Dear All:
This is off topic but I would rather try here first.
I am wondering whether Dnase could be a contaminant during the nickel
affinity purification of a his.tag protein expressed using pET29b. The
cells were disrupted using sonication only. Very high yield (30 mg/liter
of cell culture).
Dear All:
I vaguely remember a program that can predict sites for introducing a
pair of Cys residues to make a disulfide. I had once used this before
but now I had forgotten about it. Does any body know about this?
My plan is introduce Cys residues into my structure that I can use for
FRET anal
Dear All:
Thanks for the overwhelming suggestions. I had in fact used SSBOND.
However, there were other sugestions which are listed below.
ssbond: http://eagle.mmid.med.ualberta.ca/forms/ssbond.html
disulfide by design: http://www.ehscenter.org/dbd/
http://www.predictprotein.org/ - have not che
Dear All:
This is not crystallography related and does not belong to this group,
but I would like to pose this to all who are working with proteins
expressed in an E. coli BL21 (DE3) or Rosetta. Is it possible for a
protein to be phosphorylated during expression? At least my
understanding was t
Dear All:
Thanks for all your replies to this.
Phosphorylation is possible in E. coli. One article of particular
interest is
Mol Cell Proteomics 2007 Eprint
by Macek B, Gnad, F, Soufu B, Kumar C, Olsen JV, Mijakovic, I and Mann
M.
Many others have pointed out that it is possible and many likely
Dear All:
We have been trying to crystallize a protein which is large - > 100 kDa.
This is soluble but the best we can get is about 1 mg/mL.
It did crystallize but did not diffract well. Efforts to increase the
concentration has been unsuccessful. I am wondering whether there are
methods that o
Dear All:
I would like to know the literature on the crystal structure of
Leukocyte Function-Associated Antigen One (LFA-1, CD11a/CD18).
I have the structure of I domain but not for the entire molecule. I
would greatly appreciate if people can point me to the right
article/reference.
Thanks
S
On 1/19/12 5:32 AM, Tim Gruene wrote:
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Hash: SHA1
Hi Megha,
your email could hardly be more cryptic to me, and maybe you increase
the chance of getting help by explaining
- - what is R& D
- - what is MiOU?
- - what is mcg? (milli-centi-gram?)
(I understand ml,
Hi all:
I am trying to purify a protein that was expressed in a baculovirus
system. The protein elutes in the flow through in a DE52 column. Further
gel filtration gives an enriched protein but we see that some medium
components tag along (weight and activity data do not match). Another
mutant
Dear All:
What does it mean when I get negative values under Anomalous Corr column
after running XDS? I set the Friedel Law=False even though I suspect
that my signal is very very weak.
Thanks
Subbu
Dear All:
Is there a simple graphical way to generate atoms that lie on a surface
(and wite out a pdb file) for modeling surface docking experiments?
Thanks a lot
Subbu
I am looking for Cerius2 type but any code will be fine
Dear all:
I am noticing that in some of my structures, at 1.5 A resolution, 1)
there is some negative density around the C of the carboxyl groups.
2) I also notice the negative density around S in a disulfide region. It
is as though the disulfide is broken.
Could some body enlighten me on this
Thanks for the quick reply and pointing out t bunch of references.
Radiation damage has been pointed out as a cause for such feature.
The following references were pointed out.
Ravelli, R.B.G. & McSweeney, S.M. (2000). The 'fingerprint' that X-rays can leave on structures. Structure 8:315-328.
Dear all:
I have a single residue mutant whose enzyme activity is about 50% of the
wild type. Interestingly, the mutation
is in a region that involves a secondary site but not the active site.
The two structures with or without ligands
fit well (0.18 A) and the metal binding and cofactor binding
Dear All:
I am working with two enzymes (40 kDa each). The crystal structures of
the two enzymes are known.
I have made a hybrid enzyme of the two individual ones.
This chimeric/hybrid enzyme is soluble and exhibits activity of the two
individual enzymes.
