Hi Shiv,
Michael has made good points. Also I found similar case, i.e, different loop
and active site catalytic residue conformations with slightly different PEG
2000 mme concentration. In my case the catalytic residue moves away from active
site and the loop more disordered in lower PEG 2000 mme concentration (30%),
and it's ordered to show a catalytic conformation at a higher PEG 2000 mme
concentration (40%). Both PEG 2000 mme stock and original hit condition from
Hampton Research. It seems like that not only the type of PEG but also the
concentration, or potential different batches may have different effects.
I didn't observe directly the presence of phosphate at 1.4 angstrom
resolution structures in either case. So maybe a stronger precipitant condition
(higher MW or concentration of PEG) promotes the ordering or some flexible
regions for certain proteins.
Ref: Cao, H. et al 2014 J. Biol. Chem. doi: 10.1074/jbc.M114.583286
Hope this helps.
Best,Conan
Best,Hongnan Cao, Ph.D.Department of BiochemistryRice
university713-348-3913h...@rice.edu
Date: Wed, 6 Aug 2014 09:49:57 -0400
From: rmgarav...@gmail.com
Subject: Re: [ccp4bb] PEG dependent structural changes in crystal structure
To: CCP4BB@JISCMAIL.AC.UK
Shiv,
PEG1500 and PEG3350 are not the same because of the nature of their synthesis
and manufacture. They are a polydisperse and semi-purified products of an
ethylene oxide condensation. So while they are described as
H-(CH2-CH2-O)<n>-OH, the value of <n> and its ranges are quite different. Some
of the PEG molecules in both with be the same size, but PEG1500 will have a lot
more small PEGs than PEG3350; it is the small PEGs that might bind more tightly
to proteins.
On top of this, there is the manufacturing issue. All PEG is made in bulk and
then fractionated; different manufacturers have slightly different recipes and
purification schemes (which are trade secrets, so good luck in finding more
information from any company). Take a look at an old paper by Fran Jurnak (J.
Cryst, Growth, 76, 577-582, 1986) for the trials and tribulations of working
with PEG from different manufacturers of PEG (as well as how the purify it if
you really get worried). She got at least 4 different space groups, and the
key was the differing amount of phosphoric acid used in the neutralization step
in PEG production. EACH batch of PEG from the same manufacturer can be
different! So Herman's comment about pH should be considered.
But as Herman said check the enzyme activity of your protein in each and get
different batches of PEG to test. Is it a particular size of PEG or a PEG
contaminant? Remember that stock solutions you may buy from Hampton or other
X-ray specialty companies can be equally suspect because of where they get the
bulk product to begin with. Even with purified PEGs (i.e., cleaned up of metal
contaminants, aldehyde, and peroxided) age. So think about how old your stocks
are.
One last comment, just about your terminology. I would avoid the use of the
phrase "improper crystal packing." There is nothing really improper about it,
just unfortunate from your point of view. The crystal packing is just the
natural consequence of physical interactions in solution that you have
prepared. You just have to be lucky sometimes.
Good luck, Michael
****************************************************************R. Michael
Garavito, Ph.D.Professor of Biochemistry & Molecular Biology603 Wilson Rd., Rm.
513 Michigan State University East Lansing, MI 48824-1319Office: (517)
355-9724 Lab: (517) 353-9125FAX: (517) 353-9334 Email:
rmgarav...@gmail.com****************************************************************
On Aug 4, 2014, at 9:20 PM, shivendra singh <shivendr...@gmail.com> wrote:Dear
All,I have been working on a protein which initially got crystallised in
condition having PEG1500 as precipitant. The space group was P21 and got solved
with reasonable Rfree. Analysis of its structure showed large deviation and
very distinct active site architecture along with disorderedness in one of its
long loop (no density) in comparison with the expected result, based on related
homologous structures. The structure does not seem to be active with one of its
active site residue moved apart from other catalytic amino acids. Also the
substrate entry tunnel looks distorted. The purified enzyme used for
crystallisation showed optimum activity in vitro. This led us to screen it
again for some other crystallisation condition and got another crystal hit in
condition having PEG3350 as precipitant. Rest of the components of
crystallisation cocktail were same. The data belonged to P212121 space group.
The regions which were disordered or distorted in earlier case were observed to
be ordered and in their expected orientation and position. The enzyme is not
reported to be in different structural or functional states as observed.
I am wondering how the protein from the same batch showed two distinct
structural organizations in conditions with varying PEGs. What may cause it to
follow such transition. Whether it has some significant functional aspect or
just a result of improper crystal packing.
Thanks.
Shiv
