Re: [ccp4bb] Puzzling electron density upon heavy metal soaking

[Joern Krausze]

Here is a snapshot of the anomalous map:

http://imageshack.us/a/img560/1905/anomalousmap.png

The map is contoured to 2.5sigma. They are the strongest peaks in the map. 
Not all 7 positions are equally strong.


J.

**
Address:

Joern Krausze
Molecular Structural Biology
Helmholtz Centre for Infection Research
Inhoffenstrasse 7
38124 Braunschweig
Germany

Email:  joern.krau...@helmholtz-hzi.de
Phone:  +49 (0)531 6181 7023 (office)
+49 (0)531 6181 7020 (lab)
**

On Thu, 1 Nov 2012, Joern Krausze wrote:


Dear all,

I have two isomorphous crystals of the same protein. One crystal, let's
call it 'derivative', was soaked with 10 mM CoCl2 whereas the other, let's
call it 'original', was not. Both crystals were otherwise grown under
identical conditions (see below) and treated the same. In the derivative,
I see some puzzling electron density which I do not see in the original
(find two pictures under the links below; the waters in the figures are
just put in for your convenience, they were no part of the refinement).
The density forms a planar hexagon of spheres with another sphere in the
exact center. The distance from corner to corner is about 3.0 A but
varies. The distance from one corner to the center is about 3.1 A. The
hexagon is almost symmetrical as the angles enclosed by two edges are
about 120° each. This density shows up at two positions, once located
between two aspartate residues and once between an aspartate and a
tyrosine residue. These are, however, no special postions. It seems
obvious that this density is caused by the presence of Co(II) since it
only shows up in the derivative and also coincides with peaks in the
difference fourier map. I am unable to interpret it in a way that makes
sense. Could anyone of you help me figure out what to build in there?

Crystallization condition for both original and derivative was 200 mM
Tris pH 8.8, 200 mM NaCl, 21% PEG 6000, 20% glycerol.

The high-resolution limit of the dataset is 2.0 A. Data were collected at
lambda=0.981 A. Anomalous data were collected at Co peak wavelength (weak
SigAno but good enough for difference fourier map).

links:

http://imageshack.us/a/img89/9376/unknowndensity1q.png
http://imageshack.us/a/img696/7135/unknowndensity2.png

Thank you in advance!

Joern

**
Address:

Joern Krausze
Molecular Structural Biology
Helmholtz Centre for Infection Research
Inhoffenstrasse 7
38124 Braunschweig
Germany

Email:  joern.krau...@helmholtz-hzi.de
Phone:  +49 (0)531 6181 7023 (office)
   +49 (0)531 6181 7020 (lab)
**



Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124 
Braunschweig | www.helmholtz-hzi.de


Vorsitzende des Aufsichtsrates: MinDir’in Bärbel Brumme-Bothe, 
Bundesministerium für Bildung und Forschung
Stellvertreter: Rüdiger Eichel, Abteilungsleiter Niedersächsisches 
Ministerium für Wissenschaft und Kultur

Geschäftsführung: Prof. Dr. Dirk Heinz; Ulf Richter, MBA
Gesellschaft mit beschränkter Haftung (GmbH)
Sitz der Gesellschaft: Braunschweig
Handelsregister: Amtsgericht Braunschweig, HRB 477

Re: [ccp4bb] Puzzling electron density upon heavy metal soaking

[Joern Krausze]

@Partha

I have 4 polypeptide chains in the au. The density in the pictures is 
located between chain A and C'(symmetry mate of chain C), so it is not 
located on a special position


I will prepare and attach a figure of the anomalous map a.s.a.p. In my 
memory there was density in all 7 position.


J.

**
Address:

Joern Krausze
Molecular Structural Biology
Helmholtz Centre for Infection Research
Inhoffenstrasse 7
38124 Braunschweig
Germany

Email:  joern.krau...@helmholtz-hzi.de
Phone:  +49 (0)531 6181 7023 (office)
+49 (0)531 6181 7020 (lab)
**

On Thu, 1 Nov 2012, Joern Krausze wrote:


Dear all,

I have two isomorphous crystals of the same protein. One crystal, let's
call it 'derivative', was soaked with 10 mM CoCl2 whereas the other, let's
call it 'original', was not. Both crystals were otherwise grown under
identical conditions (see below) and treated the same. In the derivative,
I see some puzzling electron density which I do not see in the original
(find two pictures under the links below; the waters in the figures are
just put in for your convenience, they were no part of the refinement).
The density forms a planar hexagon of spheres with another sphere in the
exact center. The distance from corner to corner is about 3.0 A but
varies. The distance from one corner to the center is about 3.1 A. The
hexagon is almost symmetrical as the angles enclosed by two edges are
about 120° each. This density shows up at two positions, once located
between two aspartate residues and once between an aspartate and a
tyrosine residue. These are, however, no special postions. It seems
obvious that this density is caused by the presence of Co(II) since it
only shows up in the derivative and also coincides with peaks in the
difference fourier map. I am unable to interpret it in a way that makes
sense. Could anyone of you help me figure out what to build in there?

