Dear BBers,
I am trying to refine a structure using COOT and Refmac5 and I have some
concerns about overrefinement and x-ray term weight in Refmac5, based on
the fact that during refinement to let R factor to drift too far from Rfree
is not good...
So... First question about that : what is too fa
Dear Napoleão, we solved this type of problem in a paper of 2004 (J. Mol.
Biol. (2004) 342, 275–287) with the using of EPMR which use a evolutionary
search algorithm, I will send you the paper later. It was 36aa dimeric
coiled-coil and we had a lot of molecular replacement problems with other
teste
I think you should look at pubmed id 14604526, it works well for me. You should
obtain the cells if you contact the authors.
Michel.
Le 18 oct. 2010 à 17:27, Daniel Bonsor a écrit :
> Have you looked at the Keio collection. Though they are not compatible with
> T7 promoters, you can use the
If you are afraid by the possible low incorporation of selenium in your
protein, and if you have some cysteines into your sequence, you can try the
double labeling --> Structure, Vol. 11, 1359–1367, November, 2003.
It works at the first try for me and I had no change in crystallization
conditions.
A solution could be to control the solubility of your protein in different
pH and salts (you can also add some additives) by using the Thermal Shift
Assay. You may find a better buffer in which to concentrate your protein.
If you think that the reason of the low diffraction is the quality of your
I see some solutions to your problem and one works well for me on a small
protein domain.
I had exactly the same problem of crystallization during concentration and
in my case I solve the problem by heating the crystal suspension. The
validation of the protocol was made with support of 1D NMR to v
Dear all,
We recently jump from a local version of ccp4 using Refmac_5.2.0019 to
an up-to-date version of ccp4 distributed by sbgrid using
Refmac_5.2.0102. From then, refinements with alternate configurations
are blowing-up in refmac. A similar problem was solved thanks to
ccp4bb ('Refmac dictiona
Hi, I think that a good choice, if your protein accept, is to try a gel
filtration at high salt, with or without DNAse incubation before gel
filtration. If your FPLC can do it, a monitoring at 280 and 260 nm will help
you.
Michel
2007/7/12, Gebhard Schertler <[EMAIL PROTECTED]>:
I have obtained some promising X-tals in 2M Ammonium Nitrate. I am now looking
for freezing
conditions. Has anybody had experience in successfully freezing X-tals under
similar conditions
? What was the best cryo-protectant and which concent
Hi,
In our lab we have background on both instruments, 2 Biorad and 2
Aktas. We got problems on both, minor ones on Aktas, major ones on
Biorads, with expensive fixing on Aktas and cheaper fixings on
Biorads, but efficient fixing on Aktas and unsolved problems on
Biorads. Actually, first Biorad w
Hi
Is DNAse essential in your protocol? Maybe it's better to not add DNAse if
you are worried about its presence. If you really need to brake DNA, using
sonication may be enough.
Michel
2007/7/31, Dima Klenchin <[EMAIL PROTECTED]>:
>
> >I have small crystals of protein DNA complex. I am worried
First i think that your protein concentration is to low... A common rule is
: protein concentration is good when 50% of your conditions are precipitates
and 50% are clears drops. If most of your drops are clear, i think that you
must increase your protein concentration (a screen from Hampton can he
Any old references for "universal buffers"...
Johnson, and Lindsey, Analyst, 64 (1939).
Ellis, D. A., Nature, 191 (1961).
Prideaux, E. B. R. , and Ward, A. T. , J. Chem. Soc., 125, 426 (1924).
Britton, H. T. S. , and Robinson, R. A. , J. Chem. Soc., 1457 (1931).
But I never use these buffers my
> I have been trying to express a rat protein in bacteria. The MBP-fusion
> expressed at very high level (~ 40 mg/L) while the GST-fusion and His-tag
> only gave inclusion bodies. The problem is that all protein runs in the void
> volume on a size-exclusion column (s-200, hepes, pH 7.4, 200 mM NaCl
2008/1/23, Zheng, Lei <[EMAIL PROTECTED]>:
>
> Hi ccp4ers,
>
>
>
> Sorry for this out-topic question:
>
> Recently we have a membrane protein expressed, after solubilized with
> detergent and purified from IMAC, the protein looks beautiful in SEC.
> However, it completely precipitates after the 2-
One classical way to optimize expression level is to screen culture conditions.
For my proteins, I solved my expression problems by changing the
expression vector to a pET or changing a pET 20 to a pET 30 (if the
protein is toxic).
But keep in mind that a low but folded expression is better than
Hi
The low pH and its interaction with PIP1 can be conflicting. The low
pH can modify the interaction site.
Nevertheless, one good way to study protein stability is thermal shift assay.
You can read this publication too : PubMed ID 16604423. (Rapid
determination of protein solubility and stability
My short experience with coiled-coil is that molecular replacement can
be difficult for "classical software" (due to the very anysotropic
shape of the protein).
