[ccp4bb] ccp4-6.1.0-core-src.tar.gz TAR error
Dear Charles, to your information: I just downloaded the Version 6.1 source tar file to my x86_64 Linux box (SuSE 10.2, AMD64) and got the following tar error message. All other (LINUX-386 and both COOCH and PHASER) tar files were fine!! ---> SNIP ... ccp4-6.1.0/lib/data/monomers/ps.resource ccp4-6.1.0/lib/data/monomers/full_names.list ccp4-6.1.0/lib/data/monomers/mon_lib_ind.cif ccp4-6.1.0/lib/data/monomers/mon_lib_ind2.cif tar: Skipping to next header tar: Archive contains obsolescent base-64 headers gzip: stdin: invalid compressed data--format violated tar: Child returned status 1 tar: Error exit delayed from previous errors ---> SNIP May be a corrupt file? Regards Michael -- Dr. Michael Weyand mail: [EMAIL PROTECTED] Max-Planck-Institut fuer molekulare Physiologie, Otto-Hahn-Strasse 11 D-44227 Dortmund, Germany, fon: +49(0)231-133-2738, fax: +49(0)231-133-2797
Re: [ccp4bb] ccp4-6.1.0-core-src.tar.gz TAR error
Dear Michael, How did you do the download? I have just downloaded this file using the old fashioned command line ftp method, and it worked fine. I expect that there has been an error somewhere in the transmission. If you fetch the file from: ftp ftp.ccp4.ac.uk cd ccp4/6.1 get ccp4-6.1.0-core-src.tar.gz you should find that everything is fine. If not, please get in touch directly with the helpdesk at [EMAIL PROTECTED] and we will try to work out what is happening. Thanks & best wishes, Graeme -Original Message- From: CCP4 bulletin board on behalf of Michael Weyand Sent: Thu 12/4/2008 8:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] ccp4-6.1.0-core-src.tar.gz TAR error Dear Charles, to your information: I just downloaded the Version 6.1 source tar file to my x86_64 Linux box (SuSE 10.2, AMD64) and got the following tar error message. All other (LINUX-386 and both COOCH and PHASER) tar files were fine!! ---> SNIP ... ccp4-6.1.0/lib/data/monomers/ps.resource ccp4-6.1.0/lib/data/monomers/full_names.list ccp4-6.1.0/lib/data/monomers/mon_lib_ind.cif ccp4-6.1.0/lib/data/monomers/mon_lib_ind2.cif tar: Skipping to next header tar: Archive contains obsolescent base-64 headers gzip: stdin: invalid compressed data--format violated tar: Child returned status 1 tar: Error exit delayed from previous errors ---> SNIP May be a corrupt file? Regards Michael -- Dr. Michael Weyand mail: [EMAIL PROTECTED] Max-Planck-Institut fuer molekulare Physiologie, Otto-Hahn-Strasse 11 D-44227 Dortmund, Germany, fon: +49(0)231-133-2738, fax: +49(0)231-133-2797
Re: [ccp4bb] Temperature factor discrepancy
Hi Wim,
I think that the main question I would ask is how you calculate the
Wilson B factor with 3.0-3.5 A data ...
We have done a rather big study of about 12,000 structures and below
2.5 A, how you calculate the B can result to
major discrepancies. There is hardly a line to get the gradient of. If
you use some scaling to an ideal plot like BEST or ARP/wARP
(same algorithm there) the minimizer and 'spikes' can have a huge
influence.
btw, have you optimized in both cases the Bfactor restraints weight ?
I wonder if that would change things (it will change the FreeR ...)
See Ian Tickle's article
http://journals.iucr.org/d/issues/2007/12/00/gx5119/index.html
I would also expect to use tighter geometry in this resolution btw ...!
(don't be fooled if the current rmsd gives the lower Rfree: if you
change B weight this will very likely change!)
btw, why not use TLS ? For one thing you will get better FreeR and
hopefully better B factor refinement ...!
A.
PS If you want to try things overnight I tend to do something like this:
#!/bin/csh -f
#
echo "xweight bweight rfactorrfree rmszbonds" > stats.log
foreach xweight ( 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0.18 0.20 0.25 0.30)
foreach bweight ( 0.1 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.5 3.0 )
#
#Put you REFMAC script here
# As you can eg obtain it from 'Run and View Command file
# make sure it has these two lines
weight MATRIX ${xweight}
temp ${bweight} 2.0 3.0 2.0 3.0
#
###
echo -n ${xweight} ${bweight} >> stats.log
#The line below will grep the right thing if you do 20 cycles of
refinement 10 TLS, 10 normal in my case
awk '(NF=11){if ($1==20) print $2, $3, $8}' refmac_${xweight}_$
{bweight}.log >> stats.log
end
end
#
On Dec 3, 2008, at 16:59, Wim Burmeister wrote:
I should have given the precision that the problem remains
unaffected by a change of the resolution range (even if I use for
example only 4.5 to 3 A resolution).
I am not using TLS and the data are quite isotropic. Rcryst values
are as expected for such a structure.
Anopther 3.5 A dataset does not show the problem (right column).
Wilson plot B-factor [Å2]
66
43
Refinement
Rcryst
0.186 (0.259)
0.190 (0.239)
Rfree
0.268 (0.408)
0.256 (0.278)
Rms deviations from ideal bondlengths (Å)
0.020
0.018
Rms deviations from ideal bond angles (°)
2.0
1.9
Average B-factor [Å2]
39
46
Values for the highest resolution bin are given in brackets.
Cheers
Wim
Wim Burmeister a écrit :
Dear all,
I have a 3 A structure refined with REFMAC which gives consistently
average atomic B-factors of 40 A2, whereas the B factor from a
Wilson plot is about 60 A2. Is there any explanation for such a
discrepancy?
There are no obvious problems:
No twinning, spacegroup P21 with two molecules in the asu, no
proper ncs symmetry. No pathologic Wilson plot, complete and
redundant dataset (although collected on several crystals with
serious problems due to radiation damage).
Interestingly, the Wilson plot of the Fcalc values is about 60 A2
as for Fobs in the output dataset.
Yours
Wim
--
***
Wim Burmeister
Professeur, Membre de l'Institut Universitaire de France
Unit of Virus Host Cell Interactions (UVHCI) UMR5233 UJF-EMBL-CNRS
6 rue Jules Horowitz
B.P. 181, F-38042 Grenoble Cedex 9 FRANCE
E-mail: [EMAIL PROTECTED]
Tel:+33 (0) 476 20 72 82 Fax: +33 (0) 476 20 94 00
http://www.uvhci.fr
***
Re: [ccp4bb] ccp4-6.1.0-core-src.tar.gz TAR error
A fast and simple method to check whether the downloaded file is corrupt is to calculate checksums, e.g. md5 or sha - corresponding programs should be available on most Unix/ Linux systems. It depends, however, on York calculating the checksum and putting it next to the actual archive for downloading. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Thu, 4 Dec 2008, Winter, G (Graeme) wrote: Dear Michael, How did you do the download? I have just downloaded this file using the old fashioned command line ftp method, and it worked fine. I expect that there has been an error somewhere in the transmission. If you fetch the file from: ftp ftp.ccp4.ac.uk cd ccp4/6.1 get ccp4-6.1.0-core-src.tar.gz you should find that everything is fine. If not, please get in touch directly with the helpdesk at [EMAIL PROTECTED] and we will try to work out what is happening. Thanks & best wishes, Graeme -Original Message- From: CCP4 bulletin board on behalf of Michael Weyand Sent: Thu 12/4/2008 8:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] ccp4-6.1.0-core-src.tar.gz TAR error Dear Charles, to your information: I just downloaded the Version 6.1 source tar file to my x86_64 Linux box (SuSE 10.2, AMD64) and got the following tar error message. All other (LINUX-386 and both COOCH and PHASER) tar files were fine!! ---> SNIP ... ccp4-6.1.0/lib/data/monomers/ps.resource ccp4-6.1.0/lib/data/monomers/full_names.list ccp4-6.1.0/lib/data/monomers/mon_lib_ind.cif ccp4-6.1.0/lib/data/monomers/mon_lib_ind2.cif tar: Skipping to next header tar: Archive contains obsolescent base-64 headers gzip: stdin: invalid compressed data--format violated tar: Child returned status 1 tar: Error exit delayed from previous errors ---> SNIP May be a corrupt file? Regards Michael -- Dr. Michael Weyand mail: [EMAIL PROTECTED] Max-Planck-Institut fuer molekulare Physiologie, Otto-Hahn-Strasse 11 D-44227 Dortmund, Germany, fon: +49(0)231-133-2738, fax: +49(0)231-133-2797
[ccp4bb] PHD POSITION IN STRUCTURAL BIOLOGY (FPI GRANT)
PREDOCTORAL POSITION IN STRUCTURAL BIOLOGY Applications are invited for a PhD position within Prof. Miquel Colls laboratory at the Institute for Research in Biomedicine / Institut de Biologia Molecular de Barcelona (CSIC) to work on the Project Estudio de Proteínas de unión a DNA. The position is supported by a recently awarded FPI grant linked to a national project awarded by the Spanish Science & Innovation Ministry (MICINN). The Institute is located in the stimulating scientific and cultural environment of the Barcelona Science Park, which also hosts public and private research centres and several pharmaceutical and biotech enterprises as well as scientific and technological services and state-of-the-art technology platforms. Candidates should be European Citizens and hold a degree in Biology, Biotechnology or similar. Last year students who end their studies by June 2009 may also apply. Those interested should contact (preferably by email): Vanessa Llobet Secretary Structural and Computational Biology Programme Institute for Research in Biomedicine (IRB Barcelona) Parc Científic de Barcelona Baldiri Reixac 10-12 08028 Barcelona, Spain Email: [EMAIL PROTECTED] Applicants should send a copy of their academic records as well as the average grade of their finished studies as soon as possible and no later than January 15th, 2009.
