Dear CCP4BB,
The Thompson Lab (https://thompsonlab.science) at UC Merced is recruiting a
Postdoctoral Scholar and a Junior Research Specialist:
The Postdoc will work on projects related to multi-temperature and
time-resolved (T-jump) crystallography at synchrotrons and XFELs. Applicants
with e
Hello All,
I'm interested in doing some comparisons of random half data sets, inspired by
statistics like CC1/2, CC*, etc. Does CCP4 contain some tool to split unmerged
data into 2 random sets? Or is there some way to intercept this information
from something like XSCALE or Aimless, etc.? Thoug
Maybe there is a reason you haven't tried this, but can you just mutate the
cysteines that ligate the cluster? Serine would be a nice choice.
Mike
- Original Message -
From: "Yuri Pompeu"
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, October 1, 2013 11:33:50 AM GMT -08:00 US/Canada Paci
Hi Peter,
I won't claim to be an expert, however, there is a small textbook by Robert K.
Scopes called "Protein Purification: Principles and Practice," that has very
good practical and theoretical information about column chromatography (among
other methods). I have found this book useful a num
Does your protein have multiple exposed Cys residues? I have observed this
before with a protein I worked with that had many exposed Cys residues. In my
case I could add more DTT and minutes later the gel would be completely
dissipated. My hand-wavy explanation was that the protein stays folded
As a follow up to Ian's suggestion, the LABELIT software (sorry for a non-CCP4
suggestion) will create artificial precession images from your raw oscillation
images.
Documentation can be found here:
http://adder.lbl.gov/labelit/
And an article describing the functionality can be found here:
Adding 1-10mM copper sulfate is often a good way to oxidize disulfide bonds,
although some proteins cannot tolerate this treatment.
Mike
- Original Message -
From: "Jacob Keller"
To: CCP4BB@JISCMAIL.AC.UK
Sent: Thursday, February 28, 2013 3:09:18 AM GMT -08:00 US/Canada Pacific
Subject
Hi Dave,
ProteinaseK is also a good one. Crystallizes rapidly, big crystals, and
relatively high resolution data (1.0-1.5A) usually. You can also buy the
lyophilized powder from sigma and prepare the sample directly from the
commercial material. We use proK for a course here at UCLA, so if you
While neither of these references detail the "development" of protein
crystallography, they are excellent stories of its birth:
1.) A book written by Richard Dickerson, "Present at the flood"
2.) A recent review in JMB by Strandberg, Dickerson, and Rossmann: "50 years of
Protein Structure Analy
: "Bernhard Rupp (Hofkristallrat a.D.)"
To: "Michael Thompson" , CCP4BB@JISCMAIL.AC.UK
Sent: Monday, March 12, 2012 12:48:42 PM GMT -08:00 US/Canada Pacific
Subject: RE: [ccp4bb] Matthews coeff. from model
> I can't imagine the results would be very different for protein-
James,
For a detailed analysis of Matthews coefficients (Matthews probabilities), you
can use this web-based tool:
http://www.ruppweb.org/Mattprob/
There is a pull-down menu to select the sample type, and one choice is
"protein-DNA complex." Perhaps someone can correct me if I'm wrong, but I c
In addition to the book recommended by Jeff, another good one is "Protein
Structure and Function" by Petsko and Ringe. A bit less cumbersome than Branden
and Tooze, but not as complete (it's a relatively thin paperback, rather than a
full-blown textbook). If you just want a nice "protein domain
Katherine,
You are not alone. I have inadvertently destroyed a GE HisTrap column with high
concentrations of proteins that contain many exposed cysteines. In my case the
Co2+ resin turned a very dark purplish-brown and the protein appeared to have
crashed out on the column. I didn't try to stri
Hi Jacob,
There are a number of programs that can calculate the radius of a pore. The one
that comes to mind is called HOLE, and it can make a nice plot of the
y-coordinate along the pore vs. pore radius. I don't recall exactly how this
calculation is done, I think it is somehow related to the
- Forwarded Message -
From: "Michael Thompson"
To: "e dodson"
Sent: Monday, November 21, 2011 11:30:17 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase
A question regarding the plea for less MR (which I support):
The
To add to what Pat has mentioned, I work with a protein that has a number of
exposed Cys residues, and it turns into a gel at 4 degrees within a week, even
if I store it in buffer with reducing agents. This happens every time I store
it at 4 degrees. To test if your "jelly" is due to S-S crossli
Hampton makes a "Detergent Screen" that works the same way as the Additive
Screen.
http://hamptonresearch.com/product_detail.aspx?cid=1&sid=39&pid=31
It has 96 unique detergent reagents for crystal screening.
