In my experience translational NCS also can a part when one has many molecules
in the a.u. If MR is an option, modern packages are rather good in dealing with
TNCS.
We used Molrep + Refmac at 3.x A (still unpublished) for a case with 18
complexes (36 monomers) in the a.u. and things weren't as b
Dear All,
My protein is cloned in pET-15b vector, contains His- tag, thrombin
cleavage site and extra sequence of 20 amino acids from vector. I
crystallized without cleaving extra sequence and never got any crystal
hits/crystals. Once, I tried thrombin reaction and it didn't work. Here I
would lik
The problem of many monomers in the "ASU" is not restricted to
macromolecules. An interesting recent small molecule example is the
structure of L-tryptophan (http://dx.doi.org/10.1107/S0108768112033484)
which, amazingly, was not published until 2012. This is perhaps in part
due to difficulty i
Dear David,
Following the discussion I am starting to wonder if I have been doing
something wrong all these years.
I always forgot to purify the complexes, I just mixed the two
macromolecules and did the crystallization experiments.
And I will admit that I did not even worry about getting the
May be your complex components interact with column? I mean: you have
calibration plot for column, right? Does the molecular mass of proteins
calculated from elution volume equals to mass calculated by another method
(like SDS-PAGE)?
2014/1/21 Keller, Jacob
> Maybe there is something required
Hi all,
Thanks for the incredibly fast and helpful responses, both on and off list.
To provide some further information. I have tried the SEC with several
buffers, including buffers identical to the SPR and DPI experiments.
To-date all SEC and SPR experiments have been conducted at room temperat
Maybe there is something required for interaction that was in the buffer used
for the other binding studies, but not in your SEC buffer?
JPK
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David
Briggs
Sent: Tuesday, January 21, 2014 10:52 AM
To: CCP4BB@JISCMAIL.AC.UK
Subj
Hey David,
the 1 nM Kd by SPR sounds fishy to me - did you do these measurements yourself ?
Unless this is an antibody-protein complex or nanobody-protein complex or
protein-small molecule complex, the value is too low (for a normal PPI, I would
expect >50 -500 nM). Possible explanation for the
Dear Dave,
Indeed, an interaction in the nM range is strong, but its dynamic is
important. If the SPR sensorgram, if published, looks like a 'crenel',
this indicates high association/dissociation rates. In that case, your
complex can dissociate during the GF run.
You can try crystallisation at
Dear all,
sorry for the slightly off topic post,
I have 2 proteins that have been shown to interact, by multiple groups, and
by multiple techniques - namely ELISA, SPR and DPI.
The Kd of the interaction as determined by SPR is on the order of 1 nM.
I would very much like to crystallise this pro
Structural and chemical biology approaches to characterize protein-protein
interactions that regulate kinases
We are looking for a talented and highly motivated student to join us for a
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Hi
Diamond Light Source provides the UK and International community with world
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