Re: [ccp4bb] "inclusion body"

[Brenda Patterson]

Lower temperature, use chaperones (e.g. TAKARA set), refolding?


Quoting shivesh kumar <[EMAIL PROTECTED]>:


Dear all,
Sorry for the off-topic question...
What can be done to avoid a protein going inside inclusion body.The gene is
cloned in pET30a with C-ter his tag and  expressed in BL21-DE3 from 37 to
18C for 3-4 hr with .5mM of IPTG,it is going to inclusion body.All
suggestions are welcome.
Thanx in advance.
Shivesh

[ccp4bb] Compound behaviour/vulnerability/truncation software/server

[Brenda Patterson]

Dear all,

I was wondering if anyone knew of any software or server that could predict
possible points of specific venerability to physical or chemical
stresses/attack, which may lead to likely truncation of a given 
compound? Another possibility would be software that would take a 
compound as an input

and then output truncated variants of this compound?

Cheers

Brenda

Re: [ccp4bb] Thermofluor experiment

[Brenda Patterson]

Hi,

I am interested in using the thermofluor to assess the stability of my protein
in different buffers.  Can anyone recommend a vendor that supplies buffer
screens, possibly in 96 well format?

Not crystallization buffers, just ordinary storage buffers.

Thanks

brenda


Quoting Andreas Förster <[EMAIL PROTECTED]>:


Dear Kornelius,

I found the idea of doing Thermofluor on membrane proteins really
intriguing - for identifying the best buffer and detergent, secondary
detergents, even for checking crystallization drops that stayed
clear. (This latter experiment should theoretically be possible with
large drops, even though you'd be working very near the limits
claimed in the publications.)

In the end, I didn't find the method too useful because of noise
issues due to detergent and exposed hydrophobic portions of the
protein.  I always felt that, in order to get reasonable signal, I'd
have to use protein at unreasonable concentrations.

The method works much better for soluble protein, though I can't tell
you a success story where it led to crystallization that seemed
impossible before.


Andreas


Kornelius Zeth wrote:

Dear all,

a question very related to the discussion before. I have been
reading the papers about the thermofluor experiment with great
interest. I wonder what people think about the underlying
principles/ideas and the success that the method yielded in their
own labs for crystallization or related purposes?

Has anybody used this method with membrane proteins in order to find
out the stability of the protein in the presence of a second
detergent?

Is the method limited to this certain dye (sypro orange)?

Have a nice day

Kornelius

P.S.: I will make a summary of all opinions.

 --
 Kornelius Zeth
 Max Planck Institute for Developmental Biology
 Dept. Protein Evolution
 Spemannstr. 35
 72076 Tuebingen, Germany
 [EMAIL PROTECTED]
 Tel -49 7071 601 323
 Fax -49 7071 601 349

Re: [ccp4bb] finicky protein

[Brenda Patterson]

Did you filter your lysate through .45 then .22 filters?

cheers
b




Quoting James Stroud <[EMAIL PROTECTED]>:


Try refolding before purification.


On Mar 2, 2008, at 11:47 PM, Tim Gruene wrote:

Hi all

sorry, for offtopic query...

I am trying to purify my protein by Ni-NTA affinity chromatography.  After
sonication as i centrifuge bacterial lysate, soon after 10 min  
whole lysates

get precipitated during loading on the column and some time it remain
soluble too. if i get purified through the column without  
precipitation, it

gets precipitated during dialysis.
I have tried lot, by chnaging buffers, increasing salt or  deacreasing salt
or no salt at are helpless.
I do purifiaction in cold room.

can any one suggest some solution?

Thanks in advance.

