Re: [ccp4bb] Biological assembly

[Kayashree M]

Respected Sir, 

Thank you for the efforts and nice depiction of the problem. 
The Dimeric assembly that is solved and the one predicted
by PISA is depicted in the attachment. This is the arrangement
I wanted to describe. The protein overall appears like a "C".

Thanking you
With Regards
KavyaSince a crystallographic 3-fold generates the trimer (of dimers) then A1-B2 can be the ASU in this case.Just generate the symmetry mates with the A1-B1 dimer and then make a new PDB of A1-B2 then generate the symmetry mates of A1-B2 to see that the lattice is complete.I compulsively made and attached an illustration to show what I mean.James

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Dimer.pdf
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Re: [ccp4bb] Biological assembly

[Vellieux Frederic]

Hi,

If, in your case, no possible asymmetric unit can contain A1-B2, then 
you deposit A1-B1 (or I suppose A2-B2...) and indicate to the PDB (like 
placing cards in the header cards) the operator to be used (and the 
subunit it applies to) in order to generate the most likely biological 
dimer. Normally the PDB can take care of that.


Fred.

Kayashree M wrote:

Respected Sir,

The space group is H3. if I generate the symmetry,
it appears to be a dimer of trimers stacked one above
the other with a rotation of 60 deg wrt each other, like
this -  A1, A2, A3 (in one trimer) stacked upon
B1, B2, B3 (second trimer). So structure that is in the ASU
is with A1-B1 while PISA predicts A1-B2.

Thank you
With Reagrds
Kavya

-CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote: -

To: CCP4BB@JISCMAIL.AC.UK
From: James Stroud
Sent by: CCP4 bulletin board
Date: 10/19/2011 10:41PM
Subject: Re: [ccp4bb] Biological assembly

On Oct 19, 2011, at 4:36 AM, Kayashree M wrote:

We have a structure which is a homodimer in the asymmetric unit.
PISA predicts most probable assembly as a dimer but this
dimeric assembly is different from what is solved (offcourse
we can generate the symmetry equivalent molecule and get that).


This last sentence is a bit vague. Can you take the just dimer
that PISA predicts, fit this dimer to the lattice (i.e. each
monomer sitting correctly in density but retaining the dimeric
relationship predicted by PISA), and then generate the complete
lattice using just this fitted dimer and crystallographic symmetries?

If so, that means that the PISA dimer is equivalent to the ASU you
can deposit the PISA dimer as the ASU.

James



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Re: [ccp4bb] Biological assembly

[Kayashree M]

Thank you Sir for the suggestions. Hi,If, in your case, no possible asymmetric unit can contain A1-B2, then you deposit A1-B1 (or I suppose A2-B2...) and indicate to the PDB (like placing cards in the header cards) the operator to be used (and the subunit it applies to) in order to generate the most likely biological dimer. Normally the PDB can take care of that.The protein looks like the letter "C", So in one of the trimer it is arrangedas "C" while in the other trimer (stacked) it is arranged like "inverted C", SoThe dimer A1-B1 and A2-B2 are same while A1-B1 and A1-B2 are different.Thanking you With RegardsKavya-- 
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Re: [ccp4bb] Biological assembly

[Eleanor Dodson]

I am sure someone has suggested this - you can submit your coordinates 
to the PISA server which will give you a detailed analysis of contacts, 
then you choose which grouping you think makes the most reasonable 
assemblyn and take the asymmetric unit from that .


I actually like to do that while still refining - it makes coot a lot 
easier to use if you are working with a compact molecule!



Eleanor

t On 10/20/2011 05:25 AM, Kayashree M wrote:

Respected Sir,

The space group is H3. if I generate the symmetry,
it appears to be a dimer of trimers stacked one above
the other with a rotation of 60 deg wrt each other, like
this - A1, A2, A3 (in one trimer) stacked upon
B1, B2, B3 (second trimer). So structure that is in the ASU
is with A1-B1 while PISA predicts A1-B2.

