I played with this (coded from scratch, both simple algorithm and a few
tweaks) for a couple of weeks for solving heavy atom substructures. With
perfect FAs it works well and quickly. With real delta-F's it didn't
work at all. Can't remember if I tried perfect delta-F's.
Probably SUPERFLIP is
I have also played with charge flipping and my experience was the same as
Kevin's. Michael Woolfson once said that all 'direct' methods work fine with
perfect data. The method requires expansion of the data to P1 which seems
to degrade the quality of the solution; when the data are noisy, as is
Another reason why charge flipping may not work so well with real
anomalous differences is that the data tend to be rather incomplete,
for example all the centric reflections are missing. This degrades
the quality of the resulting maps, which is more serious if you are
modifying low densities
Hmm - that is odd.
You may have the wrong SG in the mtz file. Try MOLREP or PHASER with the
option to try all spacegroups consistewnt with the pointgroup.
Eleanor
intekhab alam wrote:
Hi all
I am trying to do molecular replacement with low resolution (4Å) using
Molrep and Phaser.
Overall
Salameh, Mohd A., Ph.D. wrote:
Dear All,
I'm trying to prepare an alignment figure of 2 proteins that highlight
conserved and similar residues and probably secondary structures; I will
greatly appreciate it if anybody can recommend a software that I can
use. Thanks, Mohd
We found esprit very
Hi Pavel
Phew! Lots of questions - this could take a while:
- where this formula come from and what are the grounds for this?
It's just the RMSD of the density divided by its standard uncertainty
(sigma), which we're assuming is the same for all grid points: this
isn't quite true, sigma is
Dear CCP4er's,
Sorry for the non-crystallography related question and was hoping someone on
the bulletin board might have some suggestions to overcome my peculiar
protein purification problem.
I am working on several membrane proteins (for crystallization trials) that
have a C-Terminal eGFP
Title: Re: [ccp4bb] Alignment software
I usually use Espript via the web interface.
downloadable espript: http://espript.ibcp.fr/ESPript/ESPript/
or the web server: http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi
hope it's of use,
Paul..
###
Dr. Paul A.
Yes, you can try a sequence search of the PDB. But if your sequence
matches only part of an existing entry, be aware there is a bug in the
PDB sequence search so that it reports the wrong residue numbers for the
existing entry. This bug has been reported to RCSB, but they apparently
have not
BSD
You can also use the OCA browser for FASTA searches of the PDB
oca.weizmann.ac.il
Harry
On May 23, 2010, at 10:23 PM, Paul Lindblom wrote:
Hi everybody,
I just crystallized a new project protein. How can I find a
possible model for using molecular replacement? I have the sequence
The last molrep job just finished and it found only an odd solution. So I
think I will try to get my phases elsewhere. But I am somewhat astonished
that there are still enough cases you can't solve by MR.
Thanks to all who replied. Here is a list of servers/programs to find a MR
model:
Hi Celina,
I cannot answer to your question concerning the GFP-related problem.
Fot the thrombin vs TEV protease related question I can tell you that in my
hands thrombin works really very well in most detergents (TEV is somehow
more sensitive). I am working on membrane proteins purified in very
Dear Paul,
Thank you for initiating this thread, for so carefully evaluating the
suggestions you received, and for reporting the outcome.
Your astonishment gives renewed motivation to people who believe it is
still worthwhile continuing to push the frontiers of experimental phasing
and
You've also applied BRAIN 2.0 ?
I mean looked at homologous structures, superimposed them and decided which
parts are to be removed ?
Never trust programs :-) There could be a flexible alpha helix which if you
removed it would have given you in all programs a solution.
it's Monday,
Jürgen
Dear all,
We have tested the Charge Flipping algorithm with SUPERFLIP program
on various experimental data (anomalous delta-F's, MAD FA's).
see http://www.cbs.cnrs.fr/SP/crystal/SUPERFLIP/
Dumas, van der Lee, Acta Cryst D64, 864-73
In all successfull trials, a good quality substructure is
Hello all,
I am working with a protein that is expressed as with an N-terminal domain
that is normally cleaved for activation of the protein (and
crystallization). For in vitro reasons I've needed to switch the normal site
to a TEV site. However, even though the TEV site is in the same place as
Hi Matthew,
TEV is probably the least robust protease among those commonly used for tag
removal. Here’s a common unit definition of TEV. One unit (corresponding to
0.1 ug TurboTEV) cleaves ≥85% of 3 μg control substrate in 1 hour at 30C.
