Re: [ccp4bb] [COOT] coot install on redhat
Hi Heidi, why don't you just install the latest binary distribution? That's much less hassle ... just go to http://www.ysbl.york.ac.uk/~emsley/software/binaries/nightlies/pre-release/ and grab e.g. coot-0.5-pre-1-revision-1003-binary-Linux-i386-fedora-5.tar.gz (Fedora 6 corresponds to RHEL5, thus Fedora 5 binaries are OK) Then un-tar it under /usr/local/src, and establish a symlink (ln -s) between /usr/local/bin/coot and the bin/coot of the freshly unpacked distribution. If you then run coot, and the loader complains that a certain library is missing, just ask yum whatprovides thatlibrary and install the library, again using yum. HTH, Kay Heidi Schubert schrieb: Hi All, I'm not a systems administrator ? I just play one at work. Can someone help me over these hurdles installing the newest version of coot. Coot requires the following: 1) C++ compiler ? okay got that 2) gtk+(1.2) ?requires: a) at least Glib 2.12 ? I think the problem is here, I successfully installed 2.16.3 but. glib required newer pgk-config ? okay got that now glib appears to install okay, puts a directory in /usr/local/lib b)Pango 1.13 ? okay got that c)ATK 1.9 ? problems starts here. During ./configure it gives the following: checking pkg-config is at least version 0.16... yes checking for GLIB - version = 2.0.0... *** 'pkg-config --modversion glib-2.0' returned 2.16.3, but GLIB (2.4.7) *** was found! If pkg-config was correct, then it is best *** to remove the old version of GLib. You may also be able to fix the error *** by modifying your LD_LIBRARY_PATH enviroment variable, or by editing *** /etc/ld.so.conf. Make sure you have run ldconfig if that is *** required on your system. *** If pkg-config was wrong, set the environment variable PKG_CONFIG_PATH *** to point to the correct configuration files no configure: error: *** GLIB 2.0.0 or better is required. The latest version of *** GLIB is always available from ftp://ftp.gtk.org/. If GLIB is installed *** but not in the same location as pkg-config add the location of the file *** glib-2.0.pc to the environment variable PKG_CONFIG_PATH. 1)I ran ldconfig but this didn't do anything. I have no idea how one would edit the ld.so.conf file. 2) LD_LIBRARY_PATH=/usr/local/Linux-bubbles/lib:/home/local/LINUX//ccp4-6.0.2/lib:/ usr/local/Linux-bubbles/lib:/home/local/LINUX//ccp4-6.0.2/lib:/home/local/LINUX/ /ccp4-6.0.2/extralib:/home/local/LINUX//ccp4-6.0.2/extralib ?extralib doesn't exist anyway and /ccp4-6.0.2/lib doesn't have a glib file. There is a glib directory in the /linux-bubbles/lib but it says version 1.2.10 not 2.4.7 3)I'm using REDHAT 5. I'm sitting at a desktop box but installing everything into /usr/local on our server. Where do I look for this old version of glib and will it be okay to delete it? I'm worried I'm running into versions that are loaded on the desktop and not on the server, but I can't find them. Sorry I'm so linux library illterate Heidi -- Kay Diederichshttp://strucbio.biologie.uni-konstanz.de email: [EMAIL PROTECTED]Tel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz smime.p7s Description: S/MIME Cryptographic Signature
Re: [ccp4bb] discontinuous data wedges in XDS
Is it not sufficient to first integrate the one wedge, then use this wedge as a reference data set when integrating the second one? (REFERENCE_DATA_SET= ../wedge01/XDS_ASCII.HKL) Klaus - Klaus Fütterer, Ph.D. School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: [EMAIL PROTECTED] Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/ - On 29 Apr 2008, at 22:57, Juergen Bosch wrote: You can integrate them as separate wedges (in different directories), then later merge them (XDS_ASCII.HKL) in Xscale. Make sure though you have the orientation right and starting angles etc. You'll have to first refine one wedge, then once you are happy with