[ccp4bb] Dnase activity in E. coli expressed his tag proteins

Dear All: This is off topic but I would rather try here first. I am wondering whether Dnase could be a contaminant during the nickel affinity purification of a his.tag protein expressed using pET29b. The cells were disrupted using sonication only. Very high yield (30 mg/liter of cell culture).

[ccp4bb] Predict potential sites for introducing cys to make a disulfide

Dear All: I vaguely remember a program that can predict sites for introducing a pair of Cys residues to make a disulfide. I had once used this before but now I had forgotten about it. Does any body know about this? My plan is introduce Cys residues into my structure that I can use for FRET

[ccp4bb] Predict potential sites for introducing cys : Summary

Dear All: Thanks for the overwhelming suggestions. I had in fact used SSBOND. However, there were other sugestions which are listed below. ssbond: http://eagle.mmid.med.ualberta.ca/forms/ssbond.html disulfide by design: http://www.ehscenter.org/dbd/ http://www.predictprotein.org/ - have not

[ccp4bb] Is phophorylation possible in E. coli expression system?

Dear All: This is not crystallography related and does not belong to this group, but I would like to pose this to all who are working with proteins expressed in an E. coli BL21 (DE3) or Rosetta. Is it possible for a protein to be phosphorylated during expression? At least my understanding was

[ccp4bb] Summary: Is phophorylation possible in E. coli expression system?

Dear All: Thanks for all your replies to this. Phosphorylation is possible in E. coli. One article of particular interest is Mol Cell Proteomics 2007 Eprint by Macek B, Gnad, F, Soufu B, Kumar C, Olsen JV, Mijakovic, I and Mann M. Many others have pointed out that it is possible and many

[ccp4bb] How to generate atoms lying on plane - surface generation

Dear All: Is there a simple graphical way to generate atoms that lie on a surface (and wite out a pdb file) for modeling surface docking experiments? Thanks a lot Subbu I am looking for Cerius2 type but any code will be fine

[ccp4bb] Negative density around C of COO-

Dear all: I am noticing that in some of my structures, at 1.5 A resolution, 1) there is some negative density around the C of the carboxyl groups. 2) I also notice the negative density around S in a disulfide region. It is as though the disulfide is broken. Could some body enlighten me on

[ccp4bb] Summary: Negative density around COOH

Thanks for the quick reply and pointing out t bunch of references. Radiation damage has been pointed out as a cause for such feature. The following references were pointed out. Ravelli, R.B.G. McSweeney, S.M. (2000). The 'fingerprint' that X-rays can leave on structures. Structure 8:315-328.

[ccp4bb] Activity of a mutant enzyme compared to wild type - puzzle

Dear all: I have a single residue mutant whose enzyme activity is about 50% of the wild type. Interestingly, the mutation is in a region that involves a secondary site but not the active site. The two structures with or without ligands fit well (0.18 A) and the metal binding and cofactor

[ccp4bb] Baculovirus expression and purification - off topic

Dear All: I have a peculiar problem with baculovirus expression of my protein. The native protein elutes in Tris gradient. This has no problems. However, a mutant elutes in the wash. After further purification with size exclusion, I notice that this mutant is always associated with some medium

[ccp4bb] Handling protein crystals from 20% isopropanol

Dear All: I have obtained good crystals from 20% isopropanol but this is my first time dealing with this agent. Help in handling the crystals, dealing with evaporation etc. is highly appreciated. Thank you Subbu

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

ar...@xtals.org wrote: Hi, In a case like the one Raji outlined below - after all the attempts - I would have most cerainly switched to insect cells as the next step :) If you suspect that protein of interest has large disordered regions, expression in a higher order system by itself may not

Re: [ccp4bb] Structural biology inside the cell

ar...@xtals.org wrote: Hello, 1. As long as all proteins have seventy amino-acids or less and express in E. coli in mM concentrations - we're in business. 2. As for the question below - my favorite answer is 'It will take a week and ten million dollars in unmarked bills. We begin as soon as

[ccp4bb] Non-CCP4 protein purification problem - help needed

Hi all: I am trying to purify a protein that was expressed in a baculovirus system. The protein elutes in the flow through in a DE52 column. Further gel filtration gives an enriched protein but we see that some medium components tag along (weight and activity data do not match). Another

[ccp4bb] In XDS, anomalous correction column has negative values

Dear All: What does it mean when I get negative values under Anomalous Corr column after running XDS? I set the Friedel Law=False even though I suspect that my signal is very very weak. Thanks Subbu