In our case (a short parallel dimeric coiled-coil), molecular
replacement trials using AMoRe or MOLREP were unsuccessful. We solved
the st
2008/9/25 amit sharma <[EMAIL PROTECTED]>
>
> Dear CCP4bbers,
> I have a protein molecule(~9.0 kDa) that crystallized in the presence of
> 0.15M KBr and HEPES pH 7.0. Since there is no homologuous structure present,
> I intend to perform heavy metal derivatization. I read some literature which
>
What are the buffer pHs (first and second run), what is the
isoelectric point of your protein, have you tried Q column?... You
have to give more experimental details to be more precisely helped.
Nevertheless, you can find helpful advices in GE healthcare life
science Handbooks:
http://www4.gelifes
Dear Romain,
I already ask this question to someone of the pdb staff during a deposition
process, and he answer me that it is an in house program and they don't
distribute theirs in house programs, so if this direction hit your mind,
you can forget it directly.
Meow...
2013/2/15 Talon Romain
>
Dear Praveen,
As said, Biacore3000 is not the best for small molecules, but in some case you
can do nice things with this system. I see 3 possibilities :
- You can immobilize your molecules on the chip if you have an amine on it
which is supposed to not participate to the interaction.
- You can
Dear all,
I am actually dealing with a structure containing an unnatural ligand.
I generated the pdb file by drawing it on PRODRG server, I did a manual
pre-fit in the map using coot and I manually merged the pdf file of my
protein with the one of the ligand.
After that I did few cycles of refinem
5j3HvibIuT3ZiarcKl5qtMPo&r=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ&m=2Ib8W-dQ0MHoUt4AHNaero85sr0-ZRZXc93QBAWf108&s=NX4ub_6zOoHnKdhhIKKDNhy_ubtCXCpBDqtdRB6ChmM&e=>
>> <https://urldefense.proofpoint.com/v2/url?u=https-3A__twitter.com_-3Flang-3Den-2Dgb-26lang-3Den-2Dgb&d=DwMFA
Last update...
Everything works well with the same files and the same Coot version on an
other computer. The problem may come from the os of my computer which is
too old and has to be upgraded.
Thank you for your help.
2018-03-06 9:20 GMT+01:00 M T :
> Dear all,
>
> I did something ve
ol)
>
> ** (coot-bin:2710): WARNING **: Widget not found:
> cif_dictionary_file_selector_create_molecule_checkbutton
> /programs/i386-mac/ccp4/7.0/ccp4-7.0/bin/coot: line 288: 2710
> Segmentation fault: 11 $coot_bin "$@"
> catching the crash log:
> coot-exe: "
-coot.state.scm
I restarted Coot, and loaded my files (.pdb, .mtz and .cif), Coot didn't
crash and I am now able to do a real space refinement.
It should be my first intention to remove Coot preferences.
Regards.
2018-03-12 21:39 GMT+01:00 Paul Emsley :
>
> On 12/03/18 14:05, M T wrote:
&
In coot you have also the possibility to zoom into the ramachandran, this can
helps maybe.
Michel.
> Le 22 mars 2018 à 18:57, Ashley Pike a écrit :
>
> Running phenix.molprobity yourpdbfile from command-line used to work – will
> give a molprobity_coot.py (from which when you calculate>run sc
Hello,
I am refining a structure at 2.1Å, using Refmac and manual constructions in
coot.
I have few Ca, Cl and Mg in my structure and some of them have a clearly
anisotropic distribution, then I decided to use ANISOU line in pdb file
only for these atoms.
In refinement parameters of Refmac I set "
Sorry, I moved the image before the sending of the mail and then I was not
attached.
2018-04-04 11:26 GMT+02:00 M T :
> Hello,
>
> I am refining a structure at 2.1Å, using Refmac and manual constructions
> in coot.
> I have few Ca, Cl and Mg in my structure and some of them
Dear Careina,
If you have easy access to mass spectrometry, you can try to fish/rince your «
pumpkin seeds » and send them to mass to try to identify what is inside to see
if it needs optimization or not.
Best.
> Le 8 nov. 2023 à 16:03, 02531c126adf-dmarc-requ...@jiscmail.ac.uk a écrit
>
Dear Liu,
If you are really confident in your MR solution some good tools to refine low
resolution structures are LORESTR pipeline and iSOLDE software.
Best.
Michel.
> Le 21 nov. 2023 à 13:03, Yahui Liu a écrit :
>
>
> Dear all,
> I got a protein crystal dataset of 4.3 A and would like to
Dear Cyprian,
I recommend to users to use a Potter-Elvehjem homogenizer before to use the
LM20, or even (in case of non fragile protein) to do some cycle of sonicator.
I advice also to pay attention to the anti-drop ring of the used bottle.
Because in case of old one, you may have some plastic
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