Hi all,
thanks for all your help so far, and as we ended up in a more general
discussion about temperature factor refinement at not-so-great resolution,
here is a quick summary of what I'll try out:
1.) Refine overall B's instead of isotropic B's.
2.) Use isotropic B's with the following (combi
I had a similar situation, i.e. the cells wouldn't grow past an OD of 0.1 on
minimal media, but in my case I did get protein expression. The way I got
around it was as follows. I grew the cells up in minimal media plus the
amino acids that suppress methionine biosynthesis, plus L-methionine. For
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employ a multi-discipl
Manish,
I also had a similar problem getting cells (C41 (DE3) in my case) to grow
in minimal media. To get around this problem, I took cells from an agar
plate and grew them in a small volume (5 mL) of the minimal media. Once
that culture got thick, I then inoculated 200 mL of minimal media with 0
What we usually do is to grow cells in this media:
400ml H2O milli-Q Autoclaved
120ml AAmix (5X) Filtered
60ml M9 (10X) Autocl.
6ml TES (100X) Filtr.
12ml Glucose 20% Filtr.
0.6ml MgSO4 (1M) Autocl.
0.18ml CaCl2 (1M) Autocl.
1.2ml Biotine (5mg/ml) Filtr.
0.6ml Thiamine (10mg/ml)
Are you adding vitamins to your M9 minimal? RosettaBlue is thiamin
requiring and can not grow in the absence of thiamin. The thiamin
requirement is so low that you can often get slow growth to a low OD
based on residual thiamin in the cells, but you will not get robust
growth.
Also, min
Dear all,
I'm a bit confused from the output of the CORRECT step in XDS. In one of the
first tables I can read the mean I/sigma for each resolution shell, but these
values are much different from the I/sigma reported in the table at the end of
the output files, titled "completeness and quality
Michele Lunelli wrote:
Dear all,
I'm a bit confused from the output of the CORRECT step in XDS. In one of
the first tables I can read the mean I/sigma for each resolution shell,
but these values are much different from the I/sigma reported in the
table at the end of the output files, titled "c
[Posted on behalf of Lydia Tabernero. Full text of this posting
available here: http://www.ccp4.ac.uk/martyn/MSc-Leaflet.doc ]
1 year MSc in Structural Biology and Biophysics at Manchester, UK,
starting in September 2007
Aims & Objectives: Structural and biophysical analyses are essential in
unde
Manish,
Assuming that you need the minimal medium for Se, have you tried the
regular heavy atom derivatives? You already have diffracting crystals...
M9 medium can be slightly difficult. However, no one says that you *have*
to use minimal medium such as M9 (again, assuming that you're doing
Se-Me
Manish,
In practice, we have found that it is very helpful to grow up an
overnight starter culture in minimal media to acclimatize the cells for
growth under minimal conditions. (Cells transferred from LB to M9 do not
perform well for overexpression). We inoculate 1 L of modified M9 (see
below) w
Hello,
I've got a protein that forms oil phase in almost all conditions in the
screen. No xtals thus far. I was wondering if there are general hints on
dealing with such cases. Basically, I am trying to understand what
direction to go from here. E.g. decrease or increase [protein], [salt] and
Eva,
If you do have NCS then you should be aware of a bug in the CNS1.1
release. PMB has a patch to fix this, and I hope that CNS1.2 will have
fixed it also. The problem is with the NCS restraints on B-factors in
CNS. The NCS groups, usually molecules, are restrained to have
identical B-factors
Thank Eleanor and others for making suggestions. We have tested SIGMAA in
CCP4i(1.4.4.1) again carefully. In the first run, we used "combine two sets of
MIR phases" mode to combine 2 sets of MAD phases, and output column labels
changed to PHCMBtest and WCMBtest. The second run was in "combine is
Dear all,
Could any one recommend some heavy atoms used for crystals grown in 0.1M
tri-sodium citrate pH 5.6, 1M Lithium sulfate and 0.5M ammonium sulfate? I read
from Hampton user guide of heavy atom kit that "high salt concentrations are
not the ideal medium for heavy atom reactions with mac
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