Re: [ccp4bb] FW: [ccp4bb] salt sensitive complex

[Anastassis Perrakis]

Dear all -Sorry to intervene on a 'book keeping' issue, but indeed over the last few months an increasing number of people (Jerry is not the first, so Jerry please do not take it personally) attach pictures etc. I think in a bb standard practice dictates to only use text - if illustrations are needed to explain the problem, you can put them in eg a web site.Some text like that was in the 'code of conduct' off ccp4bb in the past, but I could no longer find it.Thus apologies if I am wrong and policies have changed, but maybe the ccp4 crowd could tell us what is the suggested policy.And, if you really want to send an image please do bother to make it small. The initial posting had a 630k image, which it took me 1 min to make 20k and it still makes the point (attached so I can also violate the rules i am suggesting - I love inconsistency).Thanks, TassosOn Jan 30, 2008, at 20:11, Jerry McCully wrote: Dear All:  Thanks a lot for the prompt reply on this topic of salt sensitive complex.  Attached please find one ITC final figure done under 25mM Tris(pH8.0), 60mM NaCl.  As mentioned before, the ionization of Tris will interfere with the ITC experiments. Therefore I am sure of my binding results.  Can anyone give me some comments on this ITC experiment? Basically do these two proteins bind to each other? If so, how should I improve the ITC experiments to get a similar affinity shown by BIAcore(about 0.5uM)?    Thanks again.Jerry Hi Jerry, Tris can cause problems, you are better off using something likeHEPES, and HEPES should be ok at pH 8. (Buffers with anethane-sulphonic acid group tend to be the best - those ending in'ES', so MES, TES and HEPES) FYI, the error on your K is bigger than the actual measurement -1.49x10^5 ± 1.5x10^5.Signal to noise to is probably your enemy, which is making the curvefitting difficult. Changing buffer may help this - there may be somenon-specific component to what you're seeing  - increasing salt a bitor dropping in something like 5% glycerol may help with this. Would you be able to post a jpeg/pdf of the curve? Regards, David  On 25/01/2008, Jerry McCully  wrote:>>  Dear All:>>Firstly  I would like to thank many folks here for giving me great> ideas several days ago.>>   The following are some updates for this question.>>  I did ITC experiments again using 25mMTris(pH8), 60mM NaCl(low salt> condition).>> But things still turn out to be a little weird.>> I increased the concentration of both proteins(60uM in the cell and> 1200uM in the syringe). At the end of the ITC, I saw a little of> precipitation of both the proteins.>> Fortunately I can roughly fit the curve this time. However, the heat was> still low, around 1Kcal/mole of per injectant.  I am not sure about the> fitting statistics. N 1.10 ±0.17>> K 1.49E5 ±1.5E5>> DH   -893.5  ±213>> DS   20.7>> Was the enthalpy was offset by the ionization of Tris buffer?>> Can I use Hepes buffer around pH8 to do ITC?>>>   Welcome any comments about the statistics and suggestions on how to> improve the ITC experiments.have a nice weekend.>> Jerry>Helping your favorite cause is as easy as instant messaging. You IM, we give. Learn more. Shed those extra pounds with MSN and The Biggest Loser! Learn more.

[ccp4bb] Web site GOOD, Attachment BAD (was: salt sensitive complex)

[Frank von Delft]

Actually, it's not book keeping, it's simple courtesy -- and not only on 
a BB:  an attachment is lazy, and a large attachment is downright rude. 

I am routinely stuck with a slow connection (travelling), and others are 
*permanently* stuck with one.  So please be nice... ;)


phx.



Anastassis Perrakis wrote:

Dear all -

Sorry to intervene on a 'book keeping' issue, but indeed over the last 
few months an increasing number of people (Jerry is not the first, so 
Jerry please do not take it personally) attach pictures etc. I think 
in a bb standard practice dictates to only use text - if illustrations 
are needed to explain the problem, you can put them in eg a web site.


Some text like that was in the 'code of conduct' off ccp4bb in the 
past, but I could no longer find it.


Thus apologies if I am wrong and policies have changed, but maybe the 
ccp4 crowd could tell us what is the suggested policy.


And, if you really want to send an image please do bother to make it 
small. The initial posting had a 630k image, which it took me 1 min to 
make 20k and it still makes the point (attached so I can also violate 
the rules i am suggesting - I love inconsistency).


Thanks, Tassos




On Jan 30, 2008, at 20:11, Jerry McCully wrote:



Dear All:

  Thanks a lot for the prompt reply on this topic of salt 
sensitive complex.


  Attached please find one ITC final figure done under 25mM 
Tris(pH8.0), 60mM NaCl.


  As mentioned before, the ionization of Tris will interfere with 
the ITC experiments.


 Therefore I am sure of my binding results.

  Can anyone give me some comments on this ITC experiment? 
Basically do these two proteins bind to each other? If so, how should 
I improve the ITC experiments to get a similar affinity shown by 
BIAcore(about 0.5uM)?


Thanks again.

Jerry



Hi Jerry,
 
Tris can cause problems, you are better off using something like

HEPES, and HEPES should be ok at pH 8. (Buffers with an
ethane-sulphonic acid group tend to be the best - those ending in
'ES', so MES, TES and HEPES)
 
FYI, the error on your K is bigger than the actual measurement -

1.49x10^5 ± 1.5x10^5.
Signal to noise to is probably your enemy, which is making the curve
fitting difficult. Changing buffer may help this - there may be some
non-specific component to what you're seeing  - increasing salt a bit
or dropping in something like 5% glycerol may help with this.
 
Would you be able to post a jpeg/pdf of the curve?
 
