[ccp4bb] Anion-Pi interaction in protein

[Kavyashree Manjunath]

Dear users,

Has there been any instances where there is an
anion-pi interaction in proteina. I found a density
that could accommodate an acetate ion just above
the aromatic ring of the tyrosine residue in the
protein I am working on. I just came across an article
regarding the anion-pi interaction -
"Supramolecular chemistry: Anion transport as easy as pi"
Nature Chemistry 2, 516–517, (2010)
So has anyone come across such an interaction in proteins?

Thanking you
With Regards
kavya


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Re: [ccp4bb] determination of oligomerization state of protein

[gauri misra]

Hi,
Try glutaraldehyde crosslinking of  peaks you are obtaining from SEC
individually and see if it gives you some idea.

Best wishes
Gauri

Re: [ccp4bb] Enhancing Crystal Quality

[Sangeetha Vedula]

Lucas, if your crystals diffract worse at room temp than cryoprotected, it
looks like there may be radiation damage at room temperature, so you may
need to cryoprotect. The change in osmotic pressure may be too much for
your crystals when you're cryoprotecting the crystals by a sudden dunk.
Also, did you just add PEG 400 to the reservoir solution already in the
wells or did you make it afresh with higher precipitant concentration to
compensate for removal into a solution without any soluble protein or
crystalline protein to be in equilibrium with? I always made solutions
afresh unless the crystal was cryoprotected and if it diffracted just fine
by dragging the crystal through a reservoir+cryoprotectant solution.

My crystals did not tolerate sudden changes in osmotic pressure.

Harvest the crystal into 15 uL of reservoir solution with excess
precipitant to compensate for loss of protein to be in equilibrium with (if
I had a crystal from 8% PEG 8000, I used to use 12% PEG8000 with all of the
other components having the same concentration. It was random but I found
one that worked and stuck to it. Sometimes, there may be a solubility
issue, so I had to cut back on a salt but just enough to keep everything
soluble. So I made the solution fresh because my crystals didn't always
appear at exactly the same precipitant concentration. I always set up a
small range of concentrations.

Add 2.5 uL of cryoprotectant (reservoir solution with excess precipitant
concentration AND required concentration of precipitant present in it).
Remove 2.5 uL from a different part of the drop.

Wait 6 min (again, random. 5 min didn't work well, 6 min did).

Repeat with 2.5 uL, 3.75 uL, 5 uL, 7.5 uL. By that time, I achieved enough
cryoprotectant concentration for it to do its job.

I never mixed the drop because my crystals were very fragile and obviously,
I was always looking at it under the microscope to make sure I wasn't
smashing or sucking up my crystal.

Have you tried additive screens? I had tremendous improvement in my
crystals with that. They were very mosaic and overall resolution was only
about 3.7 A or so without additives. With 3% DMSO (from an additive
screen), the size was great, still mosaic, but overall resolution with good
outer shell completeness (I don't remember the exact numbers for data
quality but it was much better than without) improved to about 2.8-ish A.

Good luck!

S.

On Sun, Aug 12, 2012 at 3:31 PM, Yi-Liang (Lucas) Liu
wrote:

> Hi CCP4ers,
>
> I tested my crystal under room temperature. It still only has low
> resolution (<7A). Are there any way to improve this? I attached the
> diffraction as well. Thanks.
>
> Lucas
>
>
>
> On Aug 2, 2012, at 5:27 PM, Roger Rowlett wrote:
>
> Mitegen makes a nice little product that is a plastic tube that will slide
> over one of their magnetic cap/loops. If you put some well solution in the
> tube and seal the base with apiezon, you can collect quite a bit of data on
> the loop mounted crystal before it dries out.
>
> Cheers,
>
> ___
> Roger S. Rowlett
> Gordon & Dorothy Kline Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
>
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@colgate.edu
>
>
>
>
>
>
> On Thu, Aug 2, 2012 at 2:14 PM, Yi-Liang Liu  wrote:
>
>> Hi Herman and other CCP3BBers,
>>
>> Thanks for your suggestions. I didn't see any cracks in the crystal drops
>> initially. I will certainly try to shot crystals under room temperature and
>> see what happens. Does the plastic loops fit into the cryo stands Molecular
>> Dimension sells?
>>
>> LUcas
>> On Aug 2, 2012, at 2:24 AM, herman.schreu...@sanofi.com wrote:
>>
>> > Hi Lucas,
>> >
>> > The funky diffraction pattern is most likely due to a cracked crystal,
>> > resulting in a mixture of slightly differently aligned diffraction
>> > patterns. Were the cracks there before you added the cryprotectant? If
>> > not, the cryoprotectant is definitively to blame. As has mentioned
>> > before, you have to take a shot at room temperature without any
>> > cryoprotectant added, to make sure the bad quality is not due to the
>> > cryoprotectant. Mitegen sells plastic capillaries, which you can slide
>> > over your loop to prevent the crystal from drying out.
>> >
>> > Good luck!
>> > Herman
>> >
>> > -Original Message-
>> > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
>> > Yi-Liang Liu
>> > Sent: Thursday, August 02, 2012 4:15 AM
>> > To: CCP4BB@JISCMAIL.AC.UK
>> > Subject: Re: [ccp4bb] Enhancing Crystal Quality
>> >
>> > Hi,
>> >
>> > Thanks for the kindly answers from everyone. I actually haven't tried
>> > different cryoprotectants. I might will give a try next time. I usually
>> > only use mother liquor+30% PEG400. It is noticeable that it has some
>> > "patterns (cracks (?))" on the crystal. However, it didn't form icy
>> > rings or etc. The diffraction pattern looks fu

