Re: [ccp4bb] Heterogeneity during purification

[Theresa Hsu]

Dear Tony

Yes, there are four cysteines. 

Theresa


On Tue, 9 Jul 2013 06:26:19 +, Antony Oliver  
wrote:

>Theresa,
>
>Are there any cysteines in your protein?
>
>Tony.
>
>On 9 Jul 2013, at 05:01, Theresa Hsu  wrote:
>
>> Dear all
>> 
>> I am working on a 30 kDa membrane protein which forms a functional dimer. 
>> The protein is His-tagged at N-terminal. In small scale expression screening 
>> from whole cells, there is only a single band on Western blot at 30 kDa. 
>> But, after purification, additional bands appear at 60 and 120 kDa on 
>> SDS-PAGE and Western blot. On size exclusion with Superdex 200, a large 
>> proportion elute near the void volume (8 ml).
>> 
>> Detail purification
>> 
>> For small scale screening, I lysed cells in 20 mM Tris pH 8, 100 mM NaCl, 1 
>> mg/ml lysozyme, 1 % DDM and DNAse for 2 hours and then centrifuged at 16000 
>> g. I then checked the supernatant on SDS-PAGE and scale it up for 
>> purification.
>> 
>> For purification, I use the buffer 50 mM Tris pH 8, 300 mM NaCl, 20 mM 
>> imidazole, 0.05 % DDM (two times CMC of DDM).
>> 
>> Is there suggestion to get homogeneous protein?
>> 
>> Thank you.
>> 
>> Theresa

Re: [ccp4bb] Heterogeneity during purification

[Antony Oliver]

Theresa, 

I would suggest then, that you include reducing agent in your buffer (it 
doesn't appear to be to be there) - TCEP is good, as you can include in right 
from the start as it doesn't interfere with the IMAC columns.  My suspicion is 
that you are seeing multimerisation through inappropriate di-suphide formation.

Tony.


On 9 Jul 2013, at 08:18, Theresa Hsu 
 wrote:

> Dear Tony
> 
> Yes, there are four cysteines. 
> 
> Theresa
> 
> 
> On Tue, 9 Jul 2013 06:26:19 +, Antony Oliver  
> wrote:
> 
>> Theresa,
>> 
>> Are there any cysteines in your protein?
>> 
>> Tony.
>> 
>> On 9 Jul 2013, at 05:01, Theresa Hsu  wrote:
>> 
>>> Dear all
>>> 
>>> I am working on a 30 kDa membrane protein which forms a functional dimer. 
>>> The protein is His-tagged at N-terminal. In small scale expression 
>>> screening from whole cells, there is only a single band on Western blot at 
>>> 30 kDa. But, after purification, additional bands appear at 60 and 120 kDa 
>>> on SDS-PAGE and Western blot. On size exclusion with Superdex 200, a large 
>>> proportion elute near the void volume (8 ml).
>>> 
>>> Detail purification
>>> 
>>> For small scale screening, I lysed cells in 20 mM Tris pH 8, 100 mM NaCl, 1 
>>> mg/ml lysozyme, 1 % DDM and DNAse for 2 hours and then centrifuged at 16000 
>>> g. I then checked the supernatant on SDS-PAGE and scale it up for 
>>> purification.
>>> 
>>> For purification, I use the buffer 50 mM Tris pH 8, 300 mM NaCl, 20 mM 
>>> imidazole, 0.05 % DDM (two times CMC of DDM).
>>> 
>>> Is there suggestion to get homogeneous protein?
>>> 
>>> Thank you.
>>> 
>>> Theresa
> 
> 

Re: [ccp4bb] Heterogeneity during purification

[Bert Van-Den-Berg]

since your protein aggregates even in a mild detergent you may have to find an 
ortholog that is more stable.
however, there are a few things you can try before moving on (in arbitrary 
order):
1. add glycerol during purification (5-20%)
2. get rid of the imidazole as fast as possible after Ni. Many proteins do not 
like imidazole at all.
3. explore different buffer conditions during purification, especially 
regarding ionic strength and pH.
4. adding reducing agents may help, but given that your aggregates are large I 
expect this may not help that much.
5. add lipids and/or substrate/inhibitor etc during purification.

Good luck, Bert

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Theresa Hsu 
[theresah...@live.com]
Sent: Tuesday, July 09, 2013 5:01 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Heterogeneity during purification

Dear all

I am working on a 30 kDa membrane protein which forms a functional dimer. The 
protein is His-tagged at N-terminal. In small scale expression screening from 
whole cells, there is only a single band on Western blot at 30 kDa. But, after 
purification, additional bands appear at 60 and 120 kDa on SDS-PAGE and Western 
blot. On size exclusion with Superdex 200, a large proportion elute near the 
void volume (8 ml).

Detail purification

For small scale screening, I lysed cells in 20 mM Tris pH 8, 100 mM NaCl, 1 
mg/ml lysozyme, 1 % DDM and DNAse for 2 hours and then centrifuged at 16000 g. 
I then checked the supernatant on SDS-PAGE and scale it up for purification.

For purification, I use the buffer 50 mM Tris pH 8, 300 mM NaCl, 20 mM 
imidazole, 0.05 % DDM (two times CMC of DDM).

Is there suggestion to get homogeneous protein?

Thank you.

Theresa

Re: [ccp4bb] modified amino acids in the PDB

[MARTYN SYMMONS]

Hi Clemens
   I guess the reason you say 'arbitrary' is because there is no explanation of 
this rule decision? 
  It would be nice if some rationalization was available alongside the values 
given. So a sentence along the lines of 'we set the number owing to the 
following considerations' ? 

