[ccp4bb] RES: [ccp4bb] 60 kDa contamination in Rosetta cells

2021-07-13 Thread Rafael Marques 
I do agree with the others about what they said previously and I would not 
bother about the ~60kDa protein. I suppose the tag is in the 15kDa one by the 
amount of protein you have in the gel. I think you might be facing two 
different “problems”. One of your proteins (or the complex) is precipitating 
during SEC. Do you centrifuge your samples before running SEC? Do you keep them 
in 4ºC? What concentration do you get after your first elution? Your sample may 
look very clean in solution but maybe this is not really real.

The other “problem” is that maybe your proteins are forming bigger oligomers in 
solution. You can try native gel to find this out but firstly I would use 
another SEC column, a bigger one. Superdex 200 10/300 is an analytical column 
and should not be used for purification itself (although I have done this many 
times). I would try maybe S200 16/60.

Best


Rafael Marques da Silva
Mestrando em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"


De: Dilip Badgujar
Enviado:terça-feira, 13 de julho de 2021 11:23
Para: CCP4BB@JISCMAIL.AC.UK
Assunto: Re: [ccp4bb] 60 kDa contamination in Rosetta cells

Hello,
Please find the attached gel picture for the reference and some additional 
information.
Lysis buffer-50 mM Tris-pH 8.0, 300 mM NaCl, 1 mM DTT. 1X protease inhibitor 
cocktail.
SEC column – Superdex 200. 16/300
One of the proteins is 37 kDa while the other one is15 kDa. I do not think that 
they are making that strong heterodimer. The total molecular of the complex 
would be ~ 52 kDa but now it is around 60 kDa. I am currently using lower 
temperature (16 ᵒC) but can try around 12 with low IPTG conc.
Regards
Dilip Badgujar

On Mon, Jul 12, 2021 at 11:56 PM zaigham khan 
mailto:mahmood.zaig...@gmail.com>> wrote:
Hey Dilip,

There are many reasons for this observation. Rafael is right, please do share 
the image of the gel. Also what are the exact sizes of the two proteins that 
you co-expressed? I have observed the heterodimeric and pentameric proteins on 
SDS-PAGE albeit the presence of DTT in the sample buffer, and despite boiling 
of the samples. Could this be that 60 KD is actually the hetero-dimer? One can 
perform western blotting, followed by the use of anti-polyhistidine and 
anti-Streptavidin antibodies on separate blots to confirm the suspected bands. 
Likewise SEC followed by WB may confirm the identity of the eluted proteins in 
different fractions after size exclusion chromatography. You may also cleave 
the tags, and then see the magic!

Tom has correctly pointed out that induction at lower temperature is best 
achieved upon incubation of culture so that the temperature is dropped before 
the induction.

Bon Voyage!

-Z


Zaigham M Khan, PhD
Associate Scientist

Icahn School of Medicine at Mount Sinai
Department of Oncological Sciences
1470 Madison Avenue
New York
United States


On Mon, Jul 12, 2021 at 2:45 AM Dilip Badgujar 
mailto:dilip@gmail.com>> wrote:
Greetings, everyone
I am trying to co-express two mammalian proteins (less than 50 kDa MW) in 
Rosetta cells but getting a contaminating band around 60 kDa. One of the 
construct is in pET28a with His tag while the other is in pET21c with Strep tag 
and I am adding all the three selection markers during growth of pre-culture 
and during induction. Initially, cells were grown at 37 °C till OD reaches to 
0.6 then induced with 0.5mM IPTG and incubated at 16°C ON. When I do 
purification using Streptactin resin; I can see proteins of my interest bound 
to the resin along with contaminating protein at 60 kDa. I have tried 
performing size exclusion as a follow up step but they are co-eluting in void 
volume. I have also tried to wash with MgCL2-ATP solution but it co-elution 
with contaminant. I am looking for valuable suggestions to avoid the 
contamination during or after expression.
Thanks in advance.
Regards
Dilip



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--
Dilip C. Badgujar, (PhD)
Post Doctoral Fellow,
IIT Bomaby



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Re: [ccp4bb] [External] Re: [ccp4bb] 60 kDa contamination in Rosetta cells

2021-07-13 Thread Srivastava, Dhiraj 
Sorry I got confused with the wrong labeling. But the stoichiometry issue and 
poor expression is still the main problem. As Tom suggested, you should try to 
improve the expression. Poor expression often results in impurities 
copurifying. Also, if the interaction is not tight, why are you co-expressing? 
Unless coexpression is helping in some way (solubility ) you are better off 
expressing (and purifying) them separately and then mixing them. often, when 
you co-express multiple proteins, you get poor expression as well.


