Re: [ccp4bb] To solve the problem of an extremely asymmetric

[Rezaul Karim]

Exactly. Without a picture of SEC profile it's even difficult to decipher the 
meaning of "extremely asymmetric peak shape".
Beside that, one domain or multi-domain protein, extended N-term or C-term 
loops containing constructs, column, load (weight & vol) and flow rate - any 
one of these could affect separation efficiency and/or peak characteristics.
Regards,Reza

“The rule for web content is to keep it short. The rule for email content is to 
keep it ultra-short.”
— Jakob Nielsen, ‘king of usability’


Md Rezaul Karim, Ph.D.
Postdoctoral Research Fellow
Department of Drug Discovery
H. Lee Moffitt Cancer Center and Research Institute
Tampa, FL 33612
Email: reza.ka...@moffitt.org, rez...@usf.edu
Phone: (813) 745 4673 ext. 5462
https://orcid.org/-0002-0424-127X 
 
  On Wed, Dec 9, 2020 at 11:42 AM, Artem Evdokimov 
wrote:   Probably a good idea to share an image :) worth many words...
Artem
On Wed, Dec 9, 2020, 9:17 AM   wrote:

Dear All
        There is a 43kd protein purified via Ni-chelating affinity 
chromatography, anion exchange chromatography and gel filtration chromatography 
in sequence. However the chromatogram obtained showed an extremely asymmetric 
peak shape. The aggregation forms of proteins are mainly in the range of 
monomers and dimers(Hepes and low concentration of salt were used as buffers 
for gel filtration chromatography). 5% glycerinum and 1mM Benzamidine 
hydrochloride had been added in order to maintain the stability of the protein 
and prevent it from degrading. But well, all the efforts seem to be useless. We 
wonder if there are any effective measures can be taken to radically solve this 
problem. We would be much indebted for the suggestions you offer.


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Re: [ccp4bb] Coming July 29: Improved Carbohydrate Data at the PDB -- N-glycans are now separate chains if more than one residue

[Marcin Wojdyr]

Dear Jasmine,

thank you for this explanation. It's the best explanation of this
remediation I've read.

The use of IDs may confuse people, so I'd like to reiterate it and ask
for clarification.
Every residue in the mmCIF format has three (3) independent chain IDs
assigned to it (and three sequence numbers, and three residue names).

In your example:
J 4 NAG 1 I NAG 1 A NAG 1310 n

J - asym_id = _atom_site.label_asym_id
I - pdb_asym_id = _atom_site.auth_asym_id (?!)
A - auth_asym_id = n/a

(correct me if I got it wrong, but I see that _atom_site.auth_asym_id
corresponds to _pdbx_branch_scheme.pdb_asym_id and not to auth_asym_id
as one could expect).

How to call these chain IDs? When I write software documentation, I
need to refer to chain IDs (and sequence numbers), but I can't find
proper words to clearly tell which ID I'm referring to. I was using
hard to read names such as auth_asym_id, but now I see that even this
is ambiguous.

BTW, when you write that wwPDB encourages depositors to use the
wwPDB-assigned chain ID in publications, which of the two
wwPDB-assigned chain IDs do you mean?

Thank you,
Marcin



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Re: [ccp4bb] when does non-isomorphism become a habit?

[Ailong Ke]

Good thoughts, although there are always exceptions. For example, if a crystal 
contact is mediated by an extended domain that is capable of adopting multiple 
conformations (due to elbow motion, for example), then it may allow multiple 
crystal forms. It happens a lot in RNA crystals, but can also be found in 
protein crystals. 

From the user’s point of view, if crystal dehydration leads to space group 
change, it is a new crystal form. If the refinement does not kick off with 
routine fiddling of refinement parameters, it is equivalent to a new crystal 
form, because one has to go through the same procedure as to solve a new 
crystal form.  

Ailong

> On Dec 9, 2020, at 1:54 AM, Manfred S. Weiss 
>  wrote:
> 
> Dear James,
> 
> let's spin the thought a bit further. What if there is a new program
> (Phaser-II) some day,
> for which the coordinates in your "new crystal form" are all of a sudden
> within the
> radius of convergence again? Does this bring your "new crystal form"
> back to the old
> crystal form again?
> 
> I'd say it is neither cell dimensions nor space group that define a new
> crystal form. It
> is the packing of the molecules. It happens quite often in dehydration
> experiments
> that molecules in a lattice move a bit and symmetry elements are lost or
> new symmetry
> elements are created. The space group changes, as well as the unit cell
> dimensions. But
> the packing remains essentially the same. You can usually infer what
> happens, but you
> still need molecular replacement to actually solve the "new" form.
> 
> Best, Manfred
> 
> 
> Am 09.12.2020 um 01:56 schrieb James Holton:
>> I have a semantics question, and I know how much this forum loves
>> discussing semantics.
>> 
>> We've all experienced non-isomorphism, where two crystals, perhaps
>> even grown from the same drop, yield different data. Different enough
>> so that merging them makes your overall data quality worse. I'd say
>> that is a fairly reasonable definition of non-isomorphism? Most of the
>> time unit cell changes are telling, but in the end it is the I/sigma
>> and resolution limit that we care about the most.
>> 
>> Now, of course, even for non-isomorphous data sets you can usually
>> "solve" the non-isomorphous data without actually doing molecular
>> replacement.  All you usually need to do is run pointless using the
>> PDB file from the first crystal as a reference, and it will re-index
>> the data to match the model.  Then you just do a few cycles of rigid
>> body and you're off and running.  A nice side-effect of this is that
>> all your PDB files will line up when you load them into coot.  No
>> worries about indexing ambiguities, space group assignment, or origin
>> choice. Phaser is a great program, but you don't have to run it on
>> everything.
>> 
>> My question is: what about when you DO have to run Phaser to solve
>> that other crystal from the same drop?  What if the space group is the
>> same, the unit cell is kinda-sorta the same, but the coordinates have
>> moved enough so as to be outside the radius of convergence of
>> rigid-body refinement?  Does that qualify as a different "crystal
>> form" or different "crystal habit"?  Or is it the same form, and just
>> really non-isomorphous?
>> 
>> Opinions?
>> 
>> -James Holton
>> MAD Scientist
>> 
>> 
>> 
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> 
> --
> Dr. Manfred S. Weiss
> Macromolecular Crystallography
> Helmholtz-Zentrum Berlin
> Albert-Einstein-Str. 15
> D-12489 Berlin
> Germany
> 
> 
> 
> 
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> 
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> e.V.
> 
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> Thomas Frederking
> 
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This message 

Re: [ccp4bb] To solve the problem of an extremely asymmetric peak shape obtained from gel filtration chromatography

[Artem Evdokimov]

Probably a good idea to share an image :) worth many words...