My questions are as follows:
1. Could
Dear All:
I have a peculiar problem with baculovirus expression of my protein. The
native protein elutes in Tris gradient. This has no problems.
However, a mutant elutes in the wash. After further purification with
size exclusion, I notice that this mutant is always associated with
some medium c
Dear All:
I have obtained good crystals from 20% isopropanol but this is my first
time dealing with this agent.
Help in handling the crystals, dealing with evaporation etc. is highly
appreciated.
Thank you
Subbu
Dear All:
Does any one out there know of an enzyme that succinylates O3 and/or O4
in GlcNAc?
Thank you all
Subbu
ar...@xtals.org wrote:
Hi,
In a case like the one Raji outlined below - after all the attempts - I
would have most cerainly switched to insect cells as the next step :)
If you suspect that protein of interest has large disordered regions,
expression in a higher order system by itself may not be
ar...@xtals.org wrote:
Hello,
1. As long as all proteins have seventy amino-acids or less and express in
E. coli in mM concentrations - we're in business.
2. As for the question below - my favorite answer is 'It will take a week
and ten million dollars in unmarked bills. We begin as soon as the
Hi. We are working on a periplasmic protein that breaks naked glycans in
peptidoglycans. There is truncated structure available but our target is the
full length protein. The difficulty us that it strongly binds to the resin with
or without his.tag. Changing the resin to acrylamide did not help.
A postdoc position is immediately available in my laboratory
at the Oral Biology Department, UMDNJ. The post-doc will work on the elucidation of
structure of enzymes involved in modifying A. actinomycetemconitans biofilms.
Crystals of two such enzymes are available.
Experience in expression and
mb1pja wrote:
Dear Fred
A really nice video that would be great for giving non-crystallographers
(including colleagues and 1st year students, and perhaps also friends and
family) an overview of what we do. Thank you for pointing it out - and of
course very many thanks to Dominique Sauter for
Just thought this will be of interest to all.
Subbu
--- Begin Message ---
Narayanan Ramasubbu wrote:
Vellieux Frederic wrote:
Narayanan Ramasubbu wrote:
Hi:
Could someone point out the name and where to get these
crystallization plates used in the video?
By the way, this is a wonderful
Hi:
I have an old fortran code that I used on an SGI Irix system. i would
like to use it on a linux (Ubuntu).
How to compile this code which uses some ccp4 libraries? What is the
command for this?
Thanks
Subbu
Dear All:
How to increase the memory size (?) to avoid the following error?
***
* Information from CCP4Interface script
***
The program run with command:
Hi:
I tried to run arp/warp using the gui. My original mtz file (space
group p222) is read correctly and I see the AU limits in the Crystal
parameters.
I then tried to change the space group using CAD/SORTMTZ export to .sca
and manually change the space group to P2212 and then used
scalepack
Deal All:
I have a 2.0 A data for a SeMet protein (native crystal not available
yet!) that has 6 Se sites.
The cell comes out to be 65 67 101 and the angles are all very close to
90. The data set was collected in house with Cu 1.5418 A
We integrated and scale in orthorhombic and the statistics
Hi:
Sorry for the same posting in here as well but I thought may be some of
you might have encountered the same problem but may not be subscribing
to sharp list.
I am trying run sharp/autoSHARP on a mac (os x 10.4) using the guven
example file: krel1-SAD.0
I am getting an error at the CAD as
Clemens Vonrhein wrote:
Hi Jim,
On Fri, Jan 22, 2010 at 09:11:00AM -0600, Jim Pflugrath wrote:
I do not know the effect myself, but the idea of "vibration fee" has been
tossed around a while.
What? I'll have to pay for vibration now as well? Are you collecting
that personally? Or is i
Gerard DVD Kleywegt wrote:
Thanks Stephen!
I was going to suggest that, but I was afraid of the self-appointed
CCP4BB Gestapo that has been seen goose-stepping in this neighbourhood
recently (Tassos recently accused me of becoming mellow and diplomatic
in my dotage, so I hope I've set the rec
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