Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Karla J. F. Satchell]

Sorry did not mean to go off topic. Original question was requests for 
suggestions of cleavable c-term tag vectors. I only meant to provide info on 
one type recommended by another. Others may have further suggestions for Flavio.

From: CCP4 bulletin board  on behalf of Karla J. F. 
Satchell 
Date: Friday, February 16, 2024 at 7:13 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector 
for E.Coli with a cleavable his-tag at the c-ter
Hi everyone—

Thought I might chime in.
This technology was developed in my lab based on our studies of toxins and we 
have methods papers and two patent.

The CPD-tag works great for production of protein with minimal residual 
residues on the protein. Our studies indicate that it can help solubilize 
protein during expression. Protein is purified with the C-terminal CPD tag, and 
the entire CPD and 6xHis-tag are released by addition of InsP6 (phytic acid) 
which is very inexpensive.

The only restriction of the recognition sequence is small-Leu-small so a small 
residue plus Leucine does remain on your protein. There does also need to be a 
little flexibility at the end of your protein for access to the protease so our 
vectors add extra Gly-Ala so the residual is GAAL. CPD is particularly useful 
for small proteins if the additional residues do not interfere with your assays.

https://pubmed.ncbi.nlm.nih.gov/28056928/
https://pubmed.ncbi.nlm.nih.gov/31773580/

https://patents.google.com/patent/US8257946B2/en
https://patents.google.com/patent/US8383400B2/en

Here is a link to the original paper on mechanism of action of CPD
https://pubmed.ncbi.nlm.nih.gov/19620709/

These are not commercially available as despite multiple marketing attempts, 
there was little interest mid-2010s from biotech for licensing new cloning 
technologies and we abandoned advanced development

If this technology is interesting to you to add to your commercial biotech 
portfolio, please feel free to reach out to me.

Flavio, we can share our vector, but my institution does require an MTA as 
technology is patented. Most people just use synthetic DNA to generate the 
exact clone they want so we rarely get requests but happy to share sequences if 
you need for your synthetic design.

Karla Satchell


From: CCP4 bulletin board  on behalf of Srivastava, 
Dhiraj 
Date: Friday, February 16, 2024 at 6:42 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector 
for E.Coli with a cleavable his-tag at the c-ter
Hi Flavio
While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease cleavage 
site of your choice. However this strategy will results in quite a few extra 
residues from protease site.
An alternative, which is not commercial but available through addgene, is 
cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in solubility 
and expression.

https://www.addgene.org/38251/

There are other variants of this vector available on addgene that you can 
choose from. Depending on your c terminal sequence, it may leave either no 
extra residues or only one or two residues.

Dhiraj

From: CCP4 bulletin board  on behalf of Flavio Di Pisa 

Sent: Friday, February 16, 2024 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli 
with a cleavable his-tag at the c-ter

Dear community,

I'm looking for a commercial expression vector for E.Coli with a
cleavable his-tag at the c-ter. Anyone can help me?

Thank you in advance,

Flavio.

Re: [ccp4bb] About Staraniso

[vincent Chaptal]

Hi Gerard,

indeed, thank you for your clarification, I'm rusty since I'm on 
something else now.


Anisotropy might not be the right word, as it does mean the opposite of 
isotropy.
I meant 2 phenomenon at play: lack of completeness in the high 
resolution shells, and different intensity falloff compared to a soluble 
protein.


As I answered separately to some of you, I'll make the data available 
soon and will report here once it's done.


Best
Vincent

Le 16/02/2024 à 13:39, Gerard Bricogne a écrit :

L'adresse mail de l'expéditeur est extérieure :owner-ccp...@jiscmail.ac.uk   En 
cas de doute : ne répondez pas, ne cliquez pas et signalez le message au 
Support Informatique


Dear Vincent,

  Thank you for chipping in as you did, with so much useful feedback
about the difficulty of re-using PDB depositions containing anisotropic
data. It is a very useful picture of what is definitely a "bleeding edge" on
the "R" side of the wwPDB's "FAIR" ideal.

  It would be most helpful if you could share with us (off-line) the
details of the specific problems you encountered and that led to the odd
recommendation that you should use a text editor to combine the contents of
mmCIF files.