Thank you
With Reagrds
Kavya

-CCP4 bulletin boardCCP4BB@JISCMAIL.AC.UK  wrote: -

 To: CCP4BB@JISCMAIL.AC.UK
 From: James Stroud
 Sent by: CCP4 bulletin board
 Date: 10/19/2011 10:41PM
 Subject: Re: [ccp4bb] Biological assembly

 On Oct 19, 2011, at 4:36 AM, Kayashree M wrote:

 We have a structure which is a homodimer in the asymmetric unit.
 PISA predicts most probable assembly as a dimer but this
 dimeric assembly is different from what is solved (offcourse
 we can generate the symmetry equivalent molecule and get that).


 This last sentence is a bit vague. Can you take the just dimer that PISA
 predicts, fit this dimer to the lattice (i.e. each monomer sitting 
correctly
 in density but retaining the dimeric relationship predicted by PISA), and
 then generate the complete lattice using just this fitted dimer and
 crystallographic symmetries?

 If so, that means that the PISA dimer is equivalent to the ASU you can
 deposit the PISA dimer as the ASU.

 James



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Re: [ccp4bb] Biological assembly

[Kayashree M]

Respected Madam, Actually I was not aware of the fact that we have to deposit the structure which is most probablebiological assembly (since in PDB there are seperate representations for the asymmetric unit and biological unit).Also Since I dint have any experimental evidences other than PISA predictions and comparison with the structures from different organisms, I continues to refine the model PHASER predicted.In addition the PHASER refined model has 3 Cadmium ions inbetween the monomers stronglycoordinated by both the monomers. Hence I did not have any doubts upon this assembly. FinallyI submitted this assembly itself. and by mistake I mentioned most probably biological assemblysection while submitting the PDB, Which gave rise to all confusions.Thanking youWith RegardsM. Kavyashree-Eleanor Dodson c...@ysbl.york.ac.uk wrote: -To: Kayashree M ka...@ssl.serc.iisc.inFrom: Eleanor Dodson c...@ysbl.york.ac.ukDate: 10/20/2011 03:23PMCc: CCP4BB@JISCMAIL.AC.UKSubject: Re: [ccp4bb] Biological assemblyI am sure someone has suggested this - you can submit your coordinates to the PISA server which will give you a detailed analysis of contacts, then you choose which grouping you think makes the most reasonable assemblyn and take the asymmetric unit from that .I actually like to do that while still refining - it makes coot a lot easier to use if you are working with a compact molecule!Eleanort On 10/20/2011 05:25 AM, Kayashree M wrote: Respected Sir, The space group is H3. if I generate the symmetry, it appears to be a dimer of trimers stacked one above the other with a rotation of 60 deg wrt each other, like this - A1, A2, A3 (in one trimer) stacked upon B1, B2, B3 (second trimer). So structure that is in the ASU is with A1-B1 while PISA predicts A1-B2. Thank you With Reagrds Kavya -CCP4 bulletin boardCCP4BB@JISCMAIL.AC.UK wrote: -   To: CCP4BB@JISCMAIL.AC.UK   From: James Stroud   Sent by: CCP4 bulletin board   Date: 10/19/2011 10:41PM   Subject: Re: [ccp4bb] Biological assembly   On Oct 19, 2011, at 4:36 AM, Kayashree M wrote:   We have a structure which is a homodimer in the asymmetric unit.   PISA predicts most probable assembly as a dimer but this   dimeric assembly is different from what is solved (offcourse   we can generate the symmetry equivalent molecule and get that).   This last sentence is a bit vague. Can you take the just dimer that PISA   predicts, fit this dimer to the lattice (i.e. each monomer sitting correctly   in density but retaining the dimeric relationship predicted by PISA), and   then generate the complete lattice using just this fitted dimer and   crystallographic symmetries?   If so, that means that the PISA dimer is equivalent to the ASU you can   deposit the PISA dimer as the ASU.   James   --   This message has been scanned for viruses and   dangerous content by *MailScanner*http://www.mailscanner.info/, and is   believed to be clean. -- This message has been scanned for viruses and dangerous content by *MailScanner*http://www.mailscanner.info/, and is believed to be clean.-- This message has been scanned for viruses anddangerous content by MailScanner, and isbelieved to be clean.-- 
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Re: [ccp4bb] Biological assembly

