I hope this helped our Australian friends get more funding!
Ho
The TV show 'Thank God You're Here' goes to the synchrotron!
http://www.youtube.com/watch?v=N_zbySqumaA&feature=related
James Stroud wrote:
Hello,
I have a 2.45 A structure with an average b factor of 50.6. A region I
am particularly interested in has an average b factor of 87. At what
point do I say that the region is "disordered"? Does it come down to
maps? If I have reasonable simulated omit maps but the b
Hi Bryan,
Can't you choose an asymmetric unit that includes two protein
molecules and the entire non-palindromic DNA?
ho
Is your protein sticking to the membrane or crashing/aggregating from
the concentrating?
Try to find a buffer condition in which your protein is more soluble
and less aggregation prone. You can try varying salt concentration,
pH, and inclusion of additives like glycerol or detergent.
Ho
UC Berke
I incubated my pelets in 8M urea for a couple of hrs at 37C, spun and
collected the supernatant, and then refolded the protein over a Ni column,
as I had a his tag protein. To do this, you load and wash the protein
normally, but before eluting, run b-cyclodextrin (i think 0.1%, you'll
have to t
Refold protein 8M Urea:
Sanjiv,
The recommendations for rapid dilution and trying 6M GdmCl are great
recommendations. As an alternative, I had a similar problem years ago with a
membrane-associated receptor domain that was mostly found in inclusion
bodies after expression (I assume this is your
I think Pointless 1.2.23 was a broken version which escaped to the
wild in CCP4 release 6.1.0. The more recent release 6.1.1 has a fixed
version, and some more recent versions are also available from our ftp
site
eg ftp://ftp.mrc-lmb.cam.ac.uk/pub/pre/pointless-1.3.2...
sorry about this
P
A metaserver such as bioinfobank is helpful if your sequence is not
super-secret, especially if one is not an experienced modeller.
http://meta.bioinfo.pl/submit_wizard.pl
It submits your sequence to a range of sequence-based and threading-
based servers. Models (c-alpha, built with modell
In fact, I agree with Artem that detergent can be very difficult to get rid
of. It totally depend on the property of detergent. In your case that the
protein has no transmembrane region. You probably dont need much detergent
in your solution to keep your protein happy. I dont think having detergent
James,
That is a highly subjective question. However, given that your average
B-factor is 51, a B-factor of ~87 for the loop is not exceptionally
high. It is less than twice the average. If your average B was 10 and
your loop had a B-factor of 17 what would you think? I would imagine
that if y
An output file is created on hklout with or without the -copy flag. The problem is why is it picking the unity matrix (solution #2) for the reindexing operator rather than the first matrix (-h -k l) that it identifies under reindexing (which is clearly the correct answer). -Original Message-
I'm hoping someone can help me with a pointless problem. I am trying to
reindex
data into an orientation that I used to solve the structure initially. While
I can get pointless to give me the reindexing needed to make the new data match
the old data for the project, when I ask it to write the
It is a bit hard to understand why without looking at the structure..
One reason things get disordered is if the occupancies get misset so
they add up to a number > 1.0. In that case REFMAC decided this a a
"clash situation" and tries to remedy it ..
Could that have happened?
I build alterna
I have been going over some structure for a group here. When the post-
doc did the originally modeling a number of residues looked to have
alternate conformations. The resolution of the structures are between
2.0 and 1.9 angstroms. My first question is when re-refining in with
an updated
Thanks very much for the replies. Here is a list of resources:
http://www.bathsheba.com/
http://www.rpc.msoe.edu/cbm/
http://www.crystalprotein.com/
http://www.bioetch.com/
http://www.luminorum.com/
http://bioetch.com/
And if you want to roll your own:
http://www.rap-man.com/
http://homemade3dprin
Hello,
I am having difficulty using Phenix to build and refine a DNA duplex. The
issue is that my asymmetric unit consists of one protein monomer bound to
DNA, but the protein is a dimer and the DNA is not palindromic, so each
monomer is bound to a different sequence of DNA. As such, the density fo
Dear James,
I had a similar case with an active site loop which could have an open
and closed conformation. This loop had extremely high B-factors but
good, albeit low electron density. I believe this was because the loop
had this (well-defined) conformation only in say 50% of the protein
molecule
Hello
In order to know *exactly* what the reason for poor transformation outcome
was one has to do all sorts of experiments. This is not likely to be your
goal, right? Leaky expression, overload of DNA, somehow compromised cells,
or even plain old user error - and numerous other reasons can be pro
I hate to be the Negative Nancy here - but removal of detergent can be a
significantly harder task than removal of urea. Here's why:
Urea is a small molecule which diffuses freely and does not normally stick
to proteins - so removal of urea is no harder than removal of salt (we
ignore the consider
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Hello,
I have a 2.45 A structure with an average b factor of 50.6. A region I
am particularly interested in has an average b factor of 87. At what
point do I say that the region is "disordered"? Does it come down to
maps? If I have reasonable simulated omit maps but the b factor is 87,
ho
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