best Roberto On 6 Mar 2011, at 18:57, sadaf iqbal wrote: Hello everyone, Recently, i have solved a protein structure at 1.3 Å resolution. I have encountered one problem in this structure while refining in COOT. There are three disulfide bridges in my structure and if i used 2.0 Å resolution range in refmac then the density around disulfide bridges is ok but if i used 1.3 Å resolution range in refmac then red density is appeared along with blue one. Can anybody help me in this regard? I already played by increasing temperature factor of sulfur atoms but red density is still there. Thanks in advance Sadaf Iqbal PhD Scholar ICCBS, University of Karachi, Pakistan. Visiting Scientist University of Hamburg, Germany.
Re: [ccp4bb] off-topic replay: glycerol in protein stock solutions
hello Bert u can also add 50mM to 100 mM Argine and glutamate either alone or combined together..it really works fine..although it will increase the protein stability many fold but i dont know whether it will impede crystallization or not.. others comments are welcome.. Faisal SLS, JNU New Delhi India On 3/5/11, Van Den Berg, Bert lambertus.vandenb...@umassmed.edu wrote: Hi Tom, Adding glycerol to (crystallization) buffers is a very common practice when working with membrane proteins. Many membrane proteins have been crystallized (perhaps even the majority) with glycerol, even up to 30% v/v. So, at least for membrane proteins, there is no problem. Bert On 3/4/11 8:25 PM, Brett, Thomas tbr...@dom.wustl.edu wrote: Hi guys: I know this has been asked before, but I want to get (current) opinions and observations once again. Every once in a while, you work with a protein that needs to have a little something added to the buffer to keep it soluble. The most common trick is addition of glycerol (usually 5-10%). I'm looking for general observations on this. I was of the opinion that this was usually a bad thing to do with protein stock that you intend to cyrstallize because glycerol will coat the protein in a non-homogeneous manner and make your homogeneous protein prep heterogeneous (in a way). Or do people usually have good luck crystallizing proteins that have to be stored in some glycerol? Or is there a better additive? Also, when having to use glycerol, do you put it on your sizing columns, etc? I am concerned with putting glycerol on my columns I may never be able to completely wash it away. What are your thoughts, community? thanks in advance, -tom
Re: [ccp4bb] off-topic replay: glycerol in protein stock solutions
Hi Tom-- We have had good luck crystallizing proteins with 5-10% glycerol. In the majority of these cases, the glycerol was included in the buffer for the final purification step. We have also seen several cases where 1,2-propanediol worked much better than glycerol. Hope this helps! annie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Brett, Thomas Sent: Friday, March 04, 2011 8:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off-topic replay: glycerol in protein stock solutions Hi guys: I know this has been asked before, but I want to get (current) opinions and observations once again. Every once in a while, you work with a protein that needs to have a little something added to the buffer to keep it soluble. The most common trick is addition of glycerol (usually 5-10%). I'm looking for general observations on this. I was of the opinion that this was usually a bad thing to do with protein stock that you intend to cyrstallize because glycerol will coat the protein in a non-homogeneous manner and make your homogeneous protein prep heterogeneous (in a way). Or do people usually have good luck crystallizing proteins that have to be stored in some glycerol? Or is there a better additive? Also, when having to use glycerol, do you put it on your sizing columns, etc? I am concerned with putting glycerol on my columns I may never be able to completely wash it away. What are your thoughts, community? thanks in advance, -tom
Re: [ccp4bb] off-topic replay: glycerol in protein stock solutions
hello everybody how does arginine and glutamate affect crystallization..what will be the better stabilizing agent during protein purification which wont affect crystallization or have minimal affect on crystallization..example..glycerol, sorbitol, glycine, poline, arginine, glutamate..what should be opted first. regards faisal On 3/7/11, Annie Hassell annie.m.hass...