[eugene . krissinel]

Dear Bostjan,

 In case when ASU has the same multiplicity (number of chains) as the
 probable biological assembly, the latter is an ASU as well. In such a
 case, the PDB suggests to choose ASU in the form of that assembly, purely
 for simplicity. It seems to me that this is not an unreasonable
 suggestion and it would be nice if that were a common practice.
 
 Maybe I am misunderstanding what you are saying Eugene, but the ASU
 interface may not necessarily be the biological interface.

Not at all. But if anticipated biological assembly has the same size as ASU,
then one should be able to choose ASU such that it is biological assembly.
Consider that, when there is more than 1 chain in ASU, its configuration
may be chosen in many different ways (but not completely arbitrarily,
of course). E.g., in case of heterodimeric ASU, _any_ hetero-pair of molecules
in crystal is a valid ASU (even if they do not make a contact :)). By this I 
mean
that, by applying all crystal symmetry operations and unit cell translations,
one can reconstruct the whole crystal from an arbitrary hetero-pair. In case
of homodimers it is slightly more complex, e.g. one has to exclude
monomers in parallel orientations.

 I think it is
 perfectly possible that two subunits in a biological dimer, for example,
 may be related by crystallographic axis, but the NCS may be a different
 non-physiological crystal contact, therefore the ASU won't be the same as
 the biological dimer. So I am not sure if the above applies in general.

I would think (am I wrong here?) that NCS-reduced PDB entries do not
represent crystal's ASU (simply because they are reduced by
_non-crystallographic_ symmetry operations). In order to get the ASU,
one needs to apply NCS to the content of the entry. E.g., PDB entry
1stm has 5 chains, but they do not make an ASU: applying all space
group symmetry operations would not reconstruct the crystal.
NCS-expansion, however, gives a 60-chain viral capsid, which is the
ASU. For fun, you may state that your ASU in this case is 2
complementing parts of neighbouring capsids, and this would be
crystallographically correct. It is a matter of common sense (and
sense of beauty perhaps) that we choose to deposit a complete
viral capsid as an ASU here.

Eugene.


 
 Bostjan
 
 Bostjan Kobe
 NHMRC Research Fellow
 Professor of Structural Biology
 School of Chemistry and Molecular Biosciences
 
 and Institute for Molecular Bioscience (Division of Chemistry and
 Structural Biology) and Centre for Infectious Disease Research
 
 
 Cooper Road
 University of Queensland
 Brisbane, Queensland 4072
 Australia
 Phone: +61 7 3365 2132
 Fax: +61 7 3365 4699
 E-mail: b.k...@uq.edu.au
 URL: http://www.scmb.uq.edu.au/staff/bostjan-kobe
 Office: Building 76 Room 329
 Notice: If you receive this e-mail by mistake, please notify me, and do
 not make any use of its contents. I do not waive any privilege,
 confidentiality or copyright associated with it. Unless stated otherwise,
 this e-mail represents only the views of the Sender and not the views of
 The University of Queensland.
 
