[ccp4bb] rescuing crashing-out protein eluted from Nickel column
Dear all, I just ran into this problem and would like to see if I could get some helpful tips before my protein completely crashes out. I have a protein as 6His fusion and it remained bound to the Ni resin with 40 mM Imidazole wash (added to 1XPBS) but then was eluted off with 200 mM (added to 1XPBS). The protein seemed to be highly concentrated in the elution and began to get cloudy right away, with more and more precipitation produced over a matter of minutes. I felt so helpless, didn't know what to do, and then decided to add 5% of glycerol into one of the fractions but that made it even more cloudy (ohh no...). While the protein is dying in the tube, do you have some quick remedy for me? Thanks very much, -J.J.
Re: [ccp4bb] rescuing crashing-out protein eluted from Nickel column
Thank you all for the quick replies - I'm trying many of the tips in small batches and would send out a feedback as soon as appropriate, gratefully, - J.J. On 2/14/08, Jacob Wong <[EMAIL PROTECTED]> wrote: > > Dear all, > > I just ran into this problem and would like to see if I could get some > helpful tips before my protein completely crashes out. > > I have a protein as 6His fusion and it remained bound to the Ni resin with > 40 mM Imidazole wash (added to 1XPBS) but then was eluted off with 200 mM > (added to 1XPBS). The protein seemed to be highly concentrated in the > elution and began to get cloudy right away, with more and more precipitation > produced over a matter of minutes. I felt so helpless, didn't know what to > do, and then decided to add 5% of glycerol into one of the fractions but > that made it even more cloudy (ohh no...). > > > While the protein is dying in the tube, do you have some quick remedy for > me? Thanks very much, -J.J. >
Re: [ccp4bb] rescuing crashing-out protein eluted from Nickel column
Dear all, thank you so much for all the invaluable tips that were delivered faster than FedEx (and sorry for not being able to reply to individual emails). My problem was resolved by adding EDTA (to about 5 mM), spinning down at 15K for 10min to get rid of precipitation (I did have some even with the help of EDTA), and then a nice HiTrapQ column with Hepes buffer dilution (many people's favorite it turns out and apparently my protein likes it a lot since it now concentrates to 10 mg/ml at least). Since I need the protein for binding assays, I didn't try to add more salt (Q column killer also), but diluting with 1XPBS (or H2O) doesn't help clear the precipitate - so it seems that the key things here are imidazole, trace amount of nickel, and a good buffer. Thank you so much for all your help, caring and patience. This is a great community. Have a nice weekend, -J.J. On 2/14/08, Jacob Wong <[EMAIL PROTECTED]> wrote: > > Dear all, > > I just ran into this problem and would like to see if I could get some > helpful tips before my protein completely crashes out. > > I have a protein as 6His fusion and it remained bound to the Ni resin with > 40 mM Imidazole wash (added to 1XPBS) but then was eluted off with 200 mM > (added to 1XPBS). The protein seemed to be highly concentrated in the > elution and began to get cloudy right away, with more and more precipitation > produced over a matter of minutes. I felt so helpless, didn't know what to > do, and then decided to add 5% of glycerol into one of the fractions but > that made it even more cloudy (ohh no...). > > > While the protein is dying in the tube, do you have some quick remedy for > me? Thanks very much, -J.J. >
[ccp4bb] proteases for limited proteolysis, catalog numbers
Dear all, I am about to purchase/prepare some proteases for protein limited proteolysis experiments. I would therefore greatly appreciate if you could offer any tips as to what "contemporary" products you are using with good confidence/pricing (US) for trypsin, chymotrypsin, elastase, carboxypeptidase A/B/Y, aminopeptidase, Glu-C, and others that you may recommend. I wish to purchase like 1 mg for each of these so that I could make 1 mg/ml stocks and 10X series dilutions. Thanks in advance for your advice. Sincerely yours, - Jacob Wong
[ccp4bb] Singapore Research, facts and comments?
Dear All, sorry for this off-topic inquiry. I am interested in knowing about the current scientific research in Singapore but have had hard time finding the most up-to-date articles/comments on this. All I could find are the articles a few years back in Science and Nature. Would appreciate a pointer. Happy Chinese New Year! Thanks, Jacob Wong
Re: [ccp4bb] Singapore Research, facts and comments?