Dhiraj

From: CCP4 bulletin board  on behalf of Tom Huxford 

Sent: Tuesday, July 13, 2021 11:27 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] 60 kDa contamination in Rosetta cells

I agree with Dhiraj' suggestion that the 60 kDa is not your biggest concern.  
Optimize soluble protein expression and pay attention to cell lysis.  Is the 
sample remaining cold throughout the process?  Are you using enough volume of 
buffer relative to solid cell pellet?

Also, from the gel it does not look like your 37 and 15 kDa bands are 
co-eluting.  And your lane labels in the figure are not right.

Good news:  this seems like a solvable problem.  Keep after it!

Tom Huxford.

==
Tom Huxford
Structural Biochemistry Laboratory
Department of Chemistry & Biochemistry
San Diego State University
(619) 594-1606

On Jul 13, 2021, at 5:22 AM, Dilip Badgujar 
mailto:dilip@gmail.com>> wrote:


Hello,

Please find the attached gel picture for the reference and some additional 
information.

Lysis buffer-50 mM Tris-pH 8.0, 300 mM NaCl, 1 mM DTT. 1X protease inhibitor 
cocktail.

SEC column – Superdex 200. 16/300

One of the proteins is 37 kDa while the other one is15 kDa. I do not think that 
they are making that strong heterodimer. The total molecular of the complex 
would be ~ 52 kDa but now it is around 60 kDa. I am currently using lower 
temperature (16 ᵒC) but can try around 12 with low IPTG conc.

Regards

Dilip Badgujar

On Mon, Jul 12, 2021 at 11:56 PM zaigham khan 
mailto:mahmood.zaig...@gmail.com>> wrote:
Hey Dilip,

There are many reasons for this observation. Rafael is right, please do share 
the image of the gel. Also what are the exact sizes of the two proteins that 
you co-expressed? I have observed the heterodimeric and pentameric proteins on 
SDS-PAGE albeit the presence of DTT in the sample buffer, and despite boiling 
of the samples. Could this be that 60 KD is actually the hetero-dimer? One can 
perform western blotting, followed by the use of anti-polyhistidine and 
anti-Streptavidin antibodies on separate blots to confirm the suspected bands. 
Likewise SEC followed by WB may confirm the identity of the eluted proteins in 
different fractions after size exclusion chromatography. You may also cleave 
the tags, and then see the magic!

Tom has correctly pointed out that induction at lower temperature is best 
achieved upon incubation of culture so that the temperature is dropped before 
the induction.

Bon Voyage!

-Z


Zaigham M Khan, PhD
Associate Scientist

Icahn School of Medicine at Mount Sinai
Department of Oncological Sciences
1470 Madison Avenue
New York
United States


On Mon, Jul 12, 2021 at 2:45 AM Dilip Badgujar 
mailto:dilip@gmail.com>> wrote:
Greetings, everyone

I am trying to co-express two mammalian proteins (less than 50 kDa MW) in 
Rosetta cells but getting a contaminating band around 60 kDa. One of the 
construct is in pET28a with His tag while the other is in pET21c with Strep tag 
and I am adding all the three selection markers during growth of pre-culture 
and during induction. Initially, cells were grown at 37 °C till OD reaches to 
0.6 then induced with 0.5mM IPTG and incubated at 16°C ON. When I do 
purification using Streptactin resin; I can see proteins of my interest bound 
to the resin along with contaminating protein at 60 kDa. I have tried 
performing size exclusion as a follow up step but they are co-eluting in void 
volume. I have also tried to wash with MgCL2-ATP solution but it co-elution 
with contaminant. I am looking for valuable suggestions to avoid the 
contamination during or after expression.

Thanks in advance.

Regards

Dilip



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--
Dilip C. Badgujar, (PhD)
Post Doctoral Fellow,
IIT Bomaby




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This message

Re: [ccp4bb] 60 kDa contamination in Rosetta cells

2021-07-13 Thread Tom Huxford 
I agree with Dhiraj' suggestion that the 60 kDa is not your biggest concern.  
Optimize soluble protein expression and pay attention to cell lysis.  Is the 
sample remaining cold throughout the process?  Are you using enough volume of 
buffer relative to solid cell pellet?