 
 
 
 
 
 
 
 

Re: [ccp4bb] Biological assembly

[James Stroud]

Since a crystallographic 3-fold generates the trimer (of dimers) then A1-B2 can be the ASU in this case.Just generate the symmetry mates with the A1-B1 dimer and then make a new PDB of A1-B2 then generate the symmetry mates of A1-B2 to see that the lattice is complete.I compulsively made and attached an illustration to show what I mean.James

H3.pdf
Description: Adobe PDF document
On Oct 20, 2011, at 3:40 AM, Kayashree M wrote:Thank you Sir for the suggestions. Hi,If, in your case, no possible asymmetric unit can contain A1-B2, then you deposit A1-B1 (or I suppose A2-B2...) and indicate to the PDB (like placing cards in the header cards) the operator to be used (and the subunit it applies to) in order to generate the most likely biological dimer. Normally the PDB can take care of that.The protein looks like the letter "C", So in one of the trimer it is arrangedas "C" while in the other trimer (stacked) it is arranged like "inverted C", SoThe dimer A1-B1 and A2-B2 are same while A1-B1 and A1-B2 are different.Thanking you With RegardsKavya-- 
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[ccp4bb] Biological assembly

[Kayashree M]

Dear users, We have a structure which is a homodimer in the asymmetric unit.PISA predicts most probable assembly as a dimer but thisdimeric assembly is different from what is solved (offcoursewe can generate the symmetry equivalent molecule and get that).My question is - is it necessary that we deposit a structure, whichPISA predicted as most probable assembly in PDB as an asymmetric unit  biological assembly or can we deposit a dimer(asymmetric unit) and give explanation for the biological assemblyaccording to what PISA predicted.Other than such predictions what other criteria needs to be considered to say that one specific assembly is a biological assembly?Another question-In this case one of the chain has 3 MSE residues, while the otherchain has only 2 MSE (When we change MET to MSE, there will be a huge negetive density coming up).Are there any such instances in PDB, where two homodimer (or any mer)will have different percentage of MSE?Thanking youWith Regardskavya-- 
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Re: [ccp4bb] Biological assembly

[Frederic VELLIEUX]

Hi,

What you must deposit is what is present in the asymmetric unit of the crystal. 
The HEADER cards (and the publication) can describe the most likely biological 
assembly. 
Why is that: there is no reason why the crystal should conform to the 
biological function (and associated quaternary structure), there are examples 
of having the 
asymmetric unit different than the biological assembly. Such as a crystal 
asymmetric unit containing half a viral capsid.

So the PDB files contain what you see in the crystal, and there are places 
for the interpretation, such as these pictures appearing on the web page for 
the structure 
showing the most likely biological unit. Or if you (as a user) request the PDB 
to provide you with the coordinates for that most probable biological assembly.

Fred.

 Message du 19/10/11 12:36
 De : Kayashree M 
 A : CCP4BB@JISCMAIL.AC.UK
 Copie à : 
 Objet : [ccp4bb] Biological assembly
 
 Dear users, 

We have a structure which is a homodimer in the asymmetric unit.

PISA predicts most probable assembly as a dimer but this
dimeric assembly is different from what is solved (offcourse
we can generate the symmetry 
equivalent molecule and get that).

My question is - is it necessary that we deposit a structure, which
PISA predicted as most probable assembly in PDB as an 

asymmetric unit  biological assembly or can we deposit a dimer
(asymmetric unit) and give explanation for the biological assembly
according to what PISA 
predicted.

Other than such predictions what other criteria needs to be 
considered to say that one specific assembly is a biological assembly?


Another question-
In this case one of the chain has 3 MSE residues, while the other
chain has only 2 MSE (When we change MET to MSE, there will be a 

huge negetive density coming up).

Are there any such instances in PDB, where two homodimer (or any mer)
will have different percentage of MSE?


Thanking you
With Regards
kavya

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Re: [ccp4bb] Biological assembly

[eugene . krissinel]

Dear Kavya,

If I understand your question correctly, it is about the choice of asymmetric 
unit for your deposition. In case of dimeric asymmetric unit (ASU), there are, 
indeed, a few valid possibilities and you arrive to just one of them in the 
course of structure solution. What you decide to deposit, is your own choice, 
and I would think that the PDB can suggest an alternative but they would not 
really insist on it (hopefully).

