At 5:56 PM +0100 8/26/10, Hari Namboodiri wrote:
Can anyone provide insights about expressing heme containing
proteins in E.col? Does E.coli need any porphyrin precursor during
expression or you need special E.coli strains. I have references
mentioning delta-aminolevulinic acid for one ortholo
I assume you are trying to do a non-denaturing prep. Have you run your
sample on a size exclusion column to see if it is aggregated? If it is
aggregated, it can stick to a lot of contaminating proteins which will
be difficult if not impossible to separate.
ho
> On Thu, Aug 26, 2010 at 8:24 AM,
Hi,
A little danger this condition. I suggest you should firstly make sure they are
protein crystals. I know it is very difficult. I suggest you can add proteases
to the drops. If "crystals" dissolve, no problem, they are protein crystals.
Best
Yibin
2010-08-27
yybbll
发件人： rui
发送时间：
Hary,
Delta-aminolevulinic acid is the classical heme (heme b)
precursor for your experiments. As far as I know, the synthesis of
porphyrin goes very quickly in E. coli, since I tried it once with a
bacterial P450 and got a fat overexpression band after ~2hrs in the
lysate. Also the photometri
One more additional way is to apply harmonic restraint on all coordinates and
all B values apart from those you want to refine. If harmonic restraints are
strong enough then B values will not move much.
But I do not like this option.
Regards
Garib
On 26 Aug 2010, at 19:56, Hailiang Zhang wrot
Thanks a lot Ethan, I will give it a try.
Best Regards, Hailiang
> On Thursday 26 August 2010 11:56:39 am Hailiang Zhang wrote:
>> Hi,
>>
>> I want to refine B factors for several residues only (all the other B
>> factors and all coordinates fixed, I know it sounds weird but there is a
>> reason t
On Thursday 26 August 2010 11:56:39 am Hailiang Zhang wrote:
> Hi,
>
> I want to refine B factors for several residues only (all the other B
> factors and all coordinates fixed, I know it sounds weird but there is a
> reason to try that).
Maybe you could tell us what this reason is?
> Is there
Hari,
I have expressed sulfite oxidase (with a N-terminal heme) in E. coli strain
TP1000 which resulted in red protein. I did not supplement the media with iron
or a porphyrin precursor.
Sincerely,
James
On Aug 26, 2010, at 12:56 PM, Hari Namboodiri wrote:
> Hi CCPers
>
> Can anyone provi
Have you tried expression tricks like Rosetta cells? Testing different
colonies and/or starting from fresh transformants? Sometimes that matters.
If your protein is an oligomer and your contaminants are degradation products,
you might try adding some urea. If desparate, you could spike the fr
Hi,
I want to refine B factors for several residues only (all the other B
factors and all coordinates fixed, I know it sounds weird but there is a
reason to try that). Is there anyway CCP4 can do this? Thanks for any
suggestions!
Best Regards, Hailiang
I think you need a special trick to express C-type heme proteins.
For B hemes (protoheme, protoporphryn 9), which I think is
what the P450's have, E.coli is always expressing several
proteins which contain it. But I guess there may be some way
to up the production in case of an overexpresser.
Ha
Dear Hari,
You might look at Lucy Waskell's experiences with full length
mammalian Cytochrome P450 2B4. While she got >50 mg of purified
protein per liter of culture, many other heme proteins are much harder
to express in E. coli with heme assembly proteins, chaperones, etc.
You just ha
Hi CCPers
Can anyone provide insights about expressing heme containing proteins in E.col?
Does E.coli need any porphyrin precursor during expression or you need special
E.coli strains. I have references mentioning delta-aminolevulinic acid for one
ortholog but none for another. The enzyme is CY
Hi,
PDBsum can be used for not released co-ordinates as well- by using the
"generate" option (found on the left panel on the webpage).
Priyamvada
-Original Message-
From: Mark J van Raaij [mailto:mjvanra...@cnb.csic.es]
Sent: Thursday, August 26, 2010 12:46 PM
To: CCP4BB@JISCMAIL.AC.UK
Dear All,
people have pointed me to:
LigPlot+ (http://www.ebi.ac.uk/thornton-srv/software/LigPlus/)
MOE from ChemComp (not free)
PDBSUM (http://www.ebi.ac.uk/pdbsum/): prerun LIGPLOT results, only for
released pdb coordinates.
looks like I am stuck to Windows for now - at least it runs.
Mark
Hi.
What size are the impurities? If they are smaller than your protein, then
they could actually be truncation products, which will be difficult to
purify away since they maintain some of the same characteristics as the full
length protein. You can check for C-terminal truncations using a
His-a
On Thu, 2010-08-26 at 11:35 -0400, Roger Rowlett wrote:
> We routinely polish protein preps on Q-sepharose (Mono-Q should be
> even better) with at least 10 CV gradients over a narrower range of
> NaCl concentrations, maybe 0-0.5 M or even smaller.
Just wanted to add that in my experience the reso
I generally find it is possible to purify most
overexpressed proteins, even those at low levels of _expression_,
with a combination of IEX and HIC methods. We use step-gradients
to do our routine purifications, but may use gradients for
polishing. If running grad
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Hi Francis,
I might save you some time by telling you up front you should just go
back and purify your compound to remove the impurity, you dont even
need to read the rest of this, just go.
Along the lines of what Savvas was saying, with any equilibrium
binding assay between two direct co
Dear all,
I have problems in purifying a protein. The protein is 38,000 daltons and
has a N-ter His-Tag. The protein expression levels are low and as a result I
have a limit for the purification steps.
Initially I used NiNTA columns with 50 mM sodium phosphate buffer pH8, 300
mM NaCl, 20 to 250 mM
Well - that is exactly what COOT does isnt it?
Eleanor
Hailiang Zhang wrote:
Hi,
Can some utilities of CCP4 do the real-space refinement locally with the
residue range explicitly specified?
By the way, I have registered phenix bb. Just didn't realize this before,
sorry again.
Best Regards, Ha
On 25/08/10 23:24, Garib Murshudov wrote:
However 1) coot uses 2mFo-DFc maps
Typically yes, but it uses whatever Refmac (or other programs) provide
(of course)
2) you should be able to feed any map you want to coot
Yes.
so it is nice place for experimenting this kind
of calculation
H
Dear Paul!
you might want to have a look at http://salilab.org/modeller/ . The
stand alone version is free for academia.
Christian
___
Dr. Christian Rausch
Lehrstuhl für Biologische Chemie
Technische Universität München, Germany
On Thu, Aug 26, 2010 at 12:03 AM, Paul Kraft wrote:
>
> Hello,
>
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