Re: [ccp4bb] AW: Differences in a homodimer protein

2017-11-29 Thread Denis Rousseau 
Thank you Eleanor,



I did the PISA analysis and one of the "high B" chains in the region of my 
metal had a BSA ~ 10% lower than that of the "Low B" chain. I don't know if 
that is a significant difference.



Best



Denis


From: Eleanor Dodson [eleanor.dod...@york.ac.uk]
Sent: Wednesday, November 29, 2017 1:07 PM
To: Denis Rousseau
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: Differences in a homodimer protein

I would submit the coordinates to PISA to analyse the buried surface area for 
each momnomer and see if there are differences. It is pretty common that some 
copies have much lower B values and therefore are better defined..

Eleanor

On 29 November 2017 at 17:15, Denis Rousseau 
<denis.rouss...@einstein.yu.edu<mailto:denis.rouss...@einstein.yu.edu>> wrote:
I want to thank everyone on the BB for their comments about the differences in 
the homodimeric protein. I had done a superposition of the structures and found 
no differences that could account for the differences in the water occupancy I 
observed. However, I had not compared the B factors which was suggested by Mike 
Garavito.  When I did, I saw the same thing he mentioned; namely in one subunit 
the Bs were in the mid to low 40s but in the other they were in the mid to high 
50s and the metal in question went from 42 to 56. I assume these differences 
originate from crystal packing effects as suggested by several.

Best

Denis Rousseau




From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] 
on behalf of Gloria Borgstahl 
[gborgst...@gmail.com<mailto:gborgst...@gmail.com>]
Sent: Wednesday, November 29, 2017 10:32 AM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] AW: Differences in a homodimer protein

I have seen this in MnSOD.  We had a tetramer in the ASU and each
active site had different ligands in our peroxide soak. I assumed the
crystal lattice can influence these things or it is inappropriate
metal incorporation.  I would highly recommend doing ICP-MS on a
dissolved crystal and on your purified metalloprotein to make sure
your metal incorporation is 100% the metal you want.

On Wed, Nov 29, 2017 at 1:58 AM,  
<herman.schreu...@sanofi.com<mailto:herman.schreu...@sanofi.com>> wrote:
> Dear Denis,
>
>
>
> I would first superimpose both monomers to see if you can find a reason why
> one subunit has a bound water and the other not, which would in general be
> flanking side chains in (slightly) different positions. Next I would look
> for some global differences between the subunits that could be linked to
> crystal contacts explaining the non-random distribution of the subunits.
> However, these differences might be quite subtle and difficult to detect.
>
>
>
> As Emily suggested, you could also do some functional assay’s to see if
> there is any positive (or negative) cooperativity between the subunits
> providing independent support for functional differences between the
> subunits.
>
>
>
> My 2 cts,
>
> Herman
>
>
>
>
>
> Von: CCP4 bulletin board 
> [mailto:CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>] Im Auftrag von 
> Denis
> Rousseau
> Gesendet: Dienstag, 28. November 2017 20:04
> An: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
> Betreff: [EXTERNAL] [ccp4bb] Differences in a homodimer protein
>
>
>
> Hi BB members
>
>
>
> I have a homodimeric protein, which contains metal centers. In several
> different derivatives I find a water molecule on a metal center in one
> subunit which is absent on the other.  It is always the same subunit that
> contains the water molecule. The resulution is ~2.4 A. Is this an artifact
> or a functional difference? If it is truly homodimeric I would expect
> differences to be random. The space group is P212121.
>
>
>
> Thanks for the advice.
>
>
>
> Denis Rousseau
>
> 
>
>

Re: [ccp4bb] AW: Differences in a homodimer protein

2017-11-29 Thread Eleanor Dodson 
I would submit the coordinates to PISA to analyse the buried surface area
for each momnomer and see if there are differences. It is pretty common
that some copies have much lower B values and therefore are better
defined..

