Re: [ccp4bb] twinning problem ?

Dear Jacob, Measurement of the reciprocal space maps at reflections with triple axis diffractometry allows experimental separation of mosaicity and strain (variation in unit cell parameter) effects. See eg Boggon et al 2000 Acta Cryst D56, 868-880 http://dx.doi.org/10.1107/S090744495837 for

[ccp4bb] Two open PhD positions biochemistry/xtallography at the Ruhr-University Bochum / University Kaiserlautern, Germany

2 PhD positions in biochemistry/protein crystallography In a joint project we offer two PhD positions to study the evolution and mechanism of ferredoxin-dependent bilin reductases. These enzymes from cyanobacteria are involved in the biosynthesis of the linear tetrapyrrole chromophore of

[ccp4bb] EMBO Course on Macromolecular Complexes, Grenoble 2-7 June 2014

Dear all, This is the second announcment of our EMBO Practical Course on the Structural Characterization of Macromolecular Complexes, which will be offered June 2-7 in Grenoble, France. COURSE DETAILS REGISTRATION: http://events.embo.org/14-characterization/ APPLICATION DEADLINE: 4 April

[ccp4bb] twin refinement

Dear colleagues, this is a request for comments on the evaluation of crystal structures that resulted from twin refinement. From http://www.ysbl.york.ac.uk/~garib/refmac/Tutorials/refmac_tutorial.pdf: Although Rfactors are substantially smaller with twin refinement than without twin refinement

Re: [ccp4bb] twin refinement

Dear Wolfram, First of all you should make sure you have substantial twinning. Is the twin fraction close to zero, then don't use a twin target. Is twinning detected by several test, then there is a good chance your data are twinned. As for validation, if you do not completely trust your

Re: [ccp4bb] twinning problem ?

Unless you are interested in finding curious objects, what would you do with protein quasicrystal? The practices of macromolecular crystallography is about determining 3-dimensional structure of objects being crystallized. Protein quasicrystal are really unlikely to diffract to high enough

[ccp4bb] opening @ DFCI-SBC

Dear colleagues, We have an opening for a structural biologist / research scientist at DFCI's new Structural Biology Center in Boston. See link below. Please consider or forward to those who might be interested. Thank you.

Re: [ccp4bb] twinning problems ?

Hi Jacob Maybe we can get the author to repeat the study for the other usual-suspect protein crystals to find out the truth, but the score currently seems to be 1-0 in favor of cell parameter shifts versus microcrystal orientation... You don't mean me do you? The measurement you refer to was

[ccp4bb] twin refinement

Dear collegues, I'm working with a drug complexed protein structure that is having major twinning issues. The drug has a single Br atom on a benzene ring, which I'd like to use for orienting the drug in the binding site. I have various anomalous data sets, ranging from 3.0A resolution, all

Re: [ccp4bb] twinning problem ?

On 03/13/2014 10:55 AM, Keller, Jacob wrote: Unless you are interested in finding curious objects, what would you do with protein quasicrystal? The practices of macromolecular crystallography is about determining 3-dimensional structure of objects being crystallized. Protein quasicrystal are

[ccp4bb] off-topic: protein losing FAD during purification

Dear All (those dealing with wetlab stuff..), While purifying a FAD containing protein we lose part of the FAD (on the gel filtration we clearly see two bands corresponding to holoprotein and free FAD). We obtain crystals but diffracting to only about 4 A despite their beautiful look. Our

Re: [ccp4bb] off-topic: protein losing FAD during purification

Hi Stefano, Wouldn't you be better off going the other way around, that is supplementing your protein solution with FAD prior to setting up the crystallization trials? It sounds as if the FAD is quite labile in your case, but it could well stabilize the protein and hence give rise to better

Re: [ccp4bb] off-topic: protein losing FAD during purification

In cases like this, I keep FAD around during all steps starting from cell lysis, purification, dialysis and even crystallization. In one case we added FAD to protein such that the protein: FAD ratio was 1 prior to setting up crystallization trials. Hope that helps. Harkewal Sent from my iPad

Re: [ccp4bb] twinning problem ?

Hi Zbyszek I think this has deviated significantly from twinning problems! I certainly don't claim the 1998 study was typical. The crystal was large by present day standards, no cryoprotectant was used and non uniform drying/cooling rates might have occurred. The Juers et. al. paper includes

Re: [ccp4bb] off-topic: protein losing FAD during purification

And include FAD at a few uM in your column buffers. Although if you are getting two separate sharp peaks for protein and FAD, it doesn't sound like it is bleeding off during chromatography but rather already dissociated in the material you apply to the column. Take a spectrum of the material in

Re: [ccp4bb] off-topic: protein losing FAD during purification

Dr. Stefano, 1) I would add FAD in all buffers after lysis,even after GF you could perform dialysis with buffer supplemented with FAD. 2)for the second question I found this paper which could be helpful: Large-scale preparation and reconstitution of apo-flavoproteins with special reference to