In case when ASU has the same multiplicity (number of chains) as the probable 
biological assembly, the latter is an ASU as well. In such a case, the PDB 
suggests to choose ASU in the form of that assembly, purely for simplicity. It 
seems to me that this is not an unreasonable suggestion and it would be nice if 
that were a common practice. I do not want to imply here that PISA will always 
make a correct prediction, therefore, one should always be critical and use as 
much experimental evidence as possible. However, deposition of biological 
assembly as ASU, where possible, is always preferential irrespectively of 
whether the assembly agrees with PISA predictions or not. Preferential does not 
mean mandatory though :)

I hope this helps,

Eugene.


On 19 Oct 2011, at 11:36, Kayashree M wrote:

Dear users,

We have a structure which is a homodimer in the asymmetric unit.
PISA predicts most probable assembly as a dimer but this
dimeric assembly is different from what is solved (offcourse
we can generate the symmetry equivalent molecule and get that).

My question is - is it necessary that we deposit a structure, which
PISA predicted as most probable assembly in PDB as an
asymmetric unit  biological assembly or can we deposit a dimer
(asymmetric unit) and give explanation for the biological assembly
according to what PISA predicted.

Other than such predictions what other criteria needs to be
considered to say that one specific assembly is a biological assembly?

Another question-
In this case one of the chain has 3 MSE residues, while the other
chain has only 2 MSE (When we change MET to MSE, there will be a
huge negetive density coming up).

Are there any such instances in PDB, where two homodimer (or any mer)
will have different percentage of MSE?

Thanking you
With Regards
kavya

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Re: [ccp4bb] Biological assembly

[Ed Pozharski]

 My question is - is it necessary that we deposit a structure, which
 PISA predicted as most probable assembly in PDB as an 
 asymmetric unit  biological assembly or can we deposit a dimer
 (asymmetric unit) and give explanation for the biological assembly
 according to what PISA predicted.
 
As others have said - you are free to deposit whatever model you believe
is consistent with your experimental data.  If we assume that you know
what the biological assembly is (see below), you still may (and people
do that) deposit a different arrangement - it's not wrong per se, but
think about non-structural folk (there should be a better word) that
will look at your deposited model and may not have the experience to
realize what they are looking at.


 Other than such predictions what other criteria needs to be 
 considered to say that one specific assembly is a biological assembly?
 
1. You need to show that your protein is a dimer in solution.
Gel-filtration (sometimes problematic), dynamic light scattering (often
problematic), analytical ultracentrifugation (less problematic but
instruments are not widely available) as well as methods I mention in 2
are useful here.

2. You need to show somehow that the dimer you see in crystal is the
same as dimer in solution.  Many approaches are available here - I am a
recent SAXS convert, and I wholeheartedly recommend that, but your
mileage may vary.  SAXS will also unequivocally determine the
oligomerization state. Cross-linking and HD exchange, both in line of
mass-spectrometry, are good ways to get it done too.  

With that said, you may be able to get away with just the crystal
structure assuming that the PISA results are convincing (e.g. you have
one heavily hydrophobic interface that is large enough and much larger
than any other more polar alternatives.

 Another question-
 In this case one of the chain has 3 MSE residues, while the other
 chain has only 2 MSE (When we change MET to MSE, there will be a 
 huge negetive density coming up).
 
This is rather curious.  While it's definitely possible that your
protein prep had variable incorporation ratio, the fact that you see it
in a specific spot seems to suggest that selenium slightly alters the
structure directing the crystallization.

 Are there any such instances in PDB, where two homodimer (or any mer)
 will have different percentage of MSE?
 
Hopefully someone knows the answer, but you can always just mine the
database.

Cheers,

Ed.