Crystallization condition for both original and derivative was 200 mM
Tris pH 8.8, 200 mM NaCl, 21% PEG 6000, 20% glycerol.

The high-resolution limit of the dataset is 2.0 A. Data were collected at
lambda=0.981 A. Anomalous data were collected at Co peak wavelength (weak
SigAno but good enough for difference fourier map).

links:

http://imageshack.us/a/img89/9376/unknowndensity1q.png
http://imageshack.us/a/img696/7135/unknowndensity2.png

Thank you in advance!

Joern

**
Address:

Joern Krausze
Molecular Structural Biology
Helmholtz Centre for Infection Research
Inhoffenstrasse 7
38124 Braunschweig
Germany

Email:  joern.krau...@helmholtz-hzi.de
Phone:  +49 (0)531 6181 7023 (office)
   +49 (0)531 6181 7020 (lab)
**



Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124 
Braunschweig | www.helmholtz-hzi.de


Vorsitzende des Aufsichtsrates: MinDir’in Bärbel Brumme-Bothe, 
Bundesministerium für Bildung und Forschung
Stellvertreter: Rüdiger Eichel, Abteilungsleiter Niedersächsisches 
Ministerium für Wissenschaft und Kultur

Geschäftsführung: Prof. Dr. Dirk Heinz; Ulf Richter, MBA
Gesellschaft mit beschränkter Haftung (GmbH)
Sitz der Gesellschaft: Braunschweig
Handelsregister: Amtsgericht Braunschweig, HRB 477

Re: [ccp4bb] Puzzling electron density upon heavy metal soaking

[Filip Van Petegem]

Hello Joern,

do you have an image of the anomalous difference map as well? Do you see
the same 7 peaks there, or only the central blob?

Filip


On Thu, Nov 1, 2012 at 11:45 AM, Joern Krausze wrote:

> Dear all,
>
> I have two isomorphous crystals of the same protein. One crystal, let's
> call it 'derivative', was soaked with 10 mM CoCl2 whereas the other, let's
> call it 'original', was not. Both crystals were otherwise grown under
> identical conditions (see below) and treated the same. In the derivative,
> I see some puzzling electron density which I do not see in the original
> (find two pictures under the links below; the waters in the figures are
> just put in for your convenience, they were no part of the refinement).
> The density forms a planar hexagon of spheres with another sphere in the
> exact center. The distance from corner to corner is about 3.0 A but
> varies. The distance from one corner to the center is about 3.1 A. The
> hexagon is almost symmetrical as the angles enclosed by two edges are
> about 120° each. This density shows up at two positions, once located
> between two aspartate residues and once between an aspartate and a
> tyrosine residue. These are, however, no special postions. It seems
> obvious that this density is caused by the presence of Co(II) since it
> only shows up in the derivative and also coincides with peaks in the
> difference fourier map. I am unable to interpret it in a way that makes
> sense. Could anyone of you help me figure out what to build in there?
>
> Crystallization condition for both original and derivative was 200 mM
> Tris pH 8.8, 200 mM NaCl, 21% PEG 6000, 20% glycerol.
>
> The high-resolution limit of the dataset is 2.0 A. Data were collected at
> lambda=0.981 A. Anomalous data were collected at Co peak wavelength (weak
> SigAno but good enough for difference fourier map).
>
> links:
>
> http://imageshack.us/a/img89/**9376/unknowndensity1q.png
> http://imageshack.us/a/img696/**7135/unknowndensity2.png
>
> Thank you in advance!
>
> Joern
>
> 
> Address:
>
> Joern Krausze
> Molecular Structural Biology
> Helmholtz Centre for Infection Research
> Inhoffenstrasse 7
> 38124 Braunschweig
> Germany
>
> Email:  joern.krau...@helmholtz-hzi.de
> Phone:  +49 (0)531 6181 7023 (office)
> +49 (0)531 6181 7020 (lab)
> 
>
> __**__
>
> Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124
> Braunschweig | www.helmholtz-hzi.de
>
> Vorsitzende des Aufsichtsrates: MinDir’in Bärbel Brumme-Bothe,
> Bundesministerium für Bildung und Forschung
> Stellvertreter: Rüdiger Eichel, Abteilungsleiter Niedersächsisches
> Ministerium für Wissenschaft und Kultur
> Geschäftsführung: Prof. Dr. Dirk Heinz; Ulf Richter, MBA
> Gesellschaft mit beschränkter Haftung (GmbH)
> Sitz der Gesellschaft: Braunschweig
> Handelsregister: Amtsgericht Braunschweig, HRB 477
>



-- 
Filip Van Petegem, PhD
Associate Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/

Re: [ccp4bb] Puzzling electron density upon heavy metal soaking

[Dom Bellini]

Hi,

I have enocountered a similar effect with WO4- derivatives, I found a huge blob 
with some little resemblence to icosahedral features, some kind of mini 10 x W 
bucky ball.