it copy the GXPARM.XDS into the other directories (as XPARM.XDS) and input those parameters into XDS..INP Juergen Van Den Berg, Bert wrote: Hi all, is it possible to input discontinuous data wedges into XDS (obtained from for example inverse beam sweeps)? (So wedge se1 goes from 0-90 deg (image 1-90), se2 from 180-270 (image 1-90), etc). Or do I have to rename everything so that I get one data file in which the rotation ranges are continuous? Thanks, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: [EMAIL PROTECTED] http://www.umassmed.edu/pmm/faculty/vandenberg.cfm -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
Re: [ccp4bb] discontinuous data wedges in XDS
Klaus Futterer schrieb: Is it not sufficient to first integrate the one wedge, then use this wedge as a reference data set when integrating the second one? (REFERENCE_DATA_SET= ../wedge01/XDS_ASCII.HKL) Klaus no, the REFERENCE_DATA_SET (first wedge of data) would then not be merged with the second wedge (which is the data integrated in that XDS run). Specifying a REFERENCE_DATA_SET would give you R-factors of the two wedges w.r.t. each other, and would also influence the scaling (which would not be intended in this case). Merging should be done with XSCALE (as Jürgen wrote already). best, Kay -- Kay Diederichshttp://strucbio.biologie.uni-konstanz.de email: [EMAIL PROTECTED]Tel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz smime.p7s Description: S/MIME Cryptographic Signature
Re: [ccp4bb] discontinuous data wedges in XDS
Hi Bert, xia2 is your friend in cases like this. This program is a real boon for the lazy crystallographer. All you need to type is: xia2 -3d /path/to/images and xia2 will automagically index, integrate and scale all of the sweeps together. Add the -atom Se flag (atom name as appropriate) to be sure you keep anomalous signal in the final mtz file. See http://www.ccp4.ac.uk/xia/ for more info. Cheers, Stephen On 4/29/08, Van Den Berg, Bert [EMAIL PROTECTED] wrote: Hi all, is it possible to input discontinuous data wedges into XDS (obtained from for example inverse beam sweeps)? (So wedge se1 goes from 0-90 deg (image 1-90), se2 from 180-270 (image 1-90), etc). Or do I have to rename everything so that I get one data file in which the rotation ranges are continuous? Thanks, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: [EMAIL PROTECTED] http://www.umassmed.edu/pmm/faculty/vandenberg.cfm -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] xds installation
Dear Eike,
Installing XDS is in fact pretty straightforward - if you unpack the XDS
tarball containing the executables somewhere sensible - e.g.
/opt/px/xds or /usr/local/xds then put wherever you unpacked this in
your path it will work fine.
You can do this by adding this to your .bashrc file:
export PATH=${PATH}:/where/I/put/xds
If you are using bash or
setenv PATH ${PATH}:/where/I/put/xds
To your .cshrc file for c-shell (do echo $SHELL to find out which you
are using)
The errors you have here look like what you get if you run a c-shell
script with the bash shell (usually the default under linux)
Hope this helps,
Best wishes,
Graeme
-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Eike Schulz
Sent: 30 April 2008 13:39
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] xds installation
Hello everyone,
I still consider myself new to Linux so this is probably just a minor
problem but currently I can't find a way around it - I hope that
somebody can help me.
Using the xds_inst script to install XDS I get the following error
messages:
$PATH/html_doc/xds_inst: line 20: setenv: command not found
$PATH/html_doc/xds_inst: line 23: syntax error near unexpected token `('
$PATH/html_doc/xds_inst: line 23: ` set path=($XDS $path)'
, since all the errors refer to lines of the input script 'you are not
about to change' I do not know where to start.