  It is interesting that you used the turn of phrase "huge anisotropy
combined with lack of completeness in high resolution shells" as if they
were two distinct evils that had conspired to combine in order to make your
life difficult. In reality, as you are of course well aware, they are one
and the same evil, as strong anisotropy will necessarily result in low
completeness in the *spherical* shell to the highest diffraction limit. The
diagram at the beginning of the page

  https://staraniso.globalphasing.org/anisotropy_about.html

makes this totally obvious. The only way to have full completeness in the
outermost (spherical) shell is to have isotropic diffraction. Forgive me for
perhaps belabouring something you are perfectly aware of, but it provided an
opportunity to push back against "isotropic thinking" that is still so
deeply ingrained in the collective worldview.


  With best wishes,

   Gerard.

--
On Fri, Feb 16, 2024 at 09:59:59AM +0100, vincent Chaptal wrote:

Hi Clemens and all,

I've been following with a lot of interest of course, anisotropy has
taken a lot of space in my membrane-protein crystallography life.
I remember the many exchanges we've had with you and Global Phasing,
as well as many other software developpers over the years.

I would like to chip in to give the point of view of a user
struggling with it, and since 6r72 was mentioned and I'm the
author...

6r72 has been an epic battle experimentally, and afterwards with
data processing as well. The huge anisotropy combined with lack of
completeness in high resolution shells, and low resolution, made a
cocktail for not so great electron density. But since this was all I
got, I had to make something out of it. Since the quality of the
data was what it was, I definitely wanted others to be able to
re-investigate it with modern tools, or just see for themselves the
map I had to make the choices in modelling.
Remember, I asked for your help to combine all the data of the
entry, and GlobalPhasing was very helpful to make the tool for this
entry. I believe, this was the moment you created the deposition
tool you mention below.

The fact that the data is not easily retrievable is my point here.
Pavel knows about this entry as we've been exchanging about it. I
completely share with him the difficulty to parse through PDB
entries, having performed several statistical analysis of the PDB to
search for the root cause of anisotropy in membrane proteins myself
(https://pubmed.ncbi.nlm.nih.gov/36206830/).
I encountered the same issue for a recent entry (8qq7, soon
available I hope) where we tried to combine the same files. I
vaguely remembered the deposition tool you mention but I couldn't
locate it (sorry), so we engaged in a dialog with the PDB who made
us use text editors to merge the files basically. On the top of my
head, I think we labelled the blocks "original_data", "modified_SA".

A standardization of this deposition is basically what you all want,
to perform all the different downstream analysis you want. This is
not available at the moment. I've been going out of my way to
combine the files, but it would have been much easier to just give
the SA_modified file to the PDB. I thus encourage all of you
software developpers, despite your different point of views, to
design and agree on such a tool to be placed at OneDep.

To finish, in case you want to reprocess a high-anisotropy, large
difference in high-resolution limits, and low resolution, I have the
images available for 6r72, 2y5y, and 8qq7 (all of which have the
structure factors with original data, maps and modified data).

All the best
Vincent

Le 15/02/2024 à 19:25, Clemens Vonrhein a écrit :

L'adresse mail de l'expéditeur est extérieure :owner-ccp...@jiscmail.ac.uk   En 

Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Karla J. F. Satchell]

Hi everyone—

Thought I might chime in.
This technology was developed in my lab based on our studies of toxins and we 
have methods papers and two patent.

The CPD-tag works great for production of protein with minimal residual 
residues on the protein. Our studies indicate that it can help solubilize 
protein during expression. Protein is purified with the C-terminal CPD tag, and 
the entire CPD and 6xHis-tag are released by addition of InsP6 (phytic acid) 
which is very inexpensive.

The only restriction of the recognition sequence is small-Leu-small so a small 
residue plus Leucine does remain on your protein. There does also need to be a 
little flexibility at the end of your protein for access to the protease so our 
vectors add extra Gly-Ala so the residual is GAAL. CPD is particularly useful 
for small proteins if the additional residues do not interfere with your assays.

https://pubmed.ncbi.nlm.nih.gov/28056928/
https://pubmed.ncbi.nlm.nih.gov/31773580/

https://patents.google.com/patent/US8257946B2/en
https://patents.google.com/patent/US8383400B2/en

Here is a link to the original paper on mechanism of action of CPD
https://pubmed.ncbi.nlm.nih.gov/19620709/

These are not commercially available as despite multiple marketing attempts, 
there was little interest mid-2010s from biotech for licensing new cloning 
technologies and we abandoned advanced development

If this technology is interesting to you to add to your commercial biotech 
portfolio, please feel free to reach out to me.