  However a further layer of variation is that the rule does not seem to be 
consistently applied
 - just browsing CYS modifications:
   iodoacetamide treatment gives a CYS with only 4 additional atoms but it is 
split off as  ACM.
   However some ligands much larger than 10 residues have been kept with the 
cysteine ( for example CY7 in 2jiv and NPH in 1a18.  
   My betting is that it depends on whether something has been seen 'going 
solo' as a non-covalent ligand previously so that it pops up as an atomic 
structural match with a pre-defined three-letter code.
  This would explain for example the ACM case which you might expect to occur 
in a modified Cys.  But it has also been observed as a non-polymer ligand in 
its own right so goes on as a separate modification?
   However to be honest I am not sure I have ever seen the rationale for this 
written down. 

  'Non-polymer' heterogens can turn up either linked or not. Once they are in 
the residues they have to make a call on which kind of backbone they will 
feature in within the pdb.
  That is why there is  'D5M' for non-polymer deoxyAMP. Also known as ' DA' 
when it is 'DNA-linking' but so far not fessing up to life under a third code 
as 'RNA-linking' 

Now is perhaps the time to ask for explanations of these nomenclature features 
before they become hard-wired in the new pdb deposition system (however there 
may be time - I refer you to my previous posting ;). 
  
 Cheers
    Martyn 
  
Dr Martyn Symmons
Cambridge



 From: Michael Weyand 
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Monday, 8 July 2013, 10:03
Subject: [ccp4bb] modified amino acids in the PDB
 

Dear colleagues,

We deposited protein structures with modified lysine side chains and
were surprised that the PDB treats the modification as an independent
molecule, with a “LINK” record indicating the covalent bond – instead of
defining a modified residue (that’s what we had uploaded to the PDB).
Apparently, anything attached to an amino acid is considered an
independent molecule (and the lysine just called a regular lysine) if it
comprises more than 10 atoms (see below for the PDB guidelines).

I think that’s kind of arbitrary and would give all modified residue
also modified names – i.e. individual names for all modified lysines, as
it is done for acetyl- or methyl-lysines, for example. I wonder what
other people’s opinion is?!

Best regards

Clemens



This is in accordance to the wwPDB annotation guidelines
(http://www.wwpdb.org/procedure.html#toc_2).
"*Modified amino acids and nucleotides* If an amino acid or nucleotide
is modified by a chemical group greater than 10 atoms, the residue will
be split into two groups: the amino acid/nucleotide group and the
modification. A link record will be generated between the amino
acid/nucleotide group and the modification. For modified amino acids and
nucleotides that were not split will follow standard atom nomenclature."

Re: [ccp4bb] modified amino acids in the PDB

[Frances C. Bernstein]

In trying to formulate a suggested policy on het groups
versus modified side chains one needs to think about the
various cases that have arisen.

Perhaps the earliest one I can think of is a heme group.
One could view it as a very large decoration on a side
chain but, as everyone knows, one heme group makes four
links to residues.  In the early days of the PDB we decided
that heme "obviously" had to be represented as a separate group.

I would also point out that nobody would seriously suggest that
selenomethionine should be represented as a methionine with a
missing sulfur and a selenium het group bound to it.

Unfortunately all the cases that fall between selenomethionine
and heme are more difficult.  Perhaps the best that one must
hope for is that whichever representation is chosen for a
particular case, it be consistent across all entries.

  Frances

P.S. One can also have similar discussions about the representation
of microheterogeneity and of sugar chains but we should leave those
for another day.

=
Bernstein + Sons
*   *   Information Systems Consultants
5 Brewster Lane, Bellport, NY 11713-2803
*   * ***
 *Frances C. Bernstein
  *   ***  f...@bernstein-plus-sons.com
 *** *
  *   *** 1-631-286-1339FAX: 1-631-286-1999
=

On Tue, 9 Jul 2013, MARTYN SYMMONS wrote:


Hi Clemens
   I guess the reason you say 'arbitrary' is because there is no explanation of 
this
rule decision? 
  It would be nice if some rationalization was available alongside the values 
given.
So a sentence along the lines of 'we set the number owing to the following
considerations' ? 

  However a further layer of variation is that the rule does not seem to be
consistently applied
 - just browsing CYS modifications:
   iodoacetamide treatment gives a CYS with only 4 additional atoms but it is 
split
off as  ACM.
   However some ligands much larger than 10 residues have been kept with the 
cysteine
( for example CY7 in 2jiv and NPH in 1a18.  
   My betting is that it depends on whether something has been seen 'going 
solo' as a
non-covalent ligand previously so that it pops up as an atomic structural match 
with
a pre-defined three-letter code.
  This would explain for example the ACM case which you might expect to occur 
in a
modified Cys.  But it has also been observed as a non-polymer ligand in its own 
right
so goes on as a separate modification?
   However to be honest I am not sure I have ever seen the rationale for this 
written
down. 

  'Non-polymer' heterogens can turn up either linked or not. Once they are in 
the
residues they have to make a call on which kind of backbone they will feature in
within the pdb.
  That is why there is  'D5M' for non-polymer deoxyAMP. Also known as ' DA' 
when it
is 'DNA-linking' but so far not fessing up to life under a third code as
'RNA-linking' 

Now is perhaps the time to ask for explanations of these nomenclature features 
before
they become hard-wired in the new pdb deposition system (however there may be 
time -
I refer you to my previous posting ;). 
  
 Cheers
    Martyn 
  
Dr Martyn Symmons
Cambridge

_
From: Michael Weyand 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, 8 July 2013, 10:03
Subject: [ccp4bb] modified amino acids in the PDB

Dear colleagues,

We deposited protein structures with modified lysine side chains and
were surprised that the PDB treats the modification as an independent
molecule, with a ?LINK? record indicating the covalent bond ? instead of
defining a modified residue (that?s what we had uploaded to the PDB).
Apparently, anything attached to an amino acid is considered an
independent molecule (and the lysine just called a regular lysine) if it
comprises more than 10 atoms (see below for the PDB guidelines).