Thanks to those who has responded to me and asked for more specifics. I am interested in some most up-to-date (and published) facts/comments about the Biopolis, something that may follow up the article below that was published back in 2003. Singapore: filling biopolis. http://www.nature.com/nature/journal/v425/n6959/full/nj6959-746a.html Thank you, Jacob Wong On Sat, Jan 29, 2011 at 12:47 AM, Jacob Wong wrote: > Dear All, sorry for this off-topic inquiry. I am interested in knowing > about the current scientific research in Singapore but have had hard time > finding the most up-to-date articles/comments on this. All I could find are > the articles a few years back in Science and Nature. > > Would appreciate a pointer. Happy Chinese New Year! > > Thanks, Jacob Wong >
Re: [ccp4bb] Singapore Research, facts and comments?
Thanks to all who has responded - I believe I have found the *published*resources that I was looking for - please kindly refrain from making personal comments on the forum as that will go beyond my original inquiry. Thanks to all the warm-hearted colleagues who are here to offer tips and resources real-time, crystallography and beyond. With my best wishes for a wonderful and prosperous Chinese New Year - to researchers all over the world. Sincerely yours, Jacob Wong On Sat, Jan 29, 2011 at 7:11 AM, Jacob Wong wrote: > Thanks to those who has responded to me and asked for more specifics. I am > interested in some most up-to-date (and published) facts/comments about the > Biopolis, something that may follow up the article below that was published > back in 2003. > > Singapore: filling biopolis. > http://www.nature.com/nature/journal/v425/n6959/full/nj6959-746a.html > > Thank you, Jacob Wong > > > > On Sat, Jan 29, 2011 at 12:47 AM, Jacob Wong wrote: > >> Dear All, sorry for this off-topic inquiry. I am interested in knowing >> about the current scientific research in Singapore but have had hard time >> finding the most up-to-date articles/comments on this. All I could find are >> the articles a few years back in Science and Nature. >> >> Would appreciate a pointer. Happy Chinese New Year! >> >> Thanks, Jacob Wong >> > >
[ccp4bb] Coot: Residue number conflicts during model building
Dear all, I know I could use your help to work more effectively so here comes my questions. I am building a fairly long polypeptide chain (quite a few hundreds of aa) in Coot. I got the phases from Phenix, which traced much of the main chains for me already, with each fragments assigned with numbers that are apparently mismatched with the linear protein sequence. So, I begin to mutate/fit these Ala fragments (one piece at a time) into correct residues using Coot and also try to correct their numbers by doing "renumber residues". The outcome is that now I have overlapped numbers as I move along, prohibiting me to do real-space refinement effectively. How to get around with this? Thank you! Also, are there any easily accessible programs that could help me build these side-chains based on the very good experimental density map currently at 2.3A? Thanks, Jacob Wong
Re: [ccp4bb] Coot: Residue number conflicts during model building
Thanks Tim for the timely tips. I haven't tried Arp/wARP but will follow your recommendation right away. I haven't installed Resolve yet but will look into it and Buccaneer as well - I was having fun modeling some of the apparent fragments into the density but with a protein so big, I ran exhausted before long ;p)... Due to the same fatigue, I wasn't patient enough to rename the Chain ID of the updated fragments. Practically, it seems that* I have to rename the chain ID before "Renumber Residues" as they are two different entities on the menu.* Actually, would Coot authors consider adding "Change Chain ID" into the "Renumber Residues" window so that I I could do both changes on the fly? Thanks, Jacob Wong On Wed, May 4, 2011 at 3:59 AM, Tim Gruene wrote: > Dear Jacob Wong, > > have you tried arp warp for model building? It should do a reasonably good > job > at 2.3A resolution. Alternatively buccaneer or resolve might also help. > > For the overlapping: Does it help to give the subsequent fragment a > different > chain ID? > > Tim > > > On Wed, May 04, 2011 at 02:48:11AM -0400, Jacob Wong wrote: > > Dear all, I know I could use your help to work more effectively so here > > comes my questions. > > > > I am building a fairly long polypeptide chain (quite a few hundreds of > aa) > > in Coot. I got the phases from Phenix, which traced much of the main > chains > > for me already, with each fragments assigned