Re: [ccp4bb] ccp4-6.1.0-core-src.tar.gz TAR error
Hi Tim, You are the third person to make this very sensible suggestion. Once we actually release the software we will be sure to do this! The limiting step at the moment is that many small changes are going in (you will have noticed the absence of a release announcement) which of course change the checksums. It is not "York" but the CCP4 core team at Daresbury who will need to do this, and we will. Thanks & best wishes, Graeme -Original Message- From: Tim Gruene [mailto:[EMAIL PROTECTED] Sent: 04 December 2008 10:11 To: Winter, G (Graeme) Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] ccp4-6.1.0-core-src.tar.gz TAR error A fast and simple method to check whether the downloaded file is corrupt is to calculate checksums, e.g. md5 or sha - corresponding programs should be available on most Unix/ Linux systems. It depends, however, on York calculating the checksum and putting it next to the actual archive for downloading. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Thu, 4 Dec 2008, Winter, G (Graeme) wrote: > Dear Michael, > > How did you do the download? I have just downloaded this file using the old fashioned command line ftp method, and it worked fine. I expect that there has been an error somewhere in the transmission. > > If you fetch the file from: > > ftp ftp.ccp4.ac.uk > cd ccp4/6.1 > get ccp4-6.1.0-core-src.tar.gz > > you should find that everything is fine. If not, please get in touch directly with the helpdesk at [EMAIL PROTECTED] and we will try to work out what is happening. > > Thanks & best wishes, > > Graeme > > > > > -Original Message- > From: CCP4 bulletin board on behalf of Michael Weyand > Sent: Thu 12/4/2008 8:59 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] ccp4-6.1.0-core-src.tar.gz TAR error > > Dear Charles, > > to your information: > > I just downloaded the Version 6.1 source tar file to my x86_64 Linux box > (SuSE 10.2, AMD64) and got the following tar error message. > All other (LINUX-386 and both COOCH and PHASER) tar files were fine!! > > ---> SNIP > ... > ccp4-6.1.0/lib/data/monomers/ps.resource > ccp4-6.1.0/lib/data/monomers/full_names.list > ccp4-6.1.0/lib/data/monomers/mon_lib_ind.cif > ccp4-6.1.0/lib/data/monomers/mon_lib_ind2.cif > tar: Skipping to next header > tar: Archive contains obsolescent base-64 headers > > gzip: stdin: invalid compressed data--format violated > tar: Child returned status 1 > tar: Error exit delayed from previous errors > > ---> SNIP > > May be a corrupt file? > > Regards > Michael > > -- > Dr. Michael Weyand mail: [EMAIL PROTECTED] > Max-Planck-Institut fuer molekulare Physiologie, Otto-Hahn-Strasse 11 > D-44227 Dortmund, Germany, fon: +49(0)231-133-2738, fax: +49(0)231-133-2797 >
Re: [ccp4bb] ccp4-6.1.0-core-src.tar.gz TAR error
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 I have to apologise: For some reason I assumed that the ccp4-server (or the people who package the suite) was located in York, which - as I have been pointed out to - is not true. I would like to apologise for my prejudice. Anyway I did not mean to blame anyone, my comment was meant as a suggestion. Tim - -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Thu, 4 Dec 2008, Tim Gruene wrote: A fast and simple method to check whether the downloaded file is corrupt is to calculate checksums, e.g. md5 or sha - corresponding programs should be available on most Unix/ Linux systems. It depends, however, on York calculating the checksum and putting it next to the actual archive for downloading. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Thu, 4 Dec 2008, Winter, G (Graeme) wrote: Dear Michael, How did you do the download? I have just downloaded this file using the old fashioned command line ftp method, and it worked fine. I expect that there has been an error somewhere in the transmission. If you fetch the file from: ftp ftp.ccp4.ac.uk cd ccp4/6.1 get ccp4-6.1.0-core-src.tar.gz you should find that everything is fine. If not, please get in touch directly with the helpdesk at [EMAIL PROTECTED] and we will try to work out what is happening. Thanks & best wishes, Graeme -Original Message- From: CCP4 bulletin board on behalf of Michael Weyand Sent: Thu 12/4/2008 8:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] ccp4-6.1.0-core-src.tar.gz TAR error Dear Charles, to your information: I just downloaded the Version 6.1 source tar file to my x86_64 Linux box (SuSE 10.2, AMD64) and got the following tar error message. All other (LINUX-386 and both COOCH and PHASER) tar files were fine!! ---> SNIP ... ccp4-6.1.0/lib/data/monomers/ps.resource ccp4-6.1.0/lib/data/monomers/full_names.list ccp4-6.1.0/lib/data/monomers/mon_lib_ind.cif ccp4-6.1.0/lib/data/monomers/mon_lib_ind2.cif tar: Skipping to next header tar: Archive contains obsolescent base-64 headers gzip: stdin: invalid compressed data--format violated tar: Child returned status 1 tar: Error exit delayed from previous errors ---> SNIP May be a corrupt file? Regards Michael -- Dr. Michael Weyand mail: [EMAIL PROTECTED] Max-Planck-Institut fuer molekulare Physiologie, Otto-Hahn-Strasse 11 D-44227 Dortmund, Germany, fon: +49(0)231-133-2738, fax: +49(0)231-133-2797 -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.6 (GNU/Linux) iD8DBQFJN7D/UxlJ7aRr7hoRAnujAJ0T9jOf7RlUoLWOpUK1EjJGtztCMQCeMdgx ecQXQ1ez+9LdOGXNe01n8yc= =nXf+ -END PGP SIGNATURE-
Re: [ccp4bb] getting weak diffracting crystals
Dear Deb, We've seen the detrimental effects of local disorder time and tme again. So - yes it is very likely that the putative disordered loops detrimentally affect the quality of your crystals. You can try to engineer your protein to be better - it usually takes a number of internally engineered constructs to get things right (about 10 per 350aa protein, in my experience). This is different from terminal truncations - which also can have a huge effect on crystallization. You might also try some of the methods summarized here: http://www.xtals.org/pdfs/rescue_crystals.pdf Cheers, Artem > > Dear Members, > > I am getting crystals of my protein. The secondary structure prediction > implies that it has N-terminal with high degree of loop regions. I also > get some mountable crystals yielding weak diffraction pattern(10 A). The > quality of the crystals can also be assumed from its texture for it has > tortuous surface. The big crystals are achieved at very high concentration > in hanging drop method (44mg/ml) whereas the initial hit (small crystals) > is at lower concentration in sitting drop (~ 5mg/ml). > > I have some queries about it. Does the N terminal loop regions are having > any effect of the crystal quality. Any suggestions of its quality > improvement are welcome. I will be highly benefited with your generous > replies. > > Sincerely > Deb >
[ccp4bb] suggestions for UV spectrometer
Dear all, we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. We don't need anything fancy, a small, fast device would be sufficient. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] suggestions for UV spectrometer
Dear Tim -- On 4 Dec 2008, at 15:16, Tim Gruene wrote: we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. I love the Nanodrop system... I know you said you don't want anything fancy, but I like to spend as little protein as possible when measuring its concentration, and I like it fast and reproducible. Plus, in my hands, the Nanodrop system give very reproducible results. Small problem... expensive... You can find infos there: http://www.nanodrop.com/nd-1000-overview.html 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/
Re: [ccp4bb] suggestions for UV spectrometer
I would also recommend the nanodrop. It takes a whole spectra every measurement and there is no need to dilute your sample. You can demo it for a week and try it out. James M. Vergis, Ph.D. University of Virginia Molecular Physiology and Biological Physics MKWEINR 360A Snyder Building 480 Ray C. Hunt Drive PO Box 800886 Charlottesville, VA 22908-0886 phone: 434-243-2730 FAX: 434-243-8271 [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Tim Gruene Sent: Thursday, December 04, 2008 10:16 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] suggestions for UV spectrometer Dear all, we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. We don't need anything fancy, a small, fast device would be sufficient. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] tortion angle restraints in REFMAC
Acturally, I want to find a way to keep the OH tilted out of the neightbouring plane by about 10 degrees. At 2.1A resolution the REFMAC refinement tends to refine it into the plane even though I have included torsion angle restraint in the library for the ligand. I thought I could play with the weight or the esd of the target value but having trouble to achieve it. In CNS, adjusting the force constant of the target value is a way to tighten or loosen the restrain. I would like to know how to "enforce" a geometry effectively in REFMAC. Thanks for any comments. Huiying On Wed, 3 Dec 2008, Abhinav Kumar wrote: If you want to restrain the OH group to a plane, you need to include it in the plane definition, and not the torsion definition. Thanks Abhinav Stanford Synchrotron Radiation Laboratory Joint Center for Structural Genomics Mail Stop 99 Phone: (650) 926-2992 Fax: (650) 926-3292 Huiying Li wrote: I want to impose restraints during REFMAC refinement on the tortion angles that control the tilting of an OH group from a plane in a ligand bound to the protein. A few things that confused me: 1. In library cif file, should I just increase or decrease the tor.value_angle_esd if I want to loosen or tighten the restraits? 2. What is the meaning of the last column in torsion angle parameters: _chem_comp_tor.period, in cif file? In the PDB output file REFMAC also lists the RMS and WEIGHT for the torsion angles, period 1 through 4. 3. In REFMAC gui under Geometric parameters, there is only one user controlled weight for torsion. By changing the weight here, does it change the torsion weight for all 4 periods? Thanks in advance for the help. Huiying -- Huiying Li, Ph. D Department of Molecular Biology and Biochemistry Natural Sciences I, Rm 2443 University of California at Irvine Irvine, CA 92697, USA Tel: 949-824-4322(or -1953); Fax: 949-824-3280 email: [EMAIL PROTECTED]