I think that?s kind of arbitrary and would give all modified residue
also modified names ? i.e. individual names for all modified lysines, as
it is done for acetyl- or methyl-lysines, for example. I wonder what
other people?s opinion is?!

Best regards

Clemens




This is in accordance to the wwPDB annotation guidelines
(http://www.wwpdb.org/procedure.html#toc_2).
"*Modified amino acids and nucleotides* If an amino acid or nucleotide
is modified by a chemical group greater than 10 atoms, the residue will
be split into two groups: the amino acid/nucleotide group and the
modification. A link record will be generated between the amino
acid/nucleotide group and the modification. For modified amino acids and
nucleotides that were not split will follow standard atom nomenclature."

Re: [ccp4bb] Heterogeneity during purification

[R. M. Garavito]

Dear Teresa,

In addition to Bert's excellent list, I have found that many membrane proteins 
aggregate in regular SDS-PAGE sample buffer, particularly when heated.  In the 
best case, we always see "dimers" or, in the worst case, a ladder or smear of 
aggregates.  If you heat your SDS-PAGE samples, try not heating them.  
Secondly, your observation that you have protein eluting near the void volume 
on Superdex 200 may also suggests that you have either incomplete 
solubilization (i.e., big lipid-protein complexes) or, as Bert remarks, an 
unhappy protein that aggregates in the detergent you have chosen.

However, before you consider this a problem, ask yourself if this is an 
expected result due to the heterologous overexpression of a recombinant 
protein.  Unlike purifying a "native" protein, overexpression of a recombinant 
protein can led to a considerable amount of misfolded or misassembled protein 
mixed with good protein.  As you might be overloading the membrane protein 
insertion system, you may need to dial back the induction.

Finally, were you measuring the amount of high order aggregates in SEC by A280 
or by another means?  The proportion of high order aggregates observed near the 
void volume is always skewed due to light-scattering of the much larger 
particles.  Because of this, a large void volume peak (~7.5-8 mL on a Superdex 
200 10/300 column) may contain a lot less protein than you think.  

Good luck,

Michael


R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
603 Wilson Rd., Rm. 513   
Michigan State University  
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 353-9125
FAX:  (517) 353-9334Email:  rmgarav...@gmail.com





On Jul 9, 2013, at 4:47 AM, Bert Van-Den-Berg wrote:

> since your protein aggregates even in a mild detergent you may have to find 
> an ortholog that is more stable.
> however, there are a few things you can try before moving on (in arbitrary 
> order):
> 1. add glycerol during purification (5-20%)
> 2. get rid of the imidazole as fast as possible after Ni. Many proteins do 
> not like imidazole at all.
> 3. explore different buffer conditions during purification, especially 
> regarding ionic strength and pH.
> 4. adding reducing agents may help, but given that your aggregates are large 
> I expect this may not help that much.
> 5. add lipids and/or substrate/inhibitor etc during purification.
> 
> Good luck, Bert
> 
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Theresa Hsu 
> [theresah...@live.com]
> Sent: Tuesday, July 09, 2013 5:01 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Heterogeneity during purification
> 
> Dear all
> 
> I am working on a 30 kDa membrane protein which forms a functional dimer. The 
> protein is His-tagged at N-terminal. In small scale expression screening from 
> whole cells, there is only a single band on Western blot at 30 kDa. But, 
> after purification, additional bands appear at 60 and 120 kDa on SDS-PAGE and 
> Western blot. On size exclusion with Superdex 200, a large proportion elute 
> near the void volume (8 ml).
> 
> Detail purification
> 
> For small scale screening, I lysed cells in 20 mM Tris pH 8, 100 mM NaCl, 1 
> mg/ml lysozyme, 1 % DDM and DNAse for 2 hours and then centrifuged at 16000 
> g. I then checked the supernatant on SDS-PAGE and scale it up for 
> purification.
> 
> For purification, I use the buffer 50 mM Tris pH 8, 300 mM NaCl, 20 mM 
> imidazole, 0.05 % DDM (two times CMC of DDM).
> 
> Is there suggestion to get homogeneous protein?
> 
> Thank you.
> 
> Theresa

Re: [ccp4bb] modified amino acids in the PDB

[Mark J van Raaij]

- really the only complicated case would be where a group is covalently linked 
to more than one amino acid, wouldn't it? Any case where only one covalent link 
with an is present could (should?) be treated as a special amino acid, i.e. 
like selenomethionine.
- groups without any covalent links to the protein are better kept separate I 
would think (but I guess this is stating the obvious).

Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij





On 9 Jul 2013, at 12:49, Frances C. Bernstein wrote:

> In trying to formulate a suggested policy on het groups
> versus modified side chains one needs to think about the
> various cases that have arisen.
> 
> Perhaps the earliest one I can think of is a heme group.
> One could view it as a very large decoration on a side
> chain but, as everyone knows, one heme group makes four
> links to residues.  In the early days of the PDB we decided
> that heme "obviously" had to be represented as a separate group.
> 
> I would also point out that nobody would seriously suggest that
> selenomethionine should be represented as a methionine with a
> missing sulfur and a selenium het group bound to it.
> 
> Unfortunately all the cases that fall between selenomethionine
> and heme are more difficult.  Perhaps the best that one must
> hope for is that whichever representation is chosen for a
> particular case, it be consistent across all entries.
> 
>  Frances
> 
> P.S. One can also have similar discussions about the representation
> of microheterogeneity and of sugar chains but we should leave those
> for another day.
> 
> =
> Bernstein + Sons
> *   *   Information Systems Consultants
> 5 Brewster Lane, Bellport, NY 11713-2803
> *   * ***
>  *Frances C. Bernstein
>  *   ***  f...@bernstein-plus-sons.com
> *** *
>  *   *** 1-631-286-1339FAX: 1-631-286-1999
> =
> 
> On Tue, 9 Jul 2013, MARTYN SYMMONS wrote:
> 
>> Hi Clemens
>>I guess the reason you say 'arbitrary' is because there is no explanation 
>> of this
>> rule decision? 
>>   It would be nice if some rationalization was available alongside the 
>> values given.
>> So a sentence along the lines of 'we set the number owing to the following
>> considerations' ? 
>>   However a further layer of variation is that the rule does not seem to be
>> consistently applied
>>  - just browsing CYS modifications:
>>iodoacetamide treatment gives a CYS with only 4 additional atoms but it 
>> is split
>> off as  ACM.
>>However some ligands much larger than 10 residues have been kept with the 
>> cysteine
>> ( for example CY7 in 2jiv and NPH in 1a18.  
>>My betting is that it depends on whether something has been seen 'going 
>> solo' as a
>> non-covalent ligand previously so that it pops up as an atomic structural 
>> match with
>> a pre-defined three-letter code.
>>   This would explain for example the ACM case which you might expect to 
>> occur in a
>> modified Cys.  But it has also been observed as a non-polymer ligand in its 
>> own right
>> so goes on as a separate modification?
>>However to be honest I am not sure I have ever seen the rationale for 
>> this written
>> down. 
>>   'Non-polymer' heterogens can turn up either linked or not. Once they are 
>> in the
>> residues they have to make a call on which kind of backbone they will 
>> feature in
>> within the pdb.
>>   That is why there is  'D5M' for non-polymer deoxyAMP. Also known as ' DA' 
>> when it
>> is 'DNA-linking' but so far not fessing up to life under a third code as
>> 'RNA-linking' 
>> Now is perhaps the time to ask for explanations of these nomenclature 
>> features before
>> they become hard-wired in the new pdb deposition system (however there may 
>> be time -
>> I refer you to my previous posting ;). 
>>   
>>  Cheers
>> Martyn 
>>   
>> Dr Martyn Symmons
>> Cambridge
>> _
>> From: Michael Weyand 
>> To: CCP4BB@JISCMAIL.AC.UK
>> Sent: Monday, 8 July 2013, 10:03
>> Subject: [ccp4bb] modified amino acids in the PDB
>> Dear colleagues,
>> We deposited protein structures with modified lysine side chains and
>> were surprised that the PDB treats the modification as an independent
>> molecule, with a ?LINK? record indicating the covalent bond ? instead of
>> defining a modified residue (that?s what we had uploaded to the PDB).
>> Apparently, anything attached to an amino acid is considered an
>> independent molecule (and the lysine just called a regular lysine) if it
>> comprises more than 10 atoms (see below for the PDB guidelines).
>> I think that?s kind of arbitrary and would give all modified residue
>> also modified names ? i.e.

Re: [ccp4bb] modified amino acids in the PDB

[MARTYN SYMMONS]

Hiya Mark
   once again I find myself asking why not give the authors deposited file 
alongside any other 'cleaned-up' PDB files. 
 The authors' one could be foo.pdb_0 then after PDB heterogen annotation you 
get foo.pdb_1 - if the heterogens are subsequently handled differently then you 
could have foo.pdb_2 for the 'remediated' file. 
  This latter has certainly happened as there appear to be 'orphan' heterogen 
definitions in the pdb that are not currently used by any entries - most likely 
these were 'split' when atoms were taken out and associated with polymer 
entities, or in some cases, I notice, 'lumped' when they have been included 
with residues alongside to give a new larger heterogen.
  PDB file versioning would also give transparency in cases such as occupancy 
remediation when the pdb altered all occupancies that summed to >1.0 to enforce 
a  total of 1.0 (giving holes in the authors' density presumably) which may 
mystify the sharp-eyed user.    
 Currently there are the REV_DAT lines in the header but these give only the 
titles of changed cards not any detailed explanation. Likewise REVDAT only 
starts at foo.pdb_1 in my example - still missing the authors original intent.  
  all the best
   Martyn 


 From: Mark J van Raaij 
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Tuesday, 9 July 2013, 15:23
Subject: Re: [ccp4bb] modified amino acids in the PDB
 

- really the only complicated case would be where a group is covalently linked 
to more than one amino acid, wouldn't it? Any case where only one covalent link 
with an is present could (should?) be treated as a special amino acid, i.e. 
like selenomethionine.
- groups without any covalent links to the protein are better kept separate I 
would think (but I guess this is stating the obvious).

Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij





On 9 Jul 2013, at 12:49, Frances C. Bernstein wrote:

