Re: [ccp4bb] One little clash
Christine, As interesting as the di-Tyrosine idea is, from the picture you provided, it looks more like the two tyrosines share a C=C bond. Given your slightly elevated R-factors and the high symmetry space group, I would thoroughly check for even the slightest sign of twinning. I once had a case of a coiled coil that seemed to be sitting on a crystallographic two-fold. R-factors were a bit too high for the resolution. Lowering the symmetry and redefining that two-fold axis as a twinning operator lead to very reasonable R-factors, and the ends of the helices (in the original model "disordered") to adopt obviously different conformations. At the time, we did not feel too confident with that interpretation (swapping crystallographic symmetry for a twinning operator and an NCS operator at the same time) and screened for a crystal that seemed not to suffer from that malady (which we found); therefore it was - in hindsight unfortunately - never published in that form. Nowadays, I feel very strongly, that reducing the symmetry and reassigning certain "crystallographic" operators to a mix of twinning operators/NCS operators to be actually a legitimate - and often probably more correct - way of interpreting certain problematic crystals. Just 2 cents Jens On Wed, 2012-07-11 at 13:37 -0600, Lukacs, Christine wrote: > Hi all- > > > > I have a protein that crystallizes in I422, and diffracts well, > between 1.3-1.7A. Beautiful density, slightly higher final R-factors > than you might expect at this resolution (low to mid 20s). The > density is all beautiful, except that I have this one little clash, > between a few atoms from a tyrosine and its symmetry mate. In this > picture I have it modeled as an Alanine and you can see the two > tyrosine rings interlocking; and there is clearly no alternate > conformation. > > > > > > > > Since it is not near my site of interest, I have been pretty much > ignoring it, going through refinement with it as an alanine, then > changing it at the very end to a tyrosine and just minimizing B-s, no > positional. Now that I plan to publish a bunch of these, I should > probably figure out what is really going on. Any insights? > > > > Thanks > > Christine > > Christine Lukacs > Roche > > This message is intended for the use of the named recipient(s) only > and may contain confidential and/or proprietary information. If you > are not the intended recipient, please contact the sender and delete > this message. Any unauthorized use of the information contained in > this message is prohibited. > > > >
Re: [ccp4bb] One little clash
googling "dityrosine" suggests thaey can be formed by osidation by Cu or Ni ions. In structures of cytochrome oxidase, Tyrosine forms a covalent bond from the same meta carbon to NE2 of a histidine. see Fig 1 of: http://www.sciencemag.org/content/280/5370/1723.long And histidine is ligating a Cu. It is believed to be functionally relevant. James Stroud wrote: The hydroxyls were on the wrong carbons in the previous picture I sent. These are correct. James On Jul 11, 2012, at 1:37 PM, Lukacs, Christine wrote: Hi all- I have a protein that crystallizes in I422, and diffracts well, between 1.3-1.7A. Beautiful density, slightly higher final R-factors than you might expect at this resolution (low to mid 20s). The density is all beautiful, except that I have this one little clash, between a few atoms from a tyrosine and its symmetry mate. In this picture I have it modeled as an Alanine and you can see the two tyrosine rings interlocking; and there is clearly no alternate conformation. Since it is not near my site of interest, I have been pretty much ignoring it, going through refinement with it as an alanine, then changing it at the very end to a tyrosine and just minimizing B-s, no positional. Now that I plan to publish a bunch of these, I should probably figure out what is really going on. Any insights? Thanks Christine *Christine Lukacs* Roche This message is intended for the use of the named recipient(s) only and may contain confidential and/or proprietary information. If you are not the intended recipient, please contact the sender and delete this message. Any unauthorized use of the information contained in this message is prohibited.
[ccp4bb] CNS installation
Dear ccp4 users, I tried to install CNS under Debian 64bit. I followed the installation giude as reported by CNS developers but I received the following message when souurcing cns_solve_env: bash: setenv: command not found bash: setenv: command not found bash: cns_solve_env: line 32: syntax error near unexpected token `setenv' bash: cns_solve_env: line 32: ` if ( ! $?CNS_ARCH ) setenv CNS_ARCH ` $CNS_SOLVE/bin/getarch`' I know that the script would set all variables; it is indicated for csh (or tcsh) shell. I tried to run the script under a csh shell but I received a different error: Word too long The above statement is also received if a bash-optimized script is run. Does anyone have experience in CNS installation and environment setting? Any advice? Thanks in advance Fulvio Saccoccia Dept. of Biochemical Sciences Sapienza University of Rome, Italy
Re: [ccp4bb] FE-Sulfur proteins
Hi Jan, I wonder if the protein has a hexaHis tag. I recently was working on a Fe-S containing protein and noticed significant aggregation/precipitation. After I cleaving the His tag, the enzyme seems stable for days in the same buffer. HTH, Yuri
Re: [ccp4bb] CNS installation
Hi Fulvio Your scripts are for csh (or tcsh) so won't work under bash. Have you checked whether there are versions for sh (or bash) already set up? If not you will have to run them under csh, or better still tcsh. If the script doesn't work with the version of csh or tcsh you are using you should try debugging it, since the error message by itself is fairly useless without knowing which command in your script caused it. Debugging shell scripts is very simple: just type 'set verbose' or 'set echo' at the command line before sourcing your script: 'set verbose' echoes the lines of your script before variable substitution, and 'set echo' echoes them after substitution (the 'unset' command reverses the effect of 'set'). You should be able to tell from the last line echoed which is the offending command in the script that is giving the error message; then we can go from there. An unfortunate downside of using csh/tcsh is that unlike bash the syntax is not standardised, so if despite all efforts at debugging, the version you are using steadfastly refuses to work, you could try to install another version, though admittedly this may not be easy if it's not supported by your distribution. Cheers -- Ian On 12 July 2012 15:49, fulvio saccoccia wrote: > Dear ccp4 users, > I tried to install CNS under Debian 64bit. I followed the installation > giude as reported by CNS developers but I received the following > message when souurcing cns_solve_env: > > bash: setenv: command not found > bash: setenv: command not found > bash: cns_solve_env: line 32: syntax error near unexpected token > `setenv' > bash: cns_solve_env: line 32: ` if ( ! $?CNS_ARCH ) setenv CNS_ARCH ` > $CNS_SOLVE/bin/getarch`' > > I know that the script would set all variables; it is indicated for csh > (or tcsh) shell. I tried to run the script under a csh shell but I > received a different error: > > Word too long > > The above statement is also received if a bash-optimized script is run. > > Does anyone have experience in CNS installation and environment setting? > Any advice? > > Thanks in advance > > Fulvio Saccoccia > Dept. of Biochemical Sciences > Sapienza University of Rome, Italy
Re: [ccp4bb] CNS installation
Dear Fulvio Saccoccia, along the lines of Ian Tickle's reply: there should be a script "cns_solve_env_sh" using /bin/sh, which is usually a soft-link to /bin/bash. I use this script for setup of CNS under bash. Best regards, Dirk. Am 12.07.12 16:49, schrieb fulvio saccoccia: Dear ccp4 users, I tried to install CNS under Debian 64bit. I followed the installation giude as reported by CNS developers but I received the following message when souurcing cns_solve_env: bash: setenv: command not found bash: setenv: command not found bash: cns_solve_env: line 32: syntax error near unexpected token `setenv' bash: cns_solve_env: line 32: ` if ( ! $?CNS_ARCH ) setenv CNS_ARCH ` $CNS_SOLVE/bin/getarch`' I know that the script would set all variables; it is indicated for csh (or tcsh) shell. I tried to run the script under a csh shell but I received a different error: Word too long The above statement is also received if a bash-optimized script is run. Does anyone have experience in CNS installation and environment setting? Any advice? Thanks in advance Fulvio Saccoccia Dept. of Biochemical Sciences Sapienza University of Rome, Italy -- *** Dirk Kostrewa Gene Center Munich Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de ***
Re: [ccp4bb] cryo for high salt crystal