Flavio, we can share our vector, but my institution does require an MTA as 
technology is patented. Most people just use synthetic DNA to generate the 
exact clone they want so we rarely get requests but happy to share sequences if 
you need for your synthetic design.

Karla Satchell


From: CCP4 bulletin board  on behalf of Srivastava, 
Dhiraj 
Date: Friday, February 16, 2024 at 6:42 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector 
for E.Coli with a cleavable his-tag at the c-ter
Hi Flavio
While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease cleavage 
site of your choice. However this strategy will results in quite a few extra 
residues from protease site.
An alternative, which is not commercial but available through addgene, is 
cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in solubility 
and expression.

https://www.addgene.org/38251/

There are other variants of this vector available on addgene that you can 
choose from. Depending on your c terminal sequence, it may leave either no 
extra residues or only one or two residues.

Dhiraj

From: CCP4 bulletin board  on behalf of Flavio Di Pisa 

Sent: Friday, February 16, 2024 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli 
with a cleavable his-tag at the c-ter

Dear community,

I'm looking for a commercial expression vector for E.Coli with a
cleavable his-tag at the c-ter. Anyone can help me?

Thank you in advance,

Flavio.



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of 
www.jiscmail.ac.uk/CCP4BB,
 a mailing list hosted by 
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Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Srivastava, Dhiraj]

Hi Flavio
While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease cleavage 
site of your choice. However this strategy will results in quite a few extra 
residues from protease site.
An alternative, which is not commercial but available through addgene, is 
cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in solubility 
and expression.

https://www.addgene.org/38251/

There are other variants of this vector available on addgene that you can 
choose from. Depending on your c terminal sequence, it may leave either no 
extra residues or only one or two residues.

Dhiraj

From: CCP4 bulletin board  on behalf of Flavio Di Pisa 

Sent: Friday, February 16, 2024 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli 
with a cleavable his-tag at the c-ter

Dear community,

I'm looking for a commercial expression vector for E.Coli with a
cleavable his-tag at the c-ter. Anyone can help me?

Thank you in advance,

Flavio.



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

This message was issued to members of 
www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are 
available at https://www.jiscmail.ac.uk/policyandsecurity/



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

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hosted by www.jiscmail.ac.uk, terms & conditions are available at 
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Re: [ccp4bb] About Staraniso

[Gerard Bricogne]

Dear Vincent,

 Thank you for chipping in as you did, with so much useful feedback
about the difficulty of re-using PDB depositions containing anisotropic
data. It is a very useful picture of what is definitely a "bleeding edge" on
the "R" side of the wwPDB's "FAIR" ideal.

 It would be most helpful if you could share with us (off-line) the
details of the specific problems you encountered and that led to the odd
recommendation that you should use a text editor to combine the contents of
mmCIF files. 

 It is interesting that you used the turn of phrase "huge anisotropy
combined with lack of completeness in high resolution shells" as if they
were two distinct evils that had conspired to combine in order to make your
life difficult. In reality, as you are of course well aware, they are one
and the same evil, as strong anisotropy will necessarily result in low
completeness in the *spherical* shell to the highest diffraction limit. The
diagram at the beginning of the page 

 https://staraniso.globalphasing.org/anisotropy_about.html

makes this totally obvious. The only way to have full completeness in the
outermost (spherical) shell is to have isotropic diffraction. Forgive me for
perhaps belabouring something you are perfectly aware of, but it provided an
opportunity to push back against "isotropic thinking" that is still so
deeply ingrained in the collective worldview.


 With best wishes,

  Gerard.

