Re: [ccp4bb] Mg++ binding to N7 of G

William, it is not clear from your message why you think this is a Mg++, not Mn++ which has been observed in this site before? Mn++ would be visible in anomalous data from a home source, but not Mg++. The only way to distinguish Mg++ and water is the number of coordination which should be very

Re: [ccp4bb] Mg++ binding to N7 of G

Hi Bill, On Apr 2, 2008, at 10:58 PM, William G. Scott wrote: I've got what appears to be an inner-sphere interaction between Mg++ and the N7 of a G. The mode of binding is the same as what is observed at this site for Mn++, confirmed with anomalous data. Our resolution is 1.6 Å, so I am

[ccp4bb] Production of recombinant membrane protein complex in E. coli

Dear all, Dose anyone have experience about the production of recombinant membrane protein complex in E. coli? All suggestions are welcome. Thanks in advance. Yi-Wei Chang Institute of Molecular Biology Academia Sinica, Taiwan

Re: [ccp4bb] 3D model in glass

Dear Rene I have used a company called Imaage in Australia for engraving proteins in glass blocks. Their web address is http://www.imaage.com.au/ I send them VRML output from MOLSCRIPT and that usually works fine. You need to generate the image in a single color (black), though you can u

Re: [ccp4bb] Mg++ binding to N7 of G

Sorry, I should have been less cryptic: On Apr 2, 2008, at 2:13 PM, Jacob Keller wrote: Forgive the naive questions: To what do the terms "hard" and "soft" refer here? In inorganic chemistry, "hard" refers to bonding where the Coulomb potential dominates, and "soft" where orbital terms do

[ccp4bb] Mg++ binding to N7 of G

Howdie folks: I've got what appears to be an inner-sphere interaction between Mg++ and the N7 of a G. The mode of binding is the same as what is observed at this site for Mn++, confirmed with anomalous data. Our resolution is 1.6 Å, so I am reasonably confident this is right. However, my

[ccp4bb] 3D model in glass

Dear all, Anyone know about how to transfer a 3D image (ligand + protein surface) into a format that would be compatible for 3D laser engraving in plexiglass. I am aware of a few cies that can provide the final product (see below) but we are looking for file format information Luminorum (UK) Cr

Re: [ccp4bb] Co-expression plasmids

Hi Mark, Proper co-expression in trans requires that you use plasmids each with different origins of replication and antibiotic markers, such as p15 + Kan and ColE1 + Amp. I think the pET vectors of the DUET system use these two origins (p15 and ColE1). This ensures much more efficient

Re: [ccp4bb] Co-expression plasmids

Just to explain to people who've never had to do co-expression - if I remember correctly, conventional wisdom states that you can't have two different vectors with the same origin of replication in the same host (maybe someone might know more details as to why). However, as pointed out, it can

Re: [ccp4bb] Co-expression plasmids

I thought only I was having trouble with cloning into pETDuet1. But just to add to what someone just said Yes, I had hell with trying to get one of my ~2kb fragments into pETDuet1 (5.4kb). Could be a combination of vector size and insert size.. Who knows! Sometimes, I do what Tasos says: Tra

Re: [ccp4bb] Co-expression plasmids

We have had good experience with the awfully simple minded approach of using two pET vectors with different antibiotic resistance. Its the easiest thing to do, and it often works ... Apologies for the shameless plugin, since there are many good papers on the subject, but you can read some hin

Re: [ccp4bb] Co-expression plasmids

Hi, If you are expressing just two proteins, you could try a single pET vector with a pCDF vector. The only reason I'm suggesting this is that I had trouble with pET-Duet, but doing each one separately worked first time (plasmid size issue?). I used pET24-a and pCDF-1b - so I had one construct

Re: [ccp4bb] Inverse 3D profile/threading

Hi Jason, RosettaDesign server will do just that: http://rosettadesign.med.unc.edu/ You can specify that certain residues remain unchanged (say active site), that they fall into defined physico-chemical categories (polar, hyrophobic, etc), or let the server change all residues into whatever i

Re: [ccp4bb] twinned?

Hi Qiang, A normal data set has a unimodal intensity distribution with a predictable shape. When there is twinning the distribution remains unimodal but becomes sharper and this is picked up in the twinning analysis. When there is pseudo-translational symmetry, as you indicate you have, then

[ccp4bb] difference betwen blue confocal max-flux and osmic confocal max-flux

Dear CCP4 Members, Apologise for a non-ccp4 question. Could anybody give the difference between "blue confocal max-flux optics" (supplied by Rigaku) and "osmic confocal max-flux optics" (supplied by ?). Are they use same technology (i.e. max-flux) or different? Thanking you Sincerely Karthik

[ccp4bb] twinned?