Hi Jim, 25% is w/v. Thanks for the information. Will check the webinar. Thanks,Min From: jim.pflugr...@rigaku.com To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] cryo for high salt crystal Date: Tue, 10 Jul 2012 17:39:56 + Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that w/v or v/w?), try using 100% saturated in reservoir, 75% saturated in reservoir, or 50% saturated in reservoir. You will have to TEST these. See also this webinar on cryocrystallography which shows how to make these solutions: http://www.rigaku.com/node/1388 You could also try high salt solutions with similar technique. Good luck! Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m zhang [mzhang...@hotmail.com] Sent: Tuesday, July 10, 2012 11:28 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] cryo for high salt crystal regaentDear All, I am sure this question was discussed before. But I am wondering if anyone got the same experience as I do. I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, or ammonium sulfate itself: The problem is that all the cryo plus original reagents in the reservoir precipitate the salts out. And more serious problem is because of high salt in the condition, while I am trying to loop the crystal, both the drop and cryoprotectant drop form salt crystals (not sure it is KCl or ammonia sulfate) significantly and very quickly, that cause my crystal dissolved. My crystal doesn't seem to survive paraton-N oil. Does anyone here have similiar case? any suggestion will be appreciated. Thanks, Min
Re: [ccp4bb] cryo for high salt crystal
We frequently crystallize one of our proteins and variants of it in 1.6-1.8 M ammonium sulfate solutions. Cryoprotection with 25-30% glycerol or 25-30% glucose does not cause precipitation of salts. Both KCl (4.6 M) and ammonium sulfate (5.6 M) have enormous solubilities in water, so I would not expect cryoprotectant concentrations of glycerol or glucose to cause precipitation (We can save cryoprotectant solutions of at least 2 M ammonium sulfate indefinitely). How are you introducing cryprotectant? We use one of two methods: 1. Fish the crystal out of the mother liquor and place into artificial mother liquor with the same composition as the well solution + cryoprotectant. For glycerol or other liquids, you have to make this from scratch. For glucose, we just weigh out 300 mg of glucose in a microcentrifuge tube and make to the 1.0 mL mark with well solution. (Mix well of course before use. Gentle heating in a block or sonication will help dissolve the glucose. 2. Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to the drop the crystals are in. You can do this all at once, or in stages, keeping the drop hydrated by placing the hanging drop back in the well between additions. If your drops are drying out during crystal harvesting (very possible in dry conditions), you might try harvesting in the cold room, where evaporation is slower. We often have problems with crystal cracking and drop-drying in the winter months when the humidity is very low indoors. The cold room is usually humid enough and cold enough to slow evaporation to allow crystal harvesting. (I hate working in the meat locker, though.) Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 7/12/2012 12:55 PM, m zhang wrote: Hi Jim, 25% is w/v. Thanks for the information. Will check the webinar. Thanks, Min From: jim.pflugr...@rigaku.com To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] cryo for high salt crystal Date: Tue, 10 Jul 2012 17:39:56 + Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that w/v or v/w?), try using 100% saturated in reservoir, 75% saturated in reservoir, or 50% saturated in reservoir. You will have to TEST these. See also this webinar on cryocrystallography which shows how to make these solutions: http://www.rigaku.com/node/1388 You could also try high salt solutions with similar technique. Good luck! Jim *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m zhang [mzhang...@hotmail.com] *Sent:* Tuesday, July 10, 2012 11:28 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] cryo for high salt crystal regaentDear All, I am sure this question was discussed before. But I am wondering if anyone got the same experience as I do. I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, or ammonium sulfate itself: The problem is that all the cryo plus original reagents in the reservoir precipitate the salts out. And more serious problem is because of high salt in the condition, while I am trying to loop the crystal, both the drop and cryoprotectant drop form salt crystals (not sure it is KCl or ammonia sulfate) significantly and very quickly, that cause my crystal dissolved. My crystal doesn't seem to survive paraton-N oil. Does anyone here have similiar case? any suggestion will be appreciated. Thanks, Min
Re: [ccp4bb] cryo for high salt crystal
Roger's note reminded me of some older literature (old in the sense that this problem extends back into the mid-1970's). Dealing with cryopreservation of crystals grown in "high" salt can be a real problem, but as many people have pointed out, the normal cryoprotectants can work, although many salts work just as well (not only malonate, but ammonium formate, lithium citrate, etc.). I don't consider 1.6-2 M ammonium sulfate as really high salt; try working with 3 M ammonium sulfate. However, the trick is not simply the cryoprotectant, but also the exchange method, as Roger mentions. While you can make artificial mother liquors (I love these old terms) with high concentrations of salt and nonionic cryoprotectants (sugars, glycerol, ethylene glycol, etc.), that does not mean they will readily exchange with the crystal, even after soaking for hours. Bill Ray, a classical enzymologist and physical biochemist from Purdue, really wanted to determine the crystal structure of phosphoglucomutase with ligands, but there were many difficulties. Using crystals grown in 2.1 M ammonium sulfate was one of these problems. He realized that the phase interactions between the bulk solution (i.e., the mother liquor) and the interstitial salt-rich solvent was a major obstacle in the proper solvent exchange and good subsequent cryopreservation. See how he solved the problem: W. J. Ray, Jr., et al. Removal of salt from a salt-induced protein crystal without cross-linking. Preliminary examination of "desalted" crystals of phosphoglucomutase by X-ray crystallography at low temperature. Biochemistry. 1991 Jul 16;30(28):6866-75. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Jul 12, 2012, at 2:10 PM, Roger Rowlett wrote: > We frequently crystallize one of our proteins and variants of it in 1.6-1.8 M > ammonium sulfate solutions. Cryoprotection with 25-30% glycerol or 25-30% > glucose does not cause precipitation of salts. Both KCl (4.6 M) and ammonium > sulfate (5.6 M) have enormous solubilities in water, so I would not expect > cryoprotectant concentrations of glycerol or glucose to cause precipitation > (We can save cryoprotectant solutions of at least 2 M ammonium sulfate > indefinitely). How are you introducing cryprotectant? We use one of two > methods: > > Fish the crystal out of the mother liquor and place into artificial mother > liquor with the same composition as the well solution + cryoprotectant. For > glycerol or other liquids, you have to make this from scratch. For glucose, > we just weigh out 300 mg of glucose in a microcentrifuge tube and make to the > 1.0 mL mark with well solution. (Mix well of course before use. Gentle > heating in a block or sonication will help dissolve the glucose. > Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to the drop > the crystals are in. You can do this all at once, or in stages, keeping the > drop hydrated by placing the hanging drop back in the well between additions. > If your drops are drying out during crystal harvesting (very possible in dry > conditions), you might try harvesting in the cold room, where evaporation is > slower. We often have problems with crystal cracking and drop-drying in the > winter months when the humidity is very low indoors. The cold room is usually > humid enough and cold enough to slow evaporation to allow crystal harvesting. > (I hate working in the meat locker, though.) > Cheers, > ___ > Roger S. Rowlett > Gordon & Dorothy Kline Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 > fax: (315)-228-7935 > email: rrowl...@colgate.edu > > > On 7/12/2012 12:55 PM, m zhang wrote: >> Hi Jim, >> >> 25% is w/v. Thanks for the information. Will check the webinar. >> >> Thanks, >> Min >> >> From: jim.pflugr...@rigaku.com >> To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK >> Subject: RE: [ccp4bb] cryo for high salt crystal >> Date: Tue, 10 Jul 2012 17:39:56 + >> >> Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that w/v >> or v/w?), try using 100% saturated in reservoir, 75% saturated in reservoir, >> or 50% saturated in reservoir. You will have to TEST these. See also this >> webinar on cryocrystallography which shows how to make these solutions: >> http://www.rigaku.com/node/1388 >> >> You could also try high salt solutions with similar technique. >> >> Good luck! >> >> Jim >> >> >> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m zhang >> [
Re: [ccp4bb] CNS installation
I think the bash-compatible startup script has disappeared from CNS
distribution at some point. The one that was distributed with cns 1.1
still works though, and I attach my copy of it.