> In trying to formulate a suggested policy on het groups
> versus modified side chains one needs to think about the
> various cases that have arisen.
> 
> Perhaps the earliest one I can think of is a heme group.
> One could view it as a very large decoration on a side
> chain but, as everyone knows, one heme group makes four
> links to residues.  In the early days of the PDB we decided
> that heme "obviously" had to be represented as a separate group.
> 
> I would also point out that nobody would seriously suggest that
> selenomethionine should be represented as a methionine with a
> missing sulfur and a selenium het group bound to it.
> 
> Unfortunately all the cases that fall between selenomethionine
> and heme are more difficult.  Perhaps the best that one must
> hope for is that whichever representation is chosen for a
> particular case, it be consistent across all entries.
> 
>                          Frances
> 
> P.S. One can also have similar discussions about the representation
> of microheterogeneity and of sugar chains but we should leave those
> for another day.
> 
> =
>                 Bernstein + Sons
> *   *       Information Systems Consultants
>     5 Brewster Lane, Bellport, NY 11713-2803
> *   * ***
>  *            Frances C. Bernstein
>  *   ***      f...@bernstein-plus-sons.com
> ***     *
>  *   *** 1-631-286-1339    FAX: 1-631-286-1999
> =
> 
> On Tue, 9 Jul 2013, MARTYN SYMMONS wrote:
> 
>> Hi Clemens
>>    I guess the reason you say 'arbitrary' is because there is no explanation 
>>of this
>> rule decision? 
>>   It would be nice if some rationalization was available alongside the 
>>values given.
>> So a sentence along the lines of 'we set the number owing to the following
>> considerations' ? 
>>   However a further layer of variation is that the rule does not seem to be
>> consistently applied
>>  - just browsing CYS modifications:
>>    iodoacetamide treatment gives a CYS with only 4 additional atoms but it 
>>is split
>> off as  ACM.
>>    However some ligands much larger than 10 residues have been kept with the 
>>cysteine
>> ( for example CY7 in 2jiv and NPH in 1a18.  
>>    My betting is that it depends on whether something has been seen 'going 
>>solo' as a
>> non-covalent ligand previously so that it pops up as an atomic structural 
>> match with
>> a pre-defined three-letter code.
>>   This would explain for example the ACM case which you might expect to 
>>occur in a
>> modified Cys.  But it has also been observed as a non-polymer ligand in its 
>> own right
>> so goes on as a separate modification?
>>    However to be honest I am not sure I have ever seen the rationale for 
>>this written
>> down. 
>>   'Non-polymer' heterogens can turn up either linked or not. Once they are 
>>in the
>> residues they have to make a call on which ki

[ccp4bb] AW: [ccp4bb] modified amino acids in the PDB

[Herman . Schreuder]

Dear Marc (and BB),

I guess as usual, in real life the obvious is less obvious as it seems to be. 
I, and I guess many of my colleagues trying to find new drugs, have quite a few 
protein-inhibitor complexes where the inhibitor formed a covalent link with 
e.g. the active site serine. In these cases, I am perfectly happy with having 
the inhibitor being defined as a separate group, linked via a LINK record. For 
me, it does not make sense to treat these covalent inhibitors differently from 
noncovalent inhibitors.

In the end, I guess, it will boil down to some arbitrary choice, either imposed 
upon us by the pdb, or individually taken by the crystallographer who produced 
the crystal structure.

My 2 cts,
Herman
 

-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mark J 
van Raaij
Gesendet: Dienstag, 9. Juli 2013 16:23
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] modified amino acids in the PDB

- really the only complicated case would be where a group is covalently linked 
to more than one amino acid, wouldn't it? Any case where only one covalent link 
with an is present could (should?) be treated as a special amino acid, i.e. 
like selenomethionine.
- groups without any covalent links to the protein are better kept separate I 
would think (but I guess this is stating the obvious).

Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij





On 9 Jul 2013, at 12:49, Frances C. Bernstein wrote:

> In trying to formulate a suggested policy on het groups versus 
> modified side chains one needs to think about the various cases that 
> have arisen.
> 
> Perhaps the earliest one I can think of is a heme group.
> One could view it as a very large decoration on a side chain but, as 
> everyone knows, one heme group makes four links to residues.  In the 
> early days of the PDB we decided that heme "obviously" had to be 
> represented as a separate group.
> 
> I would also point out that nobody would seriously suggest that 
> selenomethionine should be represented as a methionine with a missing 
> sulfur and a selenium het group bound to it.
> 
> Unfortunately all the cases that fall between selenomethionine and 
> heme are more difficult.  Perhaps the best that one must hope for is 
> that whichever representation is chosen for a particular case, it be 
> consistent across all entries.
> 
>  Frances
> 
> P.S. One can also have similar discussions about the representation of 
> microheterogeneity and of sugar chains but we should leave those for 
> another day.
> 
> =
> Bernstein + Sons
> *   *   Information Systems Consultants
> 5 Brewster Lane, Bellport, NY 11713-2803
> *   * ***
>  *Frances C. Bernstein
>  *   ***  f...@bernstein-plus-sons.com
> *** *
>  *   *** 1-631-286-1339FAX: 1-631-286-1999
> =
> 
> On Tue, 9 Jul 2013, MARTYN SYMMONS wrote:
> 
>> Hi Clemens
>>I guess the reason you say 'arbitrary' is because there is no 
>> explanation of this rule decision?
>>   It would be nice if some rationalization was available alongside the 
>> values given.
>> So a sentence along the lines of 'we set the number owing to the 
>> following considerations' ?
>>   However a further layer of variation is that the rule does not seem 
>> to be consistently applied
>>  - just browsing CYS modifications:
>>iodoacetamide treatment gives a CYS with only 4 additional atoms 
>> but it is split off as  ACM.
>>However some ligands much larger than 10 residues have been kept 
>> with the cysteine ( for example CY7 in 2jiv and NPH in 1a18.
>>My betting is that it depends on whether something has been seen 
>> 'going solo' as a non-covalent ligand previously so that it pops up 
>> as an atomic structural match with a pre-defined three-letter code.
>>   This would explain for example the ACM case which you might expect 
>> to occur in a modified Cys.  But it has also been observed as a 
>> non-polymer ligand in its own right so goes on as a separate modification?
>>However to be honest I am not sure I have ever seen the rationale 
>> for this written down.
>>   'Non-polymer' heterogens can turn up either linked or not. Once 
>> they are in the residues they have to make a call on which kind of 
>> backbone they will feature in within the pdb.
>>   That is why there is  'D5M' for non-polymer deoxyAMP. Also known as 
>> ' DA' when it is 'DNA-linking' but so far not fessing up to life 
>> under a third code as 'RNA-linking'
>> Now is perhaps the time to ask for explanations of these nomenclature 
>> features before they become hard-wired in the new pdb deposition 
>> system (however there may be time - I refer you to my previous pos

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[Thomas Meier]

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The position is available from July 2013, and the initial appointment is 
for 1 year, with an option for extension. Consideration of applications 
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This call closes on July 15, 2013.