--
On Fri, Feb 16, 2024 at 09:59:59AM +0100, vincent Chaptal wrote:
> Hi Clemens and all,
> 
> I've been following with a lot of interest of course, anisotropy has
> taken a lot of space in my membrane-protein crystallography life.
> I remember the many exchanges we've had with you and Global Phasing,
> as well as many other software developpers over the years.
> 
> I would like to chip in to give the point of view of a user
> struggling with it, and since 6r72 was mentioned and I'm the
> author...
> 
> 6r72 has been an epic battle experimentally, and afterwards with
> data processing as well. The huge anisotropy combined with lack of
> completeness in high resolution shells, and low resolution, made a
> cocktail for not so great electron density. But since this was all I
> got, I had to make something out of it. Since the quality of the
> data was what it was, I definitely wanted others to be able to
> re-investigate it with modern tools, or just see for themselves the
> map I had to make the choices in modelling.
> Remember, I asked for your help to combine all the data of the
> entry, and GlobalPhasing was very helpful to make the tool for this
> entry. I believe, this was the moment you created the deposition
> tool you mention below.
> 
> The fact that the data is not easily retrievable is my point here.
> Pavel knows about this entry as we've been exchanging about it. I
> completely share with him the difficulty to parse through PDB
> entries, having performed several statistical analysis of the PDB to
> search for the root cause of anisotropy in membrane proteins myself
> (https://pubmed.ncbi.nlm.nih.gov/36206830/).
> I encountered the same issue for a recent entry (8qq7, soon
> available I hope) where we tried to combine the same files. I
> vaguely remembered the deposition tool you mention but I couldn't
> locate it (sorry), so we engaged in a dialog with the PDB who made
> us use text editors to merge the files basically. On the top of my
> head, I think we labelled the blocks "original_data", "modified_SA".
> 
> A standardization of this deposition is basically what you all want,
> to perform all the different downstream analysis you want. This is
> not available at the moment. I've been going out of my way to
> combine the files, but it would have been much easier to just give
> the SA_modified file to the PDB. I thus encourage all of you
> software developpers, despite your different point of views, to
> design and agree on such a tool to be placed at OneDep.
> 
> To finish, in case you want to reprocess a high-anisotropy, large
> difference in high-resolution limits, and low resolution, I have the
> images available for 6r72, 2y5y, and 8qq7 (all of which have the
> structure factors with original data, maps and modified data).
> 
> All the best
> Vincent
> 
> Le 15/02/2024 à 19:25, Clemens Vonrhein a écrit :
> >L'adresse mail de l'expéditeur est extérieure :owner-ccp...@jiscmail.ac.uk   
> >En cas de doute : ne répondez pas, ne cliquez pas et signalez le message au 
> >Support Informatique
> >
> >
> >Dear Pavel & CCP4bb readers,
> >
> >On Wed, Feb 14, 2024 at 08:28:03PM -0800, Pavel Afonine wrote:
> >>What follows below is not very specific to the particular program
> >>(STAIRSANISO) nor the original questions, but nonetheless, I believe it is
> >>relevant.
> >Thanks for joining the discussion: always good to have different viewpoints
> >or opinions made visible - especially for less knowledgeable users and
> >readers of the CCP4bb.
> >
> >And apologies to anyone 

Re: [ccp4bb] Coot SSM Superpose Problem

[Eleanor Dodson]

Isnt this what you want? output of CCP4I2 fitting buccaneer to the other..


On Fri, 16 Feb 2024 at 09:46, zx2...@connect.hku.hk 
wrote:

> Dear all,
>
> Following the advice provided by Paul and Yi Min, the "*Symm Shift
> Reference Chain Here*" function has proven successful!
>
> I am deeply grateful for your guidance.
>
> Best regards,
> Xin
> --
> *From:* zx2...@connect.hku.hk 
> *Sent:* 16 February 2024 17:26
> *To:* Paul Emsley 
> *Subject:* Re: [ccp4bb] Coot SSM Superpose Problem
>
> Dear Paul,
>
> I apologize for the confusion in my previous description. To clarify,
> there are actually 4 copies within a single ASU.
>
> Following your suggestion, I tried the "Symm Shift Reference Chain Here" ,
> and it worked successfully. I greatly appreciate your assistance.
>
> Thank you so much!
>
> Best regards,
> Xin
> --
> *From:* Paul Emsley 
> *Sent:* 16 February 2024 11:06
> *To:* zx2...@connect.hku.hk ; CCP4BB@JISCMAIL.AC.UK
> 
> *Subject:* Re: [ccp4bb] Coot SSM Superpose Problem
>
>
>
> On 16/02/2024 01:45, zx2...@connect.hku.hk wrote:
>
> Dear all,
>
> Hello Xin,
>
>
> I encountered an issue while attempting to calculate the RMSD between two
> PDB files (each containing 4 ASUs)
>
> do they really though?
>
>
> using the SSM Superpose tool in Coot. Unfortunately, the tool was unable
> to superpose all ASUs accurately.
>
>
> That's because you have mismatched bundles. It seem to me that you need
> some "Symm Shift Reference Chain Here" action.
>
>
> Paul.
>
>
>
> --
>
> 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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 ###
 ###
 ###
 ### CCP4 8.0.017: csymmatchversion 0.3 : 13/07/09##
 ###
 User: eleanor  Run date: 16/ 2/2024 Run time: 10:43:46 