[ccp4bb] Concentrating a protein solution - subbu

Dear All: We have been trying to crystallize a protein which is large - 100 kDa. This is soluble but the best we can get is about 1 mg/mL. It did crystallize but did not diffract well. Efforts to increase the concentration has been unsuccessful. I am wondering whether there are methods that

[ccp4bb] Paper describing the structure of LFA-1

Dear All: I would like to know the literature on the crystal structure of Leukocyte Function-Associated Antigen One (LFA-1, CD11a/CD18). I have the structure of I domain but not for the entire molecule. I would greatly appreciate if people can point me to the right article/reference. Thanks

Re: [ccp4bb] conversion of IU/ml to mcg

On 1/19/12 5:32 AM, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Megha, your email could hardly be more cryptic to me, and maybe you increase the chance of getting help by explaining - - what is R D - - what is MiOU? - - what is mcg? (milli-centi-gram?) (I understand ml,

[ccp4bb] Post-doctoral position available June 1st at UMDNJ

A postdoc position is immediately available in my laboratory at the Oral Biology Department, UMDNJ. The post-doc will work on the elucidation of structure of enzymes involved in modifying A. actinomycetemconitans biofilms. Crystals of two such enzymes are available. Experience in expression and

Re: [ccp4bb] video that explains, very simply, what Structural Molecular Biology is about

mb1pja wrote: Dear Fred A really nice video that would be great for giving non-crystallographers (including colleagues and 1st year students, and perhaps also friends and family) an overview of what we do. Thank you for pointing it out - and of course very many thanks to Dominique Sauter for

[ccp4bb] [Fwd: Re: [ccp4bb] video that explains, very simply, what Structural Molecular Biology is about]

Just thought this will be of interest to all. Subbu ---BeginMessage--- Narayanan Ramasubbu wrote: Vellieux Frederic wrote: Narayanan Ramasubbu wrote: Hi: Could someone point out the name and where to get these crystallization plates used in the video? By the way, this is a wonderful video

[ccp4bb] How to compile a small fortran program which uses some CCP$ modules in fortran

Hi: I have an old fortran code that I used on an SGI Irix system. i would like to use it on a linux (Ubuntu). How to compile this code which uses some ccp4 libraries? What is the command for this? Thanks Subbu

[ccp4bb] Memory allocation error in phaser - Mac os

Dear All: How to increase the memory size (?) to avoid the following error? *** * Information from CCP4Interface script *** The program run with

[ccp4bb] AU limts not read - arp/warp ccp4i interface

Hi: I tried to run arp/warp using the gui. My original mtz file (space group p222) is read correctly and I see the AU limits in the Crystal parameters. I then tried to change the space group using CAD/SORTMTZ export to .sca and manually change the space group to P2212 and then used

[ccp4bb] Difficult MR structures

Deal All: I have a 2.0 A data for a SeMet protein (native crystal not available yet!) that has 6 Se sites. The cell comes out to be 65 67 101 and the angles are all very close to 90. The data set was collected in house with Cu 1.5418 A We integrated and scale in orthorhombic and the

[ccp4bb] Error in CAD during ARP/wWARP run in sharp/autoSHARP - also posted in sharp discuss

Hi: Sorry for the same posting in here as well but I thought may be some of you might have encountered the same problem but may not be subscribing to sharp list. I am trying run sharp/autoSHARP on a mac (os x 10.4) using the guven example file: krel1-SAD.0 I am getting an error at the CAD

Re: [ccp4bb] question on the effect of a fire alarm on crystallization

Clemens Vonrhein wrote: Hi Jim, On Fri, Jan 22, 2010 at 09:11:00AM -0600, Jim Pflugrath wrote: I do not know the effect myself, but the idea of vibration fee has been tossed around a while. What? I'll have to pay for vibration now as well? Are you collecting that personally? Or is it

Re: [ccp4bb] Per-residue RMSD for multiple structures?

Gerard DVD Kleywegt wrote: Thanks Stephen! I was going to suggest that, but I was afraid of the self-appointed CCP4BB Gestapo that has been seen goose-stepping in this neighbourhood recently (Tassos recently accused me of becoming mellow and diplomatic in my dotage, so I hope I've set the

[ccp4bb] Difficult purification with imac columns

Hi. We are working on a periplasmic protein that breaks naked glycans in peptidoglycans. There is truncated structure available but our target is the full length protein. The difficulty us that it strongly binds to the resin with or without his.tag. Changing the resin to acrylamide did not