@gsk.com wrote: Hi Tom-- We have had good luck crystallizing proteins with 5-10% glycerol. In the majority of these cases, the glycerol was included in the buffer for the final purification step. We have also seen several cases where 1,2-propanediol worked much better than glycerol. Hope this helps! annie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Brett, Thomas Sent: Friday, March 04, 2011 8:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off-topic replay: glycerol in protein stock solutions Hi guys: I know this has been asked before, but I want to get (current) opinions and observations once again. Every once in a while, you work with a protein that needs to have a little something added to the buffer to keep it soluble. The most common trick is addition of glycerol (usually 5-10%). I'm looking for general observations on this. I was of the opinion that this was usually a bad thing to do with protein stock that you intend to cyrstallize because glycerol will coat the protein in a non-homogeneous manner and make your homogeneous protein prep heterogeneous (in a way). Or do people usually have good luck crystallizing proteins that have to be stored in some glycerol? Or is there a better additive? Also, when having to use glycerol, do you put it on your sizing columns, etc? I am concerned with putting glycerol on my columns I may never be able to completely wash it away. What are your thoughts, community? thanks in advance, -tom
Re: [ccp4bb] coot can't read my mtz and pdb file
It seems that your mtz doesn't have the map coefficients on it, that is why it is failing. Check if it has the columns FWT PHWT.. If it doesn't them coot will not automatically recognize it. A simple solution would be to run a cycle of rigid body refinement in Refmac and use the output mtz as the input for coot. The Refmac mtz will have the map coefficient columns on it. Good luck, Mario Sanches 2011/3/7 王瑞 wangrui...@gmail.com I have an ambiguous question that can't decide where it belongs . In fact, I was going to refine my data after molecular replacement, but coot can't read them. The mtz file was converted from a sca file by using Import Merged Data in ccp4.A screenshot is attached. -- Mario Sanches Postdoctoral Fellow Samuel Lunenfeld Research Institute Mount Sinai Hospital 600 University Ave Toronto - Ontario Canada M5G 1X5 http://ca.linkedin.com/in/mariosanches
Re: [ccp4bb] coot can't read my mtz and pdb file
Thanks for your suggestion. Your two suggestion is directing me go right to the heart of the matter.Thank you again. Best wishes Wangrui 2011/3/7 Mario Sanches mario...@gmail.com It seems that your mtz doesn't have the map coefficients on it, that is why it is failing. Check if it has the columns FWT PHWT.. If it doesn't them coot will not automatically recognize it. A simple solution would be to run a cycle of rigid body refinement in Refmac and use the output mtz as the input for coot. The Refmac mtz will have the map coefficient columns on it. Good luck, Mario Sanches 2011/3/7 王瑞 wangrui...@gmail.com I have an ambiguous question that can't decide where it belongs . In fact, I was going to refine my data after molecular replacement, but coot can't read them. The mtz file was converted from a sca file by using Import Merged Data in ccp4.A screenshot is attached. -- Mario Sanches Postdoctoral Fellow Samuel Lunenfeld Research Institute Mount Sinai Hospital 600 University Ave Toronto - Ontario Canada M5G 1X5 http://ca.linkedin.com/in/mariosanches
[ccp4bb] Software development post at Diamond Light Source, UK
Please see below details of a software development post available at Diamond. For full details please go to the web pages at: http://www.diamond.ac.uk/Home/Jobs/Current/DIA0608-TH.html Job Reference: DIA0608/TH Post Type: Full time / Permanent Division: Science Salary information: Circa £34k, dependent on assessment of previous relevant experience and qualifications. Application deadline: 31/03/2011 We are looking for a high calibre software engineer to join our scientific software team. The team have the responsibility for the provision of advanced data evaluation, analysis and visualisation software applications for both internal and external users of Diamond. These applications often exploit the very latest techniques to address the challenging requirements of a broad range of scientific disciplines including macromolecular crystallography through to nanostructures and materials science. You will operate within the scientific software team of experienced software scientists and engineers and will work with our beamline scientists to identify and define requirements for data analysis and visualisation applications and then ensure that they are implemented in a timely and effective way. It is important also to work with our data acquisition team to help optimise the whole scientific process at a Diamond beamline from data acquisition through to the experimenters leaving with high quality experimental results. The work will also involve enhancing and supporting the core features of a scientific data analysis workbench project at Diamond and to work with collaborators on other similar facilities in the world. Alun Ashton ___ Alun Ashton, alun.ash...@diamond.ac.uk Tel: +44 1235 778404 Scientific Software Team Leader, http://www.diamond.ac.uk/ Diamond Light Source, Chilton, Didcot, Oxon, OX11 0DE, U.K.
