[ccp4bb] AW: [ccp4bb] challenges in structural biology

[Kollmar, Martin]

Dear John,
the „100,000 human genes“ is a long-standing myth broad forward by the 
initiators of the U.S. human genome sequencing projects in 1990. This large 
number completely contradicted all genetics and mutation data since the 1940th, 
but the sequencing community (genome, cDNA, EST) didn’t read even the standard 
text books. Thus, the “30,000” genes published with the two human genome papers 
in 2001 are not “surprisingly low” but just in accordance with the predictions 
and the data since the 1940th. The gene number went down to about 23,000 
already in 2004, and the current numbers (depending on database) range around 
20,000 human protein-coding genes. The myth of the large numbers is only 
propagated by those who profit from larger numbers (e.g. bigger grants, papers 
in higher IF journals, big consortia).

I have written a review about the current state (and history) of the human 
protein-coding genes, which will appear online in BioEssays soon and finally in 
the November issue (will be open access). In this review there will be some 
(hopefully) useful plots showing the gene numbers since the 1940th and a 
detailed review of all the numbers and their experimental basis (most were 
actually just extrapolations from small-scale data).

Please excuse this kind of self-advertisement, but it is really more than time 
to move this myth out of science literature and communication.

Best regards,
Martin

Priv. Doz. Dr. Martin Kollmar

Group Systems Biology of Motor Proteins
Department NMR-based Structural Biology
Max-Planck-Institute for Biophysical Chemistry
Am Fassberg 11
37077 Goettingen
Deutschland

www.motorprotein.de (Homepage)
www.cymobase.org (Database of Cytoskeletal and Motor 
Proteins)
www.diark.org (diArk - a resource for eukaryotic genome 
research)
www.webscipio.org (Scipio - eukaryotic gene 
identification)

Von: CCP4 bulletin board  Im Auftrag von John R Helliwell
Gesendet: Donnerstag, 19. September 2019 08:51
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] challenges in structural biology

Dear James,
Well, 100,000 genes used to be the estimate of the size of the human genome.
(eg see 
https://physicsworld.com/a/protein-crystallography-the-human-genome-in-3-d/ )
It seems it has got easier, albeit still gargantuan, at ~30,000 genes to be 
expressed into proteins.

Meanwhile funding agencies also look out for Big Ideas:-
https://epsrc.ukri.org/research/ourportfolio/epsrcbigideas/?utm_source=Twitter&utm_medium=social&utm_campaign=SocialSignIn
and even helpfully spell out the difference between a Big Idea and a Grand 
Challenge!
Maybe an “Open Door for funding” for us all?

Today also the repertoire of methods capable of resolving in 3D protein 
structures has expanded further with the splendid development of cryoEM.

To define challenges in terms of projects, as Max Perutz taught us 
(“Haemoglobin the Molecular Lung”) avoids methods looking for problems.

Also a final thought, how we organise ourselves in different areas of the World 
varies according to our cultural traditions. So the Big Project is neutral to 
politics and can accommodate all contributions however so arrived at.

“What shall we do with it?”
As Darwin taught us, first make your Collection..

Greetings!
John

Emeritus Professor John R Helliwell DSc
https://www.crcpress.com/The-Whats-of-a-Scientific-Life/Helliwell/p/book/9780367233020



On 18 Sep 2019, at 22:15, James Holton 
mailto:jmhol...@lbl.gov>> wrote:
Thank you John, an excellent choice as always.  Here is your trillion dollars!  
Now, what are you going to do with it?

Do you think simply scaling up current technology could reach this goal?  More 
screens, more combinations, more compute cycles?  Remember, if you want the 
"genome/proteome" you need all of it, including all those super-cool human 
membrane proteins we gave up on because they were too hard.

I think we all have at least one of those projects in our past.  What was the 
show-stopper in the end?  Did they just not grow crystals? Poor diffraction? 
Weird diffraction? Twinned? Won't phase? Won't refine to a decent R factor? 
Annoying reviewer? Did you try cryoEM? NMR? and did they not work either?

I think a key question for all of us is: what new capability would make you 
decide to go back and pick up your old favorite project again?  Without your 
structure, the genome is incomplete.