Artem

On Wed, Dec 9, 2020, 9:17 AM   wrote:

> Dear All
> There is a 43kd protein purified via Ni-chelating affinity
> chromatography, anion exchange chromatography and gel filtration
> chromatography in sequence. However the chromatogram obtained showed an
> extremely asymmetric peak shape. The aggregation forms of proteins are
> mainly in the range of monomers and dimers(Hepes and low concentration of
> salt were used as buffers for gel filtration chromatography). 5% glycerinum
> and 1mM Benzamidine hydrochloride had been added in order to maintain the
> stability of the protein and prevent it from degrading. But well, all the
> efforts seem to be useless. We wonder if there are any effective measures
> can be taken to radically solve this problem. We would be much indebted for
> the suggestions you offer.
>
> --
>
> 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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[ccp4bb] Job Vacancy: Postdoctoral research fellow position available in Therapeutics Discovery Division at MD Anderson Cancer Center

[Leonard,Paul G]

Hi,

A postdoctoral position is currently available in the Structural Chemistry 
group, part of the Institute for Applied Cancer Science (IACS) within the 
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integrate novel biological insights with drug discovery expertise to accelerate 
the development of innovative, targeted, small-molecule therapeutics for unmet 
medical needs. Our work is conducted in a team science environment that brings 
together excellence in both academic and pharmaceutical discovery, as evidenced 
by recent publications.

We are seeking a highly motivated individual with a strong background in 
structural biology, including protein expression and purification, protein 
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proteins is not required, but would be advantageous. We are seeking only the 
most dedicated individuals who will join us in our quest to discover, develop 
and deliver more effective treatments to our patients. This is a unique 
opportunity to participate in project-driven drug discovery research within a 
highly respected academic and clinical setting.

Interested individuals should apply via the Postdoc Jobs website.
https://www.postdocjobs.com/posting/7072972

Paul Leonard
Senior Institute Research Scientist
MD Anderson Cancer Center
1881 East Road
Houston TX 77054
USA


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Re: [ccp4bb] To solve the problem of an extremely asymmetric peak shape obtained from gel filtration chromatography

[Ross Robinson]

It would be sensible to analyse the state of your purified protein further.

  *   (SEC in reasonable salt conc containing buffer – this is standard 
practice)
  *   Analytical ion exchange of purified protein - are there different states?
  *   SEC  (in reasonable buffer) in line with MALS – is there a monomer-dimer 
equilibrium?
  *   Analytical SEC (without further concentration) of the potential dimer 
side of the peak, repeat for monomer side – could give info on potential 
monomer-dimer equilibrium

Cheers,
Ross



From: CCP4 bulletin board  On Behalf Of Javier Gonzalez
Sent: 09 December 2020 14:52
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] To solve the problem of an extremely asymmetric peak 
shape obtained from gel filtration chromatography

[EXTERNAL SENDER]

Hello, I agree with Roger, you should definitely try to increase the salt 
concentration to get rid of non specific binding impurities. And if that 
doesn't work, you can try purifying your protein under denaturing conditions by 
adding one refolding step in the column.
Good luck,
Javier

On Wed, Dec 9, 2020 at 11:26 AM Roger Rowlett 
mailto:rrowl...@colgate.edu>> wrote:
Salt concentrations less than 100 mM can lead to nonspecific adsorption to the 
gel exclusion media, potentially leading to band broadening, and delayed 
elution.  Overloading gel exclusion columns (more than 2-4% Vt) can also lead 
to elution band artifacts. Check these issues first.

Roger Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University

On Wed, Dec 9, 2020, 9:17 AM  
mailto:sz20203020...@cau.edu.cn>> wrote:
Dear All
There is a 43kd protein purified via Ni-chelating affinity 
chromatography, anion exchange chromatography and gel filtration chromatography 
in sequence. However the chromatogram obtained showed an extremely asymmetric 
peak shape. The aggregation forms of proteins are mainly in the range of 
monomers and dimers(Hepes and low concentration of salt were used as buffers 
for gel filtration chromatography). 5% glycerinum and 1mM Benzamidine 
hydrochloride had been added in order to maintain the stability of the protein 
and prevent it from degrading. But well, all the efforts seem to be useless. We 
wonder if there are any effective measures can be taken to radically solve this 
problem. We would be much indebted for the suggestions you offer.


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



To unsubscribe from the CCP4BB list, click the following link:
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--
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352



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Re: [ccp4bb] Coming July 29: Improved Carbohydrate Data at the PDB -- N-glycans are now separate chains if more than one residue

[Jasmine Young]

Dear Robbie,

In the case that only single monosaccharide was modelled at 
glycosylation site with a known oligosaccharide sequence, technically 
the software cannot generate glycosidic linkages, linear descriptors for 
sequences, 2D SNFG images, etc. Therefore, this single monosaccharide 
cannot be represented as a branched entity. However, if an author 
provides PDB an updated coordinates with additional monosaccharides 
modeled that bounds to the original monosaccharide, the software will 
automatically detect this linkage and convert non-polymer entity of the 
original monosaccharide into a branched entity (vice versa).



Regards,

Jasmine

===
Jasmine Young, Ph.D.
Biocuration Team Lead
RCSB Protein Data Bank
Research Professor
Institute for Quantitative Biomedicine
Rutgers, The State University of New Jersey
174 Frelinghuysen Rd
Piscataway, NJ 08854-8087

Email: jasm...@rcsb.rutgers.edu
Phone: (848)445-0103 ext 4920
Fax: (732)445-4320
===

On 12/9/20 4:35 AM, Robbie Joosten wrote:


Dear Jasmine,

I have a few questions about this bit:

//

As some users pointed out, single NAG could be just a part of the 
glycan that the author chose to build, as most natural N-glycans must 
have stem of a common core of 5 monosaccharides or its fucosylated 
version, such as those modeled in the PDB ID 6WPS. However, the PDB is 
a 3D-atomic coordinate archive in which the model coordinates are 
built based on supporting experimental data. Therefore, carbohydrates 
are described as-is in the modeled structures without reference to 
missing components of the presumed oligosaccharide sequence. If the 
author only builds a monosaccharide, then this monosaccharide is 
described as a non-polymer ligand.


//

Is it technically allowed to have a single, covalently bound 
carbohydrate described as a branched entity of length 1?


