Dear CCP4 users,
An update for the CCP4-6.4.0 series has just been released, consisting
of the following changes:
* prelyscar: New program, predictor of lysine caboxylation
* CCP4i: prelyscar, new interface
* pdb2to3: fixed for new ccp4srs
* rapper: downgrade optimisation to workaround segv on
Dear CCP4-ers.
I am (trying) to use Xia2 (svn/Build 4599) to process some diffraction data.
However, I am coming across the following issue, which I don’t seem to be able
to solve...
Integrating SWEEP1
Status: error [XDS] cannot
Dear Antony,
Have you try to use the option - parralel 1, to see what hapen. Maybe it's
problem with the parallelism option. If you try this option you force to use
only one core and i think you decrease your need in memory. If this solution
help, you probably have to check the xdsInput
Hi Fellows,
my lab mates successfully expressed a glycoprotein in CHO cells in serum
free medium, and
the protein captures nicely on HisTrap Excel 1ml columns (obviously, high
yield is not my problem.).
We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole
gradient. 20mM
Well, this is of only possible relevance, but in a previous lab, we used sf9
cells/media quite a bit, and there was always an issue similar to this, due to
[we thought] ferritin being secreted into the medium, and sucking up the
metals. Many, in fact, crystallized ferritin this way by mistake!
Dear Tony,
Sorry to hear you are having trouble - I have never seen this message
before. Please could you send me (off list) the output of the last XDS job
ran before this one and (ideally) the XDS.INP file in the offending area?
There must be something very odd going on here.
Thanks best
Hi Bernhard,
I just stumbled over this patent, where they add cobalt or nickel ions:
http://www.google.com/patents/WO2013082518A1?cl=en
[0086] Supplementing cell culture media, such as CD FortiCHO and
Freestyle 293, with metal ions does prevent column stripping and improve
histidine tagged
I think the new versions of GE's HisTrap columns can address
these problems. Try contacting someone at GE.
I think a number of labs have had such problems in the past
and the culprit has been believed to be a chelator in the
media but never confirmed because the manufacturer of the
media is
Bernhard,
We have had similar, but not identical issues with some insect cell media, as
well as column interference by lipid. If we see this, we tend to run all the
material with the protein (cell lysate or media) through step-elution ion
exchange (quick on, quick off). While the
If EDTA is the issue (as a secret component) then I can see that Mg does not
help, because the Binding(Chelating) constant is
10 orders of magnitude higher for Ni than Mg. Ni or Co could however work to
saturate the EDTA.
pH (too basic) I don't think so because the cells would not react well to
Bernhard,
One other point, after rereading your email. For step-elution ion exchange
(quick on, quick off), you can use some very cheap and quite robust media the
will resist a lot of stuff. Have them pour their own columns and toss it away
when it gets too dirty. You don't have to use the
I doubt it is ferritin sucking up metals; although the problem of
crystallizing ferritin is an issue in secreted preps. See this paper,
where they made lemonade (a PNAS paper) from this lemon (High Five
ferritin crystals): Secreted ferritin was isolated from the
supernatants of
I use already the HisTrap Excel - they really do not leach under normal
treatment. Chelator is a prime suspect, agreed.
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Reza
Khayat
Sent: Montag, 19. Mai 2014 17:05
To: CCP4BB@JISCMAIL.AC.UK
Subject:
Our HEK medium has some pH-indicator dye in it, and phenol red
(phenolsulfonphtalein) is a good guess. Under the assumption that
the same indicator was in the other CHO medium, it did not cause a problem
there.
Thx! BR
-Original Message-
From: Oganesyan, Vaheh
Dear all,
I am looking for some suggestions here. I have lately collected some
datasets but the spots are very very weak... it is very difficult to
process them. At times it looks like XDS is stalled or at times it just
says that it can not interpret the lattice parameters... also while running
Dear CCP4-ers.
Many apologies for the previous post (with attachments).
However, it is true - somewhat crappy data, that I can’t seem to reprocess
with XDS / xia2.
Tony.
HI Graeme,
Please find below, what I think it is that you need? Files attached, plus
relevant text clips,.
NB:
-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1
Dear Antony,
you should check images 189, 190,... XDS only finds 2 strong
reflections (NSTRONG). As the LP-file indicates, these are not enough
to carry out refinement. My guess is these are blank images - XDS can
skip over them if they are not too
I worked for several years on mammalian cell expression of extra-cellular
domains of single spanning membrane proteins
and other membrane proteins.
Just like you did when we switched stable lines from serum containing to
serum free medium the expression drastically
reduced.
So after switching to
Hi Almudena
Have you tried Mosflm (since this is the CCP4 BB...)?
On 19 May 2014, at 17:18, Almudena Ponce Salvatierra wrote:
Dear all,
I am looking for some suggestions here. I have lately collected some datasets
but the spots are very very weak... it is very difficult to process them.