Please submit your application including CV and further information 
(list of publications, contact of three referees, etc.) per e-mail to: 
thomas.me...@biophys.mpg.de 


--
Dr. Thomas Meier
Research group leader
Max-Planck Institute of Biophysics
Department of Structural Biology
Max-von-Laue Str. 3
DE-60438 Frankfurt/Main
Phone: +49(0)6963033038
www.biophys.mpg.de/meier

Re: [ccp4bb] [ccp4bb] modified amino acids in the PDB

[Peter Artymiuk]

Dear BB

For my one cent's worth, I think it is simpler if the residue name in the 
coordinate set matches that in the sequence deposition for the protein (and 
DNA)  - i.e. before any post-translational modification is carried out. Whether 
a particular modification is actually present or absent may depend on the 
organism (or even the cell line) in which the protein was expressed… which is 
seldom the natural one.

best wishes
Pete






On 9 Jul 2013, at 16:21, herman.schreu...@sanofi.com wrote:

> Dear Marc (and BB),
> 
> I guess as usual, in real life the obvious is less obvious as it seems to be. 
> I, and I guess many of my colleagues trying to find new drugs, have quite a 
> few protein-inhibitor complexes where the inhibitor formed a covalent link 
> with e.g. the active site serine. In these cases, I am perfectly happy with 
> having the inhibitor being defined as a separate group, linked via a LINK 
> record. For me, it does not make sense to treat these covalent inhibitors 
> differently from noncovalent inhibitors.
> 
> In the end, I guess, it will boil down to some arbitrary choice, either 
> imposed upon us by the pdb, or individually taken by the crystallographer who 
> produced the crystal structure.
> 
> My 2 cts,
> Herman
> 
> 
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mark J 
> van Raaij
> Gesendet: Dienstag, 9. Juli 2013 16:23
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: Re: [ccp4bb] modified amino acids in the PDB
> 
> - really the only complicated case would be where a group is covalently 
> linked to more than one amino acid, wouldn't it? Any case where only one 
> covalent link with an is present could (should?) be treated as a special 
> amino acid, i.e. like selenomethionine.
> - groups without any covalent links to the protein are better kept separate I 
> would think (but I guess this is stating the obvious).
> 
> Mark J van Raaij
> Lab 20B
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> c/Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616
> http://www.cnb.csic.es/~mjvanraaij
> 
> 
> 
> 
> 
> On 9 Jul 2013, at 12:49, Frances C. Bernstein wrote:
> 
>> In trying to formulate a suggested policy on het groups versus 
>> modified side chains one needs to think about the various cases that 
>> have arisen.
>> 
>> Perhaps the earliest one I can think of is a heme group.
>> One could view it as a very large decoration on a side chain but, as 
>> everyone knows, one heme group makes four links to residues.  In the 
>> early days of the PDB we decided that heme "obviously" had to be 
>> represented as a separate group.
>> 
>> I would also point out that nobody would seriously suggest that 
>> selenomethionine should be represented as a methionine with a missing 
>> sulfur and a selenium het group bound to it.
>> 
>> Unfortunately all the cases that fall between selenomethionine and 
>> heme are more difficult.  Perhaps the best that one must hope for is 
>> that whichever representation is chosen for a particular case, it be 
>> consistent across all entries.
>> 
>> Frances
>> 
>> P.S. One can also have similar discussions about the representation of 
>> microheterogeneity and of sugar chains but we should leave those for 
>> another day.
>> 
>> =
>> Bernstein + Sons
>> *   *   Information Systems Consultants
>> 5 Brewster Lane, Bellport, NY 11713-2803
>> *   * ***
>>  *Frances C. Bernstein
>> *   ***  f...@bernstein-plus-sons.com
>> *** *
>> *   *** 1-631-286-1339FAX: 1-631-286-1999
>> =
>> 
>> On Tue, 9 Jul 2013, MARTYN SYMMONS wrote:
>> 
>>> Hi Clemens
>>>   I guess the reason you say 'arbitrary' is because there is no 
>>> explanation of this rule decision?
>>>  It would be nice if some rationalization was available alongside the 
>>> values given.
>>> So a sentence along the lines of 'we set the number owing to the 
>>> following considerations' ?
>>>  However a further layer of variation is that the rule does not seem 
>>> to be consistently applied
>>> - just browsing CYS modifications:
>>>   iodoacetamide treatment gives a CYS with only 4 additional atoms 
>>> but it is split off as  ACM.
>>>   However some ligands much larger than 10 residues have been kept 
>>> with the cysteine ( for example CY7 in 2jiv and NPH in 1a18.
>>>   My betting is that it depends on whether something has been seen 
>>> 'going solo' as a non-covalent ligand previously so that it pops up 
>>> as an atomic structural match with a pre-defined three-letter code.
>>>  This would explain for example the ACM case which you might expect 
>>> to occur in a modified Cys.  But it has also been observed as a 
>>> non-polymer ligand in its own right so goes on as a separate modification?
>>>   However to be honest I am not sure I

Re: [ccp4bb] ctruncate bug?

[James Holton]

On 6/28/2013 5:13 PM, Douglas Theobald wrote:
I admittedly don't understand TDS well. But I thought it was generally 
assumed that TDS contributes rather little to the conventional 
background measurement outside of the spot (so Stout and Jensen tells 
me :). So I was not even really considering TDS, which I see as a 
different problem from measuring background (am I mistaken here?). I 
thought the background we measure (in the area surrounding the spot) 
mostly came from diffuse solvent scatter, air scatter, loop scatter, 
etc. If so, then we can just consider Itrue = Ibragg + Itds, and worry 
about modeling the different components of Itrue at a different stage. 
And then it would make sense to think about blocking a reflection 
(say, with a minuscule, precisely positioned beam stop very near the 
crystal) and measuring the background in the spot where the reflection 
would hit. That background should be approximated pretty well by 
Iback, the background around the spot (especially if we move far 
enough away from the spot so that TDS is negligible there).