On 07/12/2012 11:24 AM, Dirk Kostrewa wrote:
Dear Fulvio Saccoccia,
along the lines of Ian Tickle's reply: there should be a script
"cns_solve_env_sh" using /bin/sh, which is usually a soft-link to
/bin/bash. I use this script for setup of CNS under bash.
Best regards,
Dirk.
Am 12.07.12 16:49, schrieb fulvio saccoccia:
Dear ccp4 users,
I tried to install CNS under Debian 64bit. I followed the
installation
giude as reported by CNS developers but I received the following
message when souurcing cns_solve_env:
bash: setenv: command not found
bash: setenv: command not found
bash: cns_solve_env: line 32: syntax error near unexpected token
`setenv'
bash: cns_solve_env: line 32: ` if ( ! $?CNS_ARCH ) setenv CNS_ARCH `
$CNS_SOLVE/bin/getarch`'
I know that the script would set all variables; it is indicated for csh
(or tcsh) shell. I tried to run the script under a csh shell but I
received a different error:
Word too long
The above statement is also received if a bash-optimized script is run.
Does anyone have experience in CNS installation and environment setting?
Any advice?
Thanks in advance
Fulvio Saccoccia
Dept. of Biochemical Sciences
Sapienza University of Rome, Italy
#!/bin/sh
#
# This file sets up the appropriate environmental variables and paths
# for CNSsolve. In the case of the same machines with different versions
# of the OS, backward compatibility is assumed - ie. a later version will
# be setup for a previous version of the OS if nothing else is available.
#
# written by: Paul Adams
#
# copyright Yale University
#
# ==
#
# >> Important: define the location of the CNSsolve directory <<
#
# CHANGE THE NEXT LINE TO POINT TO THE LOCATION OF THE CNSsolve DIRECTORY
CNS_SOLVE='/sware/cns'
#
# ==
#
# full expansion of the CNS_SOLVE variable prior to use.
#
export CNS_SOLVE; CNS_SOLVE=$CNS_SOLVE
#
# ==
#
# set the number of threads for SGI multiprocessors
# if this causes a problem on other systems it can be commented out
#
export MP_SET_NUMTHREADS; MP_SET_NUMTHREADS=1
#
# ==
#
# get the machine architecture
#
if [ -d $CNS_SOLVE ]; then
if [ ! "$CNS_ARCH" ]; then
export CNS_ARCH; CNS_ARCH=`$CNS_SOLVE/bin/getarch`
fi
else
export CNS_ARCH; CNS_ARCH='unknown'
fi
#
# ==
#
# general environmental variables
#
export CNS_LIB; CNS_LIB=$CNS_SOLVE/libraries
export CNS_MODULE; CNS_MODULE=$CNS_SOLVE/modules
export CNS_TOPPAR; CNS_TOPPAR=$CNS_LIB/toppar
export CNS_CONFDB; CNS_CONFDB=$CNS_LIB/confdb
export CNS_XTALLIB; CNS_XTALLIB=$CNS_LIB/xtal
export CNS_NMRLIB; CNS_NMRLIB=$CNS_LIB/nmr
export CNS_XRAYLIB; CNS_XRAYLIB=$CNS_LIB/xray
export CNS_XTALMODULE; CNS_XTALMODULE=$CNS_MODULE/xtal
export CNS_NMRMODULE; CNS_NMRMODULE=$CNS_MODULE/nmr
export CNS_HELPLIB; CNS_HELPLIB=$CNS_SOLVE/helplib
#
# general user aliases
#
cns_web () { $CNS_SOLVE/bin/cns_web; }
cns_header () { $CNS_SOLVE/bin/cns_header; }
cns_info () { cat $CNS_SOLVE/bin/cns_info; }
cns_transfer () { $CNS_SOLVE/bin/cns_transfer; }
if [ -x $CNS_SOLVE/bin/cns_edit_local ]; then
cns_edit () { $CNS_SOLVE/bin/cns_edit_local; }
else
cns_edit () { $CNS_SOLVE/bin/cns_edit; }
fi
run_tutorial () { "csh -f tutorial.csh"; }
#
# g77 compilation and use
#
g77on () { CNS_G77=ON; . $CNS_SOLVE/.cns_solve_env_sh; }
g77off () { unset CNS_G77; . $CNS_SOLVE/.cns_solve_env_sh; }
#
# developer aliases
#
run_tests () { $CNS_SOLVE/bin/run_tests; }
run_diffs () { $CNS_SOLVE/bin/run_diffs; }
maketar () { $CNS_SOLVE/bin/maketar; }
create_patch () { $CNS_SOLVE/bin/create_patch; }
#
#
# ==
#
# to do expansions - unset noglob just in case user has it otherwise
#
set +f
#
# try to set up appropriate path
#
# first strip off any trailing information (eg. _g77)
#
CNS_ARCH=`echo ${CNS_ARCH} | sed -e 's/_g77//g'`
#
cns_vendor=`echo $CNS_ARCH | awk 'BEGIN{FS="-"}{print $1}'`
cns_cpu=`echo $CNS_ARCH | awk 'BEGIN{FS="-"}{print $2}'`
cns_os=`echo $CNS_ARCH | awk 'BEGIN{FS="-"}{print $3}'`
cns_major=`echo $CNS_ARCH | awk 'BEGIN{FS="-"}{print $4}'`
cns_minor=`echo $cns_major | sed -e 's/\./ /g'`
#
# if we are looking for a specific type of setup then limit search
#
cns_dirs=""
if [ ! "$CNS_G77" ]; then
if /bin/ls -d $CNS_SOLVE/$cns_vendor-* >/dev/null 2>&1 ; then
cns_dirs="`/bin/ls -d $CNS_SOLVE/$cns_vendor-* 2>&1 | awk
'BEGIN{FS="/"}{print $NF}' | sort -t\- +3 -4 -n -r`"
fi
else
CNS_ARCH="${CNS_ARCH}_g77"
if /bi
Re: [ccp4bb] cryo for high salt crystal
Hello, Min. Lithium sulfate is a cryoprotectant at 2.0 M and sometimes even less (much lower than the concentrations needed for ammonium sulfate), so I would try replacing your ammonium sulfate with lithium sulfate, creating a cryoprotectant with 0.5-1.0 M KCl, 1.4-2.0 M LiSO4, at pH 7. You might need to transfer your crystals through a couple of intermediate drops. Regarding the reservoir precipitation - is there any chance you could control the humidity of the area in which you're working? Even filling a couple of adjacent reservoirs with water might help buy you a few extra crucial seconds. Also, working in a cold room to harvest your crystals will help reduce the evaporation rate. Good luck! Best, Anna On Tue, Jul 10, 2012 at 9:28 AM, m zhang wrote: > regaentDear All, > > I am sure this question was discussed before. But I am wondering if anyone > got the same experience as I do. > I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at > pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, > or ammonium sulfate itself: The problem is that all the cryo plus original > reagents in the reservoir precipitate the salts out. And more serious > problem is because of high salt in the condition, while I am trying to loop > the crystal, both the drop and cryoprotectant drop form salt crystals (not > sure it is KCl or ammonia sulfate) significantly and very quickly, that > cause my crystal dissolved. My crystal doesn't seem to survive paraton-N > oil. Does anyone here have similiar case? any suggestion will be > appreciated. > > Thanks, > Min >
Re: [ccp4bb] Disulphide bonds and closed conformation
Hi Jan, you will find some methods for detection of thiols and/or disulfides there: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Thiols_and_disulfides HTH, Guenter Dear all, I am working on a protein where I have to stabilize the closed conformation of the protein using disulphide bond. The strategy to design the cysteine mutants is based on the molecular dynamic simulations, and accordingly the residues were chosen. The ultimate goal is to trap the ligand in closed conformation of protein and crystallize it. I am facing few issues: Is there some reliable assay that can check the formation of disulphide bonds in protein. Additionally, does anybody knows another method(s) that can be used to trap a closed conformation. I look forward for your suggestions and discussions on this issue. Thanks very much! Jan
[ccp4bb] offtopic: AKTA prime