Also, from the gel it does not look like your 37 and 15 kDa bands are 
co-eluting.  And your lane labels in the figure are not right.

Good news:  this seems like a solvable problem.  Keep after it!

Tom Huxford.

==
Tom Huxford
Structural Biochemistry Laboratory
Department of Chemistry & Biochemistry
San Diego State University
(619) 594-1606

> On Jul 13, 2021, at 5:22 AM, Dilip Badgujar  wrote:
> 
> Hello, 
> 
> Please find the attached gel picture for the reference and some additional 
> information.
> 
> Lysis buffer-50 mM Tris-pH 8.0, 300 mM NaCl, 1 mM DTT. 1X protease inhibitor 
> cocktail.
> 
> SEC column – Superdex 200. 16/300
> 
> One of the proteins is 37 kDa while the other one is15 kDa. I do not think 
> that they are making that strong heterodimer. The total molecular of the 
> complex would be ~ 52 kDa but now it is around 60 kDa. I am currently using 
> lower temperature (16 ᵒC) but can try around 12 with low IPTG conc. 
> 
> Regards
> 
> Dilip Badgujar
> 
> 
> On Mon, Jul 12, 2021 at 11:56 PM zaigham khan  > wrote:
> Hey Dilip, 
> 
> There are many reasons for this observation. Rafael is right, please do share 
> the image of the gel. Also what are the exact sizes of the two proteins that 
> you co-expressed? I have observed the heterodimeric and pentameric proteins 
> on SDS-PAGE albeit the presence of DTT in the sample buffer, and despite 
> boiling of the samples. Could this be that 60 KD is actually the 
> hetero-dimer? One can perform western blotting, followed by the use of 
> anti-polyhistidine and anti-Streptavidin antibodies on separate blots to 
> confirm the suspected bands. Likewise SEC followed by WB may confirm the 
> identity of the eluted proteins in different fractions after size exclusion 
> chromatography. You may also cleave the tags, and then see the magic!
> 
> Tom has correctly pointed out that induction at lower temperature is best 
> achieved upon incubation of culture so that the temperature is dropped before 
> the induction.
> 
> Bon Voyage!
> 
> -Z
> 
> 
> Zaigham M Khan, PhD
> Associate Scientist
> 
> Icahn School of Medicine at Mount Sinai
> Department of Oncological Sciences
> 1470 Madison Avenue
> New York
> United States
> 
> 
> On Mon, Jul 12, 2021 at 2:45 AM Dilip Badgujar  > wrote:
> Greetings, everyone
> I am trying to co-express two mammalian proteins (less than 50 kDa MW) in 
> Rosetta cells but getting a contaminating band around 60 kDa. One of the 
> construct is in pET28a with His tag while the other is in pET21c with Strep 
> tag and I am adding all the three selection markers during growth of 
> pre-culture and during induction. Initially, cells were grown at 37 °C till 
> OD reaches to 0.6 then induced with 0.5mM IPTG and incubated at 16°C ON. When 
> I do purification using Streptactin resin; I can see proteins of my interest 
> bound to the resin along with contaminating protein at 60 kDa. I have tried 
> performing size exclusion as a follow up step but they are co-eluting in void 
> volume. I have also tried to wash with MgCL2-ATP solution but it co-elution 
> with contaminant. I am looking for valuable suggestions to avoid the 
> contamination during or after expression. 
> 
> Thanks in advance.
> 
> Regards
> 
> Dilip 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
> 
> 
> -- 
> Dilip C. Badgujar, (PhD)
> Post Doctoral Fellow,
> IIT Bomaby
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 
> 




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Re: [ccp4bb] Problems with refining covalently linked heme cofactor

2021-07-13 Thread Boaz Shaanan 
Hi Don and Vera,

Coot will handle the ligand properly without "pre-integrating" it into the PDB. 
Read the ligand (if it's not in the monomer library already) into aceDRG (in 
any form that it accepts), then in Coot, read the generated cif file -> get 
monomer (when positioned in the correct place)- > make link (click the atoms 
you want to link) and that's it. I would start only with the covalent link to 
the iron and leave the potential links to the propionates for later, just to 
see how it works.

This is all working within the Refmac environments. As for Phenix, there has 
been a recent discussion on the issue. I'm sure Phenix people would guide 
through the necessary steps to get the Coot-Phenix combination to work.