Thanks in advance for any advice
Kind regards
Eike
[ccp4bb] Rigaku X-ray Generator RUH3 for Sale
Dear Colleagues, we have an Rigaku RINT2000 Series, horizontal type rotaflex (RU-H3) rotating anode X-ray generator for sale at a nominal price plus lots of spare parts. The offer does not include the optics, the goniometer, any detector nor a water-cooling system. Included are all other units such as pumps and transformers required to run the generator . The purchaser would be responsible for collecting and transporting the generator from our site in Braunschweig, Germany prior to delivery of our new generator at the end of May 2008. Spare parts include: A complete, yet unused Cu-Target including the complete anode assembly. A complete anode assembly kit A box of 3 filaments (sealed) An unused Vacuum Gauge Tube A magnetic seal cassette (4980-3-2D) A sealed bearing (4555-0010) Various light bulbs, O-rings etc., etc. Overall, we would prefer one buyer taking the whole set, but failing this, we would pass on individual parts to interested parties. The offer is as is. The HZI will not carry any of the costs of shipping parts and will not accept any liability for defects or losses incurred by the buyer. No warranties or guarantees. The generator's history: It was installed in 1999/2000 and was in use without major problems until 2005 when metal debris in the in-house cooling water corroded one of the targets from inside leading to flooding of the evacuated regions. Following extensive cleaning and the replacement of the TMP the system has been running with increasing reliability for the last one and a half years. For the last half year the reliability is essentially back to what it was originally. Interested parties should please contact: Dr. Joachim Reichelt [EMAIL PROTECTED] tel.: +49 531 6181 7047 alternatively: Dr. Wolf-Dieter Schubert [EMAIL PROTECTED] tel.: +49 531 6181 7043 -- Wolf-Dieter Schubert, Dr.rer.nat. Molecular Host-Pathogen Interactions (MHPI) Division of Structural Biology (SB) Helmholtz-Centre for Infection Research (HZI) Inhoffenstr. 7 38124 Braunschweig Germany phone: +49-531-61817043 fax: +49-531-61817099 [EMAIL PROTECTED] www.helmholtz-hzi.de
Re: [ccp4bb] Are Calcium Citrate Crystals A Common False Positive?
Calcium citrate does have a relatively low solubility (~15 mM in the cold), and its solubility decreases as the temperature goes up. Thus, getting calcium citrate crystals is a possibility if both are at 200 mM. However, you can get sodium citrate up to ~1.4 M at least, depending on the pH. If they are harvestable crystals, just drop them into a low ionic strength buffered solution containing 0.2%-2% glutaraldehyde. Protein crystals will quickly be fixed quickly (faster than they dissolve) into a light golden, gelatinous lump. Sometimes they retain a crystal-like shape, other times they leave just a rubbery drop. In contrast, salt crystals should dissolve over time and should not be colored. You could also add a small drop of 2% glutaraldehyde to your protein drop. Protein crystals then turn a light golden color. Crystals fixed like this can then be put into a low ionic strength solution where salt crystals should dissolve. You can easily transfer glutaraldehyde into a protein drop by vapor diffusion by adding glutaraldehyde to the reservoir to make it 2-3%. Glutaraldehyde is quite volitile. If you can smell it, it is fixing your olfactory cells and corneas. Using solutions of glutaraldehyde less than 1% is generally safe. The only caveat to using this method is that there should be no free amines around other than on the protein (i.e., no ethanolamine or Tris buffer, no ammonium ions, etc.). I have never found a protein crystal I couldn't fix; I always end up with a gelatinous lump, at least. But there probably is an exception or two. I prefer this method over Izit and other dye methods. Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: [EMAIL PROTECTED] On Apr 29, 2008, at 9:42 PM, Dunten, Pete W. wrote: And how will you know if they are the calcium citrate xtals Sam asked about, and not sodium citrate xtals? Sodium citrate in the Hampton Crystal Screen condition will crystallize out at 4 degrees. It's solubiility is pH and temperature dependent. You'll need to go to your friendly neighborhood synchrotron and either look at the x-ray fluorescence emission spectrum to see if calcium is there, or collect a dataset and solve the structure. (Setting myself up here for a reply from Bruker about how easy the latter would be). Pete -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jim Pflugrath Sent: Tuesday, April 29, 2008 4:24 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Are Calcium Citrate Crystals A Common False Positive? That's an easy hypothesis to test. Simply set up your drops with the same conditions except without protein and see if you get crystals. Please let us know the results. Thanks! Jim On Tue, 29 Apr 2008, Sam Stephenson wrote: Are calcium citrate crystals a common false positive in trays with up to 200mM of each? There is absolutely no phosphate in the trays so I'm almost positive they're not calcium phosphate. Cheers, Sam
Re: [ccp4bb] xds installation
Eike Schulz schrieb:
Hello everyone,
I still consider myself new to Linux so this is probably just a minor
problem but currently I can't find a way around it - I hope that
somebody can help me.