[ccp4bb] BCA Spring Meeting 2011: Last day of Early Bird Registration
Dear All, If you are planning to come to the British Crystallographic Association Spring Meeting in Keele this year today (7th March) is your last chance for the discounted early bird registration. You can register here: http://crystallography.org.uk/spring-meeting-2011 This year's meeting is jam packed with exciting sessions including: * Protein Crystallization: dealing with low solubility proteins and protein:ligand complexes * Membrane Proteins * Radiation Damage * Bacterial Cell Walls - synthesis, virulence and inhibitors * Hot structures * Twinning and Pseudosymmetry * New developments at Diamond * Macro and small molecule crystallography - a combined future * Time-resolved structural science as well as the now traditional Young Crystallographer Satellite meeting and, this year, a YC showcase session during the main meeting. Hope you are able to join us, Arwen Programme Chair -- Dr Arwen R Pearson RCUK Academic Research Fellow Astbury Centre for Structural Molecular Biology Astbury Building The University of Leeds LS2 9JT United Kingdom (t) +44 (0)113 34 33032 (e) a.r.pear...@leeds.ac.uk
[ccp4bb] PYMOL installation in openSUSE 11.3 (64 bit)
Hello, I was trying to install PYMOL in my openSUSE 11.3 as follows: cd /usr/local hena@a:/usr/local sudo svn co https://pymol.svn.sourceforge.net/svnroot/pymol/trunk/pymolpymol cd pymol hena@a:/usr/local/pymol sudo python setup.py install And I got the following error at the end: package init file 'modules/web/javascript/__init__.py' not found (or not a regular file) package init file 'modules/web/javascript/__init__.py' not found (or not a regular file) running build_ext building 'pymol._cmd' extension creating build/temp.linux-x86_64-2.6 creating build/temp.linux-x86_64-2.6/modules creating build/temp.linux-x86_64-2.6/modules/cealign creating build/temp.linux-x86_64-2.6/modules/cealign/src creating build/temp.linux-x86_64-2.6/ov creating build/temp.linux-x86_64-2.6/ov/src creating build/temp.linux-x86_64-2.6/layer0 creating build/temp.linux-x86_64-2.6/layer1 creating build/temp.linux-x86_64-2.6/layer2 creating build/temp.linux-x86_64-2.6/layer3 creating build/temp.linux-x86_64-2.6/layer4 creating build/temp.linux-x86_64-2.6/layer5 gcc -pthread -fno-strict-aliasing -DNDEBUG -fmessage-length=0 -O2 -Wall -D_FORTIFY_SOURCE=2 -fstack-protector -funwind-tables -fasynchronous-unwind-tables -g -fPIC -D_PYMOL_MODULE -D_PYMOL_INLINE -D_PYMOL_FREETYPE -D_PYMOL_LIBPNG -Iov/src -Ilayer0 -Ilayer1 -Ilayer2 -Ilayer3 -Ilayer4 -Ilayer5 -I/usr/include/freetype2 -Imodules/cealign/src -Imodules/cealign/src/tnt -I/usr/include/python2.6 -c modules/cealign/src/ccealignmodule.cpp -o build/temp.linux-x86_64-2.6/modules/cealign/src/ccealignmodule.o -ffast-math -funroll-loops -O3 In file included from modules/cealign/src/ccealignmodule.H:57:0, from modules/cealign/src/ccealignmodule.cpp:32: layer0/os_python.h:30:19: fatal error: Python.h: No such file or directory compilation terminated. error: command 'gcc' failed with exit status 1 Can someone advise me at this point or suggest the right pakage of PYMOL that I can install in open SUSE 11.3 Thanks... Hena
Re: [ccp4bb] PYMOL installation in openSUSE 11.3 (64 bit)
Hello Hena, I was trying to install PYMOL in my openSUSE 11.3 as follows: ... In file included from modules/cealign/src/ccealignmodule.H:57:0, from modules/cealign/src/ccealignmodule.cpp:32: layer0/os_python.h:30:19: fatal error: Python.h: No such file or directory You're missing the Python header files. There should be an OS package that provides this. You should really post your PyMOL questions to the PyMOL mailing list though. -ben -- | Ben Eisenbraun | SBGrid Consortium | http://sbgrid.org | | Harvard Medical School | http://hms.harvard.edu |
Re: [ccp4bb] PYMOL installation in openSUSE 11.3 (64 bit)
Hi, If you're missing Python.h (since your compiler complains about it), I guess you're missing the development package of python, whatever that's called in SUSE. Probably python-devel, according to this: http://www.novell.com/products/linuxpackages/opensuse/python-devel.html Good luck, Mark On 7 March 2011 17:21, Hena Dutta hdutt...