-James Holton
MAD Scientist
On 9/16/2019 12:24 AM, John R Helliwell wrote:
Dear James,
Here you go, a “grand challenge” suggestion to consider for funding from the 
“James Holton Foundation for structural biology research”:-
“The human genome/proteome in 3-D”
Greetings,
John
Emeritus Professor John R Helliwell DSc




On 14 Sep 2019, at 02:39, James Holton 
mailto:jmhol...@lbl.gov>> wrote:

I would like to thank everyone who took the time to respond to my question that 
started this thread.  It is really good f

[ccp4bb] MoRDa update

[Alexey Vagin]

Dear All

MoRDa is a pipeline for molecular replacement structure solution.

A new update to the package is now available. The structure solution program is 
improved and database is extended .

Both update and installation instructions are available from Morda homepage 
.

The update is also available for existing local Morda installations and can be 
installed from command line: change to installation directory (by default 
MoRDa_DB) and type ./update_morda.

Also Morda at ccp4online  has been updated. 

Best regards
Alexey




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Re: [ccp4bb] challenges in structural biology

[Phoebe A. Rice]

Giving the vagaries of alternate splicing, is there one discrete number of 
human genes out there to be determined?
And what percentage of the encoded mass of protein is actually structured?

Another more biochemical challenge for structural biology is figuring out how 
to deal with weak cooperative interactions among multiple flexible partners.

~~~
Phoebe A. Rice
Dept. of Biochem & Mol. Biol. and
  Committee on Microbiology
https://voices.uchicago.edu/phoebericelab/


From: CCP4 bulletin board  on behalf of John R Helliwell 

Reply-To: John R Helliwell 
Date: Thursday, September 19, 2019 at 1:51 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] challenges in structural biology

Dear James,
Well, 100,000 genes used to be the estimate of the size of the human genome.
(eg see 
https://physicsworld.com/a/protein-crystallography-the-human-genome-in-3-d/ )
It seems it has got easier, albeit still gargantuan, at ~30,000 genes to be 
expressed into proteins.

Meanwhile funding agencies also look out for Big Ideas:-
https://epsrc.ukri.org/research/ourportfolio/epsrcbigideas/?utm_source=Twitter&utm_medium=social&utm_campaign=SocialSignIn
and even helpfully spell out the difference between a Big Idea and a Grand 
Challenge!
Maybe an “Open Door for funding” for us all?

Today also the repertoire of methods capable of resolving in 3D protein 
structures has expanded further with the splendid development of cryoEM.

To define challenges in terms of projects, as Max Perutz taught us 
(“Haemoglobin the Molecular Lung”) avoids methods looking for problems.

Also a final thought, how we organise ourselves in different areas of the World 
varies according to our cultural traditions. So the Big Project is neutral to 
politics and can accommodate all contributions however so arrived at.

“What shall we do with it?”
As Darwin taught us, first make your Collection..

Greetings!
John

Emeritus Professor John R Helliwell DSc
https://www.crcpress.com/The-Whats-of-a-Scientific-Life/Helliwell/p/book/9780367233020



On 18 Sep 2019, at 22:15, James Holton 
mailto:jmhol...@lbl.gov>> wrote:
Thank you John, an excellent choice as always.  Here is your trillion dollars!  
Now, what are you going to do with it?

Do you think simply scaling up current technology could reach this goal?  More 
screens, more combinations, more compute cycles?  Remember, if you want the 
"genome/proteome" you need all of it, including all those super-cool human 
membrane proteins we gave up on because they were too hard.

I think we all have at least one of those projects in our past.  What was the 
show-stopper in the end?  Did they just not grow crystals? Poor diffraction? 
Weird diffraction? Twinned? Won't phase? Won't refine to a decent R factor? 
Annoying reviewer? Did you try cryoEM? NMR? and did they not work either?

I think a key question for all of us is: what new capability would make you 
decide to go back and pick up your old favorite project again?  Without your 
structure, the genome is incomplete.

-James Holton
MAD Scientist
On 9/16/2019 12:24 AM, John R Helliwell wrote:
Dear James,
Here you go, a “grand challenge” suggestion to consider for funding from the 
“James Holton Foundation for structural biology research”:-
“The human genome/proteome in 3-D”
Greetings,
John
Emeritus Professor John R Helliwell DSc




On 14 Sep 2019, at 02:39, James Holton 
mailto:jmhol...@lbl.gov>> wrote:

I would like to thank everyone who took the time to respond to my question that 
started this thread.  It is really good for me to get a sense of the community 