Using the xds_inst script to install XDS I get the following error
messages:
$PATH/html_doc/xds_inst: line 20: setenv: command not found
$PATH/html_doc/xds_inst: line 23: syntax error near unexpected token `('
$PATH/html_doc/xds_inst: line 23: ` set path=($XDS $path)'
, since all the errors refer to lines of the input script 'you are not
about to change' I do not know where to start.
Thanks in advance for any advice
Kind regards
Eike
Eike,
the problem is probably that you use a different shell (bash ?) than
xds_inst is meant to be used with (tcsh or csh).
Someone who regularly uses bash (maybe your system admininistrator?)
should not have any difficulties in translating xds_inst into bash
language.
Even if you use tcsh or csh, you should source xds_inst (i.e. not
execute it). The only purpose of xds_inst is to put the binaries into
your $PATH. If they are already in your $PATH (like when they reside in
/usr/local/bin), then you don't really need xds_inst.
HTH,
Kay
--
Kay Diederichshttp://strucbio.biologie.uni-konstanz.de
email: [EMAIL PROTECTED]Tel +49 7531 88 4049 Fax 3183
Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz
smime.p7s
Description: S/MIME Cryptographic Signature
[ccp4bb] Bacterial induction at 18C
Hi Folks, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. I suspect (among many things) that Ampicillin+ Chloramphenicol+ Kanamycin in the medium may be the source of my woes. My colleagues have suggested growing cultures at 18C, say for 4-6h instead. Has anyone had reasonable protein expression levels by inducing cultures at 18C for 6h? From what I understand, the E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask. I am already playing with lowering and/or doing away with the antibiotics. Any suggestions wrt 18C? The protein is insoluble at 30C. Thanks. Raji
Re: [ccp4bb] Bacterial induction at 18C
Are you sure it is not a phage infection that is causing lysis of your cells? Christopher J. Law, PhD Skirball Institute of Biomolecular Medicine, Structural Biology (3-5), New York University Medical Center, 540 First Avenue, New York, NY 10016, USA. 2008/4/30 Raji Edayathumangalam [EMAIL PROTECTED]: Hi Folks, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. I suspect (among many things) that Ampicillin+ Chloramphenicol+ Kanamycin in the medium may be the source of my woes. My colleagues have suggested growing cultures at 18C, say for 4-6h instead. Has anyone had reasonable protein expression levels by inducing cultures at 18C for 6h? From what I understand, the E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask. I am already playing with lowering and/or doing away with the antibiotics. Any suggestions wrt 18C? The protein is insoluble at 30C. Thanks. Raji
Re: [ccp4bb] Bacterial induction at 18C
Raji, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. Sounds more like a phage contamination. The phage becomes active as soon as the cells energy level decreases, e.g upon induction. We had once the same trouble. If it is a phage, autoclave everything and clean the lab thoroughly. I suspect (among many things) that Ampicillin+ Chloramphenicol+ Kanamycin in the medium may be the source of my woes. My colleagues have suggested growing cultures at 18C, say for 4-6h instead. Has anyone had reasonable protein expression levels by inducing cultures at 18C for 6h? From what I understand, the E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask. Rule of the thumb is the Q10 rule, or in the case of e.coli it is a Q7 rule. Doubling decreases twofold when temperature eis decreased by 7 deg C. Godd luck, Guenter I am already playing with lowering and/or doing away with the antibiotics. Any suggestions wrt 18C? The protein is insoluble at 30C. Thanks. Raji -- *** Priv.Doz.Dr. Guenter Fritz Fachbereich Biologie Sektion Naturwissenschaften Universitaet Konstanz http://www.biologie.uni-konstanz.de/fritz Universitaetsstrasse 10 Postfach M665 D-78457 Konstanz e-mail: [EMAIL PROTECTED] Tel. Office: +49-(0)7531 88 3205 Tel. Lab : +49-(0)7531 88 3687 Fax: +49-(0)7531 88 2966
Re: [ccp4bb] Bacterial induction at 18C