I was wondering whether there could be any slightly possibility that your 
protein may be able of some kind of yet unknown chemistry that could react with 
the Co(II) and create some type of new hexagonal molecule that was not present 
in the original conditions? e.g., even perhaps an heptaCo(II) molecule or some 
other weird thing?

D



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Joern Krausze 
[jk...@helmholtz-hzi.de]
Sent: 01 November 2012 18:45
To: ccp4bb
Subject: [ccp4bb] Puzzling electron density upon heavy metal soaking

Dear all,

I have two isomorphous crystals of the same protein. One crystal, let's
call it 'derivative', was soaked with 10 mM CoCl2 whereas the other, let's
call it 'original', was not. Both crystals were otherwise grown under
identical conditions (see below) and treated the same. In the derivative,
I see some puzzling electron density which I do not see in the original
(find two pictures under the links below; the waters in the figures are
just put in for your convenience, they were no part of the refinement).
The density forms a planar hexagon of spheres with another sphere in the
exact center. The distance from corner to corner is about 3.0 A but
varies. The distance from one corner to the center is about 3.1 A. The
hexagon is almost symmetrical as the angles enclosed by two edges are
about 120° each. This density shows up at two positions, once located
between two aspartate residues and once between an aspartate and a
tyrosine residue. These are, however, no special postions. It seems
obvious that this density is caused by the presence of Co(II) since it
only shows up in the derivative and also coincides with peaks in the
difference fourier map. I am unable to interpret it in a way that makes
sense. Could anyone of you help me figure out what to build in there?

Crystallization condition for both original and derivative was 200 mM
Tris pH 8.8, 200 mM NaCl, 21% PEG 6000, 20% glycerol.

The high-resolution limit of the dataset is 2.0 A. Data were collected at
lambda=0.981 A. Anomalous data were collected at Co peak wavelength (weak
SigAno but good enough for difference fourier map).

links:

http://imageshack.us/a/img89/9376/unknowndensity1q.png
http://imageshack.us/a/img696/7135/unknowndensity2.png

Thank you in advance!

Joern

**
Address:

Joern Krausze
Molecular Structural Biology
Helmholtz Centre for Infection Research
Inhoffenstrasse 7
38124 Braunschweig
Germany

Email:  joern.krau...@helmholtz-hzi.de
Phone:  +49 (0)531 6181 7023 (office)
 +49 (0)531 6181 7020 (lab)
**



Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124 
Braunschweig | www.helmholtz-hzi.de

Vorsitzende des Aufsichtsrates: MinDir’in Bärbel Brumme-Bothe, 
Bundesministerium für Bildung und Forschung
Stellvertreter: Rüdiger Eichel, Abteilungsleiter Niedersächsisches Ministerium 
für Wissenschaft und Kultur
Geschäftsführung: Prof. Dr. Dirk Heinz; Ulf Richter, MBA
Gesellschaft mit beschränkter Haftung (GmbH)
Sitz der Gesellschaft: Braunschweig
Handelsregister: Amtsgericht Braunschweig, HRB 477


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Re: [ccp4bb] Puzzling electron density upon heavy metal soaking

[Joern Krausze]

Thank you for your efforts!

@BR: Yes, I do have anomalous data which indicate that the density stems 
from Co(II) or anything else that shows significant anomalous dispersion 
at 1.6083 A. I just cannot picture 7 Co(II) ions forming a perfectly 
planar hexagonal structure with one Co(II) in the center. Even alternating 
Co(II) and Cl- ions or anything similar make no chemical sense. I might 
simply lack the chemical understanding, though.


@bob: I checked the I6P. With a diameter of about 8.3 A is it 2 A too
wide.

Regards,
Joern

**
Address:

Joern Krausze
Molecular Structural Biology
Helmholtz Centre for Infection Research
Inhoffenstrasse 7
38124 Braunschweig
Germany

Email:  joern.krau...@helmholtz-hzi.de
Phone:  +49 (0)531 6181 7023 (office)
+49 (0)531 6181 7020 (lab)
**

On Thu, 1 Nov 2012, Joern Krausze wrote:


Dear all,

I have two isomorphous crystals of the same protein. One crystal, let's
call it 'derivative', was soaked with 10 mM CoCl2 whereas the other, let's
call it 'original', was not. Both crystals were otherwise grown under
identical conditions (see below) and treated the same. In the derivative,
I see some puzzling electron density which I do not see in the original
(find two pictures under the links below; the waters in the figures are
just put in for your convenience, they were no part of the refinement).
The density forms a planar hexagon of spheres with another sphere in the
exact center. The distance from corner to corner is about 3.0 A but
varies. The distance from one corner to the center is about 3.1 A. The
hexagon is almost symmetrical as the angles enclosed by two edges are
about 120° each. This density shows up at two positions, once located
between two aspartate residues and once between an aspartate and a
tyrosine residue. These are, however, no special postions. It seems
obvious that this density is caused by the presence of Co(II) since it
only shows up in the derivative and also coincides with peaks in the
difference fourier map. I am unable to interpret it in a way that makes
sense. Could anyone of you help me figure out what to build in there?