Actually, almost by definition, the resolution at which the disorder in 
the crystal is enough to make the Bragg peaks fade away is also the 
"resolution" where the background due to diffuse scatter is maximized.  
Basically, it's conservation of scattered photons.  The interaction 
cross section is fixed, and the photons that don't go into Bragg peaks 
have to go somewhere.  For those who like equations, the Bragg peaks 
fade with:


Ibragg = I0 * exp(-2*B*s^2)

where "B" is the average atomic B factor (aka "Wilson B"), "s" is 
sin(theta)/lambda (0.5/d), and "I0" is the spot intensity you would see 
if the B factor was zero (perfect crystal).


The background however, goes as:

Ibg = Igas * (1 - exp(-2*B*s^2))

Where Igas is the background intensity you would see if all the atoms in 
the crystal were converted into a gas (infinite B factor) but still 
somehow remained contained within the x-ray beam.  At the so-called 
"resolution limit", the 1-exp() thing is pretty much equal to 1.


In the diffuse scattering field this 1-exp() thing is called the 
"centrosymmetric term", and the first step of data processing is to 
"subtract it out".  What is left over is signatures of correlated 
motions, like "TDS", although strictly speaking TDS is the component due 
to thermally-induced motions only.  At 100K, there is not much "TDS" 
left, but there is still plenty of "diffuse scattering" (DS) due to a 
myriad of other things.


As long as the path the incident x-ray beam takes through "loop" 
(solvent, nylon, etc) is less than the path through the crystal itself, 
and the "air path" (exposed to incident beam and visible from the 
detector) is less than 1000x the path through the crystal, then most of 
the background is actually coming from the crystal "lattice" itself.  
You could put a little spot-specific beamstop up, but all that would do 
is make what we beamline scientists call a "shadow".  Best possible case 
would be to mask off everything coming from the crystal, but since most 
of the background you need to subtract is coming from the crystal itself 
anyway, the "spot specific beamstop" experiment is not really going to 
tell you much. Unless, of course, you are trying to study the diffuse 
scattering. for these experiments spots are annoying because they are 
thousands of times brighter than the effect you are trying to measure.  
Some DS studies have actually taken great pains to avoid putting any 
Bragg peaks on the Ewald sphere.  You can read all about it in T. R. 
Welberry's Oxford University Press book: "Diffuse Scattering and Models 
of Disorder".  Apparently, urea is a classic model system for DS.


A common misconception, however, is that "TDS" can somehow "build up 
under the spot" and give it some "extra" intensity that doesn't have 
anything to do with the average electron density in a unit cell. This is 
absolutely impossible.  Anything that contributes intensity to the 
regions of reciprocal space "under the spots" must have a repeat that is 
identical to the unit cell repeat, and it must also repeat many times in 
a row to make the feature "sharp" enough to hide itself "under the 
spot".  That sounds like a unit cell repeat to me.  Yes, there is such a 
thing as modulated lattices, and also something called "Huang 
scattering" where long-range correlations (cracks and other mechanical 
effects) can give Bragg spots "tails", but where the "tails" end and the 
"unit cell" begins is really just a matter of semantics.  The molecules 
don't actually care what you think the "unit cell" is.


-James Holton
MAD Scientist

Re: [ccp4bb] modified amino acids in the PDB

[Dale Tronrud]

On 07/09/2013 07:23 AM, Mark J van Raaij wrote:

- really the only complicated case would be where a group is covalently linked 
to more than one amino acid, wouldn't it? Any case where only one covalent link 
with an is present could (should?) be treated as a special amino acid, i.e. 
like selenomethionine.
- groups without any covalent links to the protein are better kept separate I 
would think (but I guess this is stating the obvious).



   Let's consider one of your "simple" cases.  Imagine a heterodimer
(chains alpha and beta) with a single disulfide link between the
peptides.  Do you prefer to have an alpha chain with one residue
being a CYS with an entire beta chain attached as a single residue,
or a beta chain with one residue being a CYS with an entire alpha
chain as a single residue.  Either way you are going to have trouble
fitting into the PDB format's five columns all the unique atom names
for the bloated residue. ;-)

   The problem with this kind of topic is that the molecule is what
it is and it doesn't care how we describe it.  People break the molecule
up into parts to help in their understanding of it and different
people have different needs.  Do you prefer to think of rhodopsin as
containing a LYS residue linked by a Schiff base linkage to retinal
or as having a single, monster, residue with a name you have probably
never heard of?  There is value in both representations depending
on the context.

   A really nice feature of the geometry definitions that have come
out of the Refmac community is that one can define the monster residue
in terms of the LYS-(Schiff linkage)-retinal breakdown.  What hasn't
been done is to create the software that will convert a model from one
form to the other, as the user needs.

   I think this is the direction we should go.  Instead of arguing if,
for example, the "B factor" column should contain the total isotropic
B factor or the residual B factor unfit by the overarching TLS model of
motion, the file supplied by the wwPDB should be a complete,
unambiguous, representation of the model and software should exist
that displays for the user whatever representation they want.  Then the
representation stored in the master repository would not be very
important.

Dale Tronrud


Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij





On 9 Jul 2013, at 12:49, Frances C. Bernstein wrote:


In trying to formulate a suggested policy on het groups
versus modified side chains one needs to think about the
various cases that have arisen.

Perhaps the earliest one I can think of is a heme group.
One could view it as a very large decoration on a side
chain but, as everyone knows, one heme group makes four
links to residues.  In the early days of the PDB we decided
that heme "obviously" had to be represented as a separate group.