Cheers,

 Boaz



Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220
Fax:   972-8-647-2992 or 972-8-646-1710





From: CCP4 bulletin board  on behalf of Dom Bellini - 
MRC LMB 
Sent: Tuesday, July 13, 2021 5:15 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Problems with refining covalently linked heme cofactor

Dear Vera,

In my experiences I had no problems to link protein atoms to a ligand in Coot. 
Did you check that you merged the cofactor to the protein molecule first? 
otherwise that could be one reason why Coot is not creating the link.

BW,

D

On 13/07/2021 14:33, Vera Pfanzagl wrote:
Hello,

I am trying to refine a heme protein which has the heme cofactor covalently 
linked to the protein at three positions (two ester linkages and a sulfilimin 
bond) (coot.png shows the atoms which should be in the sulfilimin bond). 
However as  as coot cannot covalently link atoms between a cofactor and amino 
acids the atoms are displaced during the refinement and I don't know how to fix 
this. Also the heme b disintegrates if I run it through phenix and then try to 
refine it manually in coot. The hydrogen atoms just fly off. I can delete the 
hydrogens before running a manual refinement but this problem reappears after 
adding the hydrogens during phenix refine.

I now tried to replace it with HEB from the coot monomer library (I cannot find 
the old HEM in the monomer library) and got once the same result (atoms are 
displaced) and in the second case I cannot even place HEB correctly as some 
atoms rearrange after refinement (heb_wierd.png) and two hydrogens are 
connected when I add it from the library (heb_withoutrefine.png)

Thanks, Vera



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https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1


--
Dom Bellini, Xray Crystallography Facility (1S205)
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH
Phone 01223 267839



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Re: [ccp4bb] sphere refine radius in coot 0.9.5.1

2021-07-13 Thread Paul Emsley 
On Fri, 2021-07-09 at 13:49 -0400, Ashok Nayak wrote:
> 
> Is it possible to increase the radius of real space refinement

Yes. Use sphere_refine with an argument, e.g. sphere_refine(8.5)

You can add your own button to do this using Right Mouse on the Tool bar and 
selecting "Add a User-defined button"

>  while manually adjusting models in Coot with the sphere refine option?

Let me advise you not to use "manual" when describing your work using Coot.

The is an optimizer behind almost every editing tool which means that Coot is 
deciding where there atoms go, not you.
If you want to distinguish between work in Coot and building done by (say) 
buccaneer, then call it "interactive."

Paul.



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Re: [ccp4bb] [External] Re: [ccp4bb] 60 kDa contamination in Rosetta cells

2021-07-13 Thread Srivastava, Dhiraj 
Hi Dilip
  It seems like your problem is poor stoichiometry. 37 kda protein 
is poorly expressed. I wouldn’t be worried too much about 60 kda protein. 
At-least at this stage. I might be mistaken but based on previous post, it 
seems like your protein was aggregated. If it is so, you need to improve on 
aggregation and stoichiometry. With better yield of complex, 60 kda impurity 
will be minor.

Dhiraj

From: CCP4 bulletin board  on behalf of Dilip Badgujar 

Sent: Tuesday, July 13, 2021 7:22 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] 60 kDa contamination in Rosetta cells


Hello,

Please find the attached gel picture for the reference and some additional 
information.

Lysis buffer-50 mM Tris-pH 8.0, 300 mM NaCl, 1 mM DTT. 1X protease inhibitor 
cocktail.

SEC column – Superdex 200. 16/300

One of the proteins is 37 kDa while the other one is15 kDa. I do not think that 
they are making that strong heterodimer. The total molecular of the complex 
would be ~ 52 kDa but now it is around 60 kDa. I am currently using lower 
temperature (16 ᵒC) but can try around 12 with low IPTG conc.

Regards

Dilip Badgujar

On Mon, Jul 12, 2021 at 11:56 PM zaigham khan 
mailto:mahmood.zaig...@gmail.com>> wrote:
Hey Dilip,

There are many reasons for this observation. Rafael is right, please do share 
the image of the gel. Also what are the exact sizes of the two proteins that 
you co-expressed? I have observed the heterodimeric and pentameric proteins on 
SDS-PAGE albeit the presence of DTT in the sample buffer, and despite boiling 
of the samples. Could this be that 60 KD is actually the hetero-dimer? One can 
perform western blotting, followed by the use of anti-polyhistidine and 
anti-Streptavidin antibodies on separate blots to confirm the suspected bands. 
Likewise SEC followed by WB may confirm the identity of the eluted proteins in 
different fractions after size exclusion chromatography. You may also cleave 
the tags, and then see the magic!