You need to use really a lot of TEV. Information
Why do you need a fusion protein? Can you use His-tag directly? If you have to
have a fusion protein, use one that has no hydrophobic patches on the surface,
because otherwise they will be glued to each other. For separation of your
mixture that you have now try dilute it as much as possible (
Hi Matthew,
TEV protease is very robust. I normally digest with 1:100 ratio
according to the OD280. I normally digest at 4C for overnight around 16-18
hours. Make sure your tev protease site are not inaccessible and buried
inside.
best
Xiaohu
On Mon, May 24, 2010 at 12:27 PM, Matthew Merski
Ok, to sum up for the board, a good reference for this problem is at:
http://mcl1.ncifcrf.gov/waugh_tech/faq/tev.pdf
Thanks to everyone who responded.
Matthew
On Mon, May 24, 2010 at 9:27 AM, Matthew Merski
mer...@blur.compbio.ucsf.edu wrote:
Hello all,
I am working with a protein
Hi Matthew,
By now, you have received many posts telling you both how efficient and
inefficient TEVp is. You might be confused. This seeming contradiction
can be explained by a few events, among many others: Inaccessibility of
cleavage site, absence of reducing agents, and presence of
Hi,
Phenix.model_vs_data offers a great function which prints out the sigma
level of the electron density at each residue or atom center. This is very
useful comparing the relative density between two maps at the given
region, however, it is running a little bit slow. I am just wondering
whether
Hi Hailiang,
phenix.model_vs_data should normally run fast enough. If it is not the
case please send me the inputs and I will have a look (if you decide to
send the files please do so to my email address and not to the whole BB).
Pavel.
On 5/24/10 2:14 PM, Hailiang Zhang wrote:
Hi,
Hi all,
Here is a summary of the responses to my inquiry about refrigerated shakers:
Virtually all respondents advocated shakers manufactured by New Brunswick
Scientific. I have rarely encountered such unanimity in this forum.
The only exceptions were three votes for shakers of Swiss
If you already have your map, or if you can calculate the map in CCP4,
you can print out density at atoms using the Uppsala program MAPMAN,
function PEEK:
http://xray.bmc.uu.se/usf/mapman_man.html#S30
hth
Hailiang Zhang wrote:
Hi,
Phenix.model_vs_data offers a great function which prints out
This is generally a good idea, but removal of residues is a subjective process,
and a little trial and error. If your sequence searching finds multiple search
models you can superimpose them and systematically remove the poorer fitting
regions based upon RMSD. We have built a server for this
What did you see on your ion exchange and gel filtration chromatographs?
ho
Dear Edward:
I generated two maps in the same way, load them in MAPMAN, then normalize
them and PE VA them. I think after normalization, the out put electron
density value at each atom should be equal to the sigma.
Now the problem is, the output denisty values seems squeezed in the pdb
file
Hi Victor,
Thanks for recommending ALINE.
It is not exactly true that development has stopped. It is just
glacially slow as funding for such utilities is not easily obtained!
Recommendations for improvements (to improve that potential) are
well-received and a keen user can write their own
Hailiang Zhang wrote:
Dear Edward:
I generated two maps in the same way, load them in MAPMAN, then normalize
them and PE VA them. I think after normalization, the out put electron
density value at each atom should be equal to the sigma.
That was fast!
Yes, normalization should give the readout
Edward A. Berry wrote:
peak val m1 pdb.pdb
That should be peek - not peak!
Hi Ian,
thanks for very detailed reply!
- do you think it is better than looking at three values {map CC, 2mFo-DFc,
mFo-DFc} and why?
Yes, because all the information you need is encapsulated in 1 number
per region of interest!
I agree it's a good reason.
But I don't understand
32 matches
Mail list logo