Eleanor

On 29 November 2017 at 17:15, Denis Rousseau <denis.rouss...@einstein.yu.edu
> wrote:

> I want to thank everyone on the BB for their comments about the
> differences in the homodimeric protein. I had done a superposition of the
> structures and found no differences that could account for the differences
> in the water occupancy I observed. However, I had not compared the B
> factors which was suggested by Mike Garavito.  When I did, I saw the same
> thing he mentioned; namely in one subunit the Bs were in the mid to low 40s
> but in the other they were in the mid to high 50s and the metal in question
> went from 42 to 56. I assume these differences originate from crystal
> packing effects as suggested by several.
>
> Best
>
> Denis Rousseau
>
>
>
> 
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Gloria
> Borgstahl [gborgst...@gmail.com]
> Sent: Wednesday, November 29, 2017 10:32 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] AW: Differences in a homodimer protein
>
> I have seen this in MnSOD.  We had a tetramer in the ASU and each
> active site had different ligands in our peroxide soak. I assumed the
> crystal lattice can influence these things or it is inappropriate
> metal incorporation.  I would highly recommend doing ICP-MS on a
> dissolved crystal and on your purified metalloprotein to make sure
> your metal incorporation is 100% the metal you want.
>
> On Wed, Nov 29, 2017 at 1:58 AM,  <herman.schreu...@sanofi.com> wrote:
> > Dear Denis,
> >
> >
> >
> > I would first superimpose both monomers to see if you can find a reason
> why
> > one subunit has a bound water and the other not, which would in general
> be
> > flanking side chains in (slightly) different positions. Next I would look
> > for some global differences between the subunits that could be linked to
> > crystal contacts explaining the non-random distribution of the subunits.
> > However, these differences might be quite subtle and difficult to detect.
> >
> >
> >
> > As Emily suggested, you could also do some functional assay’s to see if
> > there is any positive (or negative) cooperativity between the subunits
> > providing independent support for functional differences between the
> > subunits.
> >
> >
> >
> > My 2 cts,
> >
> > Herman
> >
> >
> >
> >
> >
> > Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von
> Denis
> > Rousseau
> > Gesendet: Dienstag, 28. November 2017 20:04
> > An: CCP4BB@JISCMAIL.AC.UK
> > Betreff: [EXTERNAL] [ccp4bb] Differences in a homodimer protein
> >
> >
> >
> > Hi BB members
> >
> >
> >
> > I have a homodimeric protein, which contains metal centers. In several
> > different derivatives I find a water molecule on a metal center in one
> > subunit which is absent on the other.  It is always the same subunit that
> > contains the water molecule. The resulution is ~2.4 A. Is this an
> artifact
> > or a functional difference? If it is truly homodimeric I would expect
> > differences to be random. The space group is P212121.
> >
> >
> >
> > Thanks for the advice.
> >
> >
> >
> > Denis Rousseau
> >
> > 
> >
> >
>

Re: [ccp4bb] AW: Differences in a homodimer protein

2017-11-29 Thread Denis Rousseau 
I want to thank everyone on the BB for their comments about the differences in 
the homodimeric protein. I had done a superposition of the structures and found 
no differences that could account for the differences in the water occupancy I 
observed. However, I had not compared the B factors which was suggested by Mike 
Garavito.  When I did, I saw the same thing he mentioned; namely in one subunit 
the Bs were in the mid to low 40s but in the other they were in the mid to high 
50s and the metal in question went from 42 to 56. I assume these differences 
originate from crystal packing effects as suggested by several.

Best

Denis Rousseau

 


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Gloria Borgstahl 
[gborgst...@gmail.com]
Sent: Wednesday, November 29, 2017 10:32 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: Differences in a homodimer protein

I have seen this in MnSOD.  We had a tetramer in the ASU and each
active site had different ligands in our peroxide soak. I assumed the
crystal lattice can influence these things or it is inappropriate
metal incorporation.  I would highly recommend doing ICP-MS on a
dissolved crystal and on your purified metalloprotein to make sure
your metal incorporation is 100% the metal you want.