Hi all, The data I am working on has a strong translation vector. The space group is C2221 and resolution is 2.3 angstrom. There are two molecules per AU with a pseudo-2-fold axis. On the cumulative intensity distribution plot, the theor and obser curves totally do not overlap. I did "detect_twinn

Re: [ccp4bb] Co-expression plasmids

I do use the Duet vectors extensively and they work just fine. I have trouble with my protein complex, but that is solely related to my proteins. My lab mate has used these vectors very successfully. I have been using pRSF-Duet and pETDuet1 more than pCDFDuet1 or pACYCDuet1 for no specific rea

Re: [ccp4bb] "inclusion body"

Dear Skivesh, My favourite media is TPB - (paper is: Moore et al Protein expression and purification (1993) 4: 160-163). I've had luck with this and BL21 (DE3) Gold cells. I'm also a fan of cold shocking cells - after initial growth at 37 deg C, put flasks in an ice water bath for 10 min, th

[ccp4bb] Co-expression plasmids

Dear All, Anyone have experience with the NovaGen Duet co-expression vectors? Or can recommend others? http://www.emdbiosciences.com/html/NVG/Duet_Spot.html Greetings, Mark Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain

Re: [ccp4bb] "inclusion body"

Also worth trying a lower IPTG level ~ 0.1mM and or different media, e.g. TB, SOC or m9 instead of LB, it's all a bit random but sometimes these things can make a difference. Adding 3% EtOH on induction worked for me once, it's supposed to shock the cells and stimulate chaperone production

Re: [ccp4bb] Inverse 3D profile/threading

Hi Jason, While it's not quite what you described, I'd recommend you check out the Secondary Structure Matching server at the EBI: http://www.ebi.ac.uk/msd-srv/ssm/ssmstart.html That will (hopefully!) give you previously deposited structures which share a similar fold to your query pdb file.

[ccp4bb] Inverse 3D profile/threading

I am looking for software or a server to run the 3D-1D profile search in reverse: that is I have a new structure (not in yet deposited in PDB) and I want to see what sequences might fit well to this 3D structure. Can someone point me in the right direction? -jason

Re: [ccp4bb] "inclusion body"

Lower temperature, use chaperones (e.g. TAKARA set), refolding? Quoting shivesh kumar <[EMAIL PROTECTED]>: Dear all, Sorry for the off-topic question... What can be done to avoid a protein going inside inclusion body.The gene is cloned in pET30a with C-ter his tag and expressed in BL21-DE3 fr

[ccp4bb] a question about CNS composite omit map

Dear all, Sorry this might be another naive, non-ccp4 question~ I was trying to make a composite omit map with CNS, but during torsion angle dynamics, it always reports an "argument out of range" error like the following: -- step= 5 at 0.02000 ps

[ccp4bb] "inclusion body"

Dear all, Sorry for the off-topic question... What can be done to avoid a protein going inside inclusion body.The gene is cloned in pET30a with C-ter his tag and expressed in BL21-DE3 from 37 to 18C for 3-4 hr with .5mM of IPTG,it is going to inclusion body.All suggestions are welcome. Thanx in ad

Re: [ccp4bb] libstdc++.so.5

Hi Adam, The symbolic link approach does not work - I hit this problem with another (C++) program. Your pointer about the compat rpm is helpful though - this is probably the most robust solution. I ended up compiling from scratch when I hit a similar problem. Best, Graeme -Original Message

Re: [ccp4bb] libstdc++.so.5

Dear Shivesh, I believe that Fedora 8 has a more updated version of libstdc++ in /usr/lib. You can install libstdc++.so.5 from compat-libstdc++-33-3.2.3-62 using yum (if you have it). An easier approach would be to make a symbolic link between libstdc++.so.5 and libstdc++.so.6 but there maybe

[ccp4bb] libstdc++.so.5

Dear all, We have installed CCP4 6.0.2 in Fedora core 8. While running the program we are getting the following error message- error while loading shared lilbraries libstdc++.so.5, cannot open shared object file: no such file or directory. Please suggest how to get this library file... Thanx in ad

Re: [ccp4bb] convenient means to find I/sigma for publications?

It is recorded by SCALA - if you use that data processing procedure. You can still do a run of scala after SCALEPACK or XDS or whatever providing you output integrated, unmerged intensities. You are right though - we need a data analysias tool.. Eleanor James Pauff wrote: Hello all, Silly