If so, if an author does specify such a single modeled residue as 
branched entity, for instance because (s)he has a good reason to 
suspect that a second residue was there, but isn’t comfortable with 
building it, then is this specification kept in annotation?


If not, do you expect model building programs to switch from branched 
to non-poly entities when a second residue is removed when a model is 
written out? And back again when a residue is added? I find this 
rather unpractical from an implementation point of view. We change 
carbohydrate trees quite regularly.


Cheers,

Robbie

*From:*CCP4 bulletin board  *On Behalf Of 
*Jasmine Young

*Sent:* Tuesday, December 8, 2020 21:01
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* Re: [ccp4bb] Coming July 29: Improved Carbohydrate Data at 
the PDB -- N-glycans are now separate chains if more than one residue


Dear PDB Data Users:

Thank you for providing feedback on the results of an archival-level 
carbohydrate remediation project that led to the re-release of over 
14,000 PDB structures in July 2020. This update includes diverse 
oligosaccharides: glycosylation; metabolites such as maltose, sucrose, 
cellulose fragments; glycosaminoglycans, such as fragments of heparin 
and heparan sulfate; epitope patterns such as A/B blood group antigens 
and the H-type or Lewis-type stems; and many artificial carbohydrates 
mimicking or counting natural products 
(https://www.wwpdb.org/documentation/carbohydrate-remediation 
).


Starting in 2017, this PDB remediation aimed to standardize the 
biochemical nomenclature of the carbohydrate components following the 
IUPAC-IUBMB recommendations established by the carbohydrate community 
(https://media.iupac.org/publications/pac/1996/pdf/6810x1919.pdf 
), 
and to provide uniform representation of oligosaccharides to improve 
the identification and searchability of oligosaccharides modeled in 
the PDB structures.  During the remediation planning, wwPDB consulted 
community users and the PDBx/mmCIF Working Group and made data files 
available on GitHub in early 2020 for community feedback. wwPDB has 
collaborated with Robert Woods at University of Georgia in US, 
researchers at The Noguchi Institute and Soka University in Japan, and 
Thomas Lutteke in Germany to generate uniform linear descriptors for 
the oligosaccharide sequences.


To achieve these community goals, each oligosaccharide is represented 
as a branched entity with complete biochemical description and each 
glycosidic linkage specified. The full representation of carbohydrates 
is provided in the mmCIF format file, but this is not possible in 
legacy PDB format files (as the format has been frozen since 2012 
(https://www.wwpdb.org/documentation/file-formats-and-the-pdb 
).


Proper indexing is necessary for branched entity representation and 
for generation of 

Re: [ccp4bb] To solve the problem of an extremely asymmetric peak shape obtained from gel filtration chromatography

[Javier Gonzalez]

Hello, I agree with Roger, you should definitely try to increase the salt
concentration to get rid of non specific binding impurities. And if that
doesn't work, you can try purifying your protein under denaturing
conditions by adding one refolding step in the column.
Good luck,
Javier

On Wed, Dec 9, 2020 at 11:26 AM Roger Rowlett  wrote:

> Salt concentrations less than 100 mM can lead to nonspecific adsorption to
> the gel exclusion media, potentially leading to band broadening, and
> delayed elution.  Overloading gel exclusion columns (more than 2-4% Vt) can
> also lead to elution band artifacts. Check these issues first.
>
> Roger Rowlett
> Gordon & Dorothy Kline Professor, Emeritus
> Department of Chemistry
> Colgate University
>
> On Wed, Dec 9, 2020, 9:17 AM   wrote:
>
>> Dear All
>> There is a 43kd protein purified via Ni-chelating affinity
>> chromatography, anion exchange chromatography and gel filtration
>> chromatography in sequence. However the chromatogram obtained showed an
>> extremely asymmetric peak shape. The aggregation forms of proteins are
>> mainly in the range of monomers and dimers(Hepes and low concentration of
>> salt were used as buffers for gel filtration chromatography). 5% glycerinum
>> and 1mM Benzamidine hydrochloride had been added in order to maintain the
>> stability of the protein and prevent it from degrading. But well, all the
>> efforts seem to be useless. We wonder if there are any effective measures
>> can be taken to radically solve this problem. We would be much indebted for
>> the suggestions you offer.
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352



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Re: [ccp4bb] To solve the problem of an extremely asymmetric peak shape obtained from gel filtration chromatography

[Roger Rowlett]

Salt concentrations less than 100 mM can lead to nonspecific adsorption to
the gel exclusion media, potentially leading to band broadening, and
delayed elution.  Overloading gel exclusion columns (more than 2-4% Vt) can
also lead to elution band artifacts. Check these issues first.

Roger Rowlett
Gordon & Dorothy Kline Professor, Emeritus
Department of Chemistry
Colgate University

On Wed, Dec 9, 2020, 9:17 AM   wrote:

> Dear All
> There is a 43kd protein purified via Ni-chelating affinity
> chromatography, anion exchange chromatography and gel filtration
> chromatography in sequence. However the chromatogram obtained showed an
> extremely asymmetric peak shape. The aggregation forms of proteins are
> mainly in the range of monomers and dimers(Hepes and low concentration of
> salt were used as buffers for gel filtration chromatography). 5% glycerinum
> and 1mM Benzamidine hydrochloride had been added in order to maintain the
> stability of the protein and prevent it from degrading. But well, all the
> efforts seem to be useless. We wonder if there are any effective measures
> can be taken to radically solve this problem. We would be much indebted for
> the suggestions you offer.
>
> --
>
> 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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[ccp4bb] To solve the problem of an extremely asymmetric peak shape obtained from gel filtration chromatography

Dear All
There is a 43kd protein purified via Ni-chelating affinity 
chromatography, anion exchange chromatography and gel filtration chromatography 
in sequence. However the chromatogram obtained showed an extremely asymmetric 
peak shape. The aggregation forms of proteins are mainly in the range of 
monomers and dimers(Hepes and low concentration of salt were used as buffers 
for gel filtration chromatography). 5% glycerinum and 1mM Benzamidine 
hydrochloride had been added in order to maintain the stability of the protein 
and prevent it from degrading. But well, all the efforts seem to be useless. We 
wonder if there are any effective measures can be taken to radically solve this 
problem. We would be much indebted for the suggestions you offer.




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

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Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Bryan Lepore]

> On Dec 9, 2020, at 07:45, Harry Powell - CCP4BB 
> <193323b1e616-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> ...  GDT_TS (Global Distance Test - Total Score - you can look it up on 
> Wikipedia

Thanks, this is helpful.

