> We have crystallized a 21KD protein with 2 disulfide bonds grown for one
> month in 0.1M tri-sodium citrate pH 5.6, 0.5M (NH4)2SO4 and 1M Li2SO4. The
> crystals look big (~0.4mm x 0.4mm x 0.3mm) and pretty (sharp edge, clean
> surface) but diffracted to only 4A in-house. The spots are quite stron
>> Have you tried DTT (or another way of keeping things in a reducing
>> environment)? This might help keep your disulfides (and hopefully
>> protein) happy.
>
>
> Surely the addition of a _reducing_ agent would _reduce_ your disulphides
> -
> i.e. break them.
Whoops...sorry, I guess I've got zin
Hi Hubing,
> I have crystallized a membrane protein at cold room temperature (4°C).
The
> protein was purified in 20mM Tris, pH8 with 1% bOG. The reservoir
solution
> contains 0.1M HEPES pH7.5, 0.05-0.2M (NH4)2SO4 and 15%-26% PEG400.
>
> The cryoprotectant was made in such a way that all the other
d thought of
suggesting a gradual change. I haven't ever tested PEG400 alone for
cryo-protection (usually mixed with glycerol, peg4000, or propandiol).
Pete
> On Fri, 2 Feb 2007, Peter Adrian Meyer wrote:
>
>> Hi Hubing,
>>
>>> I have crystallized a membrane protein a
Hi Mac,
> I have noticed that going from refmac 5 (script) to generate fo-fc and
> 2fo-fc maps in O / Pymol once and Coot in the other, the outcome is not
exactly the same electron density maps-I mean some differences are seen.
> I have used the following columns in Coot (latest version): FOFCW
> I would be very much afraid that the only way to get the masks
> properly would be to edit them interactively.
> I used to do that in the PHASES package by Bill Furey and MapViewX
> which is still alive and kicking under the BnP flagship.
>
> I am pretty sure though that Dr. DVD will be online so
Hi,
I've managed to do a pretty good job of confusing myself in my latest
attempt to understand the likelihood stuff, and was hoping that I could
get some pointers as to what I'm misunderstanding.
1. How does one determine the amplitude and phase to use from a given
likelihood surface? Some of t
> I am currently working on processing a data set with mosflm and scala
and
> I'm running into a bit of difficulty. When running MOSFLM the program
crashes at image 37, but if I integrate from 1-36 and 38-180 then
everything
> is fine. I attempted to merge them with CAD 'before' scaling but not
muc
Thanks to Petrus Zwart, Navraj Pannu, Marc Schiltz and Kevin Cowtan I've
got a clearer idea as to what's going on (at least until I manage to
confuse myself again).
>1. How does one determine the amplitude and phase to use from a given
>likelihood surface? Some of the papers I've read refer to us
> Anyway, with the improvements in software we may have reached a stage
> where the limitation of the search model is not whether or not you can
> find a MR solution, but whether or not that solution is going to help
> you determine the structure. What you can and can't get away with
> depends on t
Hi Paul,
> I just installed centos4.4 and the latest version of CCP4-6.0.2. I used
the auto install and the runnable executables went just fine. Ipmosflm
opens up just fine and can find spot, index, and refine, but I can't use
the mouse to enlarge an area, which is especially needed when you have
> structure" running mode of SIGMAA. This is the run we really wanted to
combine
> the model phases with the MAD phases before going through further
density
> modifications with SOLOMON or DM.
I would have thought that you'd want to do this the other way around
(density modification on MAD before
ime after the combination. I have not tested whether combining model
> phases with the density modified MAD phases produces good quality map
> (experts in the field can make comments). I did get significant
> improvement in the map quality with the combine-then-modify procedure.
>
>
Simon,
REFI BREF OVERALL should do something close to what you're looking for (it
will refine a single B-factor for the entire model, but there shouldn't be
variation within the model).
Not sure where (or if) this is in the gui, however.
Pete
>
> Dear all,
>
> I have a structure at fairly low r
Hopefully my offhand remark wasn't taken as a criticism of ccp4i (it
wasn't meant as such); but seeing as I don't use it, it's not a place
where I could tell someone how to find an option.
> As developers, we also have to think about long-term maintainability.
> Options, in particular little-used
> I add a fake "sigma" column for each "data" column because so many
programs require one.
This is slightly tangential, but does anyone know of a good way to
generate semi-realistic sigma values for calculated/simulated data?
The best I've been able to do is borrow from an experimental dataset of
You've most likely looked at this, but if not it might be worthwhile to
check how these ripples behave while varing the low-resolution limit used
(20-2.2,15-2.2, etc).
Pete
> Dear friends of the Fourier transform,
>
> I am refining a structure with 2 adjacent Hg atoms bound to cysteines of
> diff
> If your Phaser results show a high Z-score (> 8) AND high LLG AND your
> solution packs without clashes AND refines (even though starting R/Rfree
> is high) AND reproduces density for the model portion AND produces some
> Fo-Fc density for the missing portion, most probably your solution is
> cor
Hopefully somebody's run into this one previously, and can give me some
hints...
I've almost gotten a new stereo system setup (x64 running kubuntu feisty)
using a PNY nvidia quadro 4500 graphics card and nuvision 60GX emitter
(and a DEC CRT monitor...) with the nvidia (not nv) xorg driver.
Almost
It might be worth checking $CCP4/include/ccp4.setup (whichever one you're
using) to check on the tcl/tk/bltwish locations and versions before
reinstalling. Especially if programs are working either from a
command-line or a script (does mtzdump hklin /home/uma/cal_iodine/n_i.mtz
give a similar erro
Should anyone run into this in the future, this appears to be symptomatic
of having a graphics card with a bad emitter port.
Thanks again to everybody who had suggestions for me,
Pete
> Hopefully somebody's run into this one previously, and can give me some
> hints...
>
> I've almost gotten a ne
This raises a slightly tangential question though - how do we know how
what obs/param ratio is good enough? My understanding was that obs/param
of 1 was sufficient for linear systems; but it doesn't seem that any of
the objective functions used for refinement are linear (and I haven't been
able to
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