Dear all,
I am expressing a 6xHis tagged secreted protein in a fermentor in P. pastoris,
using the standard minimal medium described in the invitrogen manual (plus
PTM1). Following collection of the culture medium, I am having problems with
purification of the protein as only a small fraction (
Petros,
It has indeed been speculated that high concentrations of Magnesium and/or
other metals present in the cell lysate effect the binding of the
Histidine-tag, and thus specific conditions for binding and elution need to be
optimized for specific elution of your target protein. I believe t
Etros, It has indeed been speculated that high concentrations of Magnesium
and/or other metals present in the cell lysate effect the binding of the
Histidine-tag, and thus specific conditions for binding and elution need to be
optimized for specific elution of your target protein. I believe tha
I agree with Eleanor 100%...
In my biased opinion, only the atoms supported by electron density
should be included in deposited models. To satisfy the "but this will
mess up the electrostatic potential coloring" argument (a valid one, of
course), the "projected model" can be deposited alongside w
Dear all,
we are pleased to announce that applications are open for the EMBO Practical
Course
"Protein expression, purification and characterization" (PEPC8), which will be
held at
EMBL Hamburg from the 3rd of September until the 11th of September 2012.
This is a hands-on course with practi
But what about the issue of resolution? As was previously pointed out, at say
3.2 Å resolution, many side chains will fail to fit, but it doesn't seem
appropriate to trim them all down. The users need to also be aware of the
quality/resolution of the structures that they are looking at.
Greg
This is a personal preference. I do model at low sigma levels if there IS
some indication of where to put atoms, always try to keep the correct
sequence even if some atoms are missing, and just for coot convenience keep
atoms with occ = 0, rather than delete them altogether. (COOT will refine a
I fully agree. Unfortunately, the perfect model does not exist (at least not
for protein crystal structures). It is like with Heisenbergs uncertainty
principle. Either one has a complete model with a number of atoms having a
coordinate uncertainty of 4-6 Å, or one has a model where the uncertain
On Mon, 2012-03-26 at 10:17 -0400, Gregory Bowman wrote:
> But what about the issue of resolution? As was previously pointed out,
> at say 3.2 Å resolution, many side chains will fail to fit, but it
> doesn't seem appropriate to trim them all down.
Why is it inappropriate to trim them down? Some
On Mon, 2012-03-26 at 16:30 +0200, herman.schreu...@sanofi.com wrote:
> It is like with Heisenbergs uncertainty principle. Either one has a
> complete model with a number of atoms having a coordinate uncertainty
> of 4-6 Å, or one has a model where the uncertainty of all atoms is
> below say 0.5 Å,
Dear Ed,
In the end it boils down to personal preferences. With the number of crystal
structures I refine each year, I am not going to go over every flexible surface
residue to decide whether to truncate the side chain or whether there may be
some low level density justifying to keep the side c
I agree with Herman about personal preference but it also boils to our job
as crystallographers to educate non-structural end-users. The fact of the
matter is that a lot of researchers use structures without looking at the
nuances of the PDB. It's actually pretty common among biologists to
download
Hi all,
I was wondering if anyone had problems with drawing molecule using JME
editor in PRODRG? If yes then how do I fix it? I could not draw molecule in
the JME editor in the PRODRG webpage recently and JAVA is already installed
in my computer.
thanks,
Shya
Hi All,
Has anyone had any luck purifying membrane proteins with DDM
(n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC
is very low: 0.009%). I would like to keep it as low as possible, so I don't
have too much DDM around when I get to the crystallization step. I wond
Hi,
I used 0.017% or 0.012%. My protein is very stable at this concentration.
Good luck.
Lin
2012-03-26
yybbll
Hi All,
Has anyone had any luck purifying membrane proteins with DDM
(n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ? (Its CMC
is very low: 0.009%).
I generally use 1 - 2% DDM for extraction only, but lower the concentration
to 0.01% for following steps (i.e. NiNTA and gel filtration). The excess
DDM is washed away by using a lower concentration in your wash and elution
buffers.
Kelly
***
Ke
Hi Katarzyna,
Yes, membrane proteins can be purified in DDM at ~1-2xCMC. Since its CMC value
is very low, at the extraction step you need to use a much higher concentration
up to ~100x CMC. During different rounds of purification, you can bring the CMC
level down to 1-2x CMC and even try deterg
Kasia,
A lot of people uses DDM to purify membrane proteins, not a lot of people
crystallises them.
If you want to crystallise a protein purified in DDM, then you should use LCP.