Re: [ccp4bb] suggestions for UV spectrometer
We use a Beckman Coulter DU730. It has a small footprint if lab space is an issue. It will do single-wavelength, multi-wavelength, or take an entire spectrum in 0.5 nm steps if you desire. It comes with the standard 1 ml cuvette holder, but we also purchased the microcuvette accessory for volumes in the 100 uL range. We've been using the instrument for over a year now with no problems. Results for determining protein concentrations using the method of Pace et al * *Protein Sci.1995 Nov;4(11):2411-23 are very reproducible using this spec. On Thu, Dec 4, 2008 at 10:37 AM, James M. Vergis <[EMAIL PROTECTED]>wrote: > I would also recommend the nanodrop. It takes a whole spectra every > measurement and there is no need to dilute your sample. You can demo it > for > a week and try it out. > > > > James M. Vergis, Ph.D. > University of Virginia Molecular Physiology and Biological Physics > MKWEINR 360A Snyder Building > 480 Ray C. Hunt Drive > PO Box 800886 > Charlottesville, VA 22908-0886 > phone: 434-243-2730 FAX: 434-243-8271 > [EMAIL PROTECTED] > > > > -Original Message- > From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Tim > Gruene > Sent: Thursday, December 04, 2008 10:16 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] suggestions for UV spectrometer > > Dear all, > > we would like to purchase a UV spectrometer for measuring protein > concentrations (280nm), and I would like to here your comments and > especially recommendations. > > We don't need anything fancy, a small, fast device would be sufficient. > > Tim > > > -- > Tim Gruene > Institut fuer anorganische Chemie > Tammannstr. 4 > D-37077 Goettingen > > GPG Key ID = A46BEE1A > -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 [EMAIL PROTECTED] [EMAIL PROTECTED]
Re: [ccp4bb] suggestions for UV spectrometer
Tim - I would recommend a spectrometer that records entire spectra, instead of one that takes readings at just 280 nm. Contributions from light scattering can be very strong and can give results that deviate from the true value by a factor of two or more. One cannot detect scattering without recording spectra. The most severe case we have had was someone who thought the protein concentration was 10 mg/mL (based on 280) when in reality (after subtraction of the scattering contribution) it was only 4 mg/mL. Lots of other, less severe cases as well. Hope that helps. Best - MM Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 On Dec 4, 2008, at 9:16 AM, Tim Gruene wrote: Dear all, we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. We don't need anything fancy, a small, fast device would be sufficient. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] suggestions for UV spectrometer
CCP4 bulletin board wrote on 12/04/2008 10:16:02 AM: > Dear all, > > we would like to purchase a UV spectrometer for measuring protein > concentrations (280nm), and I would like to here your comments and > especially recommendations. > > We don't need anything fancy, a small, fast device would be sufficient. > > Tim > > Hi Tim - I'm going to add my voice to the chorus for the Nanodrop. It's small - the size of a Kleenex box (plus a computer to run it). It's fast - you'll have a reading within two minutes of walking up to the bench, and that includes cleaning, initializing, and blanking the instrument. You can probably process 2-3 samples a minute if you're efficient since the actual measurement takes only a few seconds. There are no quartz cuvettes to wash, indeed no cuvettes at all - you just pipet 2 ul of your sample onto the measurement pedestal. Cleanup is a breeze - just wipe with a damp Kimwipe and you're done. The light source (xenon flash lamp) doesn't need to warm up and I've never had to change the bulb. The software is efficient and simple - you can do a table of measurements at a single wavelength, or full spectra in whatever wavelength range you want (from 190-720 nm). It measures 1 mm and 0.1 mm path lengths then combines the spectra, so you can use a broad range of sample concentrations without dilution - very useful for 50 mg/ml crystallization stocks! It can also do more sophisticated analysis, such as analyzing dye labeling efficiency by deconvoluting the dye absorbance spectrum from the protein absorbance spectrum. If you get one, you'll never want to go back to the old way. (And no, they didn't pay me to say this!) - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] tortion angle restraints in REFMAC
Hi Huiying At 2.1A I would be very surprised if you see any density for the H atom in which case the refinement is not going to move it out of the plane whatever weight you give the torsion restraint. To answer your earlier questions the period of a torsion restraint is the number of energy minima in a complete rotation of the angle, so the OH bond will have a period of 2, and yes the same overall weight is applied to all torsions regardless of the period, though individual torsions will also be weighted by 1/sd^2. Cheers -- Ian > -Original Message- > From: [EMAIL PROTECTED] > [mailto:[EMAIL PROTECTED] On Behalf Of Huiying Li > Sent: 04 December 2008 16:35 > To: Abhinav Kumar > Cc: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] tortion angle restraints in REFMAC > > Acturally, I want to find a way to keep the OH tilted out of the > neightbouring plane by about 10 degrees. At 2.1A resolution > the REFMAC > refinement tends to refine it into the plane even though I > have included > torsion angle restraint in the library for the ligand. I > thought I could > play with the weight or the esd of the target value but > having trouble to > achieve it. In CNS, adjusting the force constant of the > target value is a > way to tighten or loosen the restrain. I would like to know how to > "enforce" a geometry effectively in REFMAC. > > Thanks for any comments. > > Huiying > > On Wed, 3 Dec 2008, Abhinav Kumar wrote: > > > If you want to restrain the OH group to a plane, you need > to include it in > > the plane definition, and not the torsion definition. > > > > Thanks Abhinav > > Stanford Synchrotron Radiation Laboratory Joint Center for > Structural > > Genomics Mail Stop 99 Phone: (650) 926-2992 Fax: (650) 926-3292 > > > > > > Huiying Li wrote: > >> I want to impose restraints during REFMAC refinement on > the tortion angles > >> that control the tilting of an OH group from a plane in a > ligand bound to > >> the protein. A few things that confused me: > >> > >> 1. In library cif file, should I just increase or decrease the > >> tor.value_angle_esd if I want to loosen or tighten the restraits? > >> > >> 2. What is the meaning of the last column in torsion angle > parameters: > >> _chem_comp_tor.period, in cif file? In the PDB output file > REFMAC also > >> lists the RMS and WEIGHT for the torsion angles, period 1 > through 4. > >> > >> 3. In REFMAC gui under Geometric parameters, there is only > one user > >> controlled weight for torsion. By changing the weight > here, does it change > >> the torsion weight for all 4 periods? > >> > >> Thanks in advance for the help. > >> > >> Huiying > > > > -- > Huiying Li, Ph. D > Department of Molecular Biology and Biochemistry > Natural Sciences I, Rm 2443 > University of California at Irvine > Irvine, CA 92697, USA > Tel: 949-824-4322(or -1953); Fax: 949-824-3280 > email: [EMAIL PROTECTED] > > Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing [EMAIL PROTECTED] and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
[ccp4bb] Postdoctoral Position in Macromolecular Crystallography