 Please reference: Collaborative Computational Project, Number 4. 2011.
 "Overview of the CCP4 suite and current developments". Acta Cryst. D67, 
235-242.
 as well as any specific reference in the program write-up.



pdbin   
/Users/eleanor/CCP4I2_PROJECTS/jim-24948v162/CCP4_JOBS/job_308/XYZIN_QUERY-coordinates.pdb
pdbin-ref   
/Users/eleanor/CCP4I2_PROJECTS/jim-24948v162/CCP4_JOBS/job_308/XYZIN_TARGET-coordinates.pdb
origin-hand
pdbout  
/Users/eleanor/CCP4I2_PROJECTS/jim-24948v162/CCP4_JOBS/job_309/309_jim-24948v162_xyzout_csymmatch.pdb

Reference molecule:
  Spacegroup: P 1 21 1
  Unit cell:  Cell (52.932,160.535,95.563,90, 95.91,90)
Moving molecule:
  Spacegroup: P 1 21 1
  Unit cell:  Cell ( 52.96,160.61, 95.61,90, 95.92,90)


 Change of hand  : NO
 Change of origin: uvw = (   0.5,  0.25, 0)

Chain: C1-  43 will be transformed as follows:
  Symmetry operator: x, y, z
  Lattice shift: uvw = ( 0, 0, 0)
  with normalised score:  0.557587
Chain: C   45- 100 will be transformed as follows:
  Symmetry operator: x, y, z
  Lattice shift: uvw = ( 0, 0, 0)
  with normalised score:  0.498482
Chain: C  102- 363 will be transformed as follows:
  Symmetry operator: x, y, z
  Lattice shift: uvw = ( 0, 0, 0)
  with normalised score:  0.610847
Chain: A5-  44 will be transformed as follows:
  Symmetry operator: -x, y+1/2, -z
  Lattice shift: uvw = ( 1,-1, 0)
  with normalised score:  0.593516
Chain: A   48-  96 will be transformed as follows:
  Symmetry operator: -x, y+1/2, -z
  Lattice shift: uvw = ( 1,-1, 0)
  with normalised score:  0.55637
Chain: A  100- 361 will be transformed as follows:
  Symmetry operator: -x, y+1/2, -z
  Lattice shift: uvw = ( 1,-1, 0)
  with normalised score:  0.622536
Chain: D1-  43 will be transformed as follows:
  Symmetry operator: x, y, z
  Lattice shift: uvw = ( 0, 0,-1)
  with normalised score:  0.615694
Chain: D   45-  46 will be transformed as follows:
  Symmetry operator: x, y, z
  Lattice shift: uvw = ( 0, 0,-1)
  with normalised score:  0.276082
Chain: D   48-  94 will be transformed as follows:
  Symmetry operator: x, y, z
  Lattice shift: uvw = ( 0, 0,-1)
  with normalised score:  0.540653
Chain: D  102- 360 will be transformed as 

Re: [ccp4bb] Coot SSM Superpose Problem

[zx2...@connect.hku.hk]

Dear all,

Following the advice provided by Paul and Yi Min, the "Symm Shift Reference 
Chain Here" function has proven successful!

I am deeply grateful for your guidance.

Best regards,
Xin

From: zx2...@connect.hku.hk 
Sent: 16 February 2024 17:26
To: Paul Emsley 
Subject: Re: [ccp4bb] Coot SSM Superpose Problem

Dear Paul,

I apologize for the confusion in my previous description. To clarify, there are 
actually 4 copies within a single ASU.

Following your suggestion, I tried the "Symm Shift Reference Chain Here" , and 
it worked successfully. I greatly appreciate your assistance.

Thank you so much!

Best regards,
Xin

From: Paul Emsley 
Sent: 16 February 2024 11:06
To: zx2...@connect.hku.hk ; CCP4BB@JISCMAIL.AC.UK 

Subject: Re: [ccp4bb] Coot SSM Superpose Problem



On 16/02/2024 01:45, zx2...@connect.hku.hk wrote:
Dear all,
Hello Xin,

I encountered an issue while attempting to calculate the RMSD between two PDB 
files (each containing 4 ASUs)

do they really though?


using the SSM Superpose tool in Coot. Unfortunately, the tool was unable to 
superpose all ASUs accurately.


That's because you have mismatched bundles. It seem to me that you need some 
"Symm Shift Reference Chain Here" action.


Paul.




