Dear all,
I have two questions. I would like to find out the community consensus of (1)
best practices in refining against lower resolution data (~4 angstrom) to
achieve the best model, and also (2) what manuscript referees should ask for in
this regard. One might encounter a hypothetical
I agree with Engin’s suggestions.
Our group crystallized a complex of mesotypsin with BPTI, where we measured an
inhibition constant (approximating Kd) of 14 micromolar, by mixing the two pure
proteins together in 1:1 stoichiometry. Actually we do that for a lot of our
complexes with higher
We had a similar situation with a catalytically dead serine protease.
Initially I was excited to think we might be seeing residual catalytic activity
of the mutant enzyme on a highly specific substrate; however, the activity
turned out to result from contamination with a very small amount of
Bovine trypsin works well. You can buy it pretty cheap from Sigma and it
crystallizes without further purification, within a week. Crystals diffract to
1.1-1.3 A and are quite robust to handling and soaking. Conditions that I used
are described in this ref:
Dear bb,
This morning as I scanned an accepted manuscript from a
well-respected-but-not-particularly-glamorous journal that publishes
many macromolecular structures, I came across a brief mention of
homology and rmsd with a published structure listed by PDB accession
number, but no citation of
AM
To: Radisky, Evette S., Ph.D.
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Somewhat OT: question of professional courtesy
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Dear Evette,
the PDB lists the citation when you enter the PDB-ID in the search mask
of any of the web-interfaces, which
Yes, I think this is reasonable.
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of R.
M. Garavito
Sent: Wednesday, July 25, 2012 9:56 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Somewhat OT: question of professional courtesy
Evette,
I think the primary issue
As I recall, I used to wear latex gloves, add extra padding to the handle end
of the wand (and other tools) with bits of tubing, and take my tools out of the
cold room every 20 min or so to de-ice and dry them. I didn't really have a
choice about the cold room because it was the only place my
Several have mentioned harvesting in the cold room to reduce
evaporation. I used to do this also as a postdoc, but I worried whether
I risked nitrogen gas poisoning from liquid N2 boil-off, since the cold
room did not seem very well-ventilated. I've also hesitated to
recommend it to trainees in
Thanks much. It helps a lot.
On Jul 13, 2012, at 5:31 PM, Henry Bellamy hbell...@lsu.edu wrote:
liquid N2 expands about 600 fold to RT gas. The minimum O2 concentration is
19% (per OSHA I think) so if the amount of vaporized N2 is greater than 2%
of the cold room volume you could have
Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Radisky,
Evette S., Ph.D. [radisky.eve...@mayo.edu]
Sent: Saturday, July 14, 2012 7:19 AM
To: CCP4BB
This is very common among the small GTP-binding proteins of the Ras
superfamily. For an interesting analysis (although doubtless there are
lots of more recent examples) you might look at:
Corbett and Alber, TIBS 26 (12) 710-716 (2001)
Evette S. Radisky, Ph.D.
Assistant Professor
Mayo Clinic
Just create a new tag, say _atom_site.imaginary_site, which is either
true or false for every atom. Then everyone would be able to either
filter out the fake atoms or leave them in, without ambiguity or
confusion.
Aside from being a binary rather than continuous parameter, how exactly
does
bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Radisky, Evette S., Ph.D.
Sent: Monday, November 01, 2010 4:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] finding I/Sigma(I) from HKL Scalepack
Dear all,
I have previously used SCALA for data reduction, and in publications and
pdb
Dear all,
I have previously used SCALA for data reduction, and in publications and
pdb depositions, reported the Mn(I/sd) output from SCALA for the whole
data set and for the highest resolution shell.
We now have some data that has instead been reduced using the HKL suite,
and I am confused
Try a Bradford-type assay, scaled down to microplate format. You can
buy reagents pre-made; Pierce makes a good one . It is fast-- you pipet
10 microliters or so from each chromatography fraction into a well with
the detection reagent, and wait 5 minutes. If protein concentrations
are moderate
Thanks to all for your help and suggestions. We are still working out
the best protocol for our specific protein, but I list here the
suggestions received, since they may prove helpful to others as well.
1. I assume that your native (12 Cys) protein runs as monomer on
non-reducing SDS-PAGE and
Dear all,
Suppose I want to data mine the PDB to find a set of structures
containing a motif that is characterized by an unusual conformation of
backbone atoms over a short stretch of residues. This structural motif
does not correspond to any specific amino acid sequence; the residues
could be
What protein concentration are you using in your screen? It sounds like
you need to try lower protein concentration, or a screen with lower
precipitant concentration, or both. Hampton makes a Crystal Screen Lite
with the precipitant concentrations half of what they are in the normal
screen (or
Dear all,
We have a recombinant secreted glycoprotein produced in a mammalian
culture system; the native protein has 12 cysteines which form 6
intramolecular disulfide bonds. We have introduced a new cysteine
residue at a surface position, with the intention of targeting this
residue for an in
Another question re: Amicon stirred cells...
I also seem to recall seeing 1L size stirred cells in older labs of my
youth. My current lab has acquired one of 400 mL, but looking to
purchase a bigger one, I can't find any. Any ideas about where we might
find one?
Evette S. Radisky, Ph.D.
Another anecdote for you: I had crystals that grew in 15% PEG 2000 and
15% isopropanol. Cryos with glycerol melted the crystals. The best
cryo I found was with 15% PEG 2000, 15% isopropanol, and 20% PEG 400.
It's hard to predict how your crystals will behave with different
cryoprotectants--
Thanks to everyone for the great suggestions so far. To clarify and
answer a few questions, with the mutated construct we get no protein
either secreted or in the pellet. The protein in question is about 20
Kda; with glycosylation at both sites it is around 29 kDa (both in
mammalian cells and
PROTECTED]
Sent: Wednesday, March 05, 2008 10:43 AM
To: CCP4BB@JISCMAIL.AC.UK; Radisky, Evette S., Ph.D.
Subject: Re: Removal of glycosylation sites in Picha expression
construct
Evette,
This is quite intriguing. At the outset I want to say that just
because glycoslyation is not essential
Dear all,
Our lab is new to working with Pichia pastoris, also new to working with
glycosylated proteins. We have a construct for a secreted protein that
expresses pretty well in Picha, but upon mutation of the 2 N-linked
glycosylation sites to Ala, we get no expression at all, nada. The
I do not know about the affinity required for gel filtration, but I have
had success crystallizing a number of protease-inhibitor complexes just
by incubating and setting up drops with equimolar amounts of the two
components. It is probably to our advantage that we have functional
inhibition
Dear All,
I have 1.4 A data and a molecular replacement solution for a crystal
indexed as C2, with beta approximately equal to 90. Refinement with
refmac is progressing poorly, and intensity statistics (Truncate) and
other twinning tests (xtriage) suggest pseudo-merohedral twinning with a
twin
In addition to trying lower concentrations of PEG and protein, you might
consider whether aggregation in your protein stock or vibration in your
crystallization environment are contributing to the problem, and how
they might be minimized. It might be obvious, but we have found that it
is
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