It is a bit expensive, but if you don't want to purchase the whole kit you
could ju
Another option is DTNB (aka Ellman's Reagent). This compound reacts with free
thiols and produces a yellow color. The reaction is stoichiometric, so if you
have access to a very basic spectrophotometer and know your protein
concentration you can quantify the free thiols quite easily.
Basic pH d
Hello,
Regarding your empty Tb site: How similar are the two copies of the protein in
the ASU? You can overlay the two individual copies from the ASU to check and
see if the binding site without density has been distorted somehow so that it
can no longer accomodate the Tb, maybe due to crystal
Hello ccp4 & phenix BB members,
I would like to view the intensity-weighted reciprocal lattice for several data
sets that I have collected. (The data have been indexed, integrated and scaled
with Denzo and Scalepack.) I was wondering if anyone could offer some advice on
what might be the best a
I have attempted to use the RosettaDock webserver for protein-protein docking.
If I recall correctly, the webserver is only capable of doing fixed backbone
docking simulations. If you are docking a small peptide with a protein I
suspect you want the peptide to be somewhat flexible. Although my r
Hi Paul,
While crystal contacts are typically of a non-covalent nature, there are some
exceptions. A disulfide bond can act as a crystal contact, which is a covalent
interaction. A technique called synthetic symmetrization involves the
engineering of single cysteine mutants, followed by oxidati
It's not a protein, but here's another example: the PreQ1 Riboswitch. In this
case the two methods revealed different structures, but the NMR group was able
to determine that the differences were due to calcium in the crystallization
condition.
Crystal Structure:
Nat Struct Mol Biol. 2009 Mar;
Israel,
Here is a paper that describes a phenomenon like what you have observed:
Structure. 2003 Feb;11(2):139-45.
Dehydration converts DsbG crystal diffraction from low to high resolution.
Heras B, Edeling MA, Byriel KA, Jones A, Raina S, Martin JL.
And another review that touches briefly on t
Ramanuj,
This is definitely possible. Cases of "intertwined homodimers" are rare, but
there are several known structures that demonstrate this phenomenon, and they
are very interesting (especially with respect to studying knotted proteins -
see reference below). Examples are pdb IDs: 2ouf, 1myk
Ramanuj,
This is definitely possible. Cases of "intertwined homodimers" are rare, but
there are several known structures that demonstrate this phenomenon, and they
are very interesting (especially with respect to studying knotted proteins -
see reference below). Examples are pdb IDs: 2ouf, 1myk
Hi All,
I was wondering if anyone knew whether or not it is possible for reducing
agents with thiol groups, such as DTT or beta-mercaptoethanol (BME), to form
covalent S-S bonds with Cys residues, particularly solvent-exposed Cys? I have
some puzzling biochemical results, and in the absence of
Bei,
I had a former labmate who had the same situation and would load somewhere
between 6-8L of media directly onto a column. I don't remember what type of
column it was, ion exchange may not be ideal if the ionic strength of your
medium is high. I think it may have been a phenyl sepharose colu
otein which dont have ne aromatic
residue
At 9:47 AM -0700 4/9/11, Michael Thompson wrote:
>Bradford dye binds to hydrophobic residues, mainly aromatics,
The statement above is not accurate.
Compton and Jones. Anal. Biochem. 151(2): 369-374, 1985
- John
--
--
Michael C. Thompson
Gra
It is not surprising that your bradford and BCA assays don't agree if you have
no aromatic amino acids in your protein. Bradford dye binds to hydrophobic
residues, mainly aromatics, so I would guess your bradford is consistantly
giving lower measurements than the BCA assay. I also wouldn't be su
Alex,
You should check out this webserver: http://services.mbi.ucla.edu/anisoscale/
If it cannot solve your problem, it will at least point you in the direction of
some answers.
Good luck,
Mike
- Original Message -
From: "Alex Barth"
To: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, April 5,
Thanks for the explanation Ed, that makes sense.
- Original Message -
From: "Edward A. Berry"
To: mi...@chem.ucla.edu
Cc: CCP4BB@JISCMAIL.AC.UK
Sent: Tuesday, April 5, 2011 12:22:21 PM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?
W
dengzq1987,
I am also working on a data set that has this exact alignment/mosaicity problem
noted by others. If you make a movie of your dataset that displays your images
in series, or if you just click through them quickly in adxv or your program of
choice, you will see that very few of the sp
Hello All,
I am looking for some advice from some experienced RNA crystallographers. I
would like to order some relatively short (<90 bases) synthetic RNAs for
crystallization trials. I was wondering if anyone could comment on the use of
synthetic RNAs for crystallization. Specifically, what is
Hi Rashmi,
The concentration of your stock solution will depend on how you plan to mix
your screen conditions. It's not really that important as long as: 1.)