NSH



--
James Stroud
UCLA-DOE Institute for Genomics and Proteomics
Box 951570
Los Angeles, CA  90095

http://www.jamesstroud.com

[ccp4bb] torsion restraints COOT

[Brenda Patterson]

Dear all

I am doing molecular replacement with a model of sequence identity 42%. Have
been trying numerous combinations of weighting in refmac of CCP4. However the
geometry of the model is not being maintained. I am then trying manual fitting
of the model in COOT. However when torsion restraints are applied in the
refine/regularisation option the model comes out of the density. how would you
tackle this please? i also have developed CIS peptides in places and have found
that trying to revert these to trans is again causing the model to come out of
the density severely.  Does anyone think it's ok not to use the torsion
restraints in COOT and simply use real space refine/regularisation as it is in
COOT?

Cheers

Brenda

[ccp4bb] Cocrystals - should they pop up under native conditions?

[Brenda Patterson]

Hello all,

I am expecting a somewhat homogeneous reply to this one, but that is fine and
welcomed as are anecdotal experiences.

I am running co crystallisation experiments and have thus far been trying under
oil screens with some success (i.e. various hits in various conditions
resulting in crystal growth).  These conditions have varied from the native
condition up until now.  I have recently peered at some fairly old plates I set
up under hanging drop under native conditions when running a co crystal trial
and have found crystals.  Their morphology is very similar to the 'native'
form.  I bit punier than them if I had to push.

So,

1.  Should I be surprised that co crystals pop up under native conditions or
does it entirely depend on the ligand?

2.  Is it probably not going to be a co crystal as usually they don't pop up
under native conditions?

3.  It totally depends and if I don't like it then I should get out of this
beautiful game?

I am going to shoot them either way, but just thought I would ask.


cheers

Brenda

[ccp4bb] Difference Map in COOT - Possible lignad but clash with structure?

[Brenda Patterson]

Hello,

I am fairly new to this lark so please forgive me if this question is unclear,
but it is really puzzling me.

I have used phaser to generate a molecular replacement structure of my target
(which has 100% identity to my template) and this particular crystal I had
soaked with a ligand.

I have a density in my difference map which resembles my ligand.  The 
thing is,

it overlaps the density map of my structure and if I were to place the ligand
in that map, then there would be a steric clash.  Obviously I would expect the
ligand to be near to the structure, but not overlapping it?  I am 
uncertain how

to proceed?

Any helpful suggestions please?


Thanks in advance

Brenda

Re: [ccp4bb] How to make a structure-based multiple sequence alignment on DALI server?

[Brenda Patterson]

POSA at the Godzik lab does exactly what you are after, flexibly!

http://fatcat.burnham.org/POSA/



Hi all=A3=AC

I want to produce structure-based multiple sequence alignment of my =20=



protein with five of its homologs on DALI server. However, when I =20
tried the "Database Search Form", only one homolog was picked up =20
from PDB. If I align my protein with each homolog by the "DaliLite =20
Pairwise comparison", how can I combine them together?

Thanks in advance.


Best regards,
=09
Sincerely,

Dalei Wu
Drug Discovery and Design Center
Shanghai Institute of Materia Medica
Shanghai Institutes for Biological Sciences
Chinese Academy of Sciences
555 Zu Chongzhi Road, Shanghai, 201203, China
[EMAIL PROTECTED]
2007-12-05

[ccp4bb] I have my bound ligand (I think), now what?

[Brenda Patterson]

Hello all,

right, I think I have found a ligand bound to my protein.

I used the COOT utility to find the ligands after reading in a model 
and library

file generated by sketcher of my ligand.  Now I am a bit unsure as to how to
proceed?  How can I 'accept' a state and/or refine it?

Maybe there is a better way to model my ligand into the electron density not
accounted for by my protein?


Any general suggestions or comments would be appreciated as I am very
inexperienced.

cheers

Brenda

Re: [ccp4bb] how to change a membrane protein into a water soluble protein?

[Brenda Patterson]

Another option is refolding which can increase soluble protein content and is
used routinely to achieve soluble protein such as the TIMPs

http://peds.oxfordjournals.org/cgi/content/abstract/7/8/1035

http://www.proteinscience.org/cgi/reprint/11/10/2493.pdf?ck=nck


that said, this is not true of all membrane proteins.