On Wed, Nov 29, 2017 at 1:58 AM,  <herman.schreu...@sanofi.com> wrote:
> Dear Denis,
>
>
>
> I would first superimpose both monomers to see if you can find a reason why
> one subunit has a bound water and the other not, which would in general be
> flanking side chains in (slightly) different positions. Next I would look
> for some global differences between the subunits that could be linked to
> crystal contacts explaining the non-random distribution of the subunits.
> However, these differences might be quite subtle and difficult to detect.
>
>
>
> As Emily suggested, you could also do some functional assay’s to see if
> there is any positive (or negative) cooperativity between the subunits
> providing independent support for functional differences between the
> subunits.
>
>
>
> My 2 cts,
>
> Herman
>
>
>
>
>
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Denis
> Rousseau
> Gesendet: Dienstag, 28. November 2017 20:04
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [EXTERNAL] [ccp4bb] Differences in a homodimer protein
>
>
>
> Hi BB members
>
>
>
> I have a homodimeric protein, which contains metal centers. In several
> different derivatives I find a water molecule on a metal center in one
> subunit which is absent on the other.  It is always the same subunit that
> contains the water molecule. The resulution is ~2.4 A. Is this an artifact
> or a functional difference? If it is truly homodimeric I would expect
> differences to be random. The space group is P212121.
>
>
>
> Thanks for the advice.
>
>
>
> Denis Rousseau
>
> 
>
>

Re: [ccp4bb] AW: Differences in a homodimer protein

2017-11-29 Thread Gloria Borgstahl 
I have seen this in MnSOD.  We had a tetramer in the ASU and each
active site had different ligands in our peroxide soak. I assumed the
crystal lattice can influence these things or it is inappropriate
metal incorporation.  I would highly recommend doing ICP-MS on a
dissolved crystal and on your purified metalloprotein to make sure
your metal incorporation is 100% the metal you want.

On Wed, Nov 29, 2017 at 1:58 AM,   wrote:
> Dear Denis,
>
>
>
> I would first superimpose both monomers to see if you can find a reason why
> one subunit has a bound water and the other not, which would in general be
> flanking side chains in (slightly) different positions. Next I would look
> for some global differences between the subunits that could be linked to
> crystal contacts explaining the non-random distribution of the subunits.
> However, these differences might be quite subtle and difficult to detect.
>
>
>
> As Emily suggested, you could also do some functional assay’s to see if
> there is any positive (or negative) cooperativity between the subunits
> providing independent support for functional differences between the
> subunits.
>
>
>
> My 2 cts,
>
> Herman
>
>
>
>
>
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Denis
> Rousseau
> Gesendet: Dienstag, 28. November 2017 20:04
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [EXTERNAL] [ccp4bb] Differences in a homodimer protein
>
>
>
> Hi BB members
>
>
>
> I have a homodimeric protein, which contains metal centers. In several
> different derivatives I find a water molecule on a metal center in one
> subunit which is absent on the other.  It is always the same subunit that
> contains the water molecule. The resulution is ~2.4 A. Is this an artifact
> or a functional difference? If it is truly homodimeric I would expect
> differences to be random. The space group is P212121.
>
>
>
> Thanks for the advice.
>
>
>
> Denis Rousseau
>
> 
>
>

[ccp4bb] AW: Differences in a homodimer protein

2017-11-28 Thread Herman . Schreuder 
Dear Denis,

I would first superimpose both monomers to see if you can find a reason why one 
subunit has a bound water and the other not, which would in general be flanking 
side chains in (slightly) different positions. Next I would look for some 
global differences between the subunits that could be linked to crystal 
contacts explaining the non-random distribution of the subunits. However, these 
differences might be quite subtle and difficult to detect.

As Emily suggested, you could also do some functional assay's to see if there 
is any positive (or negative) cooperativity between the subunits providing 
independent support for functional differences between the subunits.

My 2 cts,
Herman


Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Denis 
Rousseau
Gesendet: Dienstag, 28. November 2017 20:04
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Differences in a homodimer protein

Hi BB members

I have a homodimeric protein, which contains metal centers. In several 
different derivatives I find a water molecule on a metal center in one subunit 
which is absent on the other.  It is always the same subunit that contains the 
water molecule. The resulution is ~2.4 A. Is this an artifact or a functional 
difference? If it is truly homodimeric I would expect differences to be random. 
The space group is P212121.

Thanks for the advice.

Denis Rousseau