@gmail.com wrote: Hello, I was trying to install PYMOL in my openSUSE 11.3 as follows: cd /usr/local hena@a:/usr/local sudo svn co https://pymol.svn.sourceforge.net/svnroot/pymol/trunk/pymolpymol cd pymol hena@a:/usr/local/pymol sudo python setup.py install And I got the following error at the end: package init file 'modules/web/javascript/__init__.py' not found (or not a regular file) package init file 'modules/web/javascript/__init__.py' not found (or not a regular file) running build_ext building 'pymol._cmd' extension creating build/temp.linux-x86_64-2.6 creating build/temp.linux-x86_64-2.6/modules creating build/temp.linux-x86_64-2.6/modules/cealign creating build/temp.linux-x86_64-2.6/modules/cealign/src creating build/temp.linux-x86_64-2.6/ov creating build/temp.linux-x86_64-2.6/ov/src creating build/temp.linux-x86_64-2.6/layer0 creating build/temp.linux-x86_64-2.6/layer1 creating build/temp.linux-x86_64-2.6/layer2 creating build/temp.linux-x86_64-2.6/layer3 creating build/temp.linux-x86_64-2.6/layer4 creating build/temp.linux-x86_64-2.6/layer5 gcc -pthread -fno-strict-aliasing -DNDEBUG -fmessage-length=0 -O2 -Wall -D_FORTIFY_SOURCE=2 -fstack-protector -funwind-tables -fasynchronous-unwind-tables -g -fPIC -D_PYMOL_MODULE -D_PYMOL_INLINE -D_PYMOL_FREETYPE -D_PYMOL_LIBPNG -Iov/src -Ilayer0 -Ilayer1 -Ilayer2 -Ilayer3 -Ilayer4 -Ilayer5 -I/usr/include/freetype2 -Imodules/cealign/src -Imodules/cealign/src/tnt -I/usr/include/python2.6 -c modules/cealign/src/ccealignmodule.cpp -o build/temp.linux-x86_64-2.6/modules/cealign/src/ccealignmodule.o -ffast-math -funroll-loops -O3 In file included from modules/cealign/src/ccealignmodule.H:57:0, from modules/cealign/src/ccealignmodule.cpp:32: layer0/os_python.h:30:19: fatal error: Python.h: No such file or directory compilation terminated. error: command 'gcc' failed with exit status 1 Can someone advise me at this point or suggest the right pakage of PYMOL that I can install in open SUSE 11.3 Thanks... Hena
Re: [ccp4bb] PYMOL installation in openSUSE 11.3 (64 bit)
Sorry for my earlier non CCP4 posting in CCP4BB. hena On Mon, Mar 7, 2011 at 9:30 AM, Ben Eisenbraun b...@hkl.hms.harvard.eduwrote: Hello Hena, I was trying to install PYMOL in my openSUSE 11.3 as follows: ... In file included from modules/cealign/src/ccealignmodule.H:57:0, from modules/cealign/src/ccealignmodule.cpp:32: layer0/os_python.h:30:19: fatal error: Python.h: No such file or directory You're missing the Python header files. There should be an OS package that provides this. You should really post your PyMOL questions to the PyMOL mailing list though. -ben -- | Ben Eisenbraun | SBGrid Consortium | http://sbgrid.org | | Harvard Medical School | http://hms.harvard.edu |
Re: [ccp4bb] determining the domain for overexpression and crystallization
Hi, To start with it would be great if you look in to the secondary structure prediction of the sequence using any of the standard servers like PSIPRED, JPRED etc. Many more available at expasy site http://ca.expasy.org/tools/. Whatever construct you finally choose to make just remember the standard rule that we generally follow is to avoid deleting the alpha helices and beta sheets. You can design your initial primers so as to obtain the complete amplification of these secondary structures from any part within the protein. You can even use the various modules of the following online available server http://scratch.proteomics.ics.uci.edu/ to have an idea of the intrinsically disordered regions in the protein, transmemebrane regions and disulfide bonds that would certainly help you in initiating in the right direction. Best wishes Gauri On Mon, Mar 7, 2011 at 4:10 AM, Xianhui Wu wuxian...@gmail.com wrote: Dear all, Before we try to study the crystal structure of an unknown protein, we need to determine the sequence that can fold into a compact and stable 3D domain. What kinds of methods can we choose? -- Best regards, XH Wu
Re: [ccp4bb] determining the domain for overexpression and crystallization
for an experimental way to determine soluble domains see the following paper: ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets. Yumerefendi H, Tarendeau F, Mas PJ, Hart DJ. J Struct Biol. 2010 Oct;172(1):66-74. Epub 2010 Mar 4. Review. PMID: 20206698 [PubMed - indexed for MEDLINE] Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij/index.php?l=1 On 7 Mar 2011, at 19:18, gauri misra wrote: Hi, To start with it would be great if you look in to the secondary structure prediction of the sequence using any of the standard servers like PSIPRED, JPRED etc. Many more available at expasy site http://ca.expasy.org/tools/. Whatever construct you finally choose to make just remember the standard rule that we generally follow is to avoid deleting the alpha helices and beta sheets. You can design your initial primers so as to obtain the complete amplification of these secondary structures from any part within the protein. You can even use the various modules of the following online available server http://scratch.proteomics.ics.uci.edu/ to have an idea of the intrinsically disordered regions in the protein, transmemebrane regions and disulfide bonds that would certainly help you in initiating in the right direction. Best wishes Gauri On Mon, Mar 7, 2011 at 4:10 AM, Xianhui Wu wuxian...@gmail.com wrote: Dear all, Before we try to study the crystal structure of an unknown protein, we need to determine the sequence that can fold into a compact and stable 3D domain. What kinds of methods can we choose? -- Best regards, XH Wu
