: [ccp4bb] Cleaved peptide density!
Dear Crystallographers,
I am working with a cysteine protease. I co-crystallized the protease with some
small chemically synthesised peptides of 7 amino acid residues. I mutated the
active Cysteine residue with Alanine to avoid the peptide cleavage so that I
can
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Cleaved peptide density!
Dear Bonsor,
Thanks for your suggestions!
It need 2-3 weeks to get the fully grown crystals. And harvest, freeze and
data collection just take usually next 3-5 days. I usually incubated
substrate overnight. Initially I
hello Dipankar,
Your queries have generated much interest. It would be helpful if you told
us which clan the cysteine
peptidase you are working with is from. Also it would be very helpful if
you could show us the
electron density of the cleaved peptide in the active site. One presumes
that you
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From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar
Manna
Sent: Monday, April 20, 2015 2:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cleaved peptide density!
Dear
@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Cleaved peptide density!
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
@JISCMAIL.AC.UK
*Subject:* [ccp4bb] Cleaved peptide density!
Dear Crystallographers,
I am working with a cysteine protease. I co-crystallized the protease
with some small chemically synthesised peptides of 7 amino acid
residues. I mutated the active Cysteine residue with Alanine to avoid
with the cleaved peptide is also
interesting, since it represents the product complex!
Good luck!
Herman
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Dipankar
Manna
Gesendet: Montag, 20. April 2015 21:22
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Cleaved peptide density
Thank you Rajesh,
That could be a possibility though. I am planning to do some MS analysis
from the extra gel bands, if I get any by running SDS-PAGE on the purified
protein.
Best,
Dipankar
On Mon, Apr 20, 2015 at 10:42 PM, rajesh ponnusamy
ponnusam...@googlemail.com wrote:
just to add up:
Dipankar,
It is possible that the peptide was cleaved by other proteases present in the
soaking solution as impurities. The crystallized protein simply selected that
fragment that binds the best. You can try soaking with a lower amount of the
peptide (use a smaller drop and concentration).
Dear Barbel,
Thank you!
Yes you are right that I did the SDS-PAGE with bigger substrate. Regarding
peptide, we did check the MS and HPLC profile for the peptides which
clearly shows that there should not be any cleaved peptides!
Best,
Dipankar
On Mon, Apr 20, 2015 at 9:46 PM, Bärbel Blaum
First of all, you don't say how long it took to first set up crystals, for them
to grow, harvest, freeze and collect data on. Secondly how long did leave the
peptide/substrate for your SDS PAGE experiment? If they are of a different time
scale e.g. 6 hours v.s. 30 days, it may be that your
Dear Crystallographers,
I am working with a cysteine protease. I co-crystallized the protease with
some small chemically synthesised peptides of 7 amino acid residues. I
mutated the active Cysteine residue with Alanine to avoid the peptide
cleavage so that I can get the whole peptide bound with
Dear Bonsor,
Thanks for your suggestions!
It need 2-3 weeks to get the fully grown crystals. And harvest, freeze and
data collection just take usually next 3-5 days. I usually incubated
substrate overnight. Initially I was purifying with the same column as the
WT but in the next batch I used new
I suppose you do the SDS PAGE test not with the peptide but some
bigger substrate. Are you sure your peptide is intact *before*
soaking? I.e. have you checked the batch yourself with MS or NMR? We
regularly get small compounds (sugars) that turn out not to be what
the label says.
Bärbel
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