Most of the labs sharing our Phoenix have had enough trouble with exactly
this that our standard procedure is to now to use 1 µl drops (.5/.5
protein/well) in our initial screens those scale up much more reliably to
the 24-well format and seem less finicky in general, reducing the chances of
an
It is important to distinguish between the solubilisation and the
purification steps:
1) During the solubisation step you need to care about the
lipid/detergent ratio. The amount (not the concentration) of detergent
(i.e. in non monomeric form, the detergent above the cmc) is important.
You
Dear All,
A bit offtopic question,
Does any body know how can I get the PIR output of the aligned sequence
from clustal omega.
I have a full length protein solved, to give weightage during comparitive
modelling, I use domains of the same/similar protein solved before.
I expect an alignment
Dear colleagues,
On behalf of the organizers, Maria Armenia Carrondo and Thomas R.
Schneider I have the following course announcement:
*COURSE ANNOUNCEMENT - BIOCRYS 2012*
FEBS Practical Lecture Course
Fundamentals of Modern Methods in Biocrystallography
*20th - 27th October 2012 at the
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Dear S. Jayashankar,
have you tried the clustalw options '-convert -output=PIR'? This should
result in a pir-formatted output file.
Tim
On 03/27/12 10:18, Jayashankar wrote:
Dear All,
A bit offtopic question,
Does any body know how can I get
Dear Tim,
I try to model a full length protein based on various domains solved
already.
In that case, alignment of multiple sequence should approximately locate to
the corresponding domain region.
Clustalw results in an unexpected alignment file.
Whereas the output from clustal omega is perfect
Hi ccp4bb,
I've been having trouble with Coot. Specifically the scripting to change
the maximum number of residues to refine.
So if I try to refine more than 20 residues I get this neat little warning
message in the terminal:
WARNING:: Hit heuristic fencepost! Too many residues to refine
works here on 0.7-pre-1 rev 3713
so try downloading the latest version if yours is 3713
On Tue, 2012-03-27 at 14:50 +0100, Morten Grøftehauge wrote:
set-refine-max-residues
--
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Hi,
I was wondering whether there was a way to assign a keyboard shortcut to
the real space refine button in COOT. Atleast this way one does not have
to keep looking back to where the real space refine button is to click
it if you want to carry out the refinement.
Cheers,
Chris
--
Dr.
Hi All,
I have set up initial screen in hanging drop trays with a protein of
theoritical pI of 8.5. The protein is in acetate buffer 10mM, KCl 100mM and
2% glycerol
pH 5 . In 85-90% of the conditions I see granular precipitate in 1 day. I
tried to open the coverslip, and touch few drops, They had
Dear Tim,
Thanks, The Jalview, output to textbox in PIR worked thr trick.
S.Jayashankar
Research Student
Institute for Biophysical Chemistry
Hannover Medical School
Germany.
On Tue, Mar 27, 2012 at 3:52 PM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote:
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Hi Brad,
I am afraid that there is no alternate source for these plates. The screw
cap system , I believe, was patented by the canadian company NEXTAL that
was then assimilated by Q...N and the patent is probably still holding.
Pascal
--
Pascal F. Egea, PhD
Assistant Professor
UCLA, David
When you lyse the cells and spin down cellular debris, is the pellet large
and white (indicating inclusion bodies)? Is your protein soluble or
membrane? What temperature did you use for expression? What vector are you
using? Providing more details allows us to better answer your questions.
Off
Hi Brad,
See below my attempts on this in 2010.
Regards,
Mathews
-Original Message-
From: Mathews, Irimpan I.
Sent: Friday, May 21, 2010 8:23 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: RE: For 24 well Netxal plates (non ccp4 question) == SUMMARY
Dear All,
Thank you for the several
I guess you already run SDS-PAGE to check the pellets before and after
sonication. Not only the media.
On Tue, Mar 27, 2012 at 7:35 AM, rana ibd rna19792...@yahoo.com wrote:
Dear all
I am expressing a 6xHis tagged in a dHBx protein in E.coli BL21 using LB
madia, I am having problems with the
Yes, look here:
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Coot#Example_11:_Paul_Emsley.27s_Key_Bindings
and the key bindings for r, x, t, R.
JED.
--
AstraZeneca UK Limited is a company incorporated
I suspect Chris is asking for the shortcut to the zone refinement
button, i.e. invoking the manual zone selection. Not sure if there is a
scripting way to do this, nothing obvious.
On Tue, 2012-03-27 at 17:03 +0100, Debreczeni, Judit wrote:
Yes, look here:
Hi Matt
Rajesh asked a similar question last week, below
Essentially, you have to *reduce *the protein concentration when you scale
up because you lose proportionally more protein from smaller drops.
This usually works very well and we see no reason to use more than 0.3 +
0.3 for initial
Try this:
1) take your favorite PDB file and set all the B factors to ~80 (reduces
series-termination errors)
2) use sfall/fft in CCP4 to calculate structure factors to 4A resolution
3) use sftools to add a SIGF column (0.1 will do) to make refmac5 happy
4) refine the perfect model against
Please excuse me for bringing up an old issue. I have an interesting
example of a difference seen when DFc was substituted for missing
reflections versus when it wasn¹t. Maybe others had this experience. I had
a structure in which the electron density showed two overlapping¹ ligands
bound in
Dear CCP4BBers and PhenixBBers (cross posting here, since we all read both
anyhow)
to the experts out there here's my question:
We have a P21 dataset with 2 molecules in the asu and a refined twin fraction
of 38% according to phenix.refine using a twin law operator.
My gut feeling tells me
[Snipped from the full message, which is appended below]
The program that kept showing me two forms bound was not
substituting Fcalc for unobserved reflections. So, I turned on the option
to substitute Fcalc, and the minor form disappeared � the density looked
like it did in the second
Thanks Garib for your input. And yes we do see some split spots. We used XDS to
overcome (I hope) most problems but still intensities of perfectly overlapping
reflections will be too large. Would you think it's safer to integrate the data
in P1 as symmetry mates will not be merged and then
I would say that you should use ncs restraints in any case. NCS relates atoms
and twin relates intensities. In some sense presence of twinning reduces
information contents (in the limiting case the number of (effective)
observervations becomes twice less) of the data and using NCS decreases the
Unless you have very good reason it is better to use highest possible space
group (without going over). Then you do not have problem of related reflections
and covariances between them, If you go to P1 then reflection will be related
with crystallographic as well as NCS as well as twin
I would be concerned about the completeness of the data if adding Fcalc values
has such a large effect on the appearance of this electron density.
Hi James,
my understanding is that phenix.refine allows any number of alternate
conformers. There may have been a limit of 4 some time in the past, but no
longer. So your idea could be tested.
Cheers,
Paul
On Mar 27, 2012, at 12:33 PM, James Holton wrote:
Try this:
1) take
phenix.refine allows any number of alternate conformers.
Hmm. quoting our old friends from the validation circuit: Where freedom
is given, liberties will be taken
BR
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