[ccp4bb] Ubuntu 10.10 64-bit and COOT
Hi, I just installed CCP4-6.1.13 on my ubuntu (10.10) 64 -bit laptop. I can run CCP4i successfully. But when i try to run coot, i get error : No command 'coot' found. Can someone help me to fix the problem? with regards, Pranjal
Re: [ccp4bb] Ubuntu 10.10 64-bit and COOT
coot has to be installed separately, it is not included in ccp4 6.1.13. It is best to get coot binaries from Paul Emsley directly, just google on coot emsley and look for download pages, then choose ubuntu tarball. Best, Eugene. On 7 Mar 2011, at 18:27, Pranjal Mahanta wrote: Hi, I just installed CCP4-6.1.13 on my ubuntu (10.10) 64 -bit laptop. I can run CCP4i successfully. But when i try to run coot, i get error : No command 'coot' found. Can someone help me to fix the problem? with regards, Pranjal
Re: [ccp4bb] Ubuntu 10.10 64-bit and COOT
I install this version of coot: coot-Linux-x86_64-ubuntu-9.04-gtk2 for ubuntu and it works fine Best Lionel Le 07/03/2011 19:35, Eugene Krissinel a écrit : coot has to be installed separately, it is not included in ccp4 6.1.13. It is best to get coot binaries from Paul Emsley directly, just google on coot emsley and look for download pages, then choose ubuntu tarball. Best, Eugene. On 7 Mar 2011, at 18:27, Pranjal Mahanta wrote: Hi, I just installed CCP4-6.1.13 on my ubuntu (10.10) 64 -bit laptop. I can run CCP4i successfully. But when i try to run coot, i get error : No command 'coot' found. Can someone help me to fix the problem? with regards, Pranjal
[ccp4bb] Question about movie making
All, Pardon the slightly off-topic question. We would like to use Pymol and generate movies with it on a WINDOWS computer. We are very familiar with Pymol and how to make the correct views etc. We write the individual frames out into PNG files. So what is left to do, is to stitch together the PNG images to an MPEG file. On Linux you could do this with mencoder. But we would like to do this on Windows and installing mencoder on windows is possible but not easy. We have found videomach, which costs a very small amount of money to obtain. Similarly, Adobe Premiere is affordable for an educational institution. We don't mind paying, but before we go there, does anyone have experience with making MPEG movies from PNG files on windows? What is your experience with quality of product and especially with user friendliness? If you have any insight, we would appreciate your comments. Thanks! Mark van der Woerd Colorado State University
Re: [ccp4bb] Question about movie making
Hey Mark, In Window PC, I used ImageReady, which comes with Photoshop. see this: http://sage.ucsc.edu/~wgscott/xtal/movie/making_movie.html Alternatively, using Mac version of Pymol, you can easily make a movie and save it in quick time format see this: www.chem.umass.edu/.../*BMS2006*Files/.../PymolMorphingMovie.pdf HTH, Matt On Mon, Mar 7, 2011 at 1:21 PM, mjvdwo...@netscape.net wrote: All, Pardon the slightly off-topic question. We would like to use Pymol and generate movies with it on a WINDOWS computer. We are very familiar with Pymol and how to make the correct views etc. We write the individual frames out into PNG files. So what is left to do, is to stitch together the PNG images to an MPEG file. On Linux you could do this with mencoder. But we would like to do this on Windows and installing mencoder on windows is possible but not easy. We have found videomach, which costs a very small amount of money to obtain. Similarly, Adobe Premiere is affordable for an educational institution. We don't mind paying, but before we go there, does anyone have experience with making MPEG movies from PNG files on windows? What is your experience with quality of product and especially with user friendliness? If you have any insight, we would appreciate your comments. Thanks! Mark van der Woerd Colorado State University -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Fairchild D143, MC 5126 Stanford School of Medicine Stanford, CA 94305-5432 Lab: 650-724-3306 Alternative Email: matt...@stanford.edu
Re: [ccp4bb] Question about movie making