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[ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Flavio Di Pisa]

Dear community,

I'm looking for a commercial expression vector for E.Coli with a 
cleavable his-tag at the c-ter. Anyone can help me?


Thank you in advance,

Flavio.



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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hosted by www.jiscmail.ac.uk, terms & conditions are available at 
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Re: [ccp4bb] About Staraniso

[vincent Chaptal]

Hi Clemens and all,

I've been following with a lot of interest of course, anisotropy has 
taken a lot of space in my membrane-protein crystallography life.
I remember the many exchanges we've had with you and Global Phasing, as 
well as many other software developpers over the years.


I would like to chip in to give the point of view of a user struggling 
with it, and since 6r72 was mentioned and I'm the author...


6r72 has been an epic battle experimentally, and afterwards with data 
processing as well. The huge anisotropy combined with lack of 
completeness in high resolution shells, and low resolution, made a 
cocktail for not so great electron density. But since this was all I 
got, I had to make something out of it. Since the quality of the data 
was what it was, I definitely wanted others to be able to re-investigate 
it with modern tools, or just see for themselves the map I had to make 
the choices in modelling.
Remember, I asked for your help to combine all the data of the entry, 
and GlobalPhasing was very helpful to make the tool for this entry. I 
believe, this was the moment you created the deposition tool you mention 
below.


The fact that the data is not easily retrievable is my point here. Pavel 
knows about this entry as we've been exchanging about it. I completely 
share with him the difficulty to parse through PDB entries, having 
performed several statistical analysis of the PDB to search for the root 
cause of anisotropy in membrane proteins myself 
(https://pubmed.ncbi.nlm.nih.gov/36206830/).
I encountered the same issue for a recent entry (8qq7, soon available I 
hope) where we tried to combine the same files. I vaguely remembered the 
deposition tool you mention but I couldn't locate it (sorry), so we 
engaged in a dialog with the PDB who made us use text editors to merge 
the files basically. On the top of my head, I think we labelled the 
blocks "original_data", "modified_SA".


A standardization of this deposition is basically what you all want, to 
perform all the different downstream analysis you want. This is not 
available at the moment. I've been going out of my way to combine the 
files, but it would have been much easier to just give the SA_modified 
file to the PDB. I thus encourage all of you software developpers, 
despite your different point of views, to design and agree on such a 
tool to be placed at OneDep.


To finish, in case you want to reprocess a high-anisotropy, large 
difference in high-resolution limits, and low resolution, I have the 
images available for 6r72, 2y5y, and 8qq7 (all of which have the 
structure factors with original data, maps and modified data).


All the best
Vincent

Le 15/02/2024 à 19:25, Clemens Vonrhein a écrit :

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Dear Pavel & CCP4bb readers,

On Wed, Feb 14, 2024 at 08:28:03PM -0800, Pavel Afonine wrote:

What follows below is not very specific to the particular program
(STAIRSANISO) nor the original questions, but nonetheless, I believe it is
relevant.

Thanks for joining the discussion: always good to have different viewpoints
or opinions made visible - especially for less knowledgeable users and
readers of the CCP4bb.

And apologies to anyone getting tired of "another long post" here, but
some remarks do require follow-ups that hopefully will help keep the
discussion at a level useful to all readers.


In the past, performing any adjustments to the diffraction data intended
for solving and refining atomic models was more or less considered taboo.

That is a very broad statement that I have trouble making sense of: what do
you mean with "adjustments" and what do you mean with "diffraction data"?
If we are truly looking at diffraction data as it comes out of our
experiment, we are looking at the raw images, right?  Those are then
handled roughly as follows (as an example for MX):

   * initial integrated intensities (simplifying 3D pixel data)
   
   * profile fitting of integrated intensities


   * scaling (with various parametrisation models)

   * selection of data (excluding image ranges due to radiation damage or
 because a crystal moves out of the beam, excluding/handling ice-ring
 contamination, selecting datasets in SSX etc)

   * adjustment of error model (to get "meaningful" error estimates,
 i.e. sigma values)

   * outlier rejection (based largely on those sigmas)

   * merging (inverse-variance weighted)

Maybe all those "adjustments to the diffraction data" are not what you are
referring to in your remark above? So let's assume you are referring to the
merged intensity data after all of the above steps as being "the
diffraction data" ... and what we are doing after that:

   * conversion from intensities to amplitudes (using different methods and
 priors) which most often will include an adjustment of weak and
 negative