with numbers that are > > apparently mismatched with the linear protein sequence. So, I begin to > > mutate/fit these Ala fragments (one piece at a time) into correct > residues > > using Coot and also try to correct their numbers by doing "renumber > > residues". The outcome is that now I have overlapped numbers as I move > > along, prohibiting me to do real-space refinement effectively. How to get > > around with this? Thank you! > > > > Also, are there any easily accessible programs that could help me build > > these side-chains based on the very good experimental density map > currently > > at 2.3A? > > > > Thanks, Jacob Wong > > -- > -- > Tim Gruene > Institut fuer anorganische Chemie > Tammannstr. 4 > D-37077 Goettingen > > phone: +49 (0)551 39 22149 > > GPG Key ID = A46BEE1A > > > -BEGIN PGP SIGNATURE- > Version: GnuPG v1.4.10 (GNU/Linux) > > iD8DBQFNwQd5UxlJ7aRr7hoRAqBAAJwNQOkDSZjzHe5Fsrj0hxuzf75nRQCfQEZq > s1BO9IHCwb66OhbVa6Gb/Hg= > =dbO3 > -END PGP SIGNATURE- > >
[ccp4bb] Phaser MR with partial solution, 8 molecules/ASU
Dear all, I have this (3.0 A) structure that has 8 molecules per ASU - Phaser was able to find 7 molecules correctly, but not the last one, as indicated by the .sol file (TFZ=5.1) below and the resultant density map. I tried to delete the entry of the last molecule and give the truncated .sol file for another round of MR but Phaser returned with the same solution that has the 8th molecule misplaced. I am tempted to place the 8th molecule by hand but before that would like to learn from you a better way of handling it. One thing I could think of is to refine/rebuild the partial structure with the 7 molecules so as to resolve any potential packing clashes due to model/structure variations and then let Phaser find/position the 8th one for me, but it appears that Phaser doesn't accept .pdb file for a "MR with partial solution" routine? Thank you, Jacob # [No title given] SPACEGROUP P 21 21 21 SOLU SET RFZ=3.7 TFZ=8.2 PAK=0 LLG=78 RFZ=3.7 TFZ=16.0 PAK=0 LLG=248 TFZ==17.2 RFZ=3.7 TFZ=17.8 PAK=0 LLG=463 TFZ==19.7 RFZ=2.9 TFZ=23.0 PAK=0 LLG=765 TFZ==23.2 RFZ=2.8 TFZ=28.6 PAK=0 LLG=1201 TFZ==30.0 RFZ=3.9 TFZ=23.1 PAK=0 LLG=1537 TFZ==24.3 RFZ=2.6 TFZ=19.5 PAK=1 LLG=2096 TFZ==32.1 RFZ=3.0 TFZ=5.1 PAK=1 LLG=1945 TFZ==7.0 SOLU 6DIM ENSE ensemble1 EULER 333.533 143.979 14.880 FRAC -0.30547 0.22985 -0.12420 BFAC 0.0 SOLU 6DIM ENSE ensemble1 EULER 287.095 31.031 200.132 FRAC -0.01202 0.48107 -0.12591 BFAC 0.0 SOLU 6DIM ENSE ensemble1 EULER 220.563 33.756 203.275 FRAC -0.33196 0.18927 -0.27514 BFAC 0.0 SOLU 6DIM ENSE ensemble1 EULER 125.978 23.511 167.527 FRAC 0.36411 -0.45096 -0.17121 BFAC 0.0 SOLU 6DIM ENSE ensemble1 EULER 148.925 162.593 356.498 FRAC -0.41711 -0.09462 -0.07282 BFAC 0.0 SOLU 6DIM ENSE ensemble1 EULER 323.033 34.903 192.073 FRAC -0.53201 -0.04754 -0.02533 BFAC 0.0 SOLU 6DIM ENSE ensemble1 EULER 113.904 157.019 345.596 FRAC 0.09892 -0.60041 -0.17986 BFAC 0.0 SOLU 6DIM ENSE ensemble1 EULER 79.967 95.631 1.727 FRAC -0.41239 0.07915 -0.05760 BFAC 0.0
[ccp4bb] CCP4i: P212121 to P22121 conversion
Dear all, a dummy question: I had processed a data set with HKL2000 to produce a P212121.sca file. Phaser seems to find MR solution with P22121 "alternative" spacegroup option. Now I want to do rigid body refinement with Refmac in CCP4i, there is no longer option for me to choose P22121. Apparently I could have run back to the lab to reprocess the scalepack, but I would rather ask for your suggestions as a case study. I tried to modify the P212121 spacegroup label in the .sca file to P22121 (line 3 header), redo the scalepack2mtz, and then use the newer mtz file as an input for Refmac, result: Refmac complains not to have such a spacegroup. How do I get around with this, without a trip back to the HKL2000? Thank you! Jacob Wong
Re: [ccp4bb] CCP4i: P212121 to P22121 conversion
Thank you all for the teaching, I had been warned of not to lose the H00, 0K0, and 00L reflections so I had re-scalepacked my data set and move on from there. -jacob On 11/8/08, Kay Diederichs <[EMAIL PROTECTED]> wrote: > > Jacob, > > you could use, for refinement, the FP SIGFP columns from the .mtz-file that > PHASER writes out, together with the .pdb-file of the best solution. > > One caveat: the resolution of the data in that file is what you chose for > the PHASER calculation, so you might need to re-run PHASER with the full > resolution (no need to do the full calculation again, there are shortcuts > possible using the "known solution"). > > HTH, > > Kay > >
[ccp4bb] cross-section view of (protein) molecules in pymol?