A postdoctoral position in macromolecular crystallography is available at the three Cassiopeia beamlines at Max-Lab in Lund, Sweden, for a period of two years starting as soon as possible. The Danscatt consortium (www.danscatt.dk) is funding the position, and the successful candidate will work full time at Max-Lab. The postdoc will provide general support to all Cassiopeia users for 1/3 of the working time. The remaining 2/3 will be dedicated to collaborative projects, including publications, with Danish macromolecular crystallography laboratories, and to the development of new directions within macromolecular crystallography at Cassiopeia. Max-Lab has excellent crystallization robotics, crystal imaging facilities, and structure determination tools. With respect to new directions, the post-doc is expected to participate in: * Installation and implementation of a free mounting system for MX * Proof of principle studies for a solution SAXS beamline with the option for microfludics applications * Implementation of automated sample changers for crystal mounting at MX beamlines The successful candidate will refer to professor Michael Gajhede, Biostructural Research, Department of Medicinal Chemistry, University of Copenhagen ([EMAIL PROTECTED]), from whom further details on the position can be obtained. Further details concerning the Cassiopeia beamlines are available from beamline manager Thomas Ursby [EMAIL PROTECTED], see also cassiopeia.maxlab.lu.se. On 23 October 2008, the Swedish government expressed support for the construction of a unique third-generation synchrotron, the Max-IV, which is planned to house two superb MX beamlines and one beamline for solution SAXS. The post-doc is invited to participate in the development of the detailed plans for these beamlines. In addition, the Max-IV may become the neighbour of the new European neutron spallation source ESS, which will initiate a completely new era in neutron studies in structural biology. The post-doctoral position therefore offers a unique opportunity to establish research in the future European powerhouse of structural biology. For more information regarding the Max-IV and ESS, see: www.maxlab.lu.se and www.esss.se. The Danish user community in structural biology holds a unique position in frontier structural biology with many recent publications in leading journals, see examples below. Projects include ion-pumping membrane proteins, large macromolecular complexes involved in RNA metabolism, proteins from the immune defence, ligand-gated ion channels and transporters in the central nervous system, enzymes from the barley fatty acid metabolism, bacterial carbohydrate modifying enzymes and enzymes containing iron. Homepages of Danish MX laboratories: www.farma.ku.dk/BR, www.bioxray.au.dk, www.ccs.ki.ku.dk, www.crc.dk, and www.kemi.dtu.dk. Selected recent MX publications from Danish MX laboratories: * Pedersen BP et al. Crystal structure of the plasma membrane proton pump. Nature 2007, 450(7172):-4. * Morth JP et al. Crystal structure of the sodium-potassium pump. Nature 2007, 450(7172):1043-9. * Olesen C et al. The structural basis of calcium transport by the calcium pump. Nature 2007, 450(7172):1036-42. * Andersen CB et al. Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA. Science 2006, 313(5795):1968-72. * Fredslund F et al. Structure of and influence of a tick complement inhibitor on human complement component 5. Nature Immunol. 2008, 9(7):753-60. * Weyand F et al. Structure and molecular mechanism of a nucleobase-cation-symport-1 family transporter. Science 2008, Published Online October 16, 2008. * Naur P et al. Ionotropic glutamate-like receptor delta2 binds D-serine and glycine. PNAS 2007, 104(35), 14116-21. * Vestergaard B et al. A helical structural nucleus is the primary elongating unit of insulin amyloid fibrils. PLoS Biology 2007, 5(5) e134. Candidates may apply before receiving their PhD degree, but the successful candidate must have earned a PhD degree before the start of the appointment. Applications should be marked 08-322/MPD-22. Include in five copies: a current curriculum vitae, copies of relevant diplomas, a complete list of publications indicating those articles relevant to the position and copies of same, and a statement of teaching qualifications. Electronics applications will not be accepted. Applications should be sent to: Faculty of Pharmaceutical Sciences University of Copenhagen Universitetsparken 2 DK-2100 Copenhagen Denmark Deadline for applications: 15 January 2009 at 12:00 noon. -- Professor Michael Gajhede Institute of Medicinal Chemistry University of Copenhagen Universitetsparken 2 DK-2100 Copenhagen Ø Denmark Phone: +45 35306407 Email: [EMAIL PROTECTED]
Re: [ccp4bb] suggestions for UV spectrometer
We also like the Nanodrop. Very fast, no cuvettes (breaking, washing, cleaning, uh nitric acid bath anyone?), and the .ndv data file is a delimited text file. Open in a text editor, copy and paste into a spreadsheet, and you have a convenient record of all of your stocks, including date, sample name, concentration, and full spectra. It is expensive, but so are good cuvettes. Mike Michael Giffin The Scripps Research Institute Department of Molecular and Experimental Medicine 10550 North Torrey Pines Road, MEM-131 La Jolla, CA 92037 email: [EMAIL PROTECTED] lab: 858-784-7758 On Thu, Dec 4, 2008 at 7:16 AM, Tim Gruene <[EMAIL PROTECTED]> wrote: > Dear all, > > we would like to purchase a UV spectrometer for measuring protein > concentrations (280nm), and I would like to here your comments and > especially recommendations. > > We don't need anything fancy, a small, fast device would be sufficient. > > Tim > > > -- > Tim Gruene > Institut fuer anorganische Chemie > Tammannstr. 4 > D-37077 Goettingen > > GPG Key ID = A46BEE1A >
Re: [ccp4bb] suggestions for UV spectrometer
At the risk of dragging this discussion even further afield from crystallography: How can you get realistic numbers for concentrated solutions using the Nanodrop? I understand that the instrument reduces absorbance by using a very short path length. However, I thought that in order for the Beer-Lambert formalism to be applicable, the solution needs to be sufficiently dilute so that the chance of molecules "shadowing" one another is negligible. Isn't this condition violated for concentrated solutions (even with short path lengths)? Pat On 4 Dec 2008, at 1:27 PM, Michael Giffin wrote: We also like the Nanodrop... --- Patrick J. Loll, Ph. D. Professor of Biochemistry & Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 [EMAIL PROTECTED]
Re: [ccp4bb] suggestions for UV spectrometer
Wow, that's like putting a pool in your backyard so you don't have to pay the $3.00 admission every day (I'm kidding). In any event, Ocean Optics has some very nice, small, and portable units that would run around $3000 total. These connect to the USB port in a computer and produce data that can be manipulated easily via any spreadsheet manipulation program (open office, whatever). They are diode array, and so they take quick, continuous spectrum. We use these in our intro chem classes (sorry, small University, we can't afford the nanodrop) and they work great. I believe it's the USB2000 if you go to the Ocean Optics website. And, for those who find cuvettes as expensive as a nanodrop system, you can purchase polyacrylate ones that are good down to around 250 nm or so. They are marketed as disposable, but work well with several washings. They seem to be consistent from batch to batch, and we do use them for simple 280's on things (though of course we pull out the lock and key and get the quartz ones for those 2 special readings we take every year). Now, the specs you get are good and reliable (even though we use them with undergrads). They are not high end Cary's, but I don't think you need that sort of system for simple 280's. These detectors work as good cheap fluorimeter sources as well with some modifications on the light source (again, look around if that's something you are interested in). We use the Vernier system here, which allows us to connect spectrophotometers (mentioned above), drop counters, pH probes, temperature probes, and a whole variety of ion selective probes to a computer for data collection using almost any device. It's quite nice actually (vernier.com I believe). Good luck with that Dave Michael Giffin wrote: We also like the Nanodrop. Very fast, no cuvettes (breaking, washing, cleaning, uh nitric acid bath anyone?), and the .ndv data file is a delimited text file. Open in a text editor, copy and paste into a spreadsheet, and you have a convenient record of all of your stocks, including date, sample name, concentration, and full spectra. It is expensive, but so are good cuvettes. Mike Michael Giffin The Scripps Research Institute Department of Molecular and Experimental Medicine 10550 North Torrey Pines Road, MEM-131 La Jolla, CA 92037 email: [EMAIL PROTECTED] lab: 858-784-7758 On Thu, Dec 4, 2008 at 7:16 AM, Tim Gruene <[EMAIL PROTECTED]> wrote: Dear all, we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. We don't need anything fancy, a small, fast device would be sufficient. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] suggestions for UV spectrometer
We too have a nano-drop. We really like it , but have not yet fully switched over. I agree with all the good things said about it , but here are the few times the nano drop falls short: 1) We still use the old spec ( 1 cm path length ) for things at a very low concentration , i.e for the monitoring free thiols in protein with ellman reagent , the absorbances are very low and give poor reproducibility on the nanodrop because of small path-length . Of course this can be overcome by using a lot of protein at a higher concentration and modifying the assay. 2) For very concentrated membrane protein samples which tend to have a large concentration of detergent . The reproducibility is not very good because of the high concentration of deteregent preventing a proper meniscus from forming. The solution to this is to dilute your sample so the dteergent concentration is manageable ( a few times the CMC instead of tens of times the CMC Other than for these issues we almost entirely use the nanodrop and would gladly recommend it Hari On Thu, Dec 4, 2008 at 1:27 PM, Michael Giffin <[EMAIL PROTECTED]> wrote: > We also like the Nanodrop. Very fast, no cuvettes (breaking, washing, > cleaning, uh nitric acid bath anyone?), and the .ndv data file is a > delimited text file. Open in a text editor, copy and paste into a > spreadsheet, and you have a convenient record of all of your stocks, > including date, sample name, concentration, and full spectra. > > It is expensive, but so are good cuvettes. > > > Mike > > > Michael Giffin > The Scripps Research Institute > Department of Molecular and Experimental Medicine > 10550 North Torrey Pines Road, MEM-131 > La Jolla, CA 92037 > email: [EMAIL PROTECTED] > lab: 858-784-7758 > > On Thu, Dec 4, 2008 at 7:16 AM, Tim Gruene <[EMAIL PROTECTED]> > wrote: > > Dear all, > > > > we would like to purchase a UV spectrometer for measuring protein > > concentrations (280nm), and I would like to here your comments and > > especially recommendations. > > > > We don't need anything fancy, a small, fast device would be sufficient. > > > > Tim > > > > > > -- > > Tim Gruene > > Institut fuer anorganische Chemie > > Tammannstr. 4 > > D-37077 Goettingen > > > > GPG Key ID = A46BEE1A > > >