Wikipedia:

“The primary GDT assessment uses only the alpha carbon atoms.”

Then there’s GDT_sc that incorporates side chains in a soecific way, then 
GDC_all.

References: Zemla A (2003). "LGA: A method for finding 3D similarities in 
protein structures". Nucleic Acids Research. 31(13): 3370–3374. 
doi:10.1093/nar/gkg571. PMC 168977. PMID 12824330.

Keedy, D.A.; Williams, CJ; Headd, JJ; Arendall, WB; Chen, VB; Kapral, GJ; 
Gillespie, RA; Block, JN; Zemla, A; Richardson, DC; Richardson, JS (2009). "The 
other 90% of the protein: Assessment beyond the α-carbon for CASP8 
template-based and high-accuracy models". Proteins. 77 (Suppl 9): 29–49. 
doi:10.1002/prot.22551. PMC 2877634. PMID 19731372.

Modi V, Xu QF, Adhikari S, Dunbrack RL (2016). "Assessment of template‐based 
modeling of protein structure in CASP11". Proteins. 84: 200–220. 
doi:10.1002/prot.25049. PMC 5030193. PMID 27081927.
...

The “C-alpha-IDDT” cited in the AlphaFold abstract was published in 2013:

Mariani et. al., Bioinformatics, 29(21), 2722-2728, 2013

The top scores increased after 2013.

-Bryan W. Lepore




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Re: [ccp4bb] AW: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Patrick Shaw Stewart]

>they can maintain an advantage through several routes - they can
> publish in patents (so people can see what they’ve done, but not legally
> implement it )


In Europe and I think some other countries, inventions can only be patented
if they have *industrial applicability.*

In any case, academics all over the world tend to ignore them.



On Wed, Dec 9, 2020 at 12:18 PM Harry Powell - CCP4BB <
193323b1e616-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hi
>
> Actually, since Deep Mind is a commercial organization (funded by
> shareholders and people who buy their services), I don’t think they are
> subject to the same rules as academia as regards making their source code
> public. It would be very nice if they would (could?) make their code
> public, but I don’t see any obligation to do so. Their responsibility is
> primarily to their shareholders (you can argue the rights and wrongs of
> that until the cows come home).
>
> Commercially, they can maintain an advantage through several routes - they
> can publish in patents (so people can see what they’ve done, but not
> legally implement it without a licence), they can keep it all confidential
> and hope that no-one manages to reverse engineer and implement it (at the
> risk of someone else publishing the details and removing their advantage),
> they can publish something that is honest but just misleading enough (or
> lacking in detail) to throw people off the scent, or…
>
> If they can provoke other developers to work out where they have gone
> wrong and produce something that competes with AlphaFold2, that would be
> great. If they can provide something like a web service that allows users
> to run their method, that would be great too, but the important thing is
> (that unless they had prior knowledge of the structures in CASP14) they’ve
> done something that no-one else has managed to do as well in spite of years
> of trying.
>
> Just my two ha’porth.
>
> Harry
>
> > On 9 Dec 2020, at 10:36, Hughes, Jonathan <
> jon.hug...@bot3.bio.uni-giessen.de> wrote:
> >
> > i think the answer to all these doubts and questions is quite simple:
> the AlphaFold2 people must make all details of their methods public (source
> code) and, as would probably be necessary, open their system for inspection
> and use by independent experts. isn't that what peer review and
> reproducibility are all about? those rules date from the time before every
> tom, dick and henriette could publicize anything they like inside their own
> zuckerberg bubble. my opinion is that this is a virtual infectious disease
> that will cause humanity far bigger problems than corona ever will – i just
> hope i'm wrong!
> >
> > best
> >
> > jon
> >
> >
> >
> > Von: CCP4 bulletin board  Im Auftrag von Mark J
> van Raaij
> > Gesendet: Mittwoch, 9. Dezember 2020 11:14
> > An: CCP4BB@JISCMAIL.AC.UK
> > Betreff: Re: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking
> and less pipetting (?)
> >
> >
> >
> > on the day the news came out, I did wonder if the AlphaFold2 team
> somehow had access to all the preliminary PDB files sent around via Gmail
> (which belongs to the same company), but more as a joke/conspirational
> thought.
> >
> > "our" target T1052, was also predicted very well by domains and as a
> monomer. It will be interesting to see how well future iterations of the
> method can assemble the complete protein chain and the complete protein
> chains into the correct heteromer.
> >
> >
> >
> > Mark J van Raaij
> > Dpto de Estructura de Macromoleculas
> > Centro Nacional de Biotecnologia - CSIC
> > calle Darwin 3
> > E-28049 Madrid, Spain
> > tel. (+34) 91 585 4616
> >
> > Section Editor Acta Crystallographica F
> > https://journals.iucr.org/f/
> >
> >
> >
> > On 9 Dec 2020, at 10:37, Cedric Govaerts 
> wrote:
> >
> >
> >
> > Dear All
> >
> >
> >
> > After about 10 (!) years of (very) hard work we solved the structures of
> our dearest membrane transporter.  Dataset at 2.9 And resolution, fairly
> anisotropic, experimental phasing, and many long nights with Coot and
> Buster to achieve model refinement.
> >
> >
> >
> > The experimental structure had a well defined ligand nicely coordinated
> but also a lipid embedded inside the binding cavity (a complete surprise
> but biologically relevant) and two detergent molecules well defined
> (experimental/crystallisation artefact).
> >
> >
> >
> > As our paper was accepted basically when CASP organisers were calling
> for targets I offered my baby to the computing Gods. However we only
> provided the sequence to CASP, no info regarding any ligand or lipid.
> >
> >
> >
> > Less than a month after, the CASP team contacted us and send us the best
> model.  In fact it was 2 half models as the transporter is a pseudo dimer,
> with the N-lobe and C-lobe moving relative to each other during transport
> cycle, thus divided as two domains in CASP.
> >
> >
> >
> > The results were breathtaking. 0.7 And RSMD on one half, 0.6 on the
> other. And yes, 

Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Harry Powell - CCP4BB]

The “something” is what gives them their edge (and which they’ve hinted at, but 
avoided being explicit)…

The main quality score used to distinguish their results is GDT_TS (Global 
Distance Test - Total Score - you can look it up on Wikipedia like I did). 
Although it doesn’t say in Wikipedia, it seems to be normalised to 100 for a 
perfect fit. Alphafold2 was scoring >90+, the best second-placed were ~60-65. 