-- 
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   Julian, King of Lemurs

Re: [ccp4bb] Biological assembly

[Kayashree M]

Dear users,Thank you all for the suggestions.With RegardsKavya-- 
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Re: [ccp4bb] Biological assembly

[James Stroud]

On Oct 19, 2011, at 4:36 AM, Kayashree M wrote:
 We have a structure which is a homodimer in the asymmetric unit.
 PISA predicts most probable assembly as a dimer but this
 dimeric assembly is different from what is solved (offcourse
 we can generate the symmetry equivalent molecule and get that).

This last sentence is a bit vague. Can you take the just dimer that PISA 
predicts, fit this dimer to the lattice (i.e. each monomer sitting correctly in 
density but retaining the dimeric relationship predicted by PISA), and then 
generate the complete lattice using just this fitted dimer and crystallographic 
symmetries?

If so, that means that the PISA dimer is equivalent to the ASU you can deposit 
the PISA dimer as the ASU.

James

Re: [ccp4bb] Biological assembly

[Bostjan Kobe]

On 19/10/11 9:19 PM, eugene.krissi...@stfc.ac.uk
eugene.krissi...@stfc.ac.uk wrote:

In case when ASU has the same multiplicity (number of chains) as the
probable biological assembly, the latter is an ASU as well. In such a
case, the PDB suggests to choose ASU in the form of that assembly, purely
for simplicity. It seems to me that this is not an unreasonable
suggestion and it would be nice if that were a common practice.

Maybe I am misunderstanding what you are saying Eugene, but the ASU
interface may not necessarily be the biological interface. I think it is
perfectly possible that two subunits in a biological dimer, for example,
may be related by crystallographic axis, but the NCS may be a different
non-physiological crystal contact, therefore the ASU won't be the same as
the biological dimer. So I am not sure if the above applies in general.

Bostjan

Bostjan Kobe
NHMRC Research Fellow
Professor of Structural Biology
School of Chemistry and Molecular Biosciences

and Institute for Molecular Bioscience (Division of Chemistry and
Structural Biology) and Centre for Infectious Disease Research


Cooper Road
University of Queensland
Brisbane, Queensland 4072
Australia
Phone: +61 7 3365 2132
Fax: +61 7 3365 4699
E-mail: b.k...@uq.edu.au
URL: http://www.scmb.uq.edu.au/staff/bostjan-kobe
Office: Building 76 Room 329
Notice: If you receive this e-mail by mistake, please notify me, and do
not make any use of its contents. I do not waive any privilege,
confidentiality or copyright associated with it. Unless stated otherwise,
this e-mail represents only the views of the Sender and not the views of
The University of Queensland.

Re: [ccp4bb] Biological assembly

[Kayashree M]

Respected Sir, The space group is H3. if I generate the symmetry, it appears to be a dimer of trimers stacked one above the other with a rotation of 60 deg wrt each other, like this - A1, A2, A3 (in one trimer) stacked uponB1, B2, B3 (second trimer). So structure that is in the ASUis with A1-B1 while PISA predicts A1-B2.Thank youWith ReagrdsKavya-CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK wrote: -To: CCP4BB@JISCMAIL.AC.UKFrom: James Stroud Sent by: CCP4 bulletin board Date: 10/19/2011 10:41PMSubject: Re: [ccp4bb] Biological assemblyOn Oct 19, 2011, at 4:36 AM, Kayashree M wrote:We have a structure which is a homodimer in the asymmetric unit.PISA predicts most probable assembly as a dimer but thisdimeric assembly is different from what is solved (offcoursewe can generate the symmetry equivalent molecule and get that).This last sentence is a bit vague. Can you take the just dimer that PISA predicts, fit this dimer to the lattice (i.e. each monomer sitting correctly in density but retaining the dimeric relationship predicted by PISA), and then generate the complete lattice using just this fitted dimer and crystallographic symmetries?If so, that means that the PISA dimer is equivalent to the ASU you can deposit the PISA dimer as the ASU.James-- 
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