Re: [ccp4bb] suggestions for UV spectrometer
If you reduce the path by a factor of 50, can you not increase the concentration by the same factor without violating the shadowing assumption? Mike On Thu, Dec 4, 2008 at 10:48 AM, Patrick Loll <[EMAIL PROTECTED]> wrote: > At the risk of dragging this discussion even further afield from > crystallography: > How can you get realistic numbers for concentrated solutions using the > Nanodrop? I understand that the instrument reduces absorbance by using a > very short path length. However, I thought that in order for the > Beer-Lambert formalism to be applicable, the solution needs to be > sufficiently dilute so that the chance of molecules "shadowing" one another > is negligible. Isn't this condition violated for concentrated solutions > (even with short path lengths)? > Pat > On 4 Dec 2008, at 1:27 PM, Michael Giffin wrote: > > We also like the Nanodrop... > > --- > > Patrick J. Loll, Ph. D. > > Professor of Biochemistry & Molecular Biology > > Director, Biochemistry Graduate Program > > Drexel University College of Medicine > > Room 10-102 New College Building > > 245 N. 15th St., Mailstop 497 > > Philadelphia, PA 19102-1192 USA > > (215) 762-7706 > > [EMAIL PROTECTED] >
[ccp4bb] suggestions for UV spectrometer
Hi Tim, The Shimadzu UV2401PC comes at a reasonable price and has all features you might possible need in a Biochemistry lab. And if you like the option to use small sample volumes, I would suggest to by a TrayCell from Helma - you put it in a regular photometer, it guides the light through a microliter chamber and back into the photometer (it is essentially a cuvette with optics and a 1 or 5 microliter chamber). Costs a few hundred Euros . If you're sure you will never need regular cuvettes - go for Nanodrop. If you want more flexibility, Shimadzu plus TrayCell . Best Clemens --- Jun.-Prof. Dr. Clemens Steegborn Ruhr-University Bochum Physiological Chemistry, MA 2/141 Universitaetsstr. 150 44801 Bochum, Germany phone: ++49 234 32 27041 fax: ++49 234 32 14193 email: [EMAIL PROTECTED]
Re: [ccp4bb] tortion angle restraints in REFMAC
Thank you all for suggestions and explanations. I did not make it clear. The tilt angle I wanted to restrain is the one from a C-OH bond to a plane (The C is in the plane). The O atom of OH group is not in the planar restraint in the cif file. At 2.1A I can "see" the feature of tilting of this OH group from the initial density map when the ligand was absent. But the density seems not strong enough to convince REFMAC that there is a tilt there. I have used very low weight term of 0.05 in the GUI to down weight the X-ray term. Refinement still pushed OH into the plane. I wonder if there are other tricks that can impose the restraint for this torsion angle. Best, Huiying On Thu, 4 Dec 2008, Ian Tickle wrote: Hi Huiying At 2.1A I would be very surprised if you see any density for the H atom in which case the refinement is not going to move it out of the plane whatever weight you give the torsion restraint. To answer your earlier questions the period of a torsion restraint is the number of energy minima in a complete rotation of the angle, so the OH bond will have a period of 2, and yes the same overall weight is applied to all torsions regardless of the period, though individual torsions will also be weighted by 1/sd^2. Cheers -- Ian -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Huiying Li Sent: 04 December 2008 16:35 To: Abhinav Kumar Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] tortion angle restraints in REFMAC Acturally, I want to find a way to keep the OH tilted out of the neightbouring plane by about 10 degrees. At 2.1A resolution the REFMAC refinement tends to refine it into the plane even though I have included torsion angle restraint in the library for the ligand. I thought I could play with the weight or the esd of the target value but having trouble to achieve it. In CNS, adjusting the force constant of the target value is a way to tighten or loosen the restrain. I would like to know how to "enforce" a geometry effectively in REFMAC. Thanks for any comments. Huiying On Wed, 3 Dec 2008, Abhinav Kumar wrote: If you want to restrain the OH group to a plane, you need to include it in the plane definition, and not the torsion definition. Thanks Abhinav Stanford Synchrotron Radiation Laboratory Joint Center for Structural Genomics Mail Stop 99 Phone: (650) 926-2992 Fax: (650) 926-3292 Huiying Li wrote: I want to impose restraints during REFMAC refinement on the tortion angles that control the tilting of an OH group from a plane in a ligand bound to the protein. A few things that confused me: 1. In library cif file, should I just increase or decrease the tor.value_angle_esd if I want to loosen or tighten the restraits? 2. What is the meaning of the last column in torsion angle parameters: _chem_comp_tor.period, in cif file? In the PDB output file REFMAC also lists the RMS and WEIGHT for the torsion angles, period 1 through 4. 3. In REFMAC gui under Geometric parameters, there is only one user controlled weight for torsion. By changing the weight here, does it change the torsion weight for all 4 periods? Thanks in advance for the help. Huiying -- Huiying Li, Ph. D Department of Molecular Biology and Biochemistry Natural Sciences I, Rm 2443 University of California at Irvine Irvine, CA 92697, USA Tel: 949-824-4322(or -1953); Fax: 949-824-3280 email: [EMAIL PROTECTED] Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing [EMAIL PROTECTED] and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674 -- Huiying Li, Ph. D Department of Molecular Biolo
Re: [ccp4bb] suggestions for UV spectrometer
One more issue regarding the Nanodrop: one has to work quite quickly to avoid potential evaporation. Before buying such an instrument, I would strongly recommend a demo and careful comparisons between the Nanodrop and a good, conventional spectrometer with a representative range of samples. Convenience is one thing... Best - MM Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 On Dec 4, 2008, at 12:48 PM, Patrick Loll wrote: At the risk of dragging this discussion even further afield from crystallography: How can you get realistic numbers for concentrated solutions using the Nanodrop? I understand that the instrument reduces absorbance by using a very short path length. However, I thought that in order for the Beer-Lambert formalism to be applicable, the solution needs to be sufficiently dilute so that the chance of molecules "shadowing" one another is negligible. Isn't this condition violated for concentrated solutions (even with short path lengths)? Pat On 4 Dec 2008, at 1:27 PM, Michael Giffin wrote: We also like the Nanodrop... --- Patrick J. Loll, Ph. D. Professor of Biochemistry & Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 [EMAIL PROTECTED]
Re: [ccp4bb] tortion angle restraints in REFMAC
On Thursday 04 December 2008 11:50:30 Huiying Li wrote: > Thank you all for suggestions and explanations. I did not make it clear. The > tilt angle I wanted to restrain is the one from a C-OH bond to a plane > (The C is in the plane). The O atom of OH group is not in the planar > restraint in the cif file. At 2.1A I can "see" the feature of tilting of > this OH group from the initial density map when the ligand was absent. > But the density seems not strong enough to convince REFMAC that there is > a tilt there. I have used very low weight term of 0.05 in the GUI to down > weight the X-ray term. Refinement still pushed OH into the plane. I wonder > if there are other tricks that can impose the restraint for this torsion > angle. From your description, it is still likely that a planarity restraint is involved, rather than a torsion restraint. Perhaps you should post the library *.cif file that you are using for this compound. Ethan > > Best, > Huiying > > On Thu, 4 Dec 2008, Ian Tickle wrote: > > > Hi Huiying > > > > At 2.1A I would be very surprised if you see any density for the H atom > > in which case the refinement is not going to move it out of the plane > > whatever weight you give the torsion restraint. To answer your earlier > > questions the period of a torsion restraint is the number of energy > > minima in a complete rotation of the angle, so the OH bond will have a > > period of 2, and > > yes the same overall weight is applied to all torsions regardless of the > > period, though individual torsions will also be weighted by 1/sd^2. > > > > Cheers > > > > -- Ian > > > >> -Original Message- > >> From: [EMAIL PROTECTED] > >> [mailto:[EMAIL