If you go for vapour diffusion, you should exchange the DDM for a detergent
with a smaller micelle size otherwise you
If I recall correctly cytochrome oxidase, which I believe was
the first protein purified with DDM, requires about 10x cmc
in column buffers to keep it soluble. Check for papers from 1970's or 80's
by S. Ferguson-Miller.
Cytochrme bc1 complex, on the other hand, is perfectly clear in 1 cmc
and can
Dear All,
I have searched the archives and would like more information about
reproducing robot tray hits using 24-well hand trays. I reproducibly get
crystals when I use small volumes (0.5 ul) in 3-well intelliplates but only
precipitate in 1-2 ul sitting drops in 24-well hand plates.
What parame
Shya
There have been a number of issues of the releases of the JVM, particularly
with browser IE9 - most resulting in security exceptions when accessing
data files.
You should be able to update your Java on your computer as these problems
appear to have been finally resolved through the normal
90% of the protein could be aggregated and hiding the His tag from the resin.
Metal ions might be removed from the starting sample by adding up to 10 mM EDTA
in the first buffer exchange cycle, but just after reading "10% of the protein
binds" I wouldn't bet much on this. Rather increase the cu
I have seen people only use robot to optimize their crystal and get
good diffraction (~2 A). If you keep having trouble, you can try this
method instead, even though generally the case is the bigger the drop
the bigger crystal.
I remember the archive suggest us to use higher concentration of
prote
On Mon, 2012-03-26 at 11:57 -0600, Matthew Lalonde wrote:
> What parameters should I vary to reproduce crystals in hand plates?
First of all, protein concentration. It also does not hurt diluting
your reservoir since you are getting precipitates. If your goal is to
get bigger crystals (which is
Hi Petros,
It's possible but unlikely that your media contains enough metal ions
to matter. More likely options include protein issues like
overgkycosylation, proteolysis (removal of the tag) or severe
aggregation. Since you are not experiencing this in BMGY, I wonder why
not to supplement your sy
I suspect that sometimes the protein chaperones the tag, which is solvent
exposed some fraction of the time. Try very slow loading or batch binding.
Kendall Nettles
On Mar 26, 2012, at 8:15 AM, "Petros Giastas"
mailto:peg...@pasteur.gr>> wrote:
Dear all,
I am expressing a 6xHis tagged secr
Actually, DDM is the most successfully used detergent for membrane
protein crystallization. See Newstead et al, Protein Sci. 17:466. But
yes, the rule of thumb is that detergents that form smaller micelles
give better diffracting crystals, but are more destabilizing.
Ho
Ho Leung Ng
University of
Dear list,
If I take all the fasta files for proteins in the PDB,
are the sequences complete?
I mean, do they have holes sometimes (missing amino acids)?
Sorry for the maybe stupid question but I know that sometimes
the PDB files have missing residues, I am hoping that
it is not the case with t
I think that depends on what the depositor considered complete.
Just as an example the construct you cloned say from residue 20 - 380 would you
consider that complete or would you consider only the sequence complete if it
contained the first 20residues ?
Regarding the gaps in terms of missing res
On Monday, 26 March 2012, Francois Berenger wrote:
> Dear list,
>
> If I take all the fasta files for proteins in the PDB,
> are the sequences complete?
>
> I mean, do they have holes sometimes (missing amino acids)?
In theory the SEQRES records describe the sequence of the
entity that was cryst
On 03/27/2012 12:20 PM, Ethan Merritt wrote:
On Monday, 26 March 2012, Francois Berenger wrote:
Dear list,
If I take all the fasta files for proteins in the PDB,
are the sequences complete?
I mean, do they have holes sometimes (missing amino acids)?
In theory the SEQRES records describe the
The total model that fits the observable electron density should be the
standard for the PDB FASTA file with the deposited structure factors, however,
not all depositions contain a link to the expression construct details because
many are not published, and I believe that it should be explicitly
Hi Matt,
This doesn't really answer your question directly, but sidesteps
around the issue -
I wrote a little something on this exact subject not so long ago -
http://xtaldave.wordpress.com/2012/02/23/on-protein-crystallisation/
(You can ignore the first 5 paragraphs of intro - it was written with
Most of the labs sharing our Phoenix have had enough trouble with exactly
this that our standard procedure is to now to use 1 µl drops (.5/.5
protein/well) in our initial screens those scale up much more reliably to
the 24-well format and seem less finicky in general, reducing the chances of
an u
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