Re: [ccp4bb] Se-Met and Se-Cys double labelling

Absolutely. But don't bother with SIRAS: straight SAD will do the job 9/10 times these days, thanks to magnificent beamlines and algorithms. Sent from tiny silly touch screen From: "Whitley, Matthew J" Sent: 22 Jun 2017 1:09 am To:

Re: [ccp4bb] Se-Met and Se-Cys double labelling

I second (or third) the suggestion that others have given to try soaking mercury compounds into your crystal. Mercury absolutely loves free cysteines, and if you have 5, you have a great chance of getting binding. If isomorphism is maintained after soaking, the isomorphous differences will be

Re: [ccp4bb] Rcrane error, update

I figured it out. Rcrane works fine if I remove the hydrogens before going into Coot. Ursula On Wed, Jun 21, 2017 at 12:04 PM, Ursula Schulze-Gahmen < uschulze-gah...@lbl.gov> wrote: > I am trying to use Rcrane in stand-alone coot version 0.8.8. The Rcrane > window opens and I am able to

Re: [ccp4bb] Se-Met and Se-Cys double labelling

To perform double labelling on your protein in an NON auxotrophic strain may be difficult. For the seleomet side, you can shut down the biosynthesis of Met by the addition of lysine, phenylalanine, threonine, isoleucine and valine; various protocols exist online. However to shutdown

Re: [ccp4bb] Se-Met and Se-Cys double labelling

Dear Vito, for SeMet have a look here: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Expression_of_SeMet_labeled_proteins Worked like a charm for E.coli and also for other expression hosts with minor modifications

Re: [ccp4bb] off-topic:fluorescence polarization displacement assay

Looks like positive cooperativity to me. If binding at one site increases affinity at the second site, then intermediate concentrations of your unlabelled ligand can (seemingly paradoxically) *increase* binding of your tracer. At higher levels still, the unlabelled ligand outcompetes the tracer

[ccp4bb] off-topic:fluorescence polarization displacement assay

Hello All, This is an off-topic question. I have some issues regarding Fluorescence Polarization competitive displacement assay and would need some advice. I have developed an in vitro fluorescence polarization based assay using a N-terminus labelled FITC peptide. The peptide is 21 amino acids

Re: [ccp4bb] Rcrane error, update

I'm thinking a nomenclature issue. H5 ? Should be H5' no? Have you run your structure through a pdb remediator? http://kinemage.biochem.duke.edu/software/remediator.php is my favorite. You will want to play around with the flags to get the right output. I think rCrane likes old style

[ccp4bb] Rcrane error, update

I am trying to use Rcrane in stand-alone coot version 0.8.8. The Rcrane window opens and I am able to calculate new conformations for my nucleic acid. But when I am trying to accept a conformation, I am getting an error message: Traceback (most recent call last): File

[ccp4bb] rcrane key error

I am trying to use Rcrane in stand-alone coot version 0.8.8. The Rcrane window opens and I am able to calculate new conformations for my nucleic acid. But when I am trying to accept a conformation, I am getting an error message: Key Error "H5". Any suggestions what might be wrong, and how I can

[ccp4bb] VS: [ccp4bb] Se-Met and Se-Cys double labelling

Something like Balbes for MR is worth a shot? I wouldnt bother manually with MR anymore unless really clear. Or try Hg and Pt for Cys, His and Met. I would try the Pt chlorides. All depends on pH etc... Tommi Alkuperäinen viesti Lähettäjä: "Keller, Jacob"

Re: [ccp4bb] Se-Met and Se-Cys double labelling

Halide soaks anyone? Cs or NaI? Jacob From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mark J van Raaij Sent: Wednesday, June 21, 2017 12:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Se-Met and Se-Cys double labelling If your data is good enough, your SeMets alone

Re: [ccp4bb] Se-Met and Se-Cys double labelling

If your data is good enough, your SeMets alone might well be enough. Soaking native crystals in Hg compounds may also work, avoiding SeMet altogether. We have had a lot of success with methylmercury chloride binding to free Cys. You may have to experiment with different soaking times and

[ccp4bb] Se-Met and Se-Cys double labelling

I am working on a protein having 360 residues. In its sequence there are 3 Met and 5 free Cys. I will need MAD to solve the structure since based on the sequence the closest homologue has 20% identity…I suppose MR would be very unlikely to work…so I would like to express a selenium derivative to

[ccp4bb] Blend error

Hi, all I am running Blend on a new machine (Redhat 6.9 Santiago) and met the following X11 module and R code error. Thanks for your help. When I run $ X -version It returns: X.Org X Server 1.17.4 Release Date: 2015-10-28 X Protocol Version 11, Revision 0 Build ID: xorg-x11-server

Re: [ccp4bb] Weak density of RNA in a complex structure

Dear Chen, Is this an icosahedral virus crystal structure, or a highly symmetric structure where the RNA may have different binding modes to each subunit and thus averaged out in the crystal? Best wishes, Reza Reza Khayat, PhD Assistant Professor City College of New York Department of

Re: [ccp4bb] Protein or DNA crystals

We have sometimes used Michael Garavito's excellent suggestion of diffusing glutaraldehyde into the drops - mentioned on this board a few days ago. Our protein crystals either turned brown or turned into brown goo. Michael and others, what effect would you expect glutaraldehyde to have on DNA

Re: [ccp4bb] Weak density of RNA in a complex structure

Dear Chen, I will answer with some question : - How is the refinement going ? Is the RNA properly taking in count ? Which soft do you use ? - How do you solve the structure ? MR ? If it's MR did you use a model which contain both protein and RNA ? Did you try to solve with the protein only

[ccp4bb] Weak density of RNA in a complex structure

Dear All, We have get a complex crystal and the resolution can be refined to nearly 1.84 Å. But the electron density of the RNA is very weak. In some datasets we can't find any density of the RNA and in other datasets we can see more or less some RNA density. For now we can build 6-7