Mike:
The trick may be doing the assay under anaerobic condition, especially if
the FAD cofactor is sensitive to oxygen.
You need an anaerobic train and tanometers for the experiment.
Good refs: Hille, R. Biochemistry 1991 Sep 3;30(35):8522-9. Electron
transfer within xanth
was working to set up an FAD enzymatic assay. I wished to be able to use 450nM
to continuously monitor the progress of the reaction. The substrate I used is
the natural substrate of the enzyme and the protein is recombinant protein and
I assume it's active since I do see changes in TLC plate. B
(I don't remember the motivation for the original question.)
Shake-and-Bake used to generate random atoms in an asymmetric unit, and
the program kept the atoms spaced by at least a bond length. Since
PDB entry 2erl, I am not up to date on Shake-and-Bake's current set of
tricks.
The crystal fo
On Monday 01 December 2008 15:07:56 Edward A. Berry wrote:
> Thanks, Ethan,
> For your third point- I realized (after sending) that the distribution
> would be stretched along the long axis- but actually I'm having
> a hard time coming to grips with that conceptually- if there
> are n atoms in the
It helps to remember that PCR does have an upper limit of total
double-stranded DNA content (regardless of its molarity!) after which it
does not work any more (due to the competition of the polymerase for
non-specific dsDNA versus primer-substrate pairs).
Therefore the theoretical limits on this
I am curious to hear what is the longest insert anyone has cloned
using a modification of the Quikchange cloning strategy. Basically,
ligation-independent cloning by strapping on homologous regions of
the vector onto the primers which also generate the initial PCR
product. I plan to proceed with m
Yo Thierry:
The periplasmic domain of the aspartate receptor, in the absence of
ligand, 1lih, is a dimer, but crystallizes as a monomer in the sense
that there is one monomer per asymmetric unit. There is a disulphide
bond between two Cys36 that maintains it as a dimer (and indeed
reduct
Have you seen these papers:
M Geiser, R Cebe, D Drewello, and R Schmitz. Integration of pcr
fragments at any specific site within
cloning vectors without the use of restriction enzymes and dna ligase.
Biotechniques, 31(1):88–90, 2001.
W Wang and B A Malcolm. Two-stage pcr protocol allowing introdu
Thank you Patrick for the reply, as well to another person who has
replied directly to me.
Please provide with examples if you know of any (say a reference or a
PDB id ), as it would allow for comparison between published results and
my own crystallization system.
To answer your points:
Just to be a pedantic pain - Km is not necessarily Kd. I
think that assumption only holds if the chemical step
following substrate binding is rate-limiting.
Phoebe
Original message
>Date: Mon, 1 Dec 2008 15:34:59 +0100
>From: mesters <[EMAIL PROTECTED]>
>Subject: Re: [ccp4bb] co-c
Thanks, Ethan,
For your third point- I realized (after sending) that the distribution
would be stretched along the long axis- but actually I'm having
a hard time coming to grips with that conceptually- if there
are n atoms in the cell, they will necessarily be distributed
more sparsely in projecti
Dear fellow crystallographers,
This is a question which is not CCP4-related.
Is anybody aware of a protein which is known to be a dimer in solution
(say by SEC), and yet crystallizes as a monomer? Wouldn't the high
concentration in the crystallization drop further favor dimerization?
In
Hi Folks,
Sorry for the non-xtallo posting.
I am curious to hear what is the longest insert anyone has cloned
using a modification of the Quikchange cloning strategy. Basically,
ligation-independent cloning by strapping on homologous regions of
the vector onto the primers which also genera
Hi All,
I am working on a 80 KDa single amino acid mutant of a protein expressed in
E.Coli. The protein is very pure and I get very nice looking crystals in
different conditions. I used different cryo protectants and tried room temp
testing of diffraction for these crystals. I don't get any diffrac
On Monday 01 December 2008 10:28:34 Edward A. Berry wrote:
> Ethan A Merritt wrote:
> > On Friday 28 November 2008, Mueller, Juergen-Joachim wrote:
> >> Dear all,
> >> does anybody know a program to
> >> fill an unit cell a,b,c randomly by an arbitrary number
> >> of spheres (atoms)?
> >
> > First
Ethan A Merritt wrote:
On Friday 28 November 2008, Mueller, Juergen-Joachim wrote:
Dear all,
does anybody know a program to
fill an unit cell a,b,c randomly by an arbitrary number
of spheres (atoms)?
First you would need to define "random".
Uniform density throughout the lattice?
Uniform dis
mesters wrote:
Yes!, there is:
the fraction of occupied protein with substance can be calculated: S /
(S + Km) with S being the concentration of the compound.
So, if S = Km, half of the sites are occupied (it follows from
Michaelis-Menten theory).
But- one warning (perhaps obvious but I th
Dear colleagues,
like in previous years there will be the SR User Group Meeting
organised as a satellite meeting of the main CCP4 Study Weekend.
Details about the meeting programme can be found here:
http://www.cse.scitech.ac.uk/events/CCP4_2009/user_group.html
The meeting will take place on Sat
As said, your Km is different in mother liquor than in your reaction
conditions, but even that is not the end of it: You ligand/substrate might be
inducing the slightest of all conformations in your protein that interferes
with crystallization, or might block crystal contacts. Then, you can eit
The Km changes with your reservoir, so predictions are limited. In
general if you have a low Km this is favourable but not a given that
your ligand will be found in the electron density map. As a starting
point try a molar ratio of >3 of the ligand to your protein and you
can go as high as
Yes!, there is:
the fraction of occupied protein with substance can be calculated: S /
(S + Km) with S being the concentration of the compound.
So, if S = Km, half of the sites are occupied (it follows from
Michaelis-Menten theory).
In order to saturate the enzyme for 90,90909 % with the co
Hello,everyone,
I have a question for cocrystallization, is there some relationship between Km
value and substrate concentration when making cocrystallization? How can I know
the substrate is enough for binding?
Thank you very much!
liuqing
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