Addition of a fusion partner, MBP, to the normally membrane associated 
FMO3 has

been shown to generate stable, soluble protein and the addition of a fusion
protein allows purification downstream more easily.

Here is a paper where they did as the original poster suggested and tried
mutagenesis of hydrophobic regions, including a truncation of a membrane
anchor.  They achieved increased solubility with this in combination with use
of detergents.

Krueger SK, Siddens LK, Henderson MC, VanDyke JE, Karplus PA, Pereira CB,
Williams DE.
Abstract
C-Terminal truncation of rabbit flavin-containing monooxygenase isoform 2
enhances solubility.
Arch Biochem Biophys. 2006 Jun 15;450(2):149-56. Epub 2006 Mar 29.


cheers










Quoting Bil Clemons <[EMAIL PROTECTED]>:


There is also the soluble KcsA.

Computational design of water-soluble analogues of the potassium channel
KcsA. A. M. Slovic, H. Kono, J. D. Lear, J. G. Saven, and W. F. DeGrado
(2004) PNAS 101, 1828-1833


Bil


Bil Clemons, PhD
Assistant Professor of Biochemistry
Caltech
157 Broad Center
MC 114-96
Pasadena, CA 91125
(626) 395-1796
[EMAIL PROTECTED] 






From: Thomas J Magliery PhD <[EMAIL PROTECTED]>
Reply-To: Thomas J Magliery PhD <[EMAIL PROTECTED]>
Date: Mon, 3 Dec 2007 16:50:03 -0500
To: 
Subject: Re: [ccp4bb] how to change a membrane protein into a water solub=

le

protein?
=20
It's hard. See:
=20
J Mol Biol. 2005 May 6;348(3):777-87.
X-ray structure of a water-soluble analog of the membrane protein
phospholamban:=20
sequence determinants defining the topology of tetrameric and pentameric
coiled
coils.
Slovic AM, Stayrook SE, North B, Degrado WF.
=20
Slovic, A. M., Summa, C. M., Lear, J. D. & DeGrado,
W. F. (2002). Computational design of a water-soluble
analog of phospholamban. Protein Sci. 12, 337=AD348.
=20
Li, H., Cocco, M. J., Steitz, T. A. & Engelman, D. E.
(2001). Conversion of phospholamban into a soluble
pentameric helical bundle. Biochemistry, 40,
6636=AD6645.
=20
Frank, S., Kammerer, R. A., Hellstern, S., Pegoraro, S.,
Stetefeld, J., Lustig, A. et al. (2000). Toward a high resolution
structure of phospholamban: design of
soluble transmembrane domain mutants.
Biochemistry, 39, 6825=AD6831.
=20
Tom
=20
=20
Daniel Jin wrote:

Hi,
I am wondering whether there is a way to turn a membrane protein with
known crystal structure into a water soluble protein by systematic
mutagenesis. I guess it should be doable if we introduce enough
hydrophilic residues on the surface. Has anyone tested this crazy idea
before? Thank you for your help.
Best,
Chen
=20

Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try
it now.=20


=20
=20
--=20
Thomas J. Magliery, Ph.D.
Assistant Professor
Department of Chemistry
& Department of Biochemistry
The Ohio State University
1043 Evans Laboratory
100 West 18th Ave.
Columbus, OH 43210-1185
=20
(614) 247-8425 office
(614) 292-1685 fax
[EMAIL PROTECTED]
http://www.chemistry.ohio-state.edu/~magliery
=20

[ccp4bb] Import and merge data collected from TWO sources?

[Brenda Patterson]

Is this possible?

Currently I have two crystal data's.  I have been importing the scaled averaged
files using d*trek.

The only difference between the two crystals is that one has been soaked with a
ligand.  Now I want to see if it is there.

Is there a way to import both sets of data at the same time in order to see the
difference (hopefully the ligand)?

Or any other suggestions regarding attacking this problem would be appreciated.


Cheers

charlie