PROTECTED] On Behalf Of Huiying Li > >> Sent: 04 December 2008 16:35 > >> To: Abhinav Kumar > >> Cc: CCP4BB@JISCMAIL.AC.UK > >> Subject: Re: [ccp4bb] tortion angle restraints in REFMAC > >> > >> Acturally, I want to find a way to keep the OH tilted out of the > >> neightbouring plane by about 10 degrees. At 2.1A resolution > >> the REFMAC > >> refinement tends to refine it into the plane even though I > >> have included > >> torsion angle restraint in the library for the ligand. I > >> thought I could > >> play with the weight or the esd of the target value but > >> having trouble to > >> achieve it. In CNS, adjusting the force constant of the > >> target value is a > >> way to tighten or loosen the restrain. I would like to know how to > >> "enforce" a geometry effectively in REFMAC. > >> > >> Thanks for any comments. > >> > >> Huiying > >> > >> On Wed, 3 Dec 2008, Abhinav Kumar wrote: > >> > >>> If you want to restrain the OH group to a plane, you need > >> to include it in > >>> the plane definition, and not the torsion definition. > >>> > >>> Thanks Abhinav > >>> Stanford Synchrotron Radiation Laboratory Joint Center for > >> Structural > >>> Genomics Mail Stop 99 Phone: (650) 926-2992 Fax: (650) 926-3292 > >>> > >>> > >>> Huiying Li wrote: > I want to impose restraints during REFMAC refinement on > >> the tortion angles > that control the tilting of an OH group from a plane in a > >> ligand bound to > the protein. A few things that confused me: > > 1. In library cif file, should I just increase or decrease the > tor.value_angle_esd if I want to loosen or tighten the restraits? > > 2. What is the meaning of the last column in torsion angle > >> parameters: > _chem_comp_tor.period, in cif file? In the PDB output file > >> REFMAC also > lists the RMS and WEIGHT for the torsion angles, period 1 > >> through 4. > > 3. In REFMAC gui under Geometric parameters, there is only > >> one user > controlled weight for torsion. By changing the weight > >> here, does it change > the torsion weight for all 4 periods? > > Thanks in advance for the help. > > Huiying > >>> > >> > >> -- > >> Huiying Li, Ph. D > >> Department of Molecular Biology and Biochemistry > >> Natural Sciences I, Rm 2443 > >> University of California at Irvine > >> Irvine, CA 92697, USA > >> Tel: 949-824-4322(or -1953); Fax: 949-824-3280 > >> email: [EMAIL PROTECTED] > >> > >> > > > > > > Disclaimer > > This communication is confidential and may contain privileged information > > intended solely for the named addressee(s). It may not be used or disclosed > > except for the purpose for which it has been sent. If you are not the > > intended recipient you must not review, use, disclose, copy, distribute or > > take any action in reliance upon it. If you have received this > > communication > > in error, please notify Astex Therapeutics Ltd by emailing > > [EMAIL PROTECTED] and destroy all copies of the message and any > > attached documents. > > Astex Therapeutics Ltd monitors, controls and protects all its messaging > > traffic in compliance with its corporate email policy. The Company accepts > > no > > liability or responsibility for any onward tr
[ccp4bb] error message for using xfit in ccp4
Hello all, We would appreciate your help on an error message one of our graduate students has encountered using Xfit in CCP4. You can respond to him directly at: [EMAIL PROTECTED] Thank you Mona Rahman Forwarded Message --- Thanks Mona Here is error message for using the xfit in ccp4. xfit Starting XtalView 4.0 for OS: Linux version: 2.6.27.5-117.fc10.i686.PAE Copyright (C) 1992-9 Duncan McRee and The Scripps Research Institute Set LANG to C Color Table initialized with 136 values from /usr/local/XtalView/data/colors.dat XView warning: Cannot load font '-b&h-lucida-medium-r-*-*-*-120-*-*-*-*-*-*' (Font package) XView warning: Cannot load font '-b&h-lucida-medium-r-normal-sans-*-120-*-*-*-*-*-*' (Font package) XView error: Cannot open connection to window server: :0.0 (Server package) We are using Fedora 10 Thanks Jimin --- Mona N. Rahman, Ph.D. Department of Biochemistry Botterell Hall, Rooms 623 and 634 (lab) Queen's University, Kingston, ON, K7L 3N6 Phone: 613-533-2993, 613-533-6293 (lab) E-mail: [EMAIL PROTECTED]
Re: [ccp4bb] tortion angle restraints in REFMAC
Hi Huiyang OK sorry for misunderstanding. I think that kind of out-of-plane restraint is usually enforced by means of a chiral restraint (even if the group in question is not chiral). You would need to calculate the expected chiral volume of the C atom for the target position of the O atom. Cheers -- Ian > -Original Message- > From: [EMAIL PROTECTED] > [mailto:[EMAIL PROTECTED] On Behalf Of Huiying Li > Sent: 04 December 2008 19:51 > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] tortion angle restraints in REFMAC > > Thank you all for suggestions and explanations. I did not > make it clear. The > tilt angle I wanted to restrain is the one from a C-OH bond > to a plane > (The C is in the plane). The O atom of OH group is not in the planar > restraint in the cif file. At 2.1A I can "see" the feature of > tilting of > this OH group from the initial density map when the ligand > was absent. > But the density seems not strong enough to convince REFMAC > that there is > a tilt there. I have used very low weight term of 0.05 in the > GUI to down > weight the X-ray term. Refinement still pushed OH into the > plane. I wonder > if there are other tricks that can impose the restraint for > this torsion > angle. > > Best, > Huiying > > On Thu, 4 Dec 2008, Ian Tickle wrote: > > > Hi Huiying > > > > At 2.1A I would be very surprised if you see any density > for the H atom > > in which case the refinement is not going to move it out of > the plane > > whatever weight you give the torsion restraint. To answer > your earlier > > questions the period of a torsion restraint is the number of energy > > minima in a complete rotation of the angle, so the OH bond > will have a > > period of 2, and > > yes the same overall weight is applied to all torsions > regardless of the > > period, though individual torsions will also be weighted by 1/sd^2. > > > > Cheers > > > > -- Ian > > > >> -Original Message- > >> From: [EMAIL PROTECTED] > >> [mailto:[EMAIL PROTECTED] On Behalf Of Huiying Li > >> Sent: 04 December 2008 16:35 > >> To: Abhinav Kumar > >> Cc: CCP4BB@JISCMAIL.AC.UK > >> Subject: Re: [ccp4bb] tortion angle restraints in REFMAC > >> > >> Acturally, I want to find a way to keep the OH tilted out of the > >> neightbouring plane by about 10 degrees. At 2.1A resolution > >> the REFMAC > >> refinement tends to refine it into the plane even though I > >> have included > >> torsion angle restraint in the library for the ligand. I > >> thought I could > >> play with the weight or the esd of the target value but > >> having trouble to > >> achieve it. In CNS, adjusting the force constant of the > >> target value is a > >> way to tighten or loosen the restrain. I would like to know how to > >> "enforce" a geometry effectively in REFMAC. > >> > >> Thanks for any comments. > >> > >> Huiying > >> > >> On Wed, 3 Dec 2008, Abhinav Kumar wrote: > >> > >>> If you want to restrain the OH group to a plane, you need > >> to include it in > >>> the plane definition, and not the torsion definition. > >>> > >>> Thanks Abhinav > >>> Stanford Synchrotron Radiation Laboratory Joint Center for > >> Structural > >>> Genomics Mail Stop 99 Phone: (650) 926-2992 Fax: (650) 926-3292 > >>> > >>> > >>> Huiying Li wrote: > I want to impose restraints during REFMAC refinement on > >> the tortion angles > that control the tilting of an OH group from a plane in a > >> ligand bound to > the protein. A few things that confused me: > > 1. In library cif file, should I just increase or decrease the > tor.value_angle_esd if I want to loosen or tighten the restraits? > > 2. What is the meaning of the last column in torsion angle > >> parameters: > _chem_comp_tor.period, in cif file? In the PDB output file > >> REFMAC also > lists the RMS and WEIGHT for the torsion angles, period 1 > >> through 4. > > 3. In REFMAC gui under Geometric parameters, there is only > >> one user > controlled weight for torsion. By changing the weight > >> here, does it change > the torsion weight for all 4 periods? > > Thanks in advance for the help. > > Huiying > >>> > >> > >> -- > >> Huiying Li, Ph. D > >> Department of Molecular Biology and Biochemistry > >> Natural Sciences I, Rm 2443 > >> University of California at Irvine > >> Irvine, CA 92697, USA > >> Tel: 949-824-4322(or -1953); Fax: 949-824-3280 > >> email: [EMAIL PROTECTED] > >> > >> > > > > > > Disclaimer > > This communication is confidential and may contain > privileged information > > intended solely for the named addressee(s). It may not be > used or disclosed > > except for the purpose for which it has been sent. If you > are not the > > intended recipient you must not review, use, disclose, > copy, distribute or > > take any action in reliance upon it. If you have received > this communication > > in error, please notify Astex Therapeutics Ltd by em
Re: [ccp4bb] suggestions for UV spectrometer