sorry, this is the right link for the mac-pymol movie: people.chem.umass.edu/jhardy/BMS2006Files/pymol%20morphing/PymolMorphingMovie.pdf On Mon, Mar 7, 2011 at 2:07 PM, Matthew Chu linghonmatt...@gmail.comwrote: Hey Mark, In Window PC, I used ImageReady, which comes with Photoshop. see this: http://sage.ucsc.edu/~wgscott/xtal/movie/making_movie.html Alternatively, using Mac version of Pymol, you can easily make a movie and save it in quick time format see this: www.chem.umass.edu/.../*BMS2006*Files/.../PymolMorphingMovie.pdf HTH, Matt On Mon, Mar 7, 2011 at 1:21 PM, mjvdwo...@netscape.net wrote: All, Pardon the slightly off-topic question. We would like to use Pymol and generate movies with it on a WINDOWS computer. We are very familiar with Pymol and how to make the correct views etc. We write the individual frames out into PNG files. So what is left to do, is to stitch together the PNG images to an MPEG file. On Linux you could do this with mencoder. But we would like to do this on Windows and installing mencoder on windows is possible but not easy. We have found videomach, which costs a very small amount of money to obtain. Similarly, Adobe Premiere is affordable for an educational institution. We don't mind paying, but before we go there, does anyone have experience with making MPEG movies from PNG files on windows? What is your experience with quality of product and especially with user friendliness? If you have any insight, we would appreciate your comments. Thanks! Mark van der Woerd Colorado State University -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Fairchild D143, MC 5126 Stanford School of Medicine Stanford, CA 94305-5432 Lab: 650-724-3306 Alternative Email: matt...@stanford.edu -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Fairchild D143, MC 5126 Stanford School of Medicine Stanford, CA 94305-5432 Lab: 650-724-3306 Alternative Email: matt...@stanford.edu
Re: [ccp4bb] Question about movie making
On Mon, Mar 7, 2011 at 1:21 PM, mjvdwo...@netscape.net wrote: We have found videomach, which costs a very small amount of money to obtain. Similarly, Adobe Premiere is affordable for an educational institution. We don't mind paying, but before we go there, does anyone have experience with making MPEG movies from PNG files on windows? What is your experience with quality of product and especially with user friendliness? I used VideoMach for exactly this circa 2004, and it was excellent, despite only being available on Windows. I would have preferred to do everything on Linux, but I couldn't find any other (free or inexpensive) program that was that easy to use and would generate MPEGs that were well-compressed, high-quality, and ran well on any computer. But why not download the trial version and see how you like it? (It will watermark your movies, which appears to be a new feature since I used it, but at least it'll give you a feel for the interface, etc.) -Nat
Re: [ccp4bb] Question about movie making
Hi Mark, just use the Windows Movie Maker - it's easy to use and comes along with Windows (or can be downloaded via microsoft.com). This will help you to generate .wmv output files of any kind of video and / or picture files you provide as input files. The best possible quality output format in Windows Movie Maker is 1080p, which should give you a good quality. If there is still the need to convert it to .mpeg, you can use ASF Converter, which is freeware as well (see here: http://www.boilsoft.com/download.html ). Matthias Date: Mon, 7 Mar 2011 16:21:02 -0500 From: mjvdwo...@netscape.net Subject: [ccp4bb] Question about movie making To: CCP4BB@JISCMAIL.AC.UK All, Pardon the slightly off-topic question. We would like to use Pymol and generate movies with it on a WINDOWS computer. We are very familiar with Pymol and how to make the correct views etc. We write the individual frames out into PNG files. So what is left to do, is to stitch together the PNG images to an MPEG file. On Linux you could do this with mencoder. But we would like to do this on Windows and installing mencoder on windows is possible but not easy. We have found videomach, which costs a very small amount of money to obtain. Similarly, Adobe Premiere is affordable for an educational institution. We don't mind paying, but before we go there, does anyone have experience with making MPEG movies from PNG files on windows? What is your experience with quality of product and especially with user friendliness? If you have any insight, we would appreciate your comments. Thanks! Mark van der Woerd Colorado State University
Re: [ccp4bb] Question about movie making
Dear Mark, I put together a post about creating a movie using PyMOL, eMovie (free) and Adobe ImageReady CS2 to generate a Quicktime movie. http://www.p212121.com/2009/03/24/easily-make-movie-pymol/ I hope that helps. Take Care, Sean P212121 http://store.p212121.com/
