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 si
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 af
Can anyone point to some especially useful resources to help explain to kids
(pre-chemistry, ~age 10-12) how and why molecules crystallize? Maybe a good
online movie or animation? I am especially needing help with the concept of
nucleation, and why nucleation is slower and then crystal growth f
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 wt
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:
http://www.pnas.org/content/103/18/
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 i
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 is
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 the
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 c
Thanks, these are great links!
Evette
On Jul 13, 2012, at 7:39 PM, "Andrew Purkiss-Trew"
wrote:
> Quoting Jacob Keller :
>
>>>
>>> The expansion ratio of liquid to gaseous nitrogen is approximately 1:700,
>>> that is, 1 liter of liquid becomes 700 liters of gas (at room temperature).
>>> When
luck, tom
>
> Tom Peat
> Biophysics Group
> CSIRO, CMSE
> 343 Royal 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 Rad
Thanks much. It helps a lot.
On Jul 13, 2012, at 5:31 PM, "Henry Bellamy" 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 a problem.
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 m
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 Canc
>
>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
From: CCP4 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 from HKL Scalepack
Dear all,
I have previously used SCALA for data reduction, and in publications and
pdb dep
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 ab
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 t
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 a
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 yo
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 v
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.
Assis
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-- hopef
lto:[EMAIL 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 ess
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 i
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
nuclei
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 ass
Associate Consultant II
Mayo Clinic Cancer Center
Griffin Cancer Research Building, Rm 310
4500 San Pablo Road
Jacksonville, FL 32224
(904) 953-6372 (office)
(904) 953-3109 (lab)
From: Radisky, Evette S. Ph.D.
Sent: Wednesday, December 06, 2006 10:40 AM
To
ice)
(904) 953-2857 (lab)
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Radisky,
Evette S. Ph.D.
Sent: Friday, April 13, 2007 4:23 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Twin refinement with SHELXL
Dear All,
I have 1.4 A data and a molecu
Can you (or anybody else) expand upon this -- what would be a
recommended screening protocol for twinning? Collect X degrees of data,
scale, merge, run mmtbx.xtriage? How many degrees of data will it take
to give a reliable estimate, and does this depend upon crystal
orientation, SG, merohedral v
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 f
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 important
iginal Message-
From: Eleanor Dodson [mailto:[EMAIL PROTECTED]
Sent: Thu 2/22/2007 3:42 AM
To: Radisky, Evette S. Ph.D.
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] NCS Phased Refinement
Well - the idea of NCS refinement is to restrain the 2 NCS related
models to be the same so that i
I have some questions about the "NCS Phased Refinement" task run from
ccp4i:
Can one use this task if there are multiple nonidentical chains in the
NCS unit (e.g. a heterodimer), and if so, how does one define the NCS
units? I find that the default is to consider each chain as a separate
"NCS uni
Here's one:
Mattos C, Rasmussen B, Ding X, Petsko GA, Ringe D.
Analogous inhibitors of elastase do not always bind analogously.
Nat Struct Biol. 1994 Jan;1(1):55-8.
Also, I had a similar story as a postdoc, soaking a poor, nonspecific
peptide substrate into trypsin; it bound sort of backwards a
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