Some of the superpositions of models from structure solution and AlphaFold2 
looked like the errors in position of main and side chains were <<1Å. 

Since I’m quite new to the field, and haven’t really paid CASP much attention 
in the past I wouldn’t want to comment about past methods. I’ve got a lot to 
learn.

Harry



> On 9 Dec 2020, at 12:35, Bryan Lepore  wrote:
> 
> On Dec 9, 2020, at 07:16, Harry Powell wrote:
>> 
>> ...the important thing is [...] they’ve done something that no-one else has 
>> managed to do as well in spite of years of trying.
> 
> What, precisely, is the “something”?
> 
> Exactly how much better than second place? 
> 
> Was the scoring the same across all years when no-one else managed to do as 
> well?
> 
> -Bryan W. Lepore
> 
> 
> 
> 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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Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Bryan Lepore]

On Dec 9, 2020, at 07:16, Harry Powell wrote:
> 
> ...the important thing is [...] they’ve done something that no-one else has 
> managed to do as well in spite of years of trying.

What, precisely, is the “something”?

Exactly how much better than second place? 

Was the scoring the same across all years when no-one else managed to do as 
well?

-Bryan W. Lepore



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Re: [ccp4bb] AW: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Harry Powell - CCP4BB]

Hi

Actually, since Deep Mind is a commercial organization (funded by shareholders 
and people who buy their services), I don’t think they are subject to the same 
rules as academia as regards making their source code public. It would be very 
nice if they would (could?) make their code public, but I don’t see any 
obligation to do so. Their responsibility is primarily to their shareholders 
(you can argue the rights and wrongs of that until the cows come home).

Commercially, they can maintain an advantage through several routes - they can 
publish in patents (so people can see what they’ve done, but not legally 
implement it without a licence), they can keep it all confidential and hope 
that no-one manages to reverse engineer and implement it (at the risk of 
someone else publishing the details and removing their advantage), they can 
publish something that is honest but just misleading enough (or lacking in 
detail) to throw people off the scent, or…

If they can provoke other developers to work out where they have gone wrong and 
produce something that competes with AlphaFold2, that would be great. If they 
can provide something like a web service that allows users to run their method, 
that would be great too, but the important thing is (that unless they had prior 
knowledge of the structures in CASP14) they’ve done something that no-one else 
has managed to do as well in spite of years of trying.

Just my two ha’porth.

Harry

> On 9 Dec 2020, at 10:36, Hughes, Jonathan 
>  wrote:
> 
> i think the answer to all these doubts and questions is quite simple: the 
> AlphaFold2 people must make all details of their methods public (source code) 
> and, as would probably be necessary, open their system for inspection and use 
> by independent experts. isn't that what peer review and reproducibility are 
> all about? those rules date from the time before every tom, dick and 
> henriette could publicize anything they like inside their own zuckerberg 
> bubble. my opinion is that this is a virtual infectious disease that will 
> cause humanity far bigger problems than corona ever will – i just hope i'm 
> wrong!
> 
> best
> 
> jon
> 
>  
> 
> Von: CCP4 bulletin board  Im Auftrag von Mark J van 
> Raaij
> Gesendet: Mittwoch, 9. Dezember 2020 11:14
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: Re: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and 
> less pipetting (?)
> 
>  
> 
> on the day the news came out, I did wonder if the AlphaFold2 team somehow had 
> access to all the preliminary PDB files sent around via Gmail (which belongs 
> to the same company), but more as a joke/conspirational thought.
> 
> "our" target T1052, was also predicted very well by domains and as a monomer. 
> It will be interesting to see how well future iterations of the method can 
> assemble the complete protein chain and the complete protein chains into the 
> correct heteromer.
> 
>  
> 
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616
> 
> Section Editor Acta Crystallographica F
> https://journals.iucr.org/f/
> 
>  
> 
> On 9 Dec 2020, at 10:37, Cedric Govaerts  wrote:
> 
>  
> 
> Dear All
> 
>  
> 
> After about 10 (!) years of (very) hard work we solved the structures of our 
> dearest membrane transporter.  Dataset at 2.9 And resolution, fairly 
> anisotropic, experimental phasing, and many long nights with Coot and 
> Buster to achieve model refinement. 
> 
>  
> 
> The experimental structure had a well defined ligand nicely coordinated but 
> also a lipid embedded inside the binding cavity (a complete surprise but 
> biologically relevant) and two detergent molecules well defined 
> (experimental/crystallisation artefact).
> 
>  
> 
> As our paper was accepted basically when CASP organisers were calling for 
> targets I offered my baby to the computing Gods. However we only provided the 
> sequence to CASP, no info regarding any ligand or lipid.
> 
>  
> 
> Less than a month after, the CASP team contacted us and send us the best 
> model.  In fact it was 2 half models as the transporter is a pseudo dimer, 
> with the N-lobe and C-lobe moving relative to each other during transport 
> cycle, thus divided as two domains in CASP.
> 
>  
> 
> The results were breathtaking. 0.7 And RSMD on one half, 0.6 on the other. 
> And yes, group 427 was the superpower (did not know at the time that it was 
> AlphaFold).
> 
>  
> 
> We had long discussions with the CASP team, as -for us- this almost exact 
> modelling was dream-like (or science fiction) and -at some point- we were 
> even suspecting fraud, as our coordinates had travelled over the internet a 
> few times around when interacting with colleagues.  The organisers reassured 
> us that we were not the only target that had been “nailed” so no reason to 
> suspect any wrongdoing.
> 
>  
> 
> To this day I am still baffled and I would be happy to hear from 

Re: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Matthew Snee]

It seems immensely powerful, but my impression is it shows just how much 
information can be extrapolated from the PDB if a technique that can make use 
of "deep similarity" can be employed.

Obviously alphafold2 can make use of relationships that arent limited to direct 
homology, but if there is a fundamental "cellular context-free" relationship 
between sequence and structure (I'm sceptical about this) then it must be via 
the sidechains.

If the sidechains predictions are worse than the backbone, and loops are also 
imperfect, then it strongly suggests that the process is still inferring the 
structure (albeit in a very clever way that can determine and weight 
similarities that go far beyond those implied by direct homology) rather than  
"building" it de novo.

Obviously sidechain and loop positions are important when we think about the 
applications of macro molecular structures, but I'm not qualified to say 
whether there is actually enough data in the PDB to beat the law of diminishing 
returns and reliably get trustworthy "experimental quality" predictions, and 
how that will scale with complex proteins which may be very context dependent 
in their ability to fold.