I want to add I absotely hate the nanodrop. We've had a demo for it, and found the readouts to be very unreliable. Fluctuations of 20% and more. Just leaving the same drop in and measuring the sample multiple times gives different values (going in both directions, so not only due to evaportations). Sure, it's easy and fast, and maybe good to have a rough idea about your protein concentration, but I would never want to use it for exact measurements such as needed for e.g. a CD or an ITC instrument. I've heard other labs in our department have similar issues. We've also had a demo for the Nanovue from GE Healthcare: same issues - very large fluctuations from one sample to another. I suppose this is simply an inherent problem with small volumes... Cheers Filip Van Petegm On Thu, Dec 4, 2008 at 12:48 PM, Patrick Loll <[EMAIL PROTECTED]> wrote: > At the risk of dragging this discussion even further afield from > crystallography: > How can you get realistic numbers for concentrated solutions using the > Nanodrop? I understand that the instrument reduces absorbance by using a > very short path length. However, I thought that in order for the > Beer-Lambert formalism to be applicable, the solution needs to be > sufficiently dilute so that the chance of molecules "shadowing" one another > is negligible. Isn't this condition violated for concentrated solutions > (even with short path lengths)? > > Pat > > On 4 Dec 2008, at 1:27 PM, Michael Giffin wrote: > > We also like the Nanodrop... > > > --- > > Patrick J. Loll, Ph. D. > > Professor of Biochemistry & Molecular Biology > > Director, Biochemistry Graduate Program > > Drexel University College of Medicine > > Room 10-102 New College Building > > 245 N. 15th St., Mailstop 497 > > Philadelphia, PA 19102-1192 USA > > > (215) 762-7706 > > [EMAIL PROTECTED] > > -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: [EMAIL PROTECTED] http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] tortion angle restraints in REFMAC
CCP4 bulletin board wrote on 12/04/2008 02:50:30 PM: > Thank you all for suggestions and explanations. I did not make it clear. The > tilt angle I wanted to restrain is the one from a C-OH bond to a plane > (The C is in the plane). The O atom of OH group is not in the planar > restraint in the cif file. At 2.1A I can "see" the feature of tilting of > this OH group from the initial density map when the ligand was absent. > But the density seems not strong enough to convince REFMAC that there is > a tilt there. I have used very low weight term of 0.05 in the GUI to down > weight the X-ray term. Refinement still pushed OH into the plane. I wonder > if there are other tricks that can impose the restraint for this torsion > angle. > > Best, > Huiying Hi Huiying - Before you spend more time adjusting your cif files, you should try adjusting your refinement weight. As you said, you see a density feature that's inconsistent with the standard protein geometry. If you down-weight the X-ray term too much, all you will see in the final model is the standard protein geometry enforced by the cif file. Try raising the refinement weight to 0.08 or 0.1 and see if you can capture the out-of-plane OH in your model without sacrificing your R factors or other model geometry terms. You should also look at the model to see *why* your OH is coming out of the plane. Is there a steric clash pushing it, or a hydrogen bond pulling it? If there is an H-bond, perhaps you need to adjust the position of the H donor/acceptor so that Refmac recognizes the bond. Perhaps you need to flip an Asn/Gln side chain so that the N and O switch positions? Finally, re-reading your initial email, I see that this C-OH is in the ligand. Did you create the cif file for this ligand yourself, or is this in some standard dictionary? When I've made cif files for non-standard ligands (using Refmac), I've observed that the algorithm tends to put expand plane definitions (e.g. from conjugated ring systems) to include too many neighboring atoms. I needed to trim the plane definitions and delete a lot of inappropriate torsion angle restraints before my ligands would refine correctly. - Matt -- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 Confidentiality Note: This e-mail, and any attachment to it, contains privileged and confidential information intended only for the use of the individual(s) or entity named on the e-mail. If the reader of this e-mail is not the intended recipient, or the employee or agent responsible for delivering it to the intended recipient, you are hereby notified that reading it is strictly prohibited. If you have received this e-mail in error, please immediately return it to the sender and delete it from your system. Thank you.
Re: [ccp4bb] suggestions for UV spectrometer
We have an Ocean Optics USB-4000 unit in our lab. It does everything from quantifying protein and nucleic acids, spectrophotometric titrations, and metalloenzyme spectra at low volume/concentration. It's not a toy, but a diode array spectrophotometer that has excellent S/N and resolution. You can get a fully thermostatted unit for under $9000, and an integrated, bulletproof non-thermostatted unit for under $3500 for UV and visible spectra. These units are also expandable and re-configurable for different tasks. Beats the tar our of our HP diode arrays, which were reliable, but not as capable. Cells from Starna run from $150 (semimicro) to $250 (micro cells). We've never found them difficult to clean. A vacuum-based cell cleaner is a good investment that can clean and dry cells quickly. -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED] David Roberts wrote: Wow, that's like putting a pool in your backyard so you don't have to pay the $3.00 admission every day (I'm kidding). In any event, Ocean Optics has some very nice, small, and portable units that would run around $3000 total. These connect to the USB port in a computer and produce data that can be manipulated easily via any spreadsheet manipulation program (open office, whatever). They are diode array, and so they take quick, continuous spectrum. We use these in our intro chem classes (sorry, small University, we can't afford the nanodrop) and they work great. I believe it's the USB2000 if you go to the Ocean Optics website. And, for those who find cuvettes as expensive as a nanodrop system, you can purchase polyacrylate ones that are good down to around 250 nm or so. They are marketed as disposable, but work well with several washings. They seem to be consistent from batch to batch, and we do use them for simple 280's on things (though of course we pull out the lock and key and get the quartz ones for those 2 special readings we take every year). Now, the specs you get are good and reliable (even though we use them with undergrads). They are not high end Cary's, but I don't think you need that sort of system for simple 280's. These detectors work as good cheap fluorimeter sources as well with some modifications on the light source (again, look around if that's something you are interested in). We use the Vernier system here, which allows us to connect spectrophotometers (mentioned above), drop counters, pH probes, temperature probes, and a whole variety of ion selective probes to a computer for data collection using almost any device. It's quite nice actually (vernier.com I believe). Good luck with that Dave Michael Giffin wrote: We also like the Nanodrop. Very fast, no cuvettes (breaking, washing, cleaning, uh nitric acid bath anyone?), and the .ndv data file is a delimited text file. Open in a text editor, copy and paste into a spreadsheet, and you have a convenient record of all of your stocks, including date, sample name, concentration, and full spectra. It is expensive, but so are good cuvettes. Mike Michael Giffin The Scripps Research Institute Department of Molecular and Experimental Medicine 10550 North Torrey Pines Road, MEM-131 La Jolla, CA 92037 email: [EMAIL PROTECTED] lab: 858-784-7758 On Thu, Dec 4, 2008 at 7:16 AM, Tim Gruene <[EMAIL PROTECTED]> wrote: Dear all, we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. We don't need anything fancy, a small, fast device would be sufficient. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] Offtopic: FAD enzymatic assay
Dear all, I may sound stupid enough. I tried the PMS-DCPIP assay system as suggested, and I choose to observe the absorption at 600nM. However, when I initialize the reaction, I actually see the Abs slowly but steadily increasing rather than decreasing. How the FAD interact with the enzyme is really unknown. No structure is available. The only thing I know is that there is a FAD binding domain. I don't know if it's dissociable or remain bound during the reaction Another question might be how to properly dissolve lipid for assay. I currently dissolve them using hexane and then add them to Tris-Chaps buffer (pH ~7, and about 10mM Chaps). Is there a better way to dissolve them for assay. Please advise. michael nelson wrote: > Dear all, > > Thank you for all your kind replies. > > Here is a little bit more about the enzyme and how I carry out the assay at the first place. > > My enzyme is a lipid desaturase, originally from plant but overexpressed in bacteria. FAD serves as a co-factor for this enzyme, in which FAD is reduced to FADH2. > > My goal is set up an assay that would allows me to continuously monitor the progress of the reaction. And I didn't want to use HPLC to analyze the final product since that would take a lot time and we don't have an instrument readily available to us. I wish FAD could be an alternative way since FAD will have different Abs in reduced or oxidized forms. > > I set up assay is a regular lab setting (not anaerobic), add FAD, substrate, ions and incubate. I finally add enzyme to initialize the reaction. I expect to see some decrease of the Ab at 450 nM. But I didn't. > > I have several concerns, one is the autooxidisability of FAD, how fast FADH2 would be reoxidized by O2 in the air or by the O2 dissolved in solution. The second cocern is how fast the FAD reaction will go. > > Please advise. > > Thank you! > > Mike > >