Hi all, We've got an old Akta prime that I think is on the verge of kicking it. Hearing some high pitched sounds coming from the pump when we're running it. Line A seems clogged and makes a thudding noise when we try to do a pump wash through that line. Does anyone have any experience w/fixing one/replacing the pump? Or any idea how much it's going to set us back to get it fixed? Sorry for the off topic question/thanks in advance for any thoughts and ideas. Peter
Re: [ccp4bb] Chiral volume outliers SO4
Hi all, Thanks very much to all who responded so quickly. The fix is a one liner in the SO4.cif file (last line) SO4 chir_01 S O1 O2 O3both which I believe is now in the 6.3.0 release. Interestingly the chirality parameters were not in the SO4.cif file in 6.1.3 but then appeared in 6.2.0. Once again I'm very happy to get to the bottom of this and get it fixed. I do wonder if it had become over parametrised. Cheers Joel -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie Joosten Sent: Thursday, 12 July 2012 12:16 a.m. To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Chiral volume outliers SO4 Hi Ian, > > @Ian: You'd be surprised how well Refmac can flatten sulfates if you > > have a chiral volume outlier (see Figure 1d in Acta Cryst. D68: > > 484-496 > (2012)). > But this is only because the 'negative' volume sign was erroneously > used in > the chiral restraint instead of 'both' (or better still IMO no chiral restraint at > all), right? If so I don't find it surprising at all that Refmac > tried to flip the > sulphate and ended up flattening it. > Seems to be a good illustration of the GIGO (garbage in - garbage > out) principle. Just because the garbage input in this case is in the official > CCP4 distribution and not (as is of course more commonly the > case) perpetrated by the user doesn't make it any less garbage. The problem is that in the creation of chiral volume targets chemically equivalent (groups of) atoms are not recognized as such. So any new or recreated restraint files will have either 'positiv' or 'negativ' and the problem starts all over again. That is why it is better to stay consistent and choose one chirality (the same one as in the 'ideal' coordinates in the PDB ligand descriptions). This will also make it easier compare ligands after aligning them (this applies to ligands more complex than sulfate). Obviously, users should not be forced to deal with these things. Programs like Refmac and COOT should fix chiral volume inversions for the user, because it is only relevant inside the computer. That is the idea of chiron, just fix these 'problems' automatically by swapping equivalent atoms whenever Refmac gives a chiral volume inversion warning. It should make life a bit easier. > The point I was making is that in this and similar cases you don't > need a chiral > restraint at all: surely 4 bond lengths and 6 bond angles define the chiral > volume pretty well already? Or are there cases where without a chiral > restraint the refinement still tries to flip the chirality (I would > fine that hard to > believe). I agree with you for sulfate, and also for phosphate ;). I don't know what happens in other compounds at poor resolution, when bond and angle targets (and their SDs) are not equivalent. I guess that some angle might 'give way' before others. That is something that should be tested. I have a growing list of chiral centers that have this problem if you are interested. Cheers, Robbie
Re: [ccp4bb] Chiral volume outliers SO4
While this change has made your symptom go away it is stretching it a bit to call this a "fix". You have not corrected the root problem that the names you have given your atoms do not match the convention which is being applied for SO4 groups. Changing the cif means that you don't have to worry about it, but people who study such details will be forced to deal with the incorrect labels of your model in the future. Wouldn't it just be easier to swap the names of two oxygen atoms in each SO4, leaving the cif alone? Your difficulties will go away and people using your model in the future will also have a simpler life. This labeling problem is not new. The fight to standardize the labeling of the methyl groups in Valine and Leucine was raging in the 1980's. Standardizing the labels on the PO4 groups in DNA/RNA was much more recent. It helps everyone when you know you can overlay two models and have a logical solution without a "rotation matrix" with a determinate of -1. Besides, you will continue to be bitten by this problem as you use other programs, until you actually swap some labels. Dale Tronrud On 07/12/12 15:00, Joel Tyndall wrote: > Hi all, > > Thanks very much to all who responded so quickly. The fix is a one liner in > the SO4.cif file (last line) > > SO4 chir_01 S O1 O2 O3both > > which I believe is now in the 6.3.0 release. > > Interestingly the chirality parameters were not in the SO4.cif file in 6.1.3 > but then appeared in 6.2.0. > > Once again I'm very happy to get to the bottom of this and get it fixed. I do > wonder if it had become over parametrised. > > Cheers > > Joel > > > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie > Joosten > Sent: Thursday, 12 July 2012 12:16 a.m. > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Chiral volume outliers SO4 > > Hi Ian, > > >>> @Ian: You'd be surprised how well Refmac can flatten sulfates if you >>> have a chiral volume outlier (see Figure 1d in Acta Cryst. D68: >>> 484-496 >> (2012)). >> But this is only because the 'negative' volume sign was erroneously >> used > in >> the chiral restraint instead of 'both' (or better still IMO no chiral > restraint at >> all), right? If so I don't find it surprising at all that Refmac >> tried to > flip the >> sulphate and ended up flattening it. >> Seems to be a good illustration of the GIGO (garbage in - garbage >> out) principle. Just because the garbage input in this case is in the > official >> CCP4 distribution and not (as is of course more commonly the >> case) perpetrated by the user doesn't make it any less garbage. > The problem is that in the creation of chiral volume targets chemically > equivalent (groups of) atoms are not recognized as such. So any new or > recreated restraint files will have either 'positiv' or 'negativ' and the > problem starts all over again. That is why it is better to stay consistent > and choose one chirality (the same one as in the 'ideal' coordinates in the > PDB ligand descriptions). This will also make it easier compare ligands after > aligning them (this applies to ligands more complex than sulfate). > Obviously, users should not be forced to deal with these things. Programs > like Refmac and COOT should fix chiral volume inversions for the user, > because it is only relevant inside the computer. That is the idea of chiron, > just fix these 'problems' automatically by swapping equivalent atoms whenever > Refmac gives a chiral volume inversion warning. It should make life a bit > easier. > > >> The point I was making is that in this and similar cases you don't >> need a > chiral >> restraint at all: surely 4 bond lengths and 6 bond angles define the > chiral >> volume pretty well already? Or are there cases where without a chiral >> restraint the refinement still tries to flip the chirality (I would >> fine > that hard to >> believe). > I agree with you for sulfate, and also for phosphate ;). I don't know what > happens in other compounds at poor resolution, when bond and angle targets > (and their SDs) are not equivalent. I guess that some angle might 'give way' > before others. That is something that should be tested. I have a growing list > of chiral centers that have this problem if you are interested. > > Cheers, > Robbie