Regards,
 
David
 
 
On 25/01/2008, Jerry McCully  wrote:

>
>  Dear All:
>
>Firstly  I would like to thank many folks here for giving me great
> ideas several days ago.
>
>   The following are some updates for this question.
>
>  I did ITC experiments again using 25mMTris(pH8), 60mM NaCl(low salt
> condition).
>
> But things still turn out to be a little weird.
>
> I increased the concentration of both proteins(60uM in the cell and
> 1200uM in the syringe). At the end of the ITC, I saw a little of
> precipitation of both the proteins.
>
> Fortunately I can roughly fit the curve this time. However, the heat 
was
> still low, around 1Kcal/mole of per injectant.  I am not sure about the
> fitting statistics.
>
>
>
> N 1.10 ±0.17
>
> K 1.49E5 ±1.5E5
>
> DH   -893.5  ±213
>
> DS   20.7
>
> Was the enthalpy was offset by the ionization of Tris buffer?
>
> Can I use Hepes buffer around pH8 to do ITC?
>
>
>   Welcome any comments about the statistics and suggestions on how to
> improve the ITC experiments.have a nice weekend.
>
> Jerry
>




Helping your favorite cause is as easy as instant messaging. You
IM, we give. Learn more.
 




Shed those extra pounds with MSN and The Biggest Loser! Learn more. 

Re: [ccp4bb] Web site GOOD, Attachment BAD (was: salt sensitive complex)

[Clemens Vonrhein]

To not only say what not to do, maybe also some tips on how to
convert/resize/scale images - for Linux with Imagemagick tools.

 * converting between formats (PNG seems something everyone is happy
   with these days):

% convert Image.jpg Image.png

% ps2epsi Image.ps # gets rid of all the white border
   # fluff
% convert Image.epsi Image.png

 * resizing (no need for a 3000x2000 image - 400x400 to 800x800 is
   usually big enough I guess):

% convert -resize 400x400 Image.jpg Image.png

 * cutting a bit out of an image (makes it easier to concentrate on
   the important bit):

% display Image.jpg

  -> left mouse click in image
 -> Transform
-> Crop
   -> left mouse and pan (is that the right word?) to mark
  the rectangular region
  -> Crop
 -> File
-> Save (as Image.png)

  * screen shot of some picture/window/plot (to show error messages,
CCP4i input window etc etc)

a) get e.g. your CCP4i loggraph window on screen

b) open another terminal (make sure this terminal doesn't overlap
   with the CCP4i loggraph window) and run

   % import test.png

c) the mouse cursor should change to a "+" sign (kind of): just
   click into the window you want to get a picture off - it will
   be saved as test.png (control with "display test.png")

Lots of other tools I guess. For more info on ImageMagick see e.g.

  http://www.imagemagick.org/script/index.php
  http://www.imagemagick.org/Usage/

or

  % convert -help

Clemens

On Thu, Jan 31, 2008 at 08:58:04AM +, Frank von Delft wrote:
> Actually, it's not book keeping, it's simple courtesy -- and not only on 
> a BB:  an attachment is lazy, and a large attachment is downright rude. 
> 
> I am routinely stuck with a slow connection (travelling), and others are 
> *permanently* stuck with one.  So please be nice... ;)
> 
> phx.
> 
> 
> 
> Anastassis Perrakis wrote:
> >Dear all -
> >
> >Sorry to intervene on a 'book keeping' issue, but indeed over the last 
> >few months an increasing number of people (Jerry is not the first, so 
> >Jerry please do not take it personally) attach pictures etc. I think 
> >in a bb standard practice dictates to only use text - if illustrations 
> >are needed to explain the problem, you can put them in eg a web site.
> >
> >Some text like that was in the 'code of conduct' off ccp4bb in the 
> >past, but I could no longer find it.
> >
> >Thus apologies if I am wrong and policies have changed, but maybe the 
> >ccp4 crowd could tell us what is the suggested policy.
> >
> >And, if you really want to send an image please do bother to make it 
> >small. The initial posting had a 630k image, which it took me 1 min to 
> >make 20k and it still makes the point (attached so I can also violate 
> >the rules i am suggesting - I love inconsistency).
> >
> >Thanks, Tassos
> >
> >
> >
> >
> >On Jan 30, 2008, at 20:11, Jerry McCully wrote:
> >
> >>
> >>Dear All:
> >>
> >>  Thanks a lot for the prompt reply on this topic of salt 
> >>sensitive complex.
> >>
> >>  Attached please find one ITC final figure done under 25mM 
> >>Tris(pH8.0), 60mM NaCl.
> >>
> >>  As mentioned before, the ionization of Tris will interfere with 
> >>the ITC experiments.
> >>
> >> Therefore I am sure of my binding results.
> >>
> >>  Can anyone give me some comments on this ITC experiment? 
> >>Basically do these two proteins bind to each other? If so, how should 
> >>I improve the ITC experiments to get a similar affinity shown by 
> >>BIAcore(about 0.5uM)?
> >>
> >>Thanks again.
> >>
> >>Jerry
> >>
> >>
> >>
> >>Hi Jerry,
> >> 
> >>Tris can cause problems, you are better off using something like
> >>HEPES, and HEPES should be ok at pH 8. (Buffers with an
> >>ethane-sulphonic acid group tend to be the best - those ending in
> >>'ES', so MES, TES and HEPES)
> >> 
> >>FYI, the error on your K is bigger than the actual measurement -
> >>1.49x10^5 ± 1.5x10^5.
> >>Signal to noise to is probably your enemy, which is making the curve
> >>fitting difficult. Changing buffer may help this - there may be some
> >>non-specific component to what you're seeing  - increasing salt a bit
> >>or dropping in something like 5% glycerol may help with this.
> >> 
> >>Would you be able to post a jpeg/pdf of the curve?
> >> 
> >>Regards,
> >> 
> >>David
> >> 
> >> 
> >>On 25/01/2008, Jerry McCully  wrote:
> >>>
> >>>  Dear All:
> >>>
> >>>Firstly  I would like to thank many folks here for giving me 
> >>great
> >>> ideas several days ago.
> >>>
> >>>   The following are some updates for this question.
> >>>
> >>>  I did ITC experiments again using 25mMTris(pH8), 60mM NaCl(low 
>