Dear Raji, I have expressed proteins in E. coli at 18ºC (and below that) with Amp+Kan+Chl and I've never had any problem with cell lysis. I usually grow cells at 37ºC and then cool the cultures down to the desired temperature before induction. I don't recomend growing your cultures at low temperature, specially having three different antibiotic resistences, as it may take a lot of time. Regarding expression time at 18ºC, I did had a protein that was expressed during only 6 hours at 17ºC and the yeld was reasonable. But normally I need longer expression times, sometimes even days. Hope it helps, Raquel Hi Folks, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. I suspect (among many things) that Ampicillin+ Chloramphenicol+ Kanamycin in the medium may be the source of my woes. My colleagues have suggested growing cultures at 18C, say for 4-6h instead. Has anyone had reasonable protein expression levels by inducing cultures at 18C for 6h? From what I understand, the E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask. I am already playing with lowering and/or doing away with the antibiotics. Any suggestions wrt 18C? The protein is insoluble at 30C. Thanks. Raji
Re: [ccp4bb] Bacterial induction at 18C
Hi Raji, Any possibility that your protein lyzed the E. coli. Some proteins do that. It's unlikely the antibiotics are the problem. You have to find out when the bugs start to lyze and harvest before that time point. In addition, lower the induction temperature further down in case the lysis is due to the enzymatic activity of your protein. We actually had a case that making an inactive mutant solved the problem. Good luck! Chun Chun Luo, Ph.D. The Protein Expert Accelagen, Inc. 11585 Sorrento Valley Road, Suite 107 San Diego, CA 92121 TeL: 858-350-8085 ext 111 Fax: 858-350-8001 [EMAIL PROTECTED] www.accelagen.com -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Raji Edayathumangalam Sent: Wednesday, April 30, 2008 8:40 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Bacterial induction at 18C Hi Folks, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. I suspect (among many things) that Ampicillin+ Chloramphenicol+ Kanamycin in the medium may be the source of my woes. My colleagues have suggested growing cultures at 18C, say for 4-6h instead. Has anyone had reasonable protein expression levels by inducing cultures at 18C for 6h? From what I understand, the E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask. I am already playing with lowering and/or doing away with the antibiotics. Any suggestions wrt 18C? The protein is insoluble at 30C. Thanks. Raji
[ccp4bb] Synchrotron beam time at EMBL Hamburg
This is a gentle reminder of our call for beam time applications at EMBL Hamburg that we circulated a few weeks ago. We kindly ask you to complete your beam time proposal by the deadline of --- 16 May 2008 If you have already taken action, please ignore this message. --- EMBL HAMBURG UNIT Call for access to Synchrotron Beamline Facilities 2008 We announce a call for synchrotron beam time applications in biological X-ray crystallography (PX) and small-angle scattering (SAXS). Up to 12 weeks of beam time will be available at the DORIS storage ring (DESY) during the period September 2008 until December 2008. The EMBL Outstation will operate the following beamlines: BeamlineTypeWavelength Scientist responsible X11 PX 0.80 A Paul Tucker X12 PX tuneable Manfred Weiss X13 PX 0.80 A Matthew Groves X33 SAXS1.5 A Dmitri Svergun, Manfred Roessle The deadline for submission of proposals is May 16th, 2008. An external Priorities Committee will assess the proposals. Electronic beam proposal forms and detailed description of the beamline facilities are available via the web links http://www.embl-hamburg.de and http://www.embl-hamburg.de/services In parallel, EMBL-Hamburg is constructing three new beamlines for applications in biological X-ray crystallography (PX) and small-angle scattering (SAXS) at the Petra-III synchrotron storage ring, with an expected opening in 2010/11. Further information can be obtained under http://www.embl-hamburg.de/services/petra. Two of the DORIS-III beamlines (BW7A, BW7B) will be used as test beamlines for future Petra-III applications. Depending on circumstances, they may become temporarily available to the external user community. Applications to use the EMBL-Hamburg high-throughput crystallisation facility can be made at any time at http://www.embl-hamburg.de/services/crystallisation Further information can be obtained by tel. +49-40-89902-110, (fax +49-40-89902-149), Email [EMAIL PROTECTED] (PX), [EMAIL PROTECTED] (SAXS). Access to the EMBL Hamburg facilities is supported by the European Commission, Research Infrastructure Action under the FP6 'Structuring the European Research Area Specific Programme', Contract Number RII3-CT-2004-506008.