Re: [ccp4bb] Chiral volume outliers SO4
Hi Dale, Thanks for the input. I guess as a relatively transient user I am not appreciating the depth of the problem. However, by at least raising this issue, I'm hoping that the "fight" can be sorted out. Where does my "sulfate" problem stem from? Is it from importing the fragment in COOT? Is it the actual cif file which is read by COOT? As noted, software updates tend to be quicker than we can refine our structures (That albeit is relatively slow on our behalf). I am all for standardisation, but in the long run (at least for me) the sulfate(s) becomes a minor part of the structures. I'll look at swapping the oxygens in the SO4's. Cheers Joel -Original Message- From: Dale Tronrud [mailto:det...@uoxray.uoregon.edu] Sent: Friday, 13 July 2012 10:22 a.m. To: Joel Tyndall Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Chiral volume outliers SO4 While this change has made your symptom go away it is stretching it a bit to call this a "fix". You have not corrected the root problem that the names you have given your atoms do not match the convention which is being applied for SO4 groups. Changing the cif means that you don't have to worry about it, but people who study such details will be forced to deal with the incorrect labels of your model in the future. Wouldn't it just be easier to swap the names of two oxygen atoms in each SO4, leaving the cif alone? Your difficulties will go away and people using your model in the future will also have a simpler life. This labeling problem is not new. The fight to standardize the labeling of the methyl groups in Valine and Leucine was raging in the 1980's. Standardizing the labels on the PO4 groups in DNA/RNA was much more recent. It helps everyone when you know you can overlay two models and have a logical solution without a "rotation matrix" with a determinate of -1. Besides, you will continue to be bitten by this problem as you use other programs, until you actually swap some labels. Dale Tronrud On 07/12/12 15:00, Joel Tyndall wrote: > Hi all, > > Thanks very much to all who responded so quickly. The fix is a one > liner in the SO4.cif file (last line) > > SO4 chir_01 S O1 O2 O3both > > which I believe is now in the 6.3.0 release. > > Interestingly the chirality parameters were not in the SO4.cif file in 6.1.3 > but then appeared in 6.2.0. > > Once again I'm very happy to get to the bottom of this and get it fixed. I do > wonder if it had become over parametrised. > > Cheers > > Joel > > > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Robbie Joosten > Sent: Thursday, 12 July 2012 12:16 a.m. > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Chiral volume outliers SO4 > > Hi Ian, > > >>> @Ian: You'd be surprised how well Refmac can flatten sulfates if you >>> have a chiral volume outlier (see Figure 1d in Acta Cryst. D68: >>> 484-496 >> (2012)). >> But this is only because the 'negative' volume sign was erroneously >> used > in >> the chiral restraint instead of 'both' (or better still IMO no chiral > restraint at >> all), right? If so I don't find it surprising at all that Refmac >> tried to > flip the >> sulphate and ended up flattening it. >> Seems to be a good illustration of the GIGO (garbage in - garbage >> out) principle. Just because the garbage input in this case is in >> the > official >> CCP4 distribution and not (as is of course more commonly the >> case) perpetrated by the user doesn't make it any less garbage. > The problem is that in the creation of chiral volume targets chemically > equivalent (groups of) atoms are not recognized as such. So any new or > recreated restraint files will have either 'positiv' or 'negativ' and the > problem starts all over again. That is why it is better to stay consistent > and choose one chirality (the same one as in the 'ideal' coordinates in the > PDB ligand descriptions). This will also make it easier compare ligands after > aligning them (this applies to ligands more complex than sulfate). > Obviously, users should not be forced to deal with these things. Programs > like Refmac and COOT should fix chiral volume inversions for the user, > because it is only relevant inside the computer. That is the idea of chiron, > just fix these 'problems' automatically by swapping equivalent atoms whenever > Refmac gives a chiral volume inversion warning. It should make life a bit > easier. > > >> The point I was making is that in this and similar cases you don't >> need a > chiral >> restraint at all: surely 4 bond lengths and 6 bond angles define the > chiral >> volume pretty well already? Or are there cases where without a >> chiral restraint the refinement still tries to flip the chirality (I >> would fine > that hard to >> believe). > I agree with you for sulfate, and also for phosphate ;). I don't know what > happens in other compounds
[ccp4bb] Mitegen angled micromounts @microfocus
Dear CCP4BBers, just wanted to share this with you so you don't run into this issue with your crystals. We have 18mm 60degree angled tips and unfortunately they don't work with the current setup at SSRL 12-2 (this is specific to this beamline and might not apply to other microfocus lines out there). Through the angled tip the positioning of the crystal is to far off for the limits through the particular beamline setup. Since this is the first time I guess somebody tried this on the 12-2 line, beamline staff will be looking into this issue and hopefully resolve it. This does not seem to be an issue with other "normal" beamlines. Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
[ccp4bb] A little bit of sunshine from the Protein Data Bank in Europe (pdbe.org)
Hi all,
Twice a year, the Protein Data Bank in Europe (PDBe; http://pdbe.org) releases
new, improved and updated versions of its tools and resources. Below is a
brief description of new features and services that have been released this
summer (or what passes as summer in the UK). As always, the URL
http://pdbe.org will take you to the PDBe website. Many of the features can be
accessed through the "PDBe Tools" menu on the left side of the front page, or
you can use the shortcut URLs mentioned below.