Re: [ccp4bb] Web site GOOD, Attachment BAD (was: salt sensitive complex)

[Martyn Winn]

Yes, we do discourage attachments. Links are better.
This is a strong recommendation rather than a hanging offence (we are
easy-going people).
I've added a sentence to that effect at http://www.ccp4.ac.uk/ccp4bb.php

Regards
Martyn

On Thu, 2008-01-31 at 08:58 +, Frank von Delft wrote:
> Actually, it's not book keeping, it's simple courtesy -- and not only on 
> a BB:  an attachment is lazy, and a large attachment is downright rude. 
> 
> I am routinely stuck with a slow connection (travelling), and others are 
> *permanently* stuck with one.  So please be nice... ;)
> 
> phx.
> 
> 
> 
> Anastassis Perrakis wrote:
> > Dear all -
> >
> > Sorry to intervene on a 'book keeping' issue, but indeed over the last 
> > few months an increasing number of people (Jerry is not the first, so 
> > Jerry please do not take it personally) attach pictures etc. I think 
> > in a bb standard practice dictates to only use text - if illustrations 
> > are needed to explain the problem, you can put them in eg a web site.
> >
> > Some text like that was in the 'code of conduct' off ccp4bb in the 
> > past, but I could no longer find it.
> >
> > Thus apologies if I am wrong and policies have changed, but maybe the 
> > ccp4 crowd could tell us what is the suggested policy.
> >
> > And, if you really want to send an image please do bother to make it 
> > small. The initial posting had a 630k image, which it took me 1 min to 
> > make 20k and it still makes the point (attached so I can also violate 
> > the rules i am suggesting - I love inconsistency).
> >
> > Thanks, Tassos
> >
> >
> >
> >
> > On Jan 30, 2008, at 20:11, Jerry McCully wrote:
> >
> >>
> >> Dear All:
> >>
> >>   Thanks a lot for the prompt reply on this topic of salt 
> >> sensitive complex.
> >>
> >>   Attached please find one ITC final figure done under 25mM 
> >> Tris(pH8.0), 60mM NaCl.
> >>
> >>   As mentioned before, the ionization of Tris will interfere with 
> >> the ITC experiments.
> >>
> >>  Therefore I am sure of my binding results.
> >>
> >>   Can anyone give me some comments on this ITC experiment? 
> >> Basically do these two proteins bind to each other? If so, how should 
> >> I improve the ITC experiments to get a similar affinity shown by 
> >> BIAcore(about 0.5uM)?
> >>
> >> Thanks again.
> >>
> >> Jerry
> >>
> >> 
> >> 
> >>
> >> Hi Jerry,
> >>  
> >> Tris can cause problems, you are better off using something like
> >> HEPES, and HEPES should be ok at pH 8. (Buffers with an
> >> ethane-sulphonic acid group tend to be the best - those ending in
> >> 'ES', so MES, TES and HEPES)
> >>  
> >> FYI, the error on your K is bigger than the actual measurement -
> >> 1.49x10^5 ± 1.5x10^5.
> >> Signal to noise to is probably your enemy, which is making the curve
> >> fitting difficult. Changing buffer may help this - there may be some
> >> non-specific component to what you're seeing  - increasing salt a bit
> >> or dropping in something like 5% glycerol may help with this.
> >>  
> >> Would you be able to post a jpeg/pdf of the curve?
> >>  
> >> Regards,
> >>  
> >> David
> >>  
> >>  
> >> On 25/01/2008, Jerry McCully  wrote:
> >> >
> >> >  Dear All:
> >> >
> >> >Firstly  I would like to thank many folks here for giving me 
> >> great
> >> > ideas several days ago.
> >> >
> >> >   The following are some updates for this question.
> >> >
> >> >  I did ITC experiments again using 25mMTris(pH8), 60mM NaCl(low 
> >> salt
> >> > condition).
> >> >
> >> > But things still turn out to be a little weird.
> >> >
> >> > I increased the concentration of both proteins(60uM in the cell 
> >> and
> >> > 1200uM in the syringe). At the end of the ITC, I saw a little of
> >> > precipitation of both the proteins.
> >> >
> >> > Fortunately I can roughly fit the curve this time. However, the 
> >> heat was
> >> > still low, around 1Kcal/mole of per injectant.  I am not sure about 
> >> the
> >> > fitting statistics.
> >> >
> >> >
> >> >
> >> > N 1.10 ±0.17
> >> >
> >> > K 1.49E5 ±1.5E5
> >> >
> >> > DH   -893.5  ±213
> >> >
> >> > DS   20.7
> >> >
> >> > Was the enthalpy was offset by the ionization of Tris buffer?
> >> >
> >> > Can I use Hepes buffer around pH8 to do ITC?
> >> >
> >> >
> >> >   Welcome any comments about the statistics and suggestions on how to
> >> > improve the ITC experiments.have a nice weekend.
> >> >
> >> > Jerry
> >> >
> >>
> >>
> >>
> >> 
> >> 
> >> Helping your favorite cause is as easy as instant messaging. You
> >> IM, we give. Learn more.
> >> 

Re: [ccp4bb] Web site GOOD, Attachment BAD (was: salt sensitive complex)

[Anastassis Perrakis]

And I cant help adding that a line graph does not need to be in 32- 
bit color but 2-bit color (b/w) will do.
I know how to do that in PhotoShop but not in Image Magick, although  
I am sure its trivial to do.


And here some more preaching:

Screen (eg Power Point): Do 400x400 pixels, 800x800 at most if full  
screen


Submit paper: 1200x1200 is far enough to print fairly well in full  
page - most journals these days  truncate to less to send to referees  
anyway. Dont use 32-bit color for line graphs, best is to submit  
these as eps!