I would also point out that nobody would seriously suggest that
selenomethionine should be represented as a methionine with a
missing sulfur and a selenium het group bound to it.

Unfortunately all the cases that fall between selenomethionine
and heme are more difficult.  Perhaps the best that one must
hope for is that whichever representation is chosen for a
particular case, it be consistent across all entries.

  Frances

P.S. One can also have similar discussions about the representation
of microheterogeneity and of sugar chains but we should leave those
for another day.

=
Bernstein + Sons
*   *   Information Systems Consultants
5 Brewster Lane, Bellport, NY 11713-2803
*   * ***
 *Frances C. Bernstein
  *   ***  f...@bernstein-plus-sons.com
*** *
  *   *** 1-631-286-1339FAX: 1-631-286-1999
=

On Tue, 9 Jul 2013, MARTYN SYMMONS wrote:


Hi Clemens
I guess the reason you say 'arbitrary' is because there is no explanation 
of this
rule decision?
   It would be nice if some rationalization was available alongside the values 
given.
So a sentence along the lines of 'we set the number owing to the following
considerations' ?
   However a further layer of variation is that the rule does not seem to be
consistently applied
  - just browsing CYS modifications:
iodoacetamide treatment gives a CYS with only 4 additional atoms but it is 
split
off as  ACM.
However some ligands much larger than 10 residues have been kept with the 
cysteine
( for example CY7 in 2jiv and NPH in 1a18.
My betting is that it depends on whether something has been seen 'going 
solo' as a
non-covalent ligand previously so that it pops up as an atomic structural match 
with
a pre-defined three-letter code.
   This would explain for example the ACM case which you might expect to occur 
in a
modified Cys.  But it has also been observed as 

Re: [ccp4bb] AW: [ccp4bb] modified amino acids in the PDB

[Engin Özkan]

Unfortunately, not all is ever that easy. If we were to follow Mark's 
rules, glycoproteins would end up losing their asparagines (and others 
residues depending on the type of glycosylation) to numerous 
underdefined new residue types. And consider the confusion caused by the 
different versions of N-linked glycosylation that dominates yeast vs. 
insects vs. mammals, which would lead to different residue names. It 
does not even end there: due to its heterogenous nature, you would end 
up with different "modified residue names" for, say 6' fucosylated vs 3' 
and 6' fucosylated, arthropod-type N-linked glycosylated Asparagine. All 
of this, when we can barely model the basic sugars half-right, let alone 
identify them.


For covalent inhibitors, what Herman mentions - regular amino acid names 
with links, would also be clearly the more practical solution.


My gripe is, as Peter mentioned, having X's in protein sequences. Why 
can't we have MSE as M? Isn't this obvious? But as always, it does not 
end there. For example, pyroglutamic acid, a naturally occuring form of 
*both* Glu (E) and Gln (Q) ends up being labeled in sequence as E (or it 
used to be). Even when every sequence database has the residue as a Q. A 
sensible solution would be to follow the depositor-submitted sequence, 
or the obvious encoded amino acid, here.


Despite being a very ugly solution, the practical way does appear to be 
a combination of (1) very loosely following simple rules (such as a 
ten-atom rule), (2) following the established convention, when there is 
one (such as for glycosylation), (3) and rational discussion between the 
depositors and annotators. The trick to this working is being able to 
compromise and being rational. Another reason why annotators should be 
trained biochemists and chemists.


Engin

P.S. Still recovering from when I was told by PDB staff that an HPUB 
structure could not yet be released, because the publication mentioned 
was just a Nature "Letter", not an "Article".


On 7/9/13 10:21 AM, herman.schreu...@sanofi.com wrote:

Dear Marc (and BB),

I guess as usual, in real life the obvious is less obvious as it seems to be. 
I, and I guess many of my colleagues trying to find new drugs, have quite a few 
protein-inhibitor complexes where the inhibitor formed a covalent link with 
e.g. the active site serine. In these cases, I am perfectly happy with having 
the inhibitor being defined as a separate group, linked via a LINK record. For 
me, it does not make sense to treat these covalent inhibitors differently from 
noncovalent inhibitors.

In the end, I guess, it will boil down to some arbitrary choice, either imposed 
upon us by the pdb, or individually taken by the crystallographer who produced 
the crystal structure.

My 2 cts,
Herman
  


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Mark J 
van Raaij
Gesendet: Dienstag, 9. Juli 2013 16:23
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] modified amino acids in the PDB

- really the only complicated case would be where a group is covalently linked 
to more than one amino acid, wouldn't it? Any case where only one covalent link 
with an is present could (should?) be treated as a special amino acid, i.e. 
like selenomethionine.
- groups without any covalent links to the protein are better kept separate I 
would think (but I guess this is stating the obvious).

Mark J van Raaij
Lab 20B
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij





On 9 Jul 2013, at 12:49, Frances C. Bernstein wrote:


In trying to formulate a suggested policy on het groups versus
modified side chains one needs to think about the various cases that
have arisen.

Perhaps the earliest one I can think of is a heme group.
One could view it as a very large decoration on a side chain but, as
everyone knows, one heme group makes four links to residues.  In the
early days of the PDB we decided that heme "obviously" had to be
represented as a separate group.

I would also point out that nobody would seriously suggest that
selenomethionine should be represented as a methionine with a missing
sulfur and a selenium het group bound to it.

Unfortunately all the cases that fall between selenomethionine and
heme are more difficult.  Perhaps the best that one must hope for is
that whichever representation is chosen for a particular case, it be
consistent across all entries.

  Frances

P.S. One can also have similar discussions about the representation of
microheterogeneity and of sugar chains but we should leave those for
another day.

=
Bernstein + Sons
*   *   Information Systems Consultants
5 Brewster Lane, Bellport, NY 11713-2803
*   * ***
 *Frances C. Bernstein
  *   ***  f...@bernstein