Hi Almudena,
you can also try mosflm as said Harry.
And you can try different setting in the XDS.INP, you can try to reduce the
STRONG_PIXEL (because you said spots are weak) number or/and
MINIMUM_NUMBER_OF_PIXELS_IN_A_SPOT (if the spot are small).
Nicolas
Hello All,
I apologize for the non-crystal related question. I am trying to get a fully
metal-free apo enzyme. The 6x His construct is consistently purified with some
metal (20-30%). I have attempted chelating away the metal with up to 30 mM EDTA
and DFO and then dialyzing it away, but this
Are you treating all your buffers with metal chelating resin? Are you
washing all plasticware with EDTA and metal-free buffer prior to use? How
are you quantifying your metal content, and what metal ions are
contaminating your sample? You might be pulling out the metal ions, but
they get right
-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1
Dear Adam,
- - many plasmids for His-tags contain a cleavage site to remove the
tag. In fact this provides you with an additional purifiction step
which is complementary to the first Ni-column and therefore quite a
good strategy (in addition to
Hi Bernhard,
I too suspect that it is some kind of metal chelating reagent causing the
problem (possibly used in medium for carrying Fe2+, as the free Fe2+ is toxic
to cells). One simple test would be to load a liter of the unused medium to the
Ni2+ column and see what happens. Do you
The answer depends on a number of questions:
* What metal ion are you trying to eliminate?
* What kind of metal-binding site is involved?
o A peripheral or loose binding site? (e.g. surface calcium
ions)--these may respond to chelators
o An active site coordinated metal?
Thank you everyone for the comments and suggestions. To answer a few questions:
-I do not use a treated buffer system. I have just used the nano-pure water. I
have looked into Chelex, but before I bought it I wanted to see if you all
recommended it. I was trying to avoid this, but it may not be
Hi Bernhard -
Like Zhijie, we have also been using a Tangential Flow Filtration (TFF) system
to address this same issue with serum-free 293 Freestyle media.
Ours is the Minimate TFF system from Pall; there is also the Millipore Labscale
system, and I’m sure others as well. I’ve done only
Hi Adam,
I have not read all the thread as it came all at once and late (9:00pm
here).
I believe the best way to strip a protein of metals is to first adsorb
it onto a solid support (e.g. IEX) and then use a sufficiently low-pH
(say equal or below 6) buffer that contains also EDTA.
You will
Adam,
We developed a protocol (loosely based on a few previous literature
reports) for metallo-substitution of beta-carbonic anhydrase (a
zinc-metalloenzyme) with cobalt(II). The metal ion in this enzyme is
extremely refractory toward extraction with chelators, and the protein
will not
Hello all,
Thank you for you suggestions. I took a look at the crystal packing for
the solution with one molecule per asu, and the next closest molecule is 50
angstroms away, suggesting that this is not likely the correct solution. I
have also tried MR with a number of different molecules per
Did you look at the maps for extra density/molecules?
JPK
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Matthew
Bratkowski
Sent: Monday, May 19, 2014 4:48 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular
Dear All,
I have a ccp4 format map file in P1 spacegroup, I would like to manipulate
it in several ways:
1. enlarge the cell dimension . When I tried CELL keyword in MAPMAN, the
density scaled up together with the cell dimension. Does anybody know how
to do it without changing the density?
2.
Hi Niu
A long time ago I wrote a program 'maptona4' which is in CCP4. This
converts a CCP4 map to and from an editable ASCII format, or at least you
can edit the map header, I wouldn't advise editing the density values! So
editing the cell SG is easy: just be sure to keep the numbers in the
Niu, on 2nd thoughts, for your translocation vector, having modified the
start end values in the header you could probably resample the map with
extends or mapmask, provided you had a complete a.u..
Cheers
-- Ian
On 19 May 2014 22:25, Niu Tou niutou2...@gmail.com wrote:
Dear All,
I have a
I found the most versatile program to manipulate maps is MAIN
(http://www-bmb.ijs.si). You can copy and move maps from any cell to any
other cell and get immediate visual feedback to see if things went the
way you expected it.
Cheers,
Jens
On Mon, 2014-05-19 at 17:25 -0400, Niu Tou wrote:
Hi all,
This is a basic question and I'm sure the answer is widely known, but
I'm having trouble finding it.
I'm working on my first structure. I have a dataset that I processed
in XDS with a resolution cutoff of 2.35 A, although the data are
extremely weak-to-nonexistent at that resolution
Reprocessing data to lower resolution only helps if there are ice rings or
other sources of non-desired diffraction that can be eliminated as
contributors to learned profiles in profile fitting. Strong ice
diffraction occurs at 2.28A and 2.68A, so there is no indication that
reprocessing data to
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