Accepted paper: use the formula (dpi journal recommends) x (final  
intended size in inches) to decide the size in pixels.


... sorry for the trivialties.

Tassos


On Jan 31, 2008, at 10:40, Clemens Vonrhein wrote:


To not only say what not to do, maybe also some tips on how to
convert/resize/scale images - for Linux with Imagemagick tools.

 * converting between formats (PNG seems something everyone is happy
   with these days):

% convert Image.jpg Image.png

% ps2epsi Image.ps # gets rid of all the white border
   # fluff
% convert Image.epsi Image.png

 * resizing (no need for a 3000x2000 image - 400x400 to 800x800 is
   usually big enough I guess):

% convert -resize 400x400 Image.jpg Image.png

 * cutting a bit out of an image (makes it easier to concentrate on
   the important bit):

% display Image.jpg

  -> left mouse click in image
 -> Transform
-> Crop
   -> left mouse and pan (is that the right word?) to mark
  the rectangular region
  -> Crop
 -> File
-> Save (as Image.png)

  * screen shot of some picture/window/plot (to show error messages,
CCP4i input window etc etc)

a) get e.g. your CCP4i loggraph window on screen

b) open another terminal (make sure this terminal doesn't overlap
   with the CCP4i loggraph window) and run

   % import test.png

c) the mouse cursor should change to a "+" sign (kind of): just
   click into the window you want to get a picture off - it will
   be saved as test.png (control with "display test.png")

Lots of other tools I guess. For more info on ImageMagick see e.g.

  http://www.imagemagick.org/script/index.php
  http://www.imagemagick.org/Usage/

or

  % convert -help

Clemens

On Thu, Jan 31, 2008 at 08:58:04AM +, Frank von Delft wrote:
Actually, it's not book keeping, it's simple courtesy -- and not  
only on
a BB:  an attachment is lazy, and a large attachment is downright  
rude.


I am routinely stuck with a slow connection (travelling), and  
others are

*permanently* stuck with one.  So please be nice... ;)

phx.



Anastassis Perrakis wrote:

Dear all -

Sorry to intervene on a 'book keeping' issue, but indeed over the  
last
few months an increasing number of people (Jerry is not the  
first, so

Jerry please do not take it personally) attach pictures etc. I think
in a bb standard practice dictates to only use text - if  
illustrations
are needed to explain the problem, you can put them in eg a web  
site.


Some text like that was in the 'code of conduct' off ccp4bb in the
past, but I could no longer find it.

Thus apologies if I am wrong and policies have changed, but maybe  
the

ccp4 crowd could tell us what is the suggested policy.

And, if you really want to send an image please do bother to make it
small. The initial posting had a 630k image, which it took me 1  
min to
make 20k and it still makes the point (attached so I can also  
violate

the rules i am suggesting - I love inconsistency).

Thanks, Tassos




On Jan 30, 2008, at 20:11, Jerry McCully wrote:



Dear All:

 Thanks a lot for the prompt reply on this topic of salt
sensitive complex.

 Attached please find one ITC final figure done under 25mM
Tris(pH8.0), 60mM NaCl.

 As mentioned before, the ionization of Tris will interfere  
with

the ITC experiments.

Therefore I am sure of my binding results.

 Can anyone give me some comments on this ITC experiment?
Basically do these two proteins bind to each other? If so, how  
should

I improve the ITC experiments to get a similar affinity shown by
BIAcore(about 0.5uM)?

   Thanks again.

Jerry


--- 
-


   Hi Jerry,

   Tris can cause problems, you are better off using something like
   HEPES, and HEPES should be ok at pH 8. (Buffers with an
   ethane-sulphonic acid group tend to be the best - those  
ending in

   'ES', so MES, TES and HEPES)

   FYI, the error on your K is bigger than the actual measurement -
   1.49x10^5 ± 1.5x10^5.
   Signal to noise to is probably your enemy, which is making  
the curve
   fitting difficult. Changing buffer may help this - there may  
be some
   non-specific component to what you're seeing  - increasing  
salt a bit

   or dropping in something like 5% glycerol may help with this.

  

Re: [ccp4bb] Web site GOOD, Attachment BAD (was: salt sensitive complex)

[Clemens Vonrhein]

On Thu, Jan 31, 2008 at 11:02:04AM +0100, Anastassis Perrakis wrote:
> And I cant help adding that a line graph does not need to be in 32- 
> bit color but 2-bit color (b/w) will do.
> I know how to do that in PhotoShop but not in Image Magick, although  
> I am sure its trivial to do.

This seems to work:

  % convert -type Grayscale Image.png Image_gray.png

> ... sorry for the trivialties.

Sometimes the most trivial things are the most useful: never wrong
repeating sensible things several times I guess ...

Clemens

-- 

***
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
*
*  Global Phasing Ltd.
*  Sheraton House, Castle Park 
*  Cambridge CB3 0AX, UK
*--
* BUSTER Development Group  (http://www.globalphasing.com)
***

[ccp4bb] BCA Spring Meeting 4th Feb Deadline

[AT Hadfield, Biochemistry]

The deadline for submission of abstracts for poster presentations at the 
York Spring Meeting (April 8 - 10) is Monday, 4th February 2008.


This has an excellent, full programme this year with sessions on:
Membrane Proteins
Neutrons in Biology
Probing Fast Biological Reactions
Complementary Methods in Structural Biology
Ligand Binding and Drug Design

The abstract template with further instructions and the submission form are 
available at:


http://www.crystallography-meetings.org.uk/abstracts.htm

The Biological Structure Group will be awarding the David Blow poster prize 
for the best biological poster; this is a Blue John trophy, along with a 
cash prize (£100). There will also be two runner-up prizes.