Glutathione stays soluble, and 2.) You can make your condition with the desired
glutathione concentration. If your glutathione is reduced
Jacob,
Roger is correct, this concept does refer to the Pearson HSAB theory. To
summarize: This theory is applicable outside of inorganic chemistry as well,
but it is extremely useful for explaining coordination chemistry of
metal-ligand complexes. The theory states that "hard" acids interact w
Hi Phil,
Depending on the characteristics of the c-terminal region of interest, you
might try a carboxypeptidase. These enzymes cleave residues from the c-terminus
and stop at various motifs, depending on the specific enzyme. There are several
available commercially, each having a slightly diff
Hello Herman and others,
I am somewhat concerned after reading the following comment:
"In pymol there is the infamous carve option, which can be used to select
density for specific fragments and which can make bad density look good if you
carve very tightly around your fragment."
I have been u
Hi Neeraj,
A few points to add to Artem's excellent advice:
If you would like to purify an untagged construct of your kinase, you could try
an ATP affinity column. The caveats to this are 1.) the ATP resins can be
expensive, and therefore it may be impractical to make a column with a high
bind
I don't know how many of these crystals you have, but if you can spare one try
freezing it straight out of the drop without cryoprotection. Certain salts can
act as cryoprotectants at high enough concentrations. I don't know about
phosphate salts, but I've had crystals that grew in 2.5M ammonium
Dan,
You could try a denaturing purification to get rid of the binding partner.
First, take your cell lysate and spin it down at high speed to remove insoluble
contaminants. (You probably do this already.) Then take your clarified lysate
and dialyze it into buffer containing 6M Guanidine HCl. T
Hi Sally,
Crystallization of proteins with multiple phosphorylation sites is often a
difficult problem. Like you said it is often an issue of inhomogeneity. Several
suggestions that may be helpful:
First, I would use a bit of your protein sample for Mass Spec analysis. It is
possible (though m
Hi Buz,
Sorry to respond a little late, but if you are still tinkering with surface
accessibility calculations, you can use the following web server that will
calculate the "diffusion accessibility" of each atom in your input .pdb file:
http://nihserver.mbi.ucla.edu/diff_acc/
"Diffusion access
Hi Jacob,
A couple more things to think about:
1.) How to get a Nobel Prize: Try an experiment if you have the means.
Transform/transfect your favorite cell type with exogenous protein sequences.
Sequence and see if they ever appear in the genome. Go after it...
2.) I realize that the idea of
Hi Bei,
I have fought this problem myself several times. In addition to some of the
suggestions from Tim, here are some suggestions for additives:
As you mentioned Tween20 and Triton X-100 are very good for solubilizing
difficult insoluble proteins. Those two are usually my first two weapons
a
Jacob,
The idea is enticing, but don't forget that there are multiple degenerate
codons for a given amino acid. Once the protein is synthesized, the specific
codon information is lost.
I think that's a fundamental problem.
Keep the ideas coming,
Mike Thompson
- Original Message -
Hi All,
I have a question for those of you familiar with the TLSMD webserver. I am
working on a structure with slightly imperfect 3-fold rotational NCS. My most
recent .pdb file has been generated using Refmac (followed by a little
tinkering in Coot), and during refinement I have been imposing
I think that Disulfide by Design, or DbD, is geared toward monomeric proteins
(ie. for enhancing stability). I'm sure you could get it to work with an
oligomer with a little tinkering, but there is another server called sGAL that
may be more like what you're looking for:
http://bioinformatics.o
Hello All,
I am currently solving a structure at 2A resolution with phases obtained from
molecular replacement. Using the MR solution, I began refinement with Refmac
using NCS restraints. I am currently building the parts of the model that were
left out of the MR search model, and have just abo
Hello Rex,
Just a few biologically-related ideas that may support modeling the planar
conformation.
I'm not sure if the protein you're working with has enzymatic activity, but is
it possible that the ring strain is indeed real and may be a part of the
reaction mechanism? Are any parts of the p
I agree completely with Anastassis that the equilibrium will be effected by
changing the concentration of the sample during gel filtration, however I
wanted to point out that the elution volumes of the two species are independent
of their populations. I apologize if I was misleading.
Mike
---
Hello Intekhab,
Your results do not seem surprising at all. It is not uncommon for molecular
interactions such as dimerization to be more stable at lower temperatures, and
this is exactly why you are seeing the shift to higher elution volumes at lower
tempratures. At lower temperatures, both th
Hello Matthew,
I almost always charge my IMAC resin with Co instead of Ni. I find that it
gives much better purity because the selectivity is better. This is because the
coordination sphere around the Co atoms require more His residues than the Ni
coordination sphere. I believe Co requires 4 Hi
53 matches
Mail list logo