We probably dont need a universal understanding of sequence/structure to get 
there, but the claim that this is just a matter of time only really follows on 
from the assumption of a true de-novo method.  Without it, the learning set may 
need to be bigger than all solved (or even solveable) structures.

This could have been framed as something really exciting and complementary to 
experimental structural biology (trivial MR, much better denovo EM etc..) at a 
time when multi-disciplinary approaches are producing incredible insights, but 
the press that has been generated, seems  misleading, and I fear this is what 
the public and funders will base their decisions upon.

Just my two cents.

Matthew.




Get Outlook for Android


From: CCP4 bulletin board  on behalf of Cedric Govaerts 

Sent: Wednesday, December 9, 2020 9:37:17 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less 
pipetting (?)

Dear All

After about 10 (!) years of (very) hard work we solved the structures of our 
dearest membrane transporter.  Dataset at 2.9 And resolution, fairly 
anisotropic, experimental phasing, and many long nights with Coot and 
Buster to achieve model refinement.

The experimental structure had a well defined ligand nicely coordinated but 
also a lipid embedded inside the binding cavity (a complete surprise but 
biologically relevant) and two detergent molecules well defined 
(experimental/crystallisation artefact).

As our paper was accepted basically when CASP organisers were calling for 
targets I offered my baby to the computing Gods. However we only provided the 
sequence to CASP, no info regarding any ligand or lipid.

Less than a month after, the CASP team contacted us and send us the best model. 
 In fact it was 2 half models as the transporter is a pseudo dimer, with the 
N-lobe and C-lobe moving relative to each other during transport cycle, thus 
divided as two domains in CASP.

The results were breathtaking. 0.7 And RSMD on one half, 0.6 on the other. And 
yes, group 427 was the superpower (did not know at the time that it was 
AlphaFold).

We had long discussions with the CASP team, as -for us- this almost exact 
modelling was dream-like (or science fiction) and -at some point- we were even 
suspecting fraud, as our coordinates had travelled over the internet a few 
times around when interacting with colleagues.  The organisers reassured us 
that we were not the only target that had been “nailed” so no reason to suspect 
any wrongdoing.

To this day I am still baffled and I would be happy to hear from the community, 
maybe from some of the CASP participants.

The target is T024, the “perfect" models are domain-split version (T024-D1 and 
T024-D2), as AlphaFold2 did not perform so well on the complete assembly.
Deposited PDB is 6T1Z

Cedric

PS: I should also note that many other groups performed very well, much better 
than I would have dreamed, including on the full protein but just not as 
crazy-good.
—
Prof. Cedric Govaerts, Ph.D.
Universite Libre de Bruxelles
Campus Plaine. Phone :+32 2 650 53 77
Building BC, Room 1C4 203
Boulevard du Triomphe, Acces 2
1050 Brussels
Belgium
http://govaertslab.ulb.ac.be/




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 

[ccp4bb] AW: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Hughes, Jonathan]

i think the answer to all these doubts and questions is quite simple: the 
AlphaFold2 people must make all details of their methods public (source code) 
and, as would probably be necessary, open their system for inspection and use 
by independent experts. isn't that what peer review and reproducibility are all 
about? those rules date from the time before every tom, dick and henriette 
could publicize anything they like inside their own zuckerberg bubble. my 
opinion is that this is a virtual infectious disease that will cause humanity 
far bigger problems than corona ever will – i just hope i'm wrong!
best
jon

Von: CCP4 bulletin board  Im Auftrag von Mark J van Raaij
Gesendet: Mittwoch, 9. Dezember 2020 11:14
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less 
pipetting (?)

on the day the news came out, I did wonder if the AlphaFold2 team somehow had 
access to all the preliminary PDB files sent around via Gmail (which belongs to 
the same company), but more as a joke/conspirational thought.
"our" target T1052, was also predicted very well by domains and as a monomer. 
It will be interesting to see how well future iterations of the method can 
assemble the complete protein chain and the complete protein chains into the 
correct heteromer.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/

On 9 Dec 2020, at 10:37, Cedric Govaerts 
mailto:cedric.govae...@ulb.ac.be>> wrote:

Dear All

After about 10 (!) years of (very) hard work we solved the structures of our 
dearest membrane transporter.  Dataset at 2.9 And resolution, fairly 
anisotropic, experimental phasing, and many long nights with Coot and 
Buster to achieve model refinement.

The experimental structure had a well defined ligand nicely coordinated but 
also a lipid embedded inside the binding cavity (a complete surprise but 
biologically relevant) and two detergent molecules well defined 
(experimental/crystallisation artefact).

As our paper was accepted basically when CASP organisers were calling for 
targets I offered my baby to the computing Gods. However we only provided the 
sequence to CASP, no info regarding any ligand or lipid.

Less than a month after, the CASP team contacted us and send us the best model. 
 In fact it was 2 half models as the transporter is a pseudo dimer, with the 
N-lobe and C-lobe moving relative to each other during transport cycle, thus 
divided as two domains in CASP.

The results were breathtaking. 0.7 And RSMD on one half, 0.6 on the other. And 
yes, group 427 was the superpower (did not know at the time that it was 
AlphaFold).

We had long discussions with the CASP team, as -for us- this almost exact 
modelling was dream-like (or science fiction) and -at some point- we were even 
suspecting fraud, as our coordinates had travelled over the internet a few 
times around when interacting with colleagues.  The organisers reassured us 
that we were not the only target that had been “nailed” so no reason to suspect 
any wrongdoing.

To this day I am still baffled and I would be happy to hear from the community, 
maybe from some of the CASP participants.