The bursary deadline for students is also 4th February, and these cover 
registration, 2 nights accommodation, much of the food including the 
conference dinner. Travel is not included. The Biological Structure Group 
pay money into the bursary fund each year - so BCA members please take 
advantage.





Dr. Andrea Hadfield

Chairman, Biological Structure Group
Dept of Biochemistry,
School of Medical Sciences,
University of Bristol,
University Walk,
Bristol BS8 1TD

NEW PHONE NUMBER
Tel: 0117 331 2151  e-mail [EMAIL PROTECTED]
Fax: 0117 331 2168

Re: [ccp4bb] Web site GOOD, Attachment BAD (was: salt sensitive complex)

[James Stroud]

If anyone is wondering where to put his or her pictures for this  
niceness, blogspot is an option if your situation limits your ability  
to host pictures. You can set up your own blog and post pictures  
there. The "thumbnails" generated by the interface will link to the  
full sized pictures. The pictures will need to be converted to one of  
the standard web types (gif, jpeg, and I think png is OK).


James

On Jan 31, 2008, at 12:58 AM, Frank von Delft wrote:

Actually, it's not book keeping, it's simple courtesy -- and not  
only on a BB:  an attachment is lazy, and a large attachment is  
downright rude.
I am routinely stuck with a slow connection (travelling), and others  
are *permanently* stuck with one.  So please be nice... ;)


phx.



Anastassis Perrakis wrote:

Dear all -

Sorry to intervene on a 'book keeping' issue, but indeed over the  
last few months an increasing number of people (Jerry is not the  
first, so Jerry please do not take it personally) attach pictures  
etc. I think in a bb standard practice dictates to only use text -  
if illustrations are needed to explain the problem, you can put  
them in eg a web site.


Some text like that was in the 'code of conduct' off ccp4bb in the  
past, but I could no longer find it.


Thus apologies if I am wrong and policies have changed, but maybe  
the ccp4 crowd could tell us what is the suggested policy.


And, if you really want to send an image please do bother to make  
it small. The initial posting had a 630k image, which it took me 1  
min to make 20k and it still makes the point (attached so I can  
also violate the rules i am suggesting - I love inconsistency).


Thanks, Tassos




On Jan 30, 2008, at 20:11, Jerry McCully wrote:



Dear All:

 Thanks a lot for the prompt reply on this topic of salt  
sensitive complex.


 Attached please find one ITC final figure done under 25mM  
Tris(pH8.0), 60mM NaCl.


 As mentioned before, the ionization of Tris will interfere  
with the ITC experiments.


Therefore I am sure of my binding results.

 Can anyone give me some comments on this ITC experiment?  
Basically do these two proteins bind to each other? If so, how  
should I improve the ITC experiments to get a similar affinity  
shown by BIAcore(about 0.5uM)?


   Thanks again.

Jerry





   Hi Jerry,
Tris can cause problems, you are better off using  
something like

   HEPES, and HEPES should be ok at pH 8. (Buffers with an
   ethane-sulphonic acid group tend to be the best - those ending in
   'ES', so MES, TES and HEPES)
FYI, the error on your K is bigger than the actual  
measurement -

   1.49x10^5 ± 1.5x10^5.
   Signal to noise to is probably your enemy, which is making the  
curve
   fitting difficult. Changing buffer may help this - there may be  
some
   non-specific component to what you're seeing  - increasing salt  
a bit

   or dropping in something like 5% glycerol may help with this.
Would you be able to post a jpeg/pdf of the curve?
Regards,
David
 On 25/01/2008, Jerry McCully  wrote:
   >
   >  Dear All:
   >
   >Firstly  I would like to thank many folks here for  
giving me great

   > ideas several days ago.
   >
   >   The following are some updates for this question.
   >
   >  I did ITC experiments again using 25mMTris(pH8), 60mM  
NaCl(low salt

   > condition).
   >
   > But things still turn out to be a little weird.
   >
   > I increased the concentration of both proteins(60uM in  
the cell and
   > 1200uM in the syringe). At the end of the ITC, I saw a little  
of

   > precipitation of both the proteins.
   >
   > Fortunately I can roughly fit the curve this time.  
However, the heat was
   > still low, around 1Kcal/mole of per injectant.  I am not sure  
about the

   > fitting statistics.
   >
   >
   >
   > N 1.10 ±0.17
   >
   > K 1.49E5 ±1.5E5
   >
   > DH   -893.5  ±213
   >
   > DS   20.7
   >
   > Was the enthalpy was offset by the ionization of Tris buffer?
   >
   > Can I use Hepes buffer around pH8 to do ITC?
   >
   >
   >   Welcome any comments about the statistics and suggestions  
on how to

   > improve the ITC experiments.have a nice weekend.
   >
   > Jerry
   >






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Re: [ccp4bb] Web site GOOD, Attachment BAD (was: salt sensitive complex)

[Raji Edayathumangalam]

Can the CCP4BB provide something like a website to upload pictures and then 
have the BB-ers just
post the link in their email. Please!

These attachments are clogging my inbox...