--
The executive summary for the busy PI:
#1. Slice viewer for tomograms in EMDB
#2. Improved analysis and validation of NMR entries
#3. Enhanced FASTA browser for the PDB archive (http://pdbe.org/fasta)
#4. New features on some PDB entry pages
#5. Weekly release history for PDB entries, EMDB entries and PDB compounds
(http://pdbe.org/latest)
#6. Advanced SQL queries for power-users (http://pdbe.org/sqldemo)
And many other smaller (or "under-the-hood") improvements.
--
The nitty-gritty details for the eager structure user:
#1. Slice viewer for tomograms in EMDB
--
The major piece of new functionality is a slice viewer for tomograms in EMDB.
The viewer, developed in collaboration with the Open Microscopy Environment
(OME), works within the context of most common web browsers and does not
require any installation of software locally. To access the slice viewer for a
particular EMDB entry, e.g. emd-1053, navigate to the entry page, in this case
http://pdbe.org/emd-1053, select the "Visualization" page (from the menu on
the left) and click on "Slice viewer (PDBe)". Hover over the question mark
icon for a brief explanation of options and controls.
#2. Improved analysis and validation of NMR entries
---
Vivaldi (http://pdbe.org/vivaldi), our interactive server for display,
analysis and validation of NMR entries (including experimental data and
violations, where available), has been improved and enhanced, thanks in part
to user feedback. New types of data and analysis include angular restraints
and their violations and circular variance plots for main-chain torsions. You
can view your favourite NMR entry by using a shortcut URL such as
http://pdbe.org/vivaldi/2k4v or from the main Vivaldi page. If you're not an
expert in NMR and feel intimidated by ensembles and distance restraints, take
Vivaldi for a spin!
PDBe also generates OLDERADO pages for NMR ensembles
(http://pdbe.org/olderado) - these show useful information about rigid domains
and clusters of models in the ensemble. Where non-experts often take "MODEL 1"
from an ensemble for display, molecular replacement, docking, homology
modelling etc., OLDERADO will tell you which model is most representative.
These pages are accessible from the OLDERADO start page, or directly, e.g.
http://www.ebi.ac.uk/pdbe-apps/nmr/olderado/searchEntry?pdbCode=2k4v. Note how
the colouring of the rigid domains and cluster representatives is repeated in
the tables to make identification easy. There are also buttons to launch
Vivaldi showing the same information in 3D.
The "Experiment" pages for NMR entries (e.g.,
http://pdbe.org/2k4v/experimental) link to both OLDERADO and Vivaldi (among
many other things).
#3. Enhanced FASTA browser for the PDB archive
--
PDBe offers a number of (PDBeXplore) browsers that allow analysis of the
archive based on a variety of biological and chemical classification systems
(EC code, Pfam family, GO classifications, taxonomy, etc. - see
http://pdbe.org/pdbexplore). One of these browsers allows you to study all PDB
entries that contain proteins that show sequence similarity to a protein of
your interest (as identified by a FASTA search; http://pdbe.org/fasta). This
browser has now been improved and can take UniProt and PDB identifiers as
input to retrieve sequences itself - after providing one of these identifiers,
the "Fetch sequence" button will retrieve the sequence (if it can find it) and
put it in the sequence box. After that, change the E-value and/or %-identity
cut-off and hit "Submit" to retrieve any and all hits in the PDB. In addition,
there is a new tab in the browser panel ("Unreleased entries") that shows any
hits to proteins found in PDB entries that have not yet been released but
whose sequences are public.
#4. New features on some PDB entry pages
PDB entry pages can be accessed quickly with URLs like http://pdbe.org/1cbs.
For some time now, we have provided links to corresponding PDB_REDO pages
(http://www.cmbi.ru.nl/pdb_redo/), but these have been somewhat hard to find.
Now, when PDB_REDO has data about an X-ray entry, we provide an explicit link
on the downloads page of that entry, e.g. http://pdbe.org/1cbs/downloads shows
a link to
Re: [ccp4bb] offtopic: AKTA prime
Yes, it has happened to me more than once. It is not a good pump. Acta Prime Plus has a better one. They still have parts. Call GE technical support and they will tell you what to do. The part costs some money (it is not cheap - probably $500). I think the Akta Prime will keep on running as long as you maintain it. They can come to your lab to do the job for you but I do not know how much it is per hour. This team is called labcrew and they are very good. I would call them and try to arrange with them. Make sure you have your solutions elevated above the pump. Even though it is a pump you need to help it. Also do not use viscous solutions with it. (I do not use glycerol because of the pump). Finally clean it up with 20% ethanol after each use to avoid mold. On Thu, Jul 12, 2012 at 5:09 PM, Peter Hsu wrote: > Hi all, > > We've got an old Akta prime that I think is on the verge of kicking it. > Hearing some high pitched sounds coming from the pump when we're running > it. Line A seems clogged and makes a thudding noise when we try to do a > pump wash through that line. Does anyone have any experience w/fixing > one/replacing the pump? Or any idea how much it's going to set us back to > get it fixed? > > Sorry for the off topic question/thanks in advance for any thoughts and > ideas. > > Peter >
[ccp4bb] offtopic: packing gel filtration columns
Hi all, Sorry for the slew of offtopic posts, but does anyone here have any experience repacking the large 120mL Superdex75/200 columns? Any advice/tips on doing it? I've got an older column that's gotten clogged while washing w/NaOH (can't go over 0.1mL/min w/o getting overpressure alarm), and not sure the bossman would be thrilled w/buying another column. Thanks for any ideas. Peter
Re: [ccp4bb] offtopic: packing gel filtration columns
Hi Peter, Most of the time it is just a clogged top filter. While the column is running, loosen the top o-ring by loosening the black knob on the top. Once that is done, carefully unscrew the white threaded adapter several turns to back the top filter off the resin bed. You can then unscrew the red top cap and remove the top assembly. Replace the nylon filter, the part number should be with the column literature. If you think it is required you can stir up the top 1mm of the resin bed and remove it. Then add some water to the top, reassemble, run the column a little to settle the resin, then lower the adapter and tighten all connections. Here is the link to the packing reservoir for the XK columns which gives more detailed instructions in case you need to do a full repack: https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1 340181598294/litdoc28992023_20120626104814.pdf Good Luck, Chris -- Christopher L. Colbert, Ph.D. Assistant Professor Department of Chemistry and Biochemistry North Dakota State University P.O. Box 6050 Dept. 2710 Fargo, ND 58108-6050 PH: (701) 231-7946 FAX: (701) 231-8324 On 7/12/12 8:51 PM, "Peter Hsu" wrote: >Hi all, > >Sorry for the slew of offtopic posts, but does anyone here have any >experience repacking the large 120mL Superdex75/200 columns? Any >advice/tips on doing it? I've got an older column that's gotten clogged >while washing w/NaOH (can't go over 0.1mL/min w/o getting overpressure >alarm), and not sure the bossman would be thrilled w/buying another >column. > >Thanks for any ideas. > >Peter >