[ccp4bb] refmac 5.5 error with ARPwARP7.0.1
Hello, I have attached a log file from a refmac refinement output that was initiated by ARPwARP (the flexwarp program in CCP4) after an initial model building was determined good. This is the refmac model refinement when the ARPwARP program starts a separate job to refine or finalize the model while the main algorithm continues. I have not had this problem with refmac 5.4 or 5.2. This error comes up most of the time but on occasion it will not. Other than this the ARPwARP in CCP4 and refmac refinement work as usual. Thanks in advance. Refmac13.log Description: Binary data
[ccp4bb] ion exchange chromatography problem
Hello everyone, I have a question about ion exchange chromatography. - I have a membrane protein (pI 9.5), 13 KDa - It's fused to MBP (maltose binding protein pI 5.1, 44 KDa) to prevent inclusion body formation during high level expresscion. - The final protein has a pI of 6.1, 57 KDa --> The protein is solublised in 1% TX, Tris 20 mM pH 8.0 20 mM NaCl. However, it doesn't bind to the column equilibrated in the same buffer condition. Can anyone help me with this problem? Is it because of the pI difference between the membrane protein and the fusion partner? Many thanks
[ccp4bb] membrane protein not solubilised
Hello everyone, I am working on a yeast membrane protein, about 100 KDa. The protein is expressed as an inclusion body in E coli even at 18 C. The problem is solved when MBP is fused to the N terminus of the protein. The resulting protein is ~140 KDa, pI 6.0. The expression level is moderate. After cell disruption and fractionation by high speed spin (100K g), the protein land in the pellet fraction as expected for a membrane protein. The pellet is then solublised in various conditions and assessed for solubility. Buffer I used: 50 mM phosphate pH 7.5, 250 mM NaCl, 5% Glycerol With different detergents at final concentration of - 0.1% ~ 4% Trition X 100 - 4% OG - 2% DDM - 2% DM - 4% CHAPS - 2% C8En - 2% C12En - 2% Cymal-4,-5,-6 - 2% MEGA-10 - 2% HEGA-10 - 2% SDS Only SDS can completely solubilise the protein, other detergents solublise less than 10%... Can anyone offer some advice? really appreciated Kien
[ccp4bb] off-topic : detergent as cryoprotectant?
Hi there, I have trouble with normal glycerol cryoprotectant. My protein crystals seems not stable in it. However, my mother liquor has extremely high concertration of Nonyl-maltoside (estimated ~20%, this membrane protein really needs that high detergent, sounds weird.8)So, I am wondering anyone with successful experience using detergents as cryoprotant can give some comments. If they works well, that means I can directly fish some crystals out of mother liquor without adding other cryos. Thanks a lot. Deliang
[ccp4bb] Postdoc position in University of Texas Houston Medical School
Dear bbers, Sorry to post a job here... One postdoctoral position is available immediately at the Center for Membrane Biology, Department of Biochemistry and Molecular Biology, The University of Texas Health Science Center at Houston. (http://www.uth.tmc.edu/cmb/) The successful candidate will focus on crystallization and structural determination of ion transporter proteins. She/He will also have an opportunity to work with other expertise in the Center to study transporter functional characterization. The ideal applicant should be a highly motivated individual and have a recent PhD degree in structural biology, biochemistry or molecular biology. Strong background in molecular biology, protein purification and crystallization is expected. Any experience with membrane protein will be a plus. Our laboratory locates in Texas Medical Center campus which provides an excellent environment for academia and research. The lab is fully equipped with all aspects of molecular biology, protein biochemistry and protein crystallography. Instant access to ALS beamline 4.2.2 is granted as a member of Molecular Biology Consortium. To apply, please email me your current resume including the names and addresses of three referees. Thank you, Lei Lei Zheng, Ph.D Assistant professor Center for Membrane Biology Department of Biochemistry and Molecular Biology University of Texas Medical School at Houston 6431 Fannin St. MSB 5.210 Houston, TX-77030 Tel.: (713)500-6083 Fax: (713)500-0545
Re: [ccp4bb] ion exchange chromatography problem
...forgot to mention I used Q sepherose from Sigma for the IEC
Re: [ccp4bb] getting weak diffracting crystals
Hi again, Thank you for your reply. I know that the loop regions are notorious for crystallization however in this case it is yielding the crystals but with lower quality. Does it not due to the quick crystallization, the lattice has no time to form up. Thats why it is happening. Can I not opt for glycerol, or sitting drop method. Sincerely Deb On Thu, 04 Dec 2008 [EMAIL PROTECTED] wrote : >Dear Deb, > >We've seen the detrimental effects of local disorder time and tme again. >So - yes it is very likely that the putative disordered loops >detrimentally affect the quality of your crystals. > >You can try to engineer your protein to be better - it usually takes a >number of internally engineered constructs to get things right (about 10 >per 350aa protein, in my experience). This is different from terminal >truncations - which also can have a huge effect on crystallization. > >You might also try some of the methods summarized here: >http://www.xtals.org/pdfs/rescue_crystals.pdf > > >Cheers, > >Artem > > > > Dear Members, > > > > I am getting crystals of my protein. The secondary structure prediction > > implies that it has N-terminal with high degree of loop regions. I also > > get some mountable crystals yielding weak diffraction pattern(10 A). The > > quality of the crystals can also be assumed from its texture for it has > > tortuous surface. The big crystals are achieved at very high concentration > > in hanging drop method (44mg/ml) whereas the initial hit (small crystals) > > is at lower concentration in sitting drop (~ 5mg/ml). > > > > I have some queries about it. Does the N terminal loop regions are having > > any effect of the crystal quality. Any suggestions of its quality > > improvement are welcome. I will be highly benefited with your generous > > replies. > > > > Sincerely > > Deb > > >
[ccp4bb] Postdoctoral position at the University of Queensland, Brisbane, Australia
POST-DOCTORAL POSITION IN PROTEIN CRYSTALLOGRAPHY, THE UNIVERSITY OF QUEENSLAND, BRISBANE, AUSTRALIA Applications are invited for a protein crystallography post-doctoral position in the laboratory of Prof Bostjan Kobe at the School of Molecular and Microbial Sciences (SMMS) and the Institute for Molecular Bioscience (IMB), University of Queensland, Brisbane, Australia. The main area of study will involve the characterization of structures and interactions of proteins involved in the process of plant disease resistance, focusing on flax-flax rust host-pathogen interaction as the model system (for recent publications in this area see Dodds PN et al (2006) Proc Natl Acad Sci USA 103: and Wang C-IA et al (2007) Plant Cell 19: 2898). Experience in molecular biology, protein purification, protein/protein interaction analysis and protein crystallography is desirable. The salary will be according to qualifications and experience, starting at AUD$ $73,484. The position is available from January 2009. University of Queensland is one of Australia's top universities. SMMS combines the disciplines of chemistry, biochemistry & molecular biology, microbiology and parasitology into a single academic unit. SMMS and IMB are situated in the beautiful St. Lucia campus on the Brisbane River. State-of-the art equipment is available for all aspects of the work, including FR-E X-ray generator /R-axis IV++ and Saturn 944 CCD X-ray detectors, Mosquito crystallization robot and the Rock Imager imaging system. Brisbane has been voted Australia's most liveable city and has a great subtropical climate. It is only a short drive from Australia's best beaches and provides opportunities for all sorts of outdoors and cultural activities. For further information please see http://seek.com.au/users/apply/index.ascx?Sequence=45&PageNumber=1&JobID=145 72918 or contact Bostjan Kobe (telephone: +617-3365-2132, email [EMAIL PROTECTED]). Please forward applications, including a cover letter, curriculum vitae and contact details for three referees, by email or post to by 16 January 2008 to Bostjan Kobe, SMMS, University of Queensland, St. Lucia, Queensland 4072, Australia. --- Bostjan Kobe ARC Federation Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience Cooper Road University of Queensland Brisbane, Queensland 4072 Australia Phone: +61 7 3365 2132 Fax: +61 7 3365 4699 E-mail: [EMAIL PROTECTED] URL: http://profiles.bacs.uq.edu.au/Bostjan.Kobe.html Office: Building 76 Room 452 Notice: If you receive this e-mail by mistake, please notify me, and do not make any use of its contents. I do not waive any privilege, confidentiality or copyright associated with it. Unless stated otherwise, this e-mail represents only the views of the Sender and not the views of The University of Queensland.