Thanks much.
Raji



-Included Message--
>Date: 31-jan-2008 03:58:44 -0500
>From: "Frank von Delft" <[EMAIL PROTECTED]>
>To: 
>Subject: [ccp4bb] Web site GOOD, Attachment BAD (was: salt sensitive complex)
>
>Actually, it's not book keeping, it's simple courtesy -- and not only on 
>a BB:  an attachment is lazy, and a large attachment is downright rude. 
>
>I am routinely stuck with a slow connection (travelling), and others are 
>*permanently* stuck with one.  So please be nice... ;)
>
>phx.
>
>
>
>Anastassis Perrakis wrote:
>> Dear all -
>>
>> Sorry to intervene on a 'book keeping' issue, but indeed over the last 
>> few months an increasing number of people (Jerry is not the first, so 
>> Jerry please do not take it personally) attach pictures etc. I think 
>> in a bb standard practice dictates to only use text - if illustrations 
>> are needed to explain the problem, you can put them in eg a web site.
>>
>> Some text like that was in the 'code of conduct' off ccp4bb in the 
>> past, but I could no longer find it.
>>
>> Thus apologies if I am wrong and policies have changed, but maybe the 
>> ccp4 crowd could tell us what is the suggested policy.
>>
>> And, if you really want to send an image please do bother to make it 
>> small. The initial posting had a 630k image, which it took me 1 min to 
>> make 20k and it still makes the point (attached so I can also violate 
>> the rules i am suggesting - I love inconsistency).
>>
>> Thanks, Tassos
>>
>>
>>
>>
>> On Jan 30, 2008, at 20:11, Jerry McCully wrote:
>>
>>>
>>> Dear All:
>>>
>>>   Thanks a lot for the prompt reply on this topic of salt 
>>> sensitive complex.
>>>
>>>   Attached please find one ITC final figure done under 25mM 
>>> Tris(pH8.0), 60mM NaCl.
>>>
>>>   As mentioned before, the ionization of Tris will interfere with 
>>> the ITC experiments.
>>>
>>>  Therefore I am sure of my binding results.
>>>
>>>   Can anyone give me some comments on this ITC experiment? 
>>> Basically do these two proteins bind to each other? If so, how should 
>>> I improve the ITC experiments to get a similar affinity shown by 
>>> BIAcore(about 0.5uM)?
>>>
>>> Thanks again.
>>>
>>> Jerry
>>>
>>> 
>>>
>>> Hi Jerry,
>>>  
>>> Tris can cause problems, you are better off using something like
>>> HEPES, and HEPES should be ok at pH 8. (Buffers with an
>>> ethane-sulphonic acid group tend to be the best - those ending in
>>> 'ES', so MES, TES and HEPES)
>>>  
>>> FYI, the error on your K is bigger than the actual measurement -
>>> 1.49x10^5 ± 1.5x10^5.
>>> Signal to noise to is probably your enemy, which is making the curve
>>> fitting difficult. Changing buffer may help this - there may be some
>>> non-specific component to what you're seeing  - increasing salt a bit
>>> or dropping in something like 5% glycerol may help with this.
>>>  
>>> Would you be able to post a jpeg/pdf of the curve?
>>>  
>>> Regards,
>>>  
>>> David
>>>  
>>>  
>>> On 25/01/2008, Jerry McCully  wrote:
>>> >
>>> >  Dear All:
>>> >
>>> >Firstly  I would like to thank many folks here for giving me 
>>> great
>>> > ideas several days ago.
>>> >
>>> >   The following are some updates for this question.
>>> >
>>> >  I did ITC experiments again using 25mMTris(pH8), 60mM NaCl(low 
>>> salt
>>> > condition).
>>> >
>>> > But things still turn out to be a little weird.
>>> >
>>> > I increased the concentration of both proteins(60uM in the cell 
>>> and
>>> > 1200uM in the syringe). At the end of the ITC, I saw a little of
>>> > precipitation of both the proteins.
>>> >
>>> > Fortunately I can roughly fit the curve this time. However, the 
>>> heat was
>>> > still low, around 1Kcal/mole of per injectant.  I am not sure about 
>>> the
>>> > fitting statistics.
>>> >
>>> >
>>> >
>>> > N 1.10 ±0.17
>>> >
>>> > K 1.49E5 ±1.5E5
>>> >
>>> > DH   -893.5  ±213
>>> >
>>> > DS   20.7
>>> >
>>> > Was the enthalpy was offset by the ionization of Tris buffer?
>>> >
>>> > Can I use Hepes buffer around pH8 to do ITC?
>>> >
>>> >
>>> >   Welcome any comments about the statistics and suggestions on how to
>>> > improve the ITC experiments.have a nice weekend.
>>> >
>>> > Jerry
>>> >
>>>
>>>
>>>
>>> 
>>> Helping your favorite cause is as easy as instant messaging. You
>>> IM, we give. Learn more.
>>> 

Re: [ccp4bb] Web site GOOD, Attachment BAD

[David J. Schuller]

On Thu, 2008-01-31 at 08:12 -0500, Raji Edayathumangalam wrote:
> Can the CCP4BB provide something like a website to upload pictures and then 
> have the BB-ers just
> post the link in their email. Please!
> 
> These attachments are clogging my inbox...
> 
> Thanks much.
> Raji

If you're talking about an open site where anyone can post, then we
could run a contest to predict how long it takes for it to be hacked and
used for illegal file dispersal. If you're talking about a site where
CCP4 personnel could remove attachments from incoming e-mail and post
them on the site, I suspect that CCP4 personnel would prefer not to
handle that extra work load. The same for a site where CCP4 personnel
would administer private accounts for users to post. I think it's better
if each person finds their own site where they can securely post.

Cheers,
-  
===
With the single exception of Cornell, there is not a college in the
United States where truth has ever been a welcome guest - R.G. Ingersoll
===
  David J. Schuller
  modern man in a post-modern world
  MacCHESS, Cornell University
  [EMAIL PROTECTED]

[ccp4bb] ARP/wARP usage at low resolution!

[Anastassis Perrakis]

Dear all,

May I ask you, if you had cases at low resolution (lets say at least  
below 2.5 to be generous) that ARP/wARP produced a useful result for  
you, to send me (... not to the list) a very short email, also  
mentioning a publication that refers to that result?


Thanks in advance, Tassos

Re: [ccp4bb] Web site GOOD, Attachment BAD

[Winter, G (Graeme)]

On this note, some tools provide this kind of thing for a fairly small
overhead. Google run picasa on the web which is a no brainer for putting
pictures up on for discussion - this has the advantage that they can
also be "hidden" or "public". Someone has also mentioned blogger as an
option, but this has a slightly higher activation energy.