[ccp4bb] offtopic: packing gel filtration columns
> From: Yarrow Madrona > Date: July 12, 2012 7:39:57 PM PDT > To: Peter Hsu > Subject: Re: [ccp4bb] offtopic: packing gel filtration columns > > It is also likely that the clogging due to NAOH is due to crapped out > protein. Try cleaning with guanadine. > > > > On Jul 12, 2012, at 6:51 PM, Peter Hsu wrote: > >> Hi all, >> >> Sorry for the slew of offtopic posts, but does anyone here have any >> experience repacking the large 120mL Superdex75/200 columns? Any advice/tips >> on doing it? I've got an older column that's gotten clogged while washing >> w/NaOH (can't go over 0.1mL/min w/o getting overpressure alarm), and not >> sure the bossman would be thrilled w/buying another column. >> >> Thanks for any ideas. >> >> Peter >>
[ccp4bb] Fwd: [ccp4bb] offtopic: packing gel filtration columns
> Subject: Re: [ccp4bb] offtopic: packing gel filtration columns > > We do it pretty routinely in our lab with great results. To do it right you > have to invest in a reservoir sold by GE. It screws onto the end of the > column. It allows you to pour the entire slury (resin and water) into the > resin in one shot. The reservoir is capped with an adapter and the pump > turned onto 10ml/min or so (less than the hardware pressure limit of 0.5 > mPascal). > > When the resin is packed you take the water and reservoir off. Then stick in > the plunger and continue to pack. when pushing down the plunger you have to > loosen the fitting that connects the akta tubing to the plunger tubing, so > that the excess liquid has somewhere to go. When you tighten the plunger to > hold it in place you will need to screw the fitting in ASAP. > > It is much easier to see than explain. Be careful of over tightening the > plunger as this will actually unscrew a fitting inside the plunger and the > column will leak. > > GE had a tutorial video on their website at one time. I don't know if it is > still up. It's one of those things that has to be done or seen rather then > explained. > > Good luck. > > > > > On Jul 12, 2012, at 6:51 PM, Peter Hsu wrote: > >> Hi all, >> >> Sorry for the slew of offtopic posts, but does anyone here have any >> experience repacking the large 120mL Superdex75/200 columns? Any advice/tips >> on doing it? I've got an older column that's gotten clogged while washing >> w/NaOH (can't go over 0.1mL/min w/o getting overpressure alarm), and not >> sure the bossman would be thrilled w/buying another column. >> >> Thanks for any ideas. >> >> Peter >>
Re: [ccp4bb] offtopic: AKTA prime
> Try manually purging the pump with a syringe. > I have had some success running hot water followed by NOAH and guanadine > when the pump was clogged with a buffer that crapped out in the pump. If the > pump is really clogged the pressure should spike when you try to run it. If > not something else is probably wrong > > If I remember right a pump will cost at least a couple thousand dollars if > bought through GE. > > Good luck. > > > > On Jul 12, 2012, at 2:09 PM, Peter Hsu wrote: > >> Hi all, >> >> We've got an old Akta prime that I think is on the verge of kicking it. >> Hearing some high pitched sounds coming from the pump when we're running it. >> Line A seems clogged and makes a thudding noise when we try to do a pump >> wash through that line. Does anyone have any experience w/fixing >> one/replacing the pump? Or any idea how much it's going to set us back to >> get it fixed? >> >> Sorry for the off topic question/thanks in advance for any thoughts and >> ideas. >> >> Peter >>
Re: [ccp4bb] offtopic: packing gel filtration columns
Hi, Yes, it can be done by yourself. I repacked our 26/60 column a few years ago with homemade apparatus. The column has been working for us since then. Basically the loading apparatus was a tubing connecting the top of the column and bottom of a reservoir (a flask with an opening near the bottom) that contained 30%-50% suspended beads. The length of the tubing was about 2m, and I left the reservoir on the highest shelf I could find in our lab, so that the gravity of the buffer in the column and tubing will drive the flow in a constant even manner. It took overnight to get the column nearly filled. The gel filtration columns need to be tightly packed with no discontinuity. As I remember, the GE gel filtration packing manual said: push the top filter 2mm further down, after the column has been fully compressed by flowing buffer at 1cm/min flow rate. The beads need to be gently suspended in the beginning, but constant stirring is not necessary. The flow of the buffer will carry beads into to column (because my reservoir has an opening at the bottom). The difficult part is at the end, when the column is filled to the top. The beads will be compressed quite a lot when you start pumping buffer, then you need to fill in more beads. As I remember, I removed the upper filter on the inlet connector so that I could load beads through the upper inlet tubing with a 10mL or 30mL syringe, which generated enough pressure while loading the beads - so that I didn't have to take the top off again and again. There is a manual from GE on how to pack superdex columns. You should be able to find it online. The Superdex beads used in different sized columns from GE have different diameters. The bigger the column is, the larger the beads are. And the larger the beads are, the easier the packing will be. 16/60 should still be fairly easy to pack. I checked the theoretical plate number of our 26/60 after repacking, it was >11000, not as high as the 13000 stated in the 26/60 new column manual, but it was good enough for us. Zhijie -- From: "Peter Hsu" Sent: Thursday, July 12, 2012 9:51 PM To: Subject: [ccp4bb] offtopic: packing gel filtration columns Hi all, Sorry for the slew of offtopic posts, but does anyone here have any experience repacking the large 120mL Superdex75/200 columns? Any advice/tips on doing it? I've got an older column that's gotten clogged while washing w/NaOH (can't go over 0.1mL/min w/o getting overpressure alarm), and not sure the bossman would be thrilled w/buying another column. Thanks for any ideas. Peter
Re: [ccp4bb] offtopic: packing gel filtration columns