Re: [ccp4bb] Bacterial induction at 18C
Thanks to everyone for all your suggestions. I am growing the cultures as we speak and have increased the temp to 22C and plan to harvest in about 6-8 hrs. Thanks for the Q7 rule. I read it before but I couldn't remember exactly and a quick-and-dirty Google and Pubmed search did not bring it up. Let me clarify what I mean by lysis. Here are my observations: a) At the time of harvest, the final OD is lower for protein A + protein B (on two plasmids) than that for the same cells expressing only protein A or protein B (all else being similar at the time of induction). b) When the cells are spun down, the supernatant is cloudy and the pellet is smaller for A+B. The supernatant is clear for A alone or B alone. I am not sure this is a result of phage contamination since I have two other 'controls' for the same batch of competent cells in the same shaker, one containing just plasmid A and the other with only plasmid B. And, this is reproducible. Yes, I also very much suspect that my proteins may be a culprit, even though I only mentioned the antibiotics. Will see what happens this time. Thanks very much for all the helpful suggestions. Raji -Included Message-- Date: 30-apr-2008 12:30:50 -0400 From: Guenter Fritz [EMAIL PROTECTED] To: [EMAIL PROTECTED] Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Bacterial induction at 18C Raji, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. Sounds more like a phage contamination. The phage becomes active as soon as the cells energy level decreases, e.g upon induction. We had once the same trouble. If it is a phage, autoclave everything and clean the lab thoroughly. I suspect (among many things) that Ampicillin+ Chloramphenicol+ Kanamycin in the medium may be the source of my woes. My colleagues have suggested growing cultures at 18C, say for 4-6h instead. Has anyone had reasonable protein expression levels by inducing cultures at 18C for 6h? From what I understand, the E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask. Rule of the thumb is the Q10 rule, or in the case of e.coli it is a Q7 rule. Doubling decreases twofold when temperature eis decreased by 7 deg C. Godd luck, Guenter I am already playing with lowering and/or doing away with the antibiotics. Any suggestions wrt 18C? The protein is insoluble at 30C. Thanks. Raji -- *** Priv.Doz.Dr. Guenter Fritz Fachbereich Biologie Sektion Naturwissenschaften Universitaet Konstanz http://www.biologie.uni-konstanz.de/fritz Universitaetsstrasse 10 Postfach M665 D-78457 Konstanz e-mail: [EMAIL PROTECTED] Tel. Office: +49-(0)7531 88 3205 Tel. Lab : +49-(0)7531 88 3687 Fax: +49-(0)7531 88 2966 -End of Included Message--
[ccp4bb] CNS 1.2.2 binary running out of memory
Hi
Since I am not on the cnsbb yet I am posting this here.
I downloaded the cns 1.2.2 intel build and was trying to run a simulated
annealing refinement on my macbook pro ( Intel) running 10.5.2 .
However the annealing job crashes roughly 40 minutes into the refinement
with the following message
There is not enough memory available to the program.
This may be because of too little physical memory (RAM)
or too little swap space on the machine. It could also be
the result of user or system limits. On most Unix systems
the limit command can be used to check the current user
limits. Please check that the datasize, memoryuse and
vmemoryuse limits are set at a large enough value.
Unfortunately on Leopard it seems that unlimit and limit are not available
under bash
Further when I use csh , I get the following values for the limits
[mango:~/aps_04_21_2008/p10_2] hari% limit
cputime unlimited
filesize unlimited
datasize 6144 kbytes
stacksize8192 kbytes
coredumpsize 0 kbytes
memoryuseunlimited
descriptors 256
memorylocked unlimited
maxproc 266
In the same csh shell unlimit returns
[mango:~/aps_04_21_2008/p10_2] hari% unlimit
unlimit: descriptors: Can't remove limit (Invalid argument)
How can I setup cns to have free reign and use up unlimited datasize and
stacksize for all cns jobs?
Thanks for your help in advance
Hari Jayaram
The detailed error is posted below
ASSFIL: file /Users/hari/cns/cns_solve_1.2/libraries/toppar/torsionmdmods
opened.