See:

http://picasaweb.google.co.uk/graeme.winter/IllustrativeExample

For an example. This was very straightforward. The benefit of sharing a
picture will almost certainly outweigh the effort required to do the
upload. 

Cheers,

Graeme



-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
David J. Schuller
Sent: 31 January 2008 13:27
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Web site GOOD, Attachment BAD

On Thu, 2008-01-31 at 08:12 -0500, Raji Edayathumangalam wrote:
> Can the CCP4BB provide something like a website to upload pictures and

> then have the BB-ers just post the link in their email. Please!
> 
> These attachments are clogging my inbox...
> 
> Thanks much.
> Raji

If you're talking about an open site where anyone can post, then we
could run a contest to predict how long it takes for it to be hacked and
used for illegal file dispersal. If you're talking about a site where
CCP4 personnel could remove attachments from incoming e-mail and post
them on the site, I suspect that CCP4 personnel would prefer not to
handle that extra work load. The same for a site where CCP4 personnel
would administer private accounts for users to post. I think it's better
if each person finds their own site where they can securely post.

Cheers,
-
===
With the single exception of Cornell, there is not a college in the
United States where truth has ever been a welcome guest - R.G. Ingersoll
===
  David J. Schuller
  modern man in a post-modern world
  MacCHESS, Cornell University
  [EMAIL PROTECTED]

Re: [ccp4bb] Web site GOOD, Attachment BAD

[Chris Richardson]

When choosing a hosting site for images, it's good to remember that  
we many of us suffer from aggressive content filtering.  My  
organisation has fairly relaxed filtering of the web sites that we  
can visit, but nonetheless common image hosting sites find their way  
onto the banned list with distressing regularity.  Worse still, whole  
countries often have filtered access to such sites.


Sometimes a (small) attachment will reach many more people than a link.

Regards,

Chris
--
Dr Chris Richardson - sysadmin, Structural Biology Section, icr.ac.uk


The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company 
Limited by Guarantee, Registered in England under Company No. 534147 with its 
Registered Office at 123 Old Brompton Road, London SW7 3RP.

This e-mail message is confidential and for use by the addressee only.  If the 
message is received by anyone other than the addressee, please return the 
message to the sender by replying to it and then delete the message from your 
computer and network.

Re: [ccp4bb] Web site GOOD, Attachment BAD

[Martyn Winn]

To answer a few specific points (some from private replies):

1. ccp4bb is now run by jiscmail i.e. not by us directly (basically
because of the load), so we'd have to request any fancy scripting and I
can't guarantee it would be done

2. Agree with David that we want to avoid a long-term commitment to an
extra workload (automated scripts/services don't always work all the
time, believe it or not)

3. We do have plans for a CCP4 wiki, which might be suitable for
long-term material. However, there are technical problems at the moment,
and I'm not sure when (if?) it will come online.

Sorry if this sounds negative, but we are not yet the ISP for the
crystallographic community (nice idea though ;-)

Martyn

On Thu, 2008-01-31 at 08:26 -0500, David J. Schuller wrote:
> On Thu, 2008-01-31 at 08:12 -0500, Raji Edayathumangalam wrote:
> > Can the CCP4BB provide something like a website to upload pictures and then 
> > have the BB-ers just
> > post the link in their email. Please!
> > 
> > These attachments are clogging my inbox...
> > 
> > Thanks much.
> > Raji
> 
> If you're talking about an open site where anyone can post, then we
> could run a contest to predict how long it takes for it to be hacked and
> used for illegal file dispersal. If you're talking about a site where
> CCP4 personnel could remove attachments from incoming e-mail and post
> them on the site, I suspect that CCP4 personnel would prefer not to
> handle that extra work load. The same for a site where CCP4 personnel
> would administer private accounts for users to post. I think it's better
> if each person finds their own site where they can securely post.
> 
> Cheers,
> -  
> ===
> With the single exception of Cornell, there is not a college in the
> United States where truth has ever been a welcome guest - R.G. Ingersoll
> ===
>   David J. Schuller
>   modern man in a post-modern world
>   MacCHESS, Cornell University
>   [EMAIL PROTECTED]
-- 
***
* *
*   Dr. Martyn Winn   *
* *
*   STFC Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, U.K.   *
*   Tel: +44 1925 603455E-mail: [EMAIL PROTECTED] *
*   Fax: +44 1925 603825Skype name: martyn.winn   * 
* URL: http://www.ccp4.ac.uk/martyn/  *
***

[ccp4bb] glycosylation sites

[Ronnie WEi]

I was asked this question by a colleague. 

Has anyone looked into where glycosylation occurs most frequently on a protein- 
loops, alpha-helices or beta-strands?

Thanks for your input!

Best,

Ronnie


  

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Re: [ccp4bb] glycosylation sites]

[Remy Loris]

I think you need to look at this paper, although already quite old:

Imberty A, Pérez S.
Stereochemistry of the N-glycosylation sites in glycoproteins.
Protein Eng. 1995 Jul;8(7):699-709.

Remy Loris
Vrije Universiteit Brussel

Ronnie WEi wrote:

I was asked this question by a colleague.
 
Has anyone looked into where glycosylation occurs most frequently on a 
protein- loops, alpha-helices or beta-strands?
 
Thanks for your input!
 
Best,
 
Ronnie



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

Re: [ccp4bb] FW: [ccp4bb] salt sensitive complex

[Jerry McCully]

Dear All:

I am sorry that I did not know the policy.

   And thanks a lot for the kind reminder.