Dear all, http://www.nature.com/nature/journal/v458/n7234/fig_tab/nature07819_F3.html Just came across this figure Fig. 3b) and would like to know what is the general and easiest way to make things like this, as a "non-brainer". Thank you, -Jacob
[ccp4bb] Science magazine jobs RSS feed not longer available?
Dear all, sorry for this off-topic subject. I had this link below functional until late last month, with the April 31st issue of Science and issues thereafter not being fed any longer. I believe someone out there may have a quick solution for me? I tried to contact them to not avail. Thanks in advance. feed://scjobs.sciencemag.org/search/rss.ashx? -jacob
[ccp4bb] more than one ligands for CNS generate.inp and refine.inp files
Dear colleagues, please help me with a CNS syntax. In the refine.inp, I
could provide as many topology (or parameter) files as necessary:
{refine.inp}
{* topology files *}
{===>} topology_infile_1="CNS_TOPPAR:protein.top";
{===>} topology_infile_2="CNS_TOPPAR:dna-rna.top";
{===>} topology_infile_3="CNS_TOPPAR:water.top";
{===>} topology_infile_4="CNS_TOPPAR:ion.top";
{===>} topology_infile_5="../data/ligand1.top";
{===>} topology_infile_6="../data/ligand2.top";
But in the generate.inp file, there is only one input option for "ligand"
(among others like protein, water, carbohydrate:
{generate.inp}
{* ligand topology file *}
{===>} ligand_topology_infile="../data/ligand1.top";
As I'm having two ligands in the structure, how do I get around with the
generate.inp syntax?
Thank you in advance, -jacob
Re: [ccp4bb] more than one ligands for CNS generate.inp and refine.inp files
Thank you all who had kindly reply to me with tips. Basically I was
suggested to combine the two ligand files together, or to "trick" CNS to
take in my 2nd ligand file at like the "prosthetic group" section. The
generate.inp runs without apparent ERR message, but the refinement was
terminated with error like below, apparently with the newer ligand I meant
to put in - the Ethylene Glycol. I was using the Ethylene Glycol files from
HIC Up, links below. I tried putting the EDO files at the authentic ligand
spots but failed as well. Do I need to edit the files after download in
anyway as I'm using CNS v12.? Thank you very much, -jacob
http://xray.bmc.uu.se/hicup/EDO/edo_xplor_top.txt
http://xray.bmc.uu.se/hicup/EDO/edo_xplor_par.txt
MESSage=OFF
ECHO=FALSe {OFF}
Program version= 1.2 File version= 1.2
Maximum of 24848 elements = 0.
MAPResolution= 2.8000
GRIDsize= 0.0
adjusted grid parameter for solvent mask calculation. It is now : 0.321429
%NBUPDA-ERR: missing nonbonded Lennard-Jones parameters %%%
ATOM: SEGId="X ", RESId="1 ", NAME="C1 ", CHEMical="C_1 "
%NBUPDA-ERR: missing nonbonded Lennard-Jones parameters %%%
ATOM: SEGId="X ", RESId="1 ", NAME="O1 ", CHEMical="O_2 "
%NBUPDA-ERR: missing nonbonded Lennard-Jones parameters %%%
ATOM: SEGId="X ", RESId="1 ", NAME="C2 ", CHEMical="C_3 "
%NBUPDA-ERR: missing nonbonded Lennard-Jones parameters %%%
ATOM: SEGId="X ", RESId="1 ", NAME="O2 ", CHEMical="O_4 "
%NBUPDA error encountered: program will be aborted.
