[ccp4bb] half occupancy linkage

Hello All, A grad student here is working on a structure, actually a series of structures, that have in interesting feature that we are not sure how to handle. She has taken a know protein and linked the N and C terminal ends varying the linkage from being direct to a 10 residue linker.

Re: [ccp4bb] Poor electron density - polyAla or PolyGly?

Hi - "ARP/wARP Loops" (from the ccp4i interface) could also be tried. What it will do is build a few "trees" of Ca's in non-negative density and in conformations consistent with penta-peptide conformations from the PDB, then build the residues in Ramachandran allowed conformations, and th

Re: [ccp4bb] ccp4 database error

Thanks to all replied. I'm back on track. ___ Vaheh -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Friday, October 17, 2008 2:58 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] ccp4 database error -- Matthew Franklin , P

Re: [ccp4bb] ccp4 database error

-- Matthew Franklin , Ph.D. Senior Scientist, ImClone Systems 180 Varick Street, 6th floor New York, NY 10014 phone:(917)606-4116 fax:(212)645-2054 CCP4 bulletin board wrote on 10/17/2008 02:09:38 PM: > Dear CCP4 community, > > Due to power failure during ccp4i session the database file got l

[ccp4bb] ccp4 database error

Dear CCP4 community, Due to power failure during ccp4i session the database file got locked (according to message from ccp4i). This prevents me from seeing previously run jobs and from running new jobs in that same project directory. I would appreciate a help on how to overcome this condition and

[ccp4bb] Release of Mosflm version 7.0.4 & iMosflm 1.0.0

Dear all We are pleased to announce the release of Mosflm 7.0.4 and iMosflm 1.0.0. This is the first production release of iMosflm; we are confident that it represents a major step forward from the series of beta-releases that we have made over the last couple of years. It is both more

[ccp4bb] GST fusion-Expresson problem - NOT GST

A protein band around 25 kDa could be also the chloramphenicolacetyltransferase which is expressed in E.coli RIL/RP cells for the coselection of the rare codon plasmids. MS woud give the soution and it may help to identify the potential cleavage site between GST and fused protein. -- Christia

Re: [ccp4bb] high temperature factor refinement

Hi Jason, - run your model through TLSMD server to identify TLS domains (it will produce PHENIX friendly TLS groups selections); http://skuld.bmsc.washington.edu/~tlsmd/ - use these selections for TLS refinement in PHENIX: http://www.phenix-online.org/documentation/refinement.htm for example,

Re: [ccp4bb] high tempterature factor refinement problem

On Friday 17 October 2008, Carl Soja wrote: > Hi All, > > I just refined a structure at 2.95A and getting a good R-work and > R-free(0.236 and 0.274). When I checked the PDB, I found many residues have > high temperature factor over 100. How can I use Phenix or CNS fix those high > temperature fa

Re: [ccp4bb] GST fusion-Expression problem

Kumar, Sometimes a pGEX vector with thrombin site causes such a problem. Using TEV or HRV3C sites may help. Did you see any band corresponding to the protein without GST? A simple way to check expression is to induce at 37C for 0.5-2 hours and run a gel of whole cell lysate. If there is

Re: [ccp4bb] GST fusion-Expression problem - NOT GST

I've had a band in the region you describe when expressing GST tagged proteins, and ID'd it with mass spec. I seem to recall it was a "FKB-type peptidyl-prolyl-cis-trans-isomerase." http://www.uniprot.org/uniprot/P45523 2008/10/17 Mazzorana.Marco <[EMAIL PROTECTED]>: > Dear all, > may I point

Re: [ccp4bb] GST fusion-Expression problem

Dear all, may I point out at this common belief that the 'GST' Kumar wrote about is probably not GST? When trying to over-express my protein in fusion with a GST tag I have noticed that many strains did not express it, but still a large band corresponding 25 kDa protein was visible from the ge

Re: [ccp4bb] Twinning for P31

This point group can be four fold twinned so it is possible but unlikely that you have equal degrees of twinning along all possible twinning axes. More likely that you have the space group wrong. Did you test all possibilities; PG321 PG 312 PG 6 PG 622 (all this is very easily tested with p

Re: [ccp4bb] GST fusion-Expression problem

Depending on your exact question, I can see the following possible problems: - a stop codon has been introduced after the GST, either by a cloning error or spontaneous mutation (if the fusion protein is toxic, these sponateous mutations may be relatively frequent). Are you using glycerol st

[ccp4bb] GST fusion-Expression problem

Dear CCP4ers, I have a GST fusion protein (eukaryotic). When I express it in E.coli BL21 (DE3) or RIPL cells, however, I mostly get GST and very little fusion protein. I get very little "leaky expression". I normally grow my cells at 37C in LB or 2X YT and after induction at 20C for 16-18h

Re: [ccp4bb] high tempterature factor refinement problem

Hi Jason I don't know what is your refinement protocol, but I said that at this resolution you can try to refine only two B factors per each aminoacid : one for the main chain and one for the side chain. There is an option to do that in CNS_SOLVE. You can find very nice examples in the CNS_SOLVE t

[ccp4bb] high tempterature factor refinement problem

Hi All, I just refined a structure at 2.95A and getting a good R-work and R-free(0.236 and 0.274). When I checked the PDB, I found many residues have high temperature factor over 100. How can I use Phenix or CNS fix those high temperature factor for further refinment? Any suggestions are welcome

Re: [ccp4bb] Twinning for P31

Hi, Peter, I have tried to carry out MR in P3. It turned out that P31 is the right one. After MR, I refined it using Phenix.refine with or without twin law. The result is as following: Twin law R Rfree Twin fraction in phenix.refine None