Jerry

CC: CCP4BB@JISCMAIL.AC.UK
From: [EMAIL PROTECTED]
Subject: Re: [ccp4bb] FW: [ccp4bb] salt sensitive complex
Date: Thu, 31 Jan 2008 09:37:01 +0100
To: [EMAIL PROTECTED]

Dear all -Sorry to intervene on a 'book keeping' issue, but indeed over the 
last few months an increasing number of people (Jerry is not the first, so 
Jerry please do not take it personally) attach pictures etc. I think in a bb 
standard practice dictates to only use text - if illustrations are needed to 
explain the problem, you can put them in eg a web site.Some text like that was 
in the 'code of conduct' off ccp4bb in the past, but I could no longer find 
it.Thus apologies if I am wrong and policies have changed, but maybe the ccp4 
crowd could tell us what is the suggested policy.And, if you really want to 
send an image please do bother to make it small. The initial posting had a 630k 
image, which it took me 1 min to make 20k and it still makes the point 
(attached so I can also violate the rules i am suggesting - I love 
inconsistency).Thanks, Tassos


_
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[ccp4bb] Open position: Beamline scientist for the MX-beamline of BESSY (Berlin)

[Uwe Mueller]

BESSY operates one of the world's most modern synchrotron radiation 
sources for VUV and soft X-rays, delivering high quality synchrotron 
radiation to more than 1300 international users annually. Among other 
research activities, BESSY operates in collaboration with the Freie 
Universität Berlin three experimental stations for macromolecular 
crystallography.
To strengthen the BESSY-MX team and to take a leading role in the 
operation and development of our MX-beamlines we are looking for a:


Beamline Scientist Ph.D.
Ref. E-MX 08

at the next possible date. The strongly team oriented work will 
additionally require a close collaboration with the structural biology 
groups of Freie Universität Berlin and the Max-Delbrück-Center Berlin. 
Candidates are expected to hold a Ph.D. in physics, chemistry or a 
comparable qualification and have postdoc experiences in macromolecular 
crystallography with a convincing research record. Furthermore, a proven 
expertise in the operation and instrumentation of MX-beamlines is highly 
desirable.


We are offering a tenure-track position with an initially three years 
contract.


Enquiries should be directed to Dr. Christian Jung, (+49 30 6392- 2944), 
E-mail: [EMAIL PROTECTED]


Women are especially encouraged to apply. Handicapped persons will be 
given preference to other applicants with the same qualification.


E-mail applications will not be accepted. To apply please send a cover 
letter with the reference code and your complete application documents to:


Berliner Elektronenspeicherring- Gesellschaft für Synchrotronstrahlung 
m.b.H. (BESSY)

Personalverwaltung
Albert-Einstein-Str. 15
12489-Berlin

http://www.bessy.de


___

Dr. Uwe Müller
BESSY-MX group

BESSY GmbH
Albert-Einstein-Straße-15
D-12489 Berlin

Phone: +49-(0)30-6392 4974
Fax : +49-(0)30-6392 4975
email: [EMAIL PROTECTED]
url : http://www.psf.bessy.de

BESSY GmbH - Mitglied der Leibniz Gemeinschaft
Vorsitzender des Aufsichtsrates: Prof. Dr. Dr. h.c. mult Joachim Treusch
Geschäftsführer: Prof. Dr. Dr. h.c. Wolfgang Eberhardt, Prof. 
Dr.Eberhard Jaeschke

Sitz Berlin, AG Charlottenburg, HRB 14635

[ccp4bb] Postdoctoral Position Available at the University of Nebraska

[Mark Wilson]

Postdoctoral Position in the Structural Biology of Neurodegeneration at 
the University of Nebraska

A postdoctoral position is available immediately in the laboratory of Dr. 
Mark Wilson in the Department of Biochemistry, University of 
Nebraska-Lincoln.  The position is for two years and involves a 
multidisciplinary structural, biophysical, and biochemical study of 
multiple redox-active proteins involved in neurodegeneration. A Ph.D. in 
Biochemistry or an allied discipline and previous research experience in 
protein biochemistry is required. Preference will be given to applicants 
with experience in either X-ray crystallography or NMR spectroscopy. 
Salary and benefits will be provided according to current NIH guidelines. 
Interested applicants should send a C.V. and the contact information for 
three references to:

Dr. Mark Wilson
N164 Beadle Center
University of Nebraska
Lincoln, NE 68516
[EMAIL PROTECTED]

The University of Nebraska is committed to a pluralistic campus community 
through affirmative action and equal opportunity.  We assure reasonable 
accommodation under the Americans with Disabilities Act. 

Mark A. Wilson
Assistant Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N164 Beadle Center
1901 Vine Street
Lincoln, NE 68588
(402) 472-3626
[EMAIL PROTECTED]

Re: [ccp4bb] glycosylation sites]

[P.Artymiuk]

hi Ronnie

There is also a survey by
Petrescu et al (2004) Glycobiology 14(2):103-114
"Statistical analysis of the protein environment of N-glycosylation sites:
implications for occupancy, structure, and folding"

best wishes
Pete Artymiuk

Quoting Remy Loris <[EMAIL PROTECTED]>:

> I think you need to look at this paper, although already quite old:
> 
> Imberty A, Pérez S.
> Stereochemistry of the N-glycosylation sites in glycoproteins.
> Protein Eng. 1995 Jul;8(7):699-709.
> 
> Remy Loris
> Vrije Universiteit Brussel
> 
> Ronnie WEi wrote:
> > I was asked this question by a colleague.
> >  
> > Has anyone looked into where glycosylation occurs most frequently on a 
> > protein- loops, alpha-helices or beta-strands?
> >  
> > Thanks for your input!
> >  
> > Best,
> >  
> > Ronnie
> > 
> > 
> > Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try 
> > it now. 
> >
>
 
> >  >
> 

[ccp4bb] peptides for cocrystallization

[George Lountos]

Dear All:

 I would appreciate if anyone has any good suggestions or recommendations on a 
company that I can use to order high quality peptides (specifically 
phosphopeptides) for use in co-crystallization studies. 

Thanks,

George


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