Re: [ccp4bb] Question about movie making
Hi Mark (and Matthias), I'm not sure if Windows Movie Maker is the same as (or maybe the predecessor to?) Windows Live Movie Maker (http://explore.live.com/windows-live-movie-maker?os=other), but this is what I used recently to string together a series of png images from pymol to make a wmv movie of a protein motion. With Windows 7, the movie maker is no longer included by default with the OS so it must be downloaded (for free) and installed. Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Mon, 7 Mar 2011, Matthias Haffke wrote: Hi Mark, just use the Windows Movie Maker - it's easy to use and comes along with Windows (or can be downloaded via microsoft.com). This will help you to generate .wmv output files of any kind of video and / or picture files you provide as input files. The best possible quality output format in Windows Movie Maker is 1080p, which should give you a good quality. If there is still the need to convert it to .mpeg, you can use ASF Converter, which is freeware as well (see here: http://www.boilsoft.com/download.html ). Matthias _ Date: Mon, 7 Mar 2011 16:21:02 -0500 From: mjvdwo...@netscape.net Subject: [ccp4bb] Question about movie making To: CCP4BB@JISCMAIL.AC.UK All, Pardon the slightly off-topic question. We would like to use Pymol and generate movies with it on a WINDOWS computer. We are very familiar with Pymol and how to make the correct views etc. We write the individual frames out into PNG files. So what is left to do, is to stitch together the PNG images to an MPEG file. On Linux you could do this with mencoder. But we would like to do this on Windows and installing mencoder on windows is possible but not easy. We have found videomach, which costs a very small amount of money to obtain. Similarly, Adobe Premiere is affordable for an educational institution. We don't mind paying, but before we go there, does anyone have experience with making MPEG movies from PNG files on windows? What is your experience with quality of product and especially with user friendliness? If you have any insight, we would appreciate your comments. Thanks! Mark van der Woerd Colorado State University
Re: [ccp4bb] Question about movie making
I use ImageJ for this purpose. There is a flag for something like 'use virtual stacks' or 'use virtual memory' that I needed to prevent crashing. Nat On Mon, Mar 7, 2011 at 5:30 PM, Sean Seaver s...@p212121.com wrote: Dear Mark, I put together a post about creating a movie using PyMOL, eMovie (free) and Adobe ImageReady CS2 to generate a Quicktime movie. http://www.p212121.com/2009/03/24/easily-make-movie-pymol/ I hope that helps. Take Care, Sean P212121 http://store.p212121.com/
Re: [ccp4bb] Question about movie making
VirtualDub is the (absolutely free) program of choice, I'd say. Good luck, Matthias Am 07/03/2011 21:21, schrieb mjvdwo...@netscape.net: All, Pardon the slightly off-topic question. We would like to use Pymol and generate movies with it on a WINDOWS computer. We are very familiar with Pymol and how to make the correct views etc. We write the individual frames out into PNG files. So what is left to do, is to stitch together the PNG images to an MPEG file. On Linux you could do this with mencoder. But we would like to do this on Windows and installing mencoder on windows is possible but not easy. We have found videomach, which costs a very small amount of money to obtain. Similarly, Adobe Premiere is affordable for an educational institution. We don't mind paying, but before we go there, does anyone have experience with making MPEG movies from PNG files on windows? What is your experience with quality of product and especially with user friendliness? If you have any insight, we would appreciate your comments. Thanks! Mark van der Woerd Colorado State University -- Dr. Matthias Zebisch Universität Leipzig Biotechnologisch-Biomedizinisches Zentrum Strukturanalytik von Biopolymeren Deutscher Platz 5 04103 Leipzig Germany Phone: 0049-341-97-31323 (lab) -31312 (office) Fax : 0049-341-97-31319 email: matthias.zebi...@bbz.uni-leipzig.de
[ccp4bb] Structural Biology position at Monsanto