The target is T024, the “perfect" models are domain-split version (T024-D1 and 
T024-D2), as AlphaFold2 did not perform so well on the complete assembly.
Deposited PDB is 6T1Z

Cedric

PS: I should also note that many other groups performed very well, much better 
than I would have dreamed, including on the full protein but just not as 
crazy-good.
—
Prof. Cedric Govaerts, Ph.D.
Universite Libre de Bruxelles
Campus Plaine. Phone :+32 2 650 53 77
Building BC, Room 1C4 203
Boulevard du Triomphe, Acces 2
1050 Brussels
Belgium
http://govaertslab.ulb.ac.be/



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



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Re: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Mark J van Raaij]

on the day the news came out, I did wonder if the AlphaFold2 team somehow had 
access to all the preliminary PDB files sent around via Gmail (which belongs to 
the same company), but more as a joke/conspirational thought.
"our" target T1052, was also predicted very well by domains and as a monomer. 
It will be interesting to see how well future iterations of the method can 
assemble the complete protein chain and the complete protein chains into the 
correct heteromer.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/


> On 9 Dec 2020, at 10:37, Cedric Govaerts  wrote:
> 
> Dear All
> 
> After about 10 (!) years of (very) hard work we solved the structures of our 
> dearest membrane transporter.  Dataset at 2.9 And resolution, fairly 
> anisotropic, experimental phasing, and many long nights with Coot and 
> Buster to achieve model refinement. 
> 
> The experimental structure had a well defined ligand nicely coordinated but 
> also a lipid embedded inside the binding cavity (a complete surprise but 
> biologically relevant) and two detergent molecules well defined 
> (experimental/crystallisation artefact).
> 
> As our paper was accepted basically when CASP organisers were calling for 
> targets I offered my baby to the computing Gods. However we only provided the 
> sequence to CASP, no info regarding any ligand or lipid.
> 
> Less than a month after, the CASP team contacted us and send us the best 
> model.  In fact it was 2 half models as the transporter is a pseudo dimer, 
> with the N-lobe and C-lobe moving relative to each other during transport 
> cycle, thus divided as two domains in CASP.
> 
> The results were breathtaking. 0.7 And RSMD on one half, 0.6 on the other. 
> And yes, group 427 was the superpower (did not know at the time that it was 
> AlphaFold).
> 
> We had long discussions with the CASP team, as -for us- this almost exact 
> modelling was dream-like (or science fiction) and -at some point- we were 
> even suspecting fraud, as our coordinates had travelled over the internet a 
> few times around when interacting with colleagues.  The organisers reassured 
> us that we were not the only target that had been “nailed” so no reason to 
> suspect any wrongdoing.
> 
> To this day I am still baffled and I would be happy to hear from the 
> community, maybe from some of the CASP participants.
> 
> The target is T024, the “perfect" models are domain-split version (T024-D1 
> and T024-D2), as AlphaFold2 did not perform so well on the complete assembly.
> Deposited PDB is 6T1Z
> 
> Cedric
> 
> PS: I should also note that many other groups performed very well, much 
> better than I would have dreamed, including on the full protein but just not 
> as crazy-good.
> —
> Prof. Cedric Govaerts, Ph.D.
> Universite Libre de Bruxelles
> Campus Plaine. Phone :+32 2 650 53 77
> Building BC, Room 1C4 203
> Boulevard du Triomphe, Acces 2
> 1050 Brussels
> Belgium
> http://govaertslab.ulb.ac.be/ 
> 
> 
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Re: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Cedric Govaerts]

Dear All

After about 10 (!) years of (very) hard work we solved the structures of our 
dearest membrane transporter.  Dataset at 2.9 And resolution, fairly 
anisotropic, experimental phasing, and many long nights with Coot and 
Buster to achieve model refinement. 

The experimental structure had a well defined ligand nicely coordinated but 
also a lipid embedded inside the binding cavity (a complete surprise but 
biologically relevant) and two detergent molecules well defined 
(experimental/crystallisation artefact).

As our paper was accepted basically when CASP organisers were calling for 
targets I offered my baby to the computing Gods. However we only provided the 
sequence to CASP, no info regarding any ligand or lipid.

Less than a month after, the CASP team contacted us and send us the best model. 
 In fact it was 2 half models as the transporter is a pseudo dimer, with the 
N-lobe and C-lobe moving relative to each other during transport cycle, thus 
divided as two domains in CASP.

The results were breathtaking. 0.7 And RSMD on one half, 0.6 on the other. And 
yes, group 427 was the superpower (did not know at the time that it was 
AlphaFold).

We had long discussions with the CASP team, as -for us- this almost exact 
modelling was dream-like (or science fiction) and -at some point- we were even 
suspecting fraud, as our coordinates had travelled over the internet a few 
times around when interacting with colleagues.  The organisers reassured us 
that we were not the only target that had been “nailed” so no reason to suspect 
any wrongdoing.

To this day I am still baffled and I would be happy to hear from the community, 
maybe from some of the CASP participants.

The target is T024, the “perfect" models are domain-split version (T024-D1 and 
T024-D2), as AlphaFold2 did not perform so well on the complete assembly.
Deposited PDB is 6T1Z

Cedric

PS: I should also note that many other groups performed very well, much better 
than I would have dreamed, including on the full protein but just not as 
crazy-good.
—
Prof. Cedric Govaerts, Ph.D.
Universite Libre de Bruxelles
Campus Plaine. Phone :+32 2 650 53 77
Building BC, Room 1C4 203
Boulevard du Triomphe, Acces 2
1050 Brussels
Belgium
http://govaertslab.ulb.ac.be/




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Re: [ccp4bb] Coming July 29: Improved Carbohydrate Data at the PDB -- N-glycans are now separate chains if more than one residue

[Robbie Joosten]

Dear Jasmine,

I have a few questions about this bit:
//
As some users pointed out, single NAG could be just a part of the glycan that 
the author chose to build, as most natural N-glycans must have stem of a common 
core of 5 monosaccharides or its fucosylated version, such as those modeled in 
the PDB ID 6WPS. However, the PDB is a 3D-atomic coordinate archive in which 
the model coordinates are built based on supporting experimental data. 
Therefore, carbohydrates are described as-is in the modeled structures without 
reference to missing components of the presumed oligosaccharide sequence. If 
the author only builds a monosaccharide, then this monosaccharide is described 
as a non-polymer ligand.
//
Is it technically allowed to have a single, covalently bound carbohydrate 
described as a branched entity of length 1?
If so, if an author does specify such a single modeled residue as branched 
entity, for instance because (s)he has a good reason to suspect that a second 
residue was there, but isn’t comfortable with building it, then is this 
specification kept in annotation?
If not, do you expect model building programs to switch from branched to 
non-poly entities when a second residue is removed when a model is written out? 
And back again when a residue is added? I find this rather unpractical from an 
implementation point of view. We change carbohydrate trees quite regularly.

Cheers,
Robbie


From: CCP4 bulletin board  On Behalf Of Jasmine Young
Sent: Tuesday, December 8, 2020 21:01
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Coming July 29: Improved Carbohydrate Data at the PDB -- 
N-glycans are now separate chains if more than one residue

Dear PDB Data Users:

Thank you for providing feedback on the results of an archival-level 
carbohydrate remediation project that led to the re-release of over 14,000 PDB 
structures in July 2020. This update includes diverse oligosaccharides: 
glycosylation; metabolites such as maltose, sucrose, cellulose fragments; 
glycosaminoglycans, such as fragments of heparin and heparan sulfate; epitope 
patterns such as A/B blood group antigens and the H-type or Lewis-type stems; 
and many artificial carbohydrates mimicking or counting natural products 
(https://www.wwpdb.org/documentation/carbohydrate-remediation).