ad do not forget to recalibrate On Thu, Jul 12, 2012 at 10:53 PM, Zhijie Li wrote: > Hi, > > Yes, it can be done by yourself. I repacked our 26/60 column a few years > ago with homemade apparatus. The column has been working for us since then. > Basically the loading apparatus was a tubing connecting the top of the > column and bottom of a reservoir (a flask with an opening near the bottom) > that contained 30%-50% suspended beads. The length of the tubing was about > 2m, and I left the reservoir on the highest shelf I could find in our lab, > so that the gravity of the buffer in the column and tubing will drive the > flow in a constant even manner. It took overnight to get the column nearly > filled. > The gel filtration columns need to be tightly packed with no > discontinuity. As I remember, the GE gel filtration packing manual said: > push the top filter 2mm further down, after the column has been fully > compressed by flowing buffer at 1cm/min flow rate. > The beads need to be gently suspended in the beginning, but constant > stirring is not necessary. The flow of the buffer will carry beads into to > column (because my reservoir has an opening at the bottom). The difficult > part is at the end, when the column is filled to the top. The beads will be > compressed quite a lot when you start pumping buffer, then you need to fill > in more beads. As I remember, I removed the upper filter on the inlet > connector so that I could load beads through the upper inlet tubing with a > 10mL or 30mL syringe, which generated enough pressure while loading the > beads - so that I didn't have to take the top off again and again. > > There is a manual from GE on how to pack superdex columns. You should be > able to find it online. The Superdex beads used in different sized columns > from GE have different diameters. The bigger the column is, the larger the > beads are. And the larger the beads are, the easier the packing will be. > 16/60 should still be fairly easy to pack. I checked the theoretical plate > number of our 26/60 after repacking, it was >11000, not as high as the > 13000 stated in the 26/60 new column manual, but it was good enough for us. > > Zhijie > > --** > From: "Peter Hsu" > Sent: Thursday, July 12, 2012 9:51 PM > To: > Subject: [ccp4bb] offtopic: packing gel filtration columns > > > Hi all, >> >> Sorry for the slew of offtopic posts, but does anyone here have any >> experience repacking the large 120mL Superdex75/200 columns? Any >> advice/tips on doing it? I've got an older column that's gotten clogged >> while washing w/NaOH (can't go over 0.1mL/min w/o getting overpressure >> alarm), and not sure the bossman would be thrilled w/buying another column. >> >> Thanks for any ideas. >> >> Peter >> > -- Pius S Padayatti,PhD, Phone: 216-658-4528
Re: [ccp4bb] offtopic: packing gel filtration columns
Sorry for the slew of offtopic posts, but does anyone here have any experience repacking the large 120mL Superdex75/200 columns? Any advice/tips on doing it? I've got an older column that's gotten clogged while washing w/NaOH (can't go over 0.1mL/min w/o getting overpressure alarm), and not sure the bossman would be thrilled w/buying another column. There is really nothing special or magical about Superdex or Pharmacia columns. Good gel-filtration column packing can be tricky but it's been done well decades ago and can still be done today. Things to keep in mind: 1. De-fine the matrix before packing. 2. Strictly vertical column, no local heating from one side. 3. Lowering surface tension helps packing ==> add 0.05-0.1% Triton X-100. 4. Prevent electrostatic repulsion of beads through residual charges ==> use 100-200 mM NaCl in packing buffer. 5. Avoid particles sorting by size ==> have slurry that is about 65-70% matrix 6. Avoid discontinuities whenever possible ==> use packing reservoir if possible and pour definite excess of the matrix. 7. Avoid bubbles like plague ==> degas the slurry and packing buffer before pouring, pour in one nice careful motion, over a glass rod inserted inside (letting the slurry slide on it down rather then dropping and making bubbles). Do not allow sedimentation - connect to pump and start packing ASAP. 8. Pack at as high flow rate as possible ==> use absolute maximum that the matrix is spec'ed for (andthe column, of course; for Superdex prep grade it means using medium pressure columns). Pack until matrix level no longer changes. 9. Pay extra attention inserting an upper adapter without bubbles - right to the top of the matrix. Pack until matrix level no longer changes, always moving the adapter down to the matrix. 10. Compress the column: *slowly* (in several stop and go steps of compressing/running buffer through) compress by moving an upper adapter down by a total ~ 2-3% of the column height. - Dima
Re: [ccp4bb] offtopic: AKTA prime
> it is not cheap - probably $500 When we purchased the pump, it was below $300 (a year ago). Replacement took ~4 hours, but you must like doing it. You'll have to disassemble almost entire pump module, so make pictures of each step and mark tubings ends with labels. Alex On Jul 12, 2012, at 6:28 PM, Jacqueline Vitali wrote: > Yes, it has happened to me more than once. It is not a good pump. Acta > Prime Plus has a better one. They still have parts. Call GE technical > support and they will tell you what to do. > > The part costs some money (it is not cheap - probably $500). I think the > Akta Prime will keep on running as long as you maintain it. They can come to > your lab to do the job for you but I do not know how much it is per hour. > This team is called labcrew and they are very good. > > I would call them and try to arrange with them. > > Make sure you have your solutions elevated above the pump. Even though it is > a pump you need to help it. Also do not use viscous solutions with it. (I > do not use glycerol because of the pump). Finally clean it up with 20% > ethanol after each use to avoid mold. > > On Thu, Jul 12, 2012 at 5:09 PM, Peter Hsu wrote: > Hi all, > > We've got an old Akta prime that I think is on the verge of kicking it. > Hearing some high pitched sounds coming from the pump when we're running it. > Line A seems clogged and makes a thudding noise when we try to do a pump wash > through that line. Does anyone have any experience w/fixing one/replacing the > pump? Or any idea how much it's going to set us back to get it fixed? > > Sorry for the off topic question/thanks in advance for any thoughts and ideas. > > Peter >
Re: [ccp4bb] offtopic: AKTA prime
I did not replace the entire pump, only a motor. Sorry for a confusion. On Jul 12, 2012, at 9:28 PM, aaleshin wrote: >> it is not cheap - probably $500 > When we purchased the pump, it was below $300 (a year ago). Replacement took > ~4 hours, but you must like doing it. You'll have to disassemble almost > entire pump module, so make pictures of each step and mark tubings ends with > labels. > > Alex > > > On Jul 12, 2012, at 6:28 PM, Jacqueline Vitali wrote: > >> Yes, it has happened to me more than once. It is not a good pump. Acta >> Prime Plus has a better one. They still have parts. Call GE technical >> support and they will tell you what to do. >> >> The part costs some money (it is not cheap - probably $500). I think the >> Akta Prime will keep on running as long as you maintain it. They can come >> to your lab to do the job for you but I do not know how much it is per hour. >> This team is called labcrew and they are very good. >> >> I would call them and try to arrange with them. >> >> Make sure you have your solutions elevated above the pump. Even though it >> is a pump you need to help it. Also do not use viscous solutions with it. >> (I do not use glycerol because of the pump). Finally clean it up with 20% >> ethanol after each use to avoid mold. >> >> On Thu, Jul 12, 2012 at 5:09 PM, Peter Hsu wrote: >> Hi all, >> >> We've got an old Akta prime that I think is on the verge of kicking it. >> Hearing some high pitched sounds coming from the pump when we're running it. >> Line A seems clogged and makes a thudding noise when we try to do a pump >> wash through that line. Does anyone have any experience w/fixing >> one/replacing the pump? Or any idea how much it's going to set us back to >> get it fixed? >> >> Sorry for the off topic question/thanks in advance for any thoughts and >> ideas. >> >> Peter >> >