Tom has correctly pointed out that induction at lower temperature is best 
achieved upon incubation of culture so that the temperature is dropped before 
the induction.

Bon Voyage!

-Z


Zaigham M Khan, PhD
Associate Scientist

Icahn School of Medicine at Mount Sinai
Department of Oncological Sciences
1470 Madison Avenue
New York
United States


On Mon, Jul 12, 2021 at 2:45 AM Dilip Badgujar 
mailto:dilip@gmail.com>> wrote:
Greetings, everyone

I am trying to co-express two mammalian proteins (less than 50 kDa MW) in 
Rosetta cells but getting a contaminating band around 60 kDa. One of the 
construct is in pET28a with His tag while the other is in pET21c with Strep tag 
and I am adding all the three selection markers during growth of pre-culture 
and during induction. Initially, cells were grown at 37 °C till OD reaches to 
0.6 then induced with 0.5mM IPTG and incubated at 16°C ON. When I do 
purification using Streptactin resin; I can see proteins of my interest bound 
to the resin along with contaminating protein at 60 kDa. I have tried 
performing size exclusion as a follow up step but they are co-eluting in void 
volume. I have also tried to wash with MgCL2-ATP solution but it co-elution 
with contaminant. I am looking for valuable suggestions to avoid the 
contamination during or after expression.

Thanks in advance.

Regards

Dilip



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https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1


--
Dilip C. Badgujar, (PhD)
Post Doctoral Fellow,
IIT Bomaby




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Re: [ccp4bb] Problems with refining covalently linked heme cofactor

2021-07-13 Thread Roversi, Pietro (Dr.) 
Try Buster!

Pietro

Get Outlook for iOS

From: CCP4 bulletin board  on behalf of Vera Pfanzagl 

Sent: Tuesday, July 13, 2021 3:33:04 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Problems with refining covalently linked heme cofactor

Hello,

I am trying to refine a heme protein which has the heme cofactor covalently 
linked to the protein at three positions (two ester linkages and a sulfilimin 
bond) (coot.png shows the atoms which should be in the sulfilimin bond). 
However as  as coot cannot covalently link atoms between a cofactor and amino 
acids the atoms are displaced during the refinement and I don't know how to fix 
this. Also the heme b disintegrates if I run it through phenix and then try to 
refine it manually in coot. The hydrogen atoms just fly off. I can delete the 
hydrogens before running a manual refinement but this problem reappears after 
adding the hydrogens during phenix refine.

I now tried to replace it with HEB from the coot monomer library (I cannot find 
the old HEM in the monomer library) and got once the same result (atoms are 
displaced) and in the second case I cannot even place HEB correctly as some 
atoms rearrange after refinement (heb_wierd.png) and two hydrogens are 
connected when I add it from the library (heb_withoutrefine.png)

Thanks, Vera



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Re: [ccp4bb] Problems with refining covalently linked heme cofactor

2021-07-13 Thread Dom Bellini - MRC LMB 

Dear Vera,

In my experiences I had no problems to link protein atoms to a ligand in 
Coot. Did you check that you merged the cofactor to the protein molecule 
first? otherwise that could be one reason why Coot is not creating the link.


BW,

D

On 13/07/2021 14:33, Vera Pfanzagl wrote:

Hello,

I am trying to refine a heme protein which has the heme cofactor 
covalently linked to the protein at three positions (two ester 
linkages and a sulfilimin bond) (coot.png shows the atoms which should 
be in the sulfilimin bond). However as  as coot cannot covalently link 
atoms between a cofactor and amino acids the atoms are displaced 
during the refinement and I don't know how to fix this. Also the heme 
b disintegrates if I run it through phenix and then try to refine it 
manually in coot. The hydrogen atoms just fly off. I can delete the 
hydrogens before running a manual refinement but this problem 
reappears after adding the hydrogens during phenix refine.


I now tried to replace it with HEB from the coot monomer library (I 
cannot find the old HEM in the monomer library) and got once the same 
result (atoms are displaced) and in the second case I cannot even 
place HEB correctly as some atoms rearrange after refinement 
(heb_wierd.png) and two hydrogens are connected when I add it from the 
library (heb_withoutrefine.png)


Thanks, Vera



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https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 





--
Dom Bellini, Xray Crystallography Facility (1S205)
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH
Phone 01223 267839




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