Starting in 2017, this PDB remediation aimed to standardize the biochemical 
nomenclature of the carbohydrate components following the IUPAC-IUBMB 
recommendations established by the carbohydrate community 
(https://media.iupac.org/publications/pac/1996/pdf/6810x1919.pdf), and to 
provide uniform representation of oligosaccharides to improve the 
identification and searchability of oligosaccharides modeled in the PDB 
structures.  During the remediation planning, wwPDB consulted community users 
and the PDBx/mmCIF Working Group and made data files available on GitHub in 
early 2020 for community feedback. wwPDB has collaborated with Robert Woods at 
University of Georgia in US, researchers at The Noguchi Institute and Soka 
University in Japan, and Thomas Lutteke in Germany to generate uniform linear 
descriptors for the oligosaccharide sequences.

To achieve these community goals, each oligosaccharide is represented as a 
branched entity with complete biochemical description and each glycosidic 
linkage specified. The full representation of carbohydrates is provided in the 
mmCIF format file, but this is not possible in legacy PDB format files (as the 
format has been frozen since 2012 
(https://www.wwpdb.org/documentation/file-formats-and-the-pdb).

Proper indexing is necessary for branched entity representation and for 
generation of linear descriptors, hence the ordering (numbering) starts at the 
reducing end (#1), where the glycosylation occurs, to the non-reducing end in 
ascending order. Unique chain IDs are assigned to branched entities 
(oligosaccharides) to avoid residue numbering overlapped with protein residues 
and to enable consistent numbering for every oligosaccharide. For example, in 
PDB ID 6WPS, there are 5 oligosaccharides associated with the same protein 
chain A, the consistent ordering and numbering can only be retained with unique 
chain ID for each oligosaccharide in both PDBx/mmCIF and PDB format files

For archival consistency, a single-monosaccharide is defined as a non-polymer 
and treated consistently with other non-polymer ligands in the PDB. A 
single-monosaccharide occurring at a glycosylation site has a unique chain ID 
in the PDBx/mmCIF file (_atom_site.label_asym_id) but not in the PDB format 
file.

Using PDB ID 6WPS as an example, the PDBx/mmCIF data item 
_atom_site.label_asym_id corresponds to the column #7 in the atom_site 
coordinates section has an asym ID ‘Y’ for the 1st instance of 
single-monosaccharide, NAG bound to ASN 61 of protein chain ‘A’. The ‘Y’ value 
is unique for this monosaccharide. The additional chain ID 
(_atom_site.auth_asym_id) in the PDBx/mmCIF file that mapped to the PDB format 
file for 

Re: [ccp4bb] when does non-isomorphism become a habit?

[vincent Chaptal]

Dear James,

I'll second Manferd's post.
We have a membrane protein that crystallizes with a continuum of cell 
dimensions, so different that you can't merge the data to reach full 
completeness, but you could manage to gain MR solutions for some 
discrete states of the continuum and ending up in the "same" solution 
(depends on what resolution we are also talking about).
I don't have the full answer to it, but I'd gladly blam detegents 
influencing crystal packing, into what I called lack of isomorphism.


Best
Vincent

Le 09/12/2020 à 07:54, Manfred S. Weiss a écrit :

Dear James,

let's spin the thought a bit further. What if there is a new program
(Phaser-II) some day,
for which the coordinates in your "new crystal form" are all of a sudden
within the
radius of convergence again? Does this bring your "new crystal form"
back to the old
crystal form again?

I'd say it is neither cell dimensions nor space group that define a new
crystal form. It
is the packing of the molecules. It happens quite often in dehydration
experiments
that molecules in a lattice move a bit and symmetry elements are lost or
new symmetry
elements are created. The space group changes, as well as the unit cell
dimensions. But
the packing remains essentially the same. You can usually infer what
happens, but you
still need molecular replacement to actually solve the "new" form.

Best, Manfred


Am 09.12.2020 um 01:56 schrieb James Holton:

I have a semantics question, and I know how much this forum loves
discussing semantics.

We've all experienced non-isomorphism, where two crystals, perhaps
even grown from the same drop, yield different data. Different enough
so that merging them makes your overall data quality worse. I'd say
that is a fairly reasonable definition of non-isomorphism? Most of the
time unit cell changes are telling, but in the end it is the I/sigma
and resolution limit that we care about the most.

Now, of course, even for non-isomorphous data sets you can usually
"solve" the non-isomorphous data without actually doing molecular
replacement.  All you usually need to do is run pointless using the
PDB file from the first crystal as a reference, and it will re-index
the data to match the model.  Then you just do a few cycles of rigid
body and you're off and running.  A nice side-effect of this is that
all your PDB files will line up when you load them into coot. No
worries about indexing ambiguities, space group assignment, or origin
choice. Phaser is a great program, but you don't have to run it on
everything.

My question is: what about when you DO have to run Phaser to solve
that other crystal from the same drop?  What if the space group is the
same, the unit cell is kinda-sorta the same, but the coordinates have
moved enough so as to be outside the radius of convergence of
rigid-body refinement?  Does that qualify as a different "crystal
form" or different "crystal habit"?  Or is it the same form, and just
really non-isomorphous?

Opinions?

-James Holton
MAD Scientist



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--
Dr. Manfred S. Weiss
Macromolecular Crystallography
Helmholtz-Zentrum Berlin
Albert-Einstein-Str. 15
D-12489 Berlin
Germany




Helmholtz-Zentrum Berlin für Materialien und Energie GmbH

Mitglied der Hermann von Helmholtz-Gemeinschaft Deutscher 
Forschungszentren e.V.


Aufsichtsrat: Vorsitzender Dr. Volkmar Dietz, stv. Vorsitzende Dr. 
Jutta Koch-Unterseher
Geschäftsführung: Prof. Dr. Bernd Rech (Sprecher), Prof. Dr. Jan 
Lüning, Thomas Frederking


Sitz Berlin, AG Charlottenburg, 89 HRB 5583

Postadresse:
Hahn-Meitner-Platz 1
D-14109 Berlin



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--

Vincent Chaptal, PhD

Director of GdR APPICOM

Drug Resistance and Membrane Proteins Lab


MMSB -UMR5086

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