Re: [ccp4bb] crystallisation and mosaicity

I have also observed this. In my cases I have also been able to get away with using less cryoprotectant when freezing in the cryostream. --paul Daniel Pomeranz Krummel wrote: Dear Sajid, I have observed a consistent reduction in mosaicity (from approx. 0.95 to 0.45) for one case by freezing t

Re: [ccp4bb] crystallisation and mosaicity

Dear Sajid, I have observed a consistent reduction in mosaicity (from approx. 0.95 to 0.45) for one case by freezing the crystals in a cryostream (N2 vapour) as opposed to submerging in liquid nitrogen. Have others observed this? Daniel > > Dear All > > My protein size is ~30kD and crystallizes

Re: [ccp4bb] crystallisation

Indeed, if Phoebe Rice is right, and you have dimethyl arsenic bound to your cysteines, you may have a homogeneity problem, not from the protein sample, but from the chemical reaction which is occurring prior to crystallization. If you have DTT, check that it is fresh. Make sure your cacodylate is

Re: [ccp4bb] crystallisation

PEG 3350 can also provide some cryoprotection; 22% PEG 3350 with 5% glycerol has proved a good cryoprotectant in my hands. kmj Phoebe Rice wrote: If you have high [DTT] in your buffer, you might be catalyzing the addition of dimethyl arsenic (from the cacodylate) to some of your cysteines? Al

Re: [ccp4bb] Crystals grown directly on loop?

Dear Jacob, We have been working on this for an year or so. We have a paper partially in the review process. Unfortunately, the referees were not very excited about the idea. However, we are developing some automation to speed up the time consuming process of placing the drops on the loops. Th

[ccp4bb] Crystals grown directly on loop?

Dear crystallographers, Has anyone ever tried to grow crystals directly on some kind of mountable support, such as some kind of loop or film, which could be frozen directly? I understand that there are some microfluidic plates through which the crystals can be diffraction-screened, but what ab

Re: [ccp4bb] crystallisation

If you have high [DTT] in your buffer, you might be catalyzing the addition of dimethyl arsenic (from the cacodylate) to some of your cysteines? Also, 10% glyercol sounds quite low for reproducibly good freezing (at least in my experience). Phoebe Original message >Date: Mon, 2 Jun 2

Re: [ccp4bb] crystallisation

I summarized most of the points in the CCP4 wiki at Hope nobody minds! Could future contributions please be added there? - thanks! Kay -- Kay Diederichs http://strucbio.biologie.uni-konst

Re: [ccp4bb] crystallisation

Hi Sajid, congratulations to your nice looking crystals. I'm not sure if someone already sugested to mount smaller crystals ? You write they need 4-5 days, I assume you can already see them earlier before they reach that size. Am I roughly right in the assumption that these crystals are about

Re: [ccp4bb] mosaicity,was:crystallisation

Are you including beam divergence is the defition of mosaicity? if you are, what happens when you go to a synchrotron? i.e., we know what happens: the beam is less divergent and hence your spots sharper. in any case you will have probably quite a bit better resolution from the extra photons at a

Re: [ccp4bb] crystallisation

Hello Sajid, Is you crystal this mosaic in all three directions? It could be that with the longer rods, that your mosaicity is high in one direction but not so much in the other two directions. In this case I'd guess shooting down the length of the xtal might result in increased mosaicity. Thu

Re: [ccp4bb] crystallisation

Hi Sajid, with my very (!) limited experience I can recommend trying to co-crystallise with your cryo-buffer at very low concentration. It helped me (once!) to get diffraction going from about 2.7 to 1.5 ... oh, and it got rid of some nasty twinning problem as well. I guess the thinking is that if

Re: [ccp4bb] crystallisation

Hi Sajid, Just a simple test for your problem: Incubate your crystal longer in your cryo/stabilization solution. This helps sometimes to lower mosaicity. Of course, you can also try co-crystallization with glycerol (2%, 5% or 10%). Good Luck, christian sajid akthar wrote: Dear All My protein

Re: [ccp4bb] Multimer threading server

I have a sequence that I would like to model by threading onto each subunit of a homo-tetramer of a related protein (crystal structure). It doesn't work to thread each subunit individually and then 'recombine' the homodimer as a single PDB because the interfaces suffer from clashes and over

[ccp4bb] Multimer threading server

To the CCP4 community, I have a sequence that I would like to model by threading onto each subunit of a homo-tetramer of a related protein (crystal structure). It doesn't work to thread each subunit individually and then 'recombine' the homodimer as a single PDB because the interfaces suff

Re: [ccp4bb] Using DM for density averaging.

Hi! There are detailed instructions with screenshots on the CCP4 wiki page on NCS averaging with DM, here: http://ccp4wiki.org/~ccp4wiki/wiki/index.php?title=Phase_improvement_by_NCS_averaging_with_DM You want the section: Determining the NCS operators using a partial model Go through that s

[ccp4bb] Using DM for density averaging.

Hello, I have a dimer with ligands present in the binding sites (1 per monomer). The electron density in one of the monomers is pretty poor compared to the other, so I was hoping to use DM to try and average the densities. I am having problems telling DM the NCS operators. How would you go about

Re: [ccp4bb] gfp problem

Hola, If you have done everything you can to improve the refinement (like trying lower symm spacegroups, leaving out the loops in question, different kinds of NCS restraints, TLS refinement, etc.), and what you do see is sufficiently interesting, there should be no problem in publishing