I'm sure that the relative weight between your structure factors and
geometric factors is wrong. If you use the default value, 0.3, it may or
may not be too high. In CNS, the relative weight factor is calculated, but
this is not done in REFMAC. As a rough rule-of-thumb, I aim for a rmsd of
bond len
Hsp70 is very hydrophobic, and the problem mentioned by you is quite
common in many affinity based purifications. I suggest that you load your
sample on an HIC (say, phenyl sepharose) and elute with a gradient from
high-salt to no-salt. Hsp70 typically elutes close to no-salt buffers on
the H
Hi, Nick,
An R-factor of 0.16 seems low for a model refined against 2.4 A data. What
are you using for the MATRIX weight in REFMAC? Here's a guide for
selecting a proper MATRIX weight
http://www.dl.ac.uk/list-archive-public/ccp4bb/2005-10/msg00592.html For
refinement against a 2.4 A data set
Hi,
I am working on a 2.4 angstrom structure that is in its final stages of
refinement and finding that REFMAC5 is giving a large difference between R and
Rfree (10%), 0.16 and 0.29, respectively. 2Fo-Fc maps looks great and there is
very little density in the difference maps. My model cover
I have never worked at 3.2A - but I suspect that the overal temperature
factor is being determined from the slope of a Wilson plot. However,
Wilson plots only really "work" at higher resolution (2.8 or better). Look
at the output from truncate and see what it is predicting - and look at
the pl
Hello all:
I just recently collected data on initial crystals I grew of an enzyme with inhibitor. The crystals diffract to only 3.2 A but I was able to get phases by molecular replacement to see if there is any inhibitor bound. Although the data processed well in HKL2000 with good statistics and th
The old CCP4 had an "how to unsubscribe" header, can we have that back?
Flip
-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Sarat
C Sahu
Sent: Friday, March 09, 2007 20:24
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] unsubscribe ccp4bb
--
Sarat Chand
--
Sarat Chandra Sahu, Ph.D.
Research Biologist
Department of Biological Sciences
Carnegie Mellon University
4400 Fifth Avenue
Pittsburgh, PA 15213-2683
E-Mail:[EMAIL PROTECTED]
Phone: (412)268-7338/3396 (O)
(412)683-0444 (R)
(412)478-4595 (Cell)
Fax : (412)268-7083
I know of several labs where batteries of wine coolers are used, we as a
small lab have one. Works fine.
Of course also our rockimager works with peltier-based temp control, its
effective, and vibration free.
Flip
-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] O
Hi Yong,
Perhaps try one of these:
1. Wash with ATP+Mg when bound on the lMAC column (to get it to
release your protein).
2. Wash with 0.1% Triton when bound on the IMAC column.
3. Hydrophobic interaction chromatography.
4. Wash with 0.5 GuHCl while bound on the IMAC column (pray!!)
On a sad
Hey Yong...
add 10 mM ATP + 2.5 mM MgCl2 in your purification buffer and wash it off during
your affinity step...this has worked for me (of course make sure that you're
not unlucky and your protein doesn't elute during this step; don't worry it
shouldn't)
if that doesn't work, maybe try changing
Yong,
I used Tris 10mM, MgCl2 10mM, ATP 5mM (adjust to pH=8.5, w/o buffer, it would
be very difficult to adjust the pH value) to wash the resin (10-20 column
volume) during on-column wash, and it works.
-
Yeming Wang, Ph.D.
Laboratory of Structural Biology: Macromolecular
Hey Sam,
Add the ISOR and CONN command for solvent to the .ins file for approximate
isotropic behaviour for waters.
Check out the pdf form of the manual, Pg 7-25. for an explanation of ISOR.
Arti
> Hi
> I am trying to refine my structure anisotropically by shelxl.
> When I use, "ANIS", all the ato
Anyone using wine coolers with peltier thermoelectric controllers
(without auto defrost) for macromolecular crystallization in their
home labs? I remember a tiny box using similar technology that once
was sold by Hampton Research, but I can no longer find it. Is there
any good reason not
Dear CCP4BBers,
Announcing the 2nd EMBO Practical Course on High throughput Protein Production
and Crystallization
June 20-28th 2007, Oxford Protein Production Facility, Oxford.
The aim of this course will be to review the state-of-the-art of
high-throughput (HTP) protein production and crysta
Hi Sam,
The ISOR command restrains the anisotropic temperature factor
parameters to (roughly) approximate isotropic temperature factors,
and is by default specified for all your water molecules when you
generate your initial SHELX script. Usually, you can use ANIS
without parameters (i.e
RE: Removal of bacterial chaperone Hsp70 contaminant from recombinant
protein preparation
Dear all,
I have a protein expressed at 37C for 3 hours in BL21 DE3 and purified
with sub-stoichiometric amount of apparent Hsp70 contaminant even
after exhaustive affinity (GST-fusion or His-tagged), ion-e
Look up ANIS in the INDEX of the SHELX MANUAL! The SHELXL syntax is very
flexible, maybe something like
ANIS N_1 > O4_123
(where the last sulfate is residue 123) is what you need. In the next
job, anisotropic atoms are recognised by their Uij parameters and so do
not need to be set again.
G
Hi
I am trying to refine my structure anisotropically by shelxl.
When I use, "ANIS", all the atoms including water becomes anisotropic.
(a) If I want to make only protein residues and ions (SO4 and Acetate) and
not the waters, how should I declare ANIS in the .ins file.
(b) If I modify or add any
Dear All,
I have received several e-mails indicating that people are not
receiving the digests. Can people who are receiving the digests tell
me, so that I can try to figure out what is going on?
Thanks in advance
Charles Ballard
CCP4
Hello Manfred and CCP4bb,
Funny, you are not the only person trying to something like this, so I have a
CCP4mg script (attached) which does the job. You do need a recent version of
ccp4mg for it to work - contact me for details.
Liz
On Thursday 08 March 2007 15:33, Manfred S. Weiss wrote:
>
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