MESSage=NORM
EVALUATE: symbol $MESSAGE_OLD_TMOD set to NORM (string)
ECHO=FALSe {OFF}
EVALUATE: symbol $ECHO_OLD_TMOD set to FALSE (logical)
NEXTCD: condition evaluated as false
Program version= 1.2 File version= 1.2
SELRPN: 0 atoms have been selected out of 2380
cns_solve(93676) malloc: *** mmap(size=300512) failed (error code=12)
*** error: can't allocate region
*** set a breakpoint in malloc_error_break to debug
ALLHP: request for -1294967296 bytes
-
There is not enough memory available to the program.
This may be because of too little physical memory (RAM)
or too little swap space on the machine. It could also be
the result of user or system limits. On most Unix systems
the limit command can be used to check the current user
limits. Please check that the datasize, memoryuse and
vmemoryuse limits are set at a large enough value.
-
%ALLHP error encountered: not enough memory available
(CNS is in mode: SET ABORT=NORMal END)
*
ABORT mode will terminate program execution.
*
Program will stop immediately.
Maximum dynamic memory allocation: 139649464 bytes
Maximum dynamic memory overhead: 944 bytes
Program started at: 14:51:17 on 30-Apr-2008
Program stopped at: 15:09:16 on 30-Apr-2008
CPU time used:1077.7678 seconds
Re: [ccp4bb] CNS 1.2.2 binary running out of memory
You need to use the syntax: unlimit stacksize unlimit datasize unlimit memoryuse and I have these in my .cshrc I can get this under OSX 10.5 (albeit on an old G5 chip machine): cputime unlimited filesize unlimited datasize unlimited stacksize65532 kbytes coredumpsize unlimited memoryuseunlimited descriptors 256 memorylocked unlimited maxproc 266 In the above there's an undesirable unlimited core dump size because I have this account set up for debugging. On 10.4 on similar hardware I get: cputime unlimited filesizeunlimited datasizeunlimited stacksize 65536 kbytes coredumpsize0 kbytes memoryuse unlimited descriptors 256 memorylockedunlimited maxproc 100 Hope this helps, Phil Jeffrey Princeton hari jayaram wrote: Hi Since I am not on the cnsbb yet I am posting this here. I downloaded the cns 1.2.2 intel build and was trying to run a simulated annealing refinement on my macbook pro ( Intel) running 10.5.2 . However the annealing job crashes roughly 40 minutes into the refinement with the following message There is not enough memory available to the program. This may be because of too little physical memory (RAM) or too little swap space on the machine. It could also be the result of user or system limits. On most Unix systems the limit command can be used to check the current user limits. Please check that the datasize, memoryuse and vmemoryuse limits are set at a large enough value. Unfortunately on Leopard it seems that unlimit and limit are not available under bash Further when I use csh , I get the following values for the limits [mango:~/aps_04_21_2008/p10_2] hari% limit cputime unlimited filesize unlimited datasize 6144 kbytes stacksize8192 kbytes coredumpsize 0 kbytes memoryuseunlimited descriptors 256 memorylocked unlimited maxproc 266 In the same csh shell unlimit returns [mango:~/aps_04_21_2008/p10_2] hari% unlimit unlimit: descriptors: Can't remove limit (Invalid argument) [snip]
Re: [ccp4bb] Bacterial induction at 18C
Raji aside from the possibilites of toxic protein as already mentioned.. we had great results with overnight induction at 20oC for a protein that was somewhat insoluble at 37oC. One thing that might work for you is to grow the cells to OD 0.5 then lower the temperature to say 18 or 20. After an hour or so (retake OD) induce for overnight growth. Alternate is higher starting OD prior to temperature reduction (i.e greater mass of cells) and shorter growth time say 5-6 hours as you are thinking (we had protein that we only induced for 1 and a half hours too). Hope that is useful! Gina -- Hi Folks, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. I suspect (among many things) that Ampicillin+ Chloramphenicol+ Kanamycin in the medium may be the source of my woes. My colleagues have suggested growing cultures at 18C, say for 4-6h instead. Has anyone had reasonable protein expression levels by inducing cultures at 18C for 6h? From what I understand, the E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask. I am already playing with lowering and/or doing away with the antibiotics. Any suggestions wrt 18C? The protein is insoluble at 30C. Thanks.