Re: [ccp4bb] determination of oligomerization state of protein

[Jim Fairman]

Multi-Angle Light Scattering (MALS) coupled with an HPLC column attached to
an analytical SEC column is my standard "go-to" method for determining
molecular weights/oligomeric states of peaks from SEC runs.  I've only ever
used the detectors from Wyatt (
http://www.wyatt.com/solutions/hardware/hardware.html), not sure if there
are any other manufacturers in the business.

Cheers, Jim

On Mon, Aug 13, 2012 at 10:04 AM, Sangeetha Vedula
wrote:

> Hello all,
>
> I am working with a protein that is probably a hexamer based on homology
> with other proteins but when I ran it on an analytical size gel filtration
> column, I see multiple peaks. I would like to determine the exact
> oligomerization state of the mixture and have considered blue native gel
> electrophoresis and gradient centrifugation. The monomer is about 54 kDa.
> All of the peaks are active enzymatically. I wish I could test higher
> concentrations to see if the equilibrium will shift towards a hexamer (or
> whatever the naturally highest state is) but the protein crashes out.
>
> The protein was expressed in baculovirus and the multitude of states could
> be an artifact but I'd like to know what I am working with. Also, down the
> line, I'll need to pick one of the states for crystallization trials.
>
> Has anyone encountered this problem? How did you determine the
> oligomerization state(s)?
>
> I have basic biorad gel apparatus available and no gradient mixer.
>
> Thanks for any input.
>
> Best regards,
>
> Sangeetha.
>
>
>


-- 
Jim Fairman, Ph D.
Crystal Core Leader I
Emerald BioStructures 
Tel: 206-780-8914
Cell: 240-479-6575
E-mail: fairman@gmail.com jfair...@embios.com

[ccp4bb] determination of oligomerization state of protein

[Sangeetha Vedula]

Hello all,

I am working with a protein that is probably a hexamer based on homology
with other proteins but when I ran it on an analytical size gel filtration
column, I see multiple peaks. I would like to determine the exact
oligomerization state of the mixture and have considered blue native gel
electrophoresis and gradient centrifugation. The monomer is about 54 kDa.
All of the peaks are active enzymatically. I wish I could test higher
concentrations to see if the equilibrium will shift towards a hexamer (or
whatever the naturally highest state is) but the protein crashes out.

The protein was expressed in baculovirus and the multitude of states could
be an artifact but I'd like to know what I am working with. Also, down the
line, I'll need to pick one of the states for crystallization trials.

Has anyone encountered this problem? How did you determine the
oligomerization state(s)?

I have basic biorad gel apparatus available and no gradient mixer.

Thanks for any input.

Best regards,

Sangeetha.

[ccp4bb] libcheck garbled small molecule in ccp4 6.3 but not in ccp4 6.2.2

[hari jayaram]

Hi,
I just upgraded to the newest CCP4  version 6.3 and noticed that libcheck
which coot uses to produce restraints from a SMILES string produces garbled
coordinates in ccp4 version 6.3 , but the same SMILES string works just
fine with CCP4 version 6.2.

I tried to get it to fail on public molecules , but couldnt find an
illustrative example.
But consistently the old version 6.2.2 succeeds but 6.3 garbles the phenyl
rings.


Anyone else seeing this.
Thanks

Hari

Re: [ccp4bb] NADP binding protein without Rossmann fold.

[Sudipta Bhattacharyya]

Dear all,


Thank you very much for your cooperation.


Regards,
Sudipta.


Sudipta Bhattacharyya.
Senior Research Fellow,
Department of Biotechnology,
Indian Institute of Technology Kharagpur, India.