Crystallization condition for both original and derivative was 200 mM
Tris pH 8.8, 200 mM NaCl, 21% PEG 6000, 20% glycerol.

The high-resolution limit of the dataset is 2.0 A. Data were collected at
lambda=0.981 A. Anomalous data were collected at Co peak wavelength (weak
SigAno but good enough for difference fourier map).

links:

http://imageshack.us/a/img89/9376/unknowndensity1q.png
http://imageshack.us/a/img696/7135/unknowndensity2.png

Thank you in advance!

Joern

**
Address:

Joern Krausze
Molecular Structural Biology
Helmholtz Centre for Infection Research
Inhoffenstrasse 7
38124 Braunschweig
Germany

Email:  joern.krau...@helmholtz-hzi.de
Phone:  +49 (0)531 6181 7023 (office)
   +49 (0)531 6181 7020 (lab)
**



Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124 
Braunschweig | www.helmholtz-hzi.de


Vorsitzende des Aufsichtsrates: MinDir’in Bärbel Brumme-Bothe, 
Bundesministerium für Bildung und Forschung
Stellvertreter: Rüdiger Eichel, Abteilungsleiter Niedersächsisches 
Ministerium für Wissenschaft und Kultur

Geschäftsführung: Prof. Dr. Dirk Heinz; Ulf Richter, MBA
Gesellschaft mit beschränkter Haftung (GmbH)
Sitz der Gesellschaft: Braunschweig
Handelsregister: Amtsgericht Braunschweig, HRB 477

[ccp4bb] Puzzling electron density upon heavy metal soaking

[Joern Krausze]

Dear all,

I have two isomorphous crystals of the same protein. One crystal, let's
call it 'derivative', was soaked with 10 mM CoCl2 whereas the other, let's
call it 'original', was not. Both crystals were otherwise grown under
identical conditions (see below) and treated the same. In the derivative,
I see some puzzling electron density which I do not see in the original
(find two pictures under the links below; the waters in the figures are
just put in for your convenience, they were no part of the refinement).
The density forms a planar hexagon of spheres with another sphere in the
exact center. The distance from corner to corner is about 3.0 A but
varies. The distance from one corner to the center is about 3.1 A. The
hexagon is almost symmetrical as the angles enclosed by two edges are
about 120° each. This density shows up at two positions, once located
between two aspartate residues and once between an aspartate and a
tyrosine residue. These are, however, no special postions. It seems
obvious that this density is caused by the presence of Co(II) since it
only shows up in the derivative and also coincides with peaks in the
difference fourier map. I am unable to interpret it in a way that makes
sense. Could anyone of you help me figure out what to build in there?

Crystallization condition for both original and derivative was 200 mM
Tris pH 8.8, 200 mM NaCl, 21% PEG 6000, 20% glycerol.

The high-resolution limit of the dataset is 2.0 A. Data were collected at
lambda=0.981 A. Anomalous data were collected at Co peak wavelength (weak
SigAno but good enough for difference fourier map).

links:

http://imageshack.us/a/img89/9376/unknowndensity1q.png
http://imageshack.us/a/img696/7135/unknowndensity2.png

Thank you in advance!

Joern

**
Address:

Joern Krausze
Molecular Structural Biology
Helmholtz Centre for Infection Research
Inhoffenstrasse 7
38124 Braunschweig
Germany

Email:  joern.krau...@helmholtz-hzi.de
Phone:  +49 (0)531 6181 7023 (office)
+49 (0)531 6181 7020 (lab)
**



Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124 
Braunschweig | www.helmholtz-hzi.de

Vorsitzende des Aufsichtsrates: MinDir’in Bärbel Brumme-Bothe, 
Bundesministerium für Bildung und Forschung
Stellvertreter: Rüdiger Eichel, Abteilungsleiter Niedersächsisches Ministerium 
für Wissenschaft und Kultur
Geschäftsführung: Prof. Dr. Dirk Heinz; Ulf Richter, MBA
Gesellschaft mit beschränkter Haftung (GmbH)
Sitz der Gesellschaft: Braunschweig
Handelsregister: Amtsgericht Braunschweig, HRB 477