Friends, I would like to draw your attention to the announcement below. Informal inquiries are welcome (directed to me) however actual applications should be directed to the web site listed in the announcement (use requisition number 003Y4 to find the position and apply). Thank you for your consideration, Artem Structural Biologist/ Protein Crystallization Scientist- Req. # 003Y4 Crystallization Specialist - Chesterfield, MO Monsanto Structural Biology team is interested in receiving applications for the position of a Crystallization Specialist. The ideal candidate is a practicing crystallization enthusiast with hands-on, current expertise in all modern techniques used to produce diffraction-quality crystals of challenging macromolecules. Expertise in both robotic and manual screening, follow-up, and condition optimization is a must; the ideal candidate has also demonstrated artful use of protein chemistry, rational mutagenesis and other sample modification techniques to accomplish the ultimate goal of producing useful crystals of the target molecule. Familiarity with a scripting language (PERL, Python) and experience with molecular biology/protein purification are highly desirable. Duties associated with this position include: - sustained efforts on parallel crystallization of multiple novel proteins, protein complexes, and other macromolecular entities - development and testing of new approaches to successful and speedy crystallization of macromolecules - support of diverse crystallization and imaging robotics - active participation in structure-driven protein discovery and protein optimization projects - gene-to-structure ownership of select structural projects - record-keeping, notebooking, and other IP associated activities Structural Biology group at Monsanto is an inter-disciplinary nexus in a highly collaborative team-based environment. We interact with numerous project teams in areas spanning the entire spectrum of Discovery activities - together we catalyze the discovery and optimization of protein leads for novel agronomic traits and the development of innovative biotechnologies. Our expression, purification, automated crystallization/imaging, data collection and structure solution facilities are state of the art and are updated on a regular basis. Qualifications - 2 plus years experience of demonstrated and sustained practical expertise in crystallization of macromolecules (several years practicing crystallization in a multi-project environment) - demonstrated interest in developing/improving protein crystallization technologies - The ideal candidate will have successfully crystallized at least 20 proteins - diverse basic science background: molecular structure, protein crystallization/crystallography, molecular biology, biophysics, rudimentary programming - M.Sc. or Ph.D. in a relevant discipline. Strong B.Sc. candidates can also be considered Please note that *only applications submitted through the Monsanto career web site will be considered for this position*. To view a more complete and detailed job description of this exciting position, please visit our website at www.monsanto.com. We offer very competitive salaries and an extensive benefits package. Monsanto values diversity and is an equal opportunity employer. M/F/D/V
[ccp4bb] Post-doc position at IBS, Grenoble
Dear colleagues, A post-doctoral position will be opened at the IBS in Grenoble (see below). I would be very grateful if you could forward the information below to potential candidates. Post-doc position at the Institut de Biologie Structurale, Grenoble, France *Structural Biology of DNA repair enzymes* A post-doctoral position in Structural Biology is available at the IBS (www.ibs.fr http://www.ibs.fr/) in Grenoble. This position is funded for two years as part of a ATIP-AVENIR grant. I will be moving to IBS to start up my new research team in May 2011 and the post-doc position would be expected to start mid-2011. Our research will focus on the study of essential DNA repair enzymes and more particularly on the dynamics of DNA repair and the mechanisms involved in DNA damage recognition. The research will entail the expression, purification and crystallisation of these key proteins, but also biochemical studies of these enzymes /in vitro/. Low and high-resolution studies will then be carried out on the proteins alone and in complex with their DNA substrates. We are seeking an enthusiastic and dedicated individual to carry out this project. The ideal candidate has a PhD in Structural Biology, Biochemistry or equivalent with extensive experience in protein purification and protein crystallography. Additional experience in the study of protein-DNA interactions and/or eukaryotic cell expression would be a clear advantage. Informal enquiries and applications in the form of a CV including publication list, short summary of research activities and contact details of two academic referees should be sent to the following address: timm...@esrf.fr mailto:timm...@esrf.fr. Deadline: 31^st March 2011. Joanna TIMMINS -- Dr. Joanna Timmins Structural Biology Group ESRF B.P. 220, 6 rue Jules Horowitz, F-38043 Grenoble Cedex, France Tel: (33) 4 76 88 25 63