(CNS is in mode: SET ABORT=NORMal END)
On 7/21/09, Jacob Wong wrote:
>
> Dear colleagues, please help me with a CNS syntax. In the refine.inp, I
> could provide as many topology (or parameter) files as necessary:
>
>
> {refine.inp}
> {* topology files *}
> {===>} topology_infile_1="CNS_TOPPAR:protein.top";
> {===>} topology_infile_2="CNS_TOPPAR:dna-rna.top";
> {===>} topology_infile_3="CNS_TOPPAR:water.top";
> {===>} topology_infile_4="CNS_TOPPAR:ion.top";
> {===>} topology_infile_5="../data/ligand1.top";
> {===>} topology_infile_6="../data/ligand2.top";
>
> But in the generate.inp file, there is only one input option for "ligand"
> (among others like protein, water, carbohydrate:
> {generate.inp}
> {* ligand topology file *}
> {===>} ligand_topology_infile="../data/ligand1.top";
>
> As I'm having two ligands in the structure, how do I get around with the
> generate.inp syntax?
>
>
> Thank you in advance, -jacob
>
>
>
[ccp4bb] surface charge residues prediction server
Dear all, I came across an online program long way back that I could not longer remember (embarrassing...). But there should be a program that will take in my protein sequence and generate a list of charge residues that may be solvent exposed for potential mutation to improve solubility or crystallization. Does anybody has a quick pointer to that? Thanks very much, -jacob
Re: [ccp4bb] surface charge residues prediction server
Got answers in a matter of minutes, thanks to all!!! -jacob http://nihserver.mbi.ucla.edu/SER/ On 2/12/10, Jacob Wong wrote: > > Dear all, I came across an online program long way back that I could not > longer remember (embarrassing...). But there should be a program that will > take in my protein sequence and generate a list of charge residues that may > be solvent exposed for potential mutation to improve solubility or > crystallization. Does anybody has a quick pointer to that? Thanks very > much, -jacob
[ccp4bb] .cif file generator for compound stereochemical restraints
Dear all, Just would love to know what else is out there in addition to ELBOW in Phenix? My utility is to generate the .pdb and .cif files from a SMILES string that would allow me to do compound fitting and real-space refinement in coot, and then reciprocal refinement elsewhere (assuming I won't have access to Phenix). Any pointer is much appreciated! Sincerely, Jacob To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
Re: [ccp4bb] .cif file generator for compound stereochemical restraints
Thanks Paul - I love the ccp4 suite. I'm interested in knowing if there is any other solution out there that may be more economical in licensing, especially if I only go as far as coot as a "commercial user". Are you suggesting that coot may not be a freeware anymore in the near future? Yours, Jacob On Sun, Mar 22, 2020 at 10:54 PM Paul Emsley wrote: > > > Just would love to know what else is out there in addition to ELBOW in > Phenix? > > Acedrg is the modern dictionary generator distributed by CCP4. > > > My utility is to generate the .pdb and .cif files from a SMILES string > that would allow me to do compound > > fitting and real-space refinement in coot, > > I recommend that you pick up 0.9-pre (at the moment) if you are going to > do that. This will be folded into > CCP4 soon (I believe). > > > and then reciprocal refinement elsewhere (assuming I won't have access > to Phenix). > > Refmac? > > Paul. > > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
Re: [ccp4bb] .cif file generator for compound stereochemical restraints
Thanks Paul for addressing my inquiry late hours. Kudos to your huge contributions to the vast crystallography community. All my best wishes, Jacob On Sun, Mar 22, 2020 at 11:15 PM Paul Emsley wrote: > On 23/03/2020 03:05, Jacob Wong wrote: > > I love the ccp4 suite. I'm interested in knowing if there is any other > solution out there that > > may be more economical in licensing, especially if I only go as far as > coot as a "commercial user". > > > > Are you suggesting that coot may not be a freeware anymore in the near > future? > > > > Some of the above terms are ambiguous, so let me just say that AceDrg and > Coot are have been and will > continue to be (at least for the next year) released under liberal > licenses and don't require a fee for use > by anyone. I don't think that there is a reciprocal space refinement > program for which one could say the same. > > Paul. > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