perspective.  Some debates were predictable, others not.  Many ideas I agree 
with, some not so much.  All were thought-provoking. I think this is going to 
be a really good GRC!

Something I did not expect to distill from all the responses is that the 
dominant challenge in structural biology is financial. The most common strategy 
suggested for addressing this challenge was torpedoing other scientists in 
similar fields, perhaps expecting to benefit from the flotsam.  Historically, 
this strategy is often counterproductive and at best inefficient. The good news 
is there is a lot of room for improvement. In reality, we are all on the same 
ship, and the people in our funding agencies fighting to get us what we need 
can be much more effective when armed with positive ideas and clear plans.  
That is a better strategy for overcoming this challenge.

To this end, my first GRC session title is going to be:

"If I had a trillion dollars for structural biology"

I think we can all agree that science in general is vastly under-funded 
relative to the impact it has on the human condition.  For example, I estimate 
the value of a general cure for cancer to be at least a trillion dollars.  This 
is based on the lives claimed every year, multiplied by how much one person 
would gladly pay after being diagnosed (amortized over the rest of their much 
longer life). This is only ~

Re: [ccp4bb] Optimisation of 2D very thin plates to thick plates

[Anastassis Perrakis]

Microseeding for new conditions?

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118815/



On Sep 19, 2019, at 17:30, William Richardson 
mailto:william.richard...@nottingham.ac.uk>>
 wrote:

Dear colleagues,
I would like some advice on crystal growth optimisation. I have been trying to 
crystallise a DNA regulator. I got microcrystals in 1.3M Lithium Sulphate and 
50 mM Cacodylate pH = 6. Through a combination of condition refinement and 
seeding I have attained crystals with a plate-like habit of dimension 50micron, 
50micron, <1micron. These have very weak (8Å) anisotropic diffraction at DLS 
i24 but as they’re so wafer thin there has been no improvement. Any suggestions 
to make them more 3D would be great. The space group is C2221
Any advice?
Thanks in advance
Regards,

Will





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[ccp4bb] XChem fragment screening - call for proposals - 2nd October 5pm

[Frank Von Delft]

Dear all,
This is a reminder that Diamond Light Source is accepting proposals for its 
XChem fragment screening facility, for the allocation period April to September 
2020.
The deadline is Wednesday 2nd October at 5pm (2 weeks from now).
Details on how to apply are here - we're oversubscribed, so please follow the 
instructions!

https://www.diamond.ac.uk/Instruments/Mx/Fragment-Screening/Applying-for-XChem.html

XChem is a platform for high-throughput crystal-based fragment screening, 
operational since 2015. We provide fragment libraries (or you can bring your 
own) and the pipeline entails:  rapid crystal soaking with an Echo acoustic 
liquid handler; high-throughput harvesting of hundreds of pre-soaked crystals 
using a Shifter; fully unattended data collection using dedicated beamtime on 
I04-1 beamline; and automated data integration along with tools for ligand 
detection (PanDDA) and streamlined model building (XChemExplorer).
Since April 2019, we offer both standard and Block Allocation Group (BAG) 
access.   Standard access accommodates both Tier 1 and Tier 2 proposals, for 
exploratory and ready-to-go projects respectively.
Since this summer, the FragLites library from Newcastle university (J. Med. 
Chem.20196273741-3752) is also available for users.
More information is on the XChem website:
https://www.diamond.ac.uk/Instruments/Mx/Fragment-Screening
https://www.diamond.ac.uk/Instruments/Mx/Fragment-Screening/Methods-for-Fragment-Screening.html
https://www.diamond.ac.uk/Instruments/Mx/Fragment-Screening/Fragment-Libraries.html
For more questions, please ask Alice (copied) or myself.
We are looking forward to collaborating with you!
The XChem team.




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Re: [ccp4bb] Optimisation of 2D very thin plates to thick plates

[Quyen Hoang]

Hi Will,
We had a similar case recently.
Microseeding gave us single crystals (originally clusters), but still very thin 
plates as yours.
We then mutated a couple surface lysine residues to alanine and that led to 
thicker crystals.
The thin crystals were indexed as C2221, but the thicker ones revealed that it 
was actually P21.

Hope this helps.

Cheers,
Quyen

Quyen Hoang, PhD
Associate Professor of Biochemistry and Molecular Biology
Adjunct Associate Professor of Neurology
Primary Investigator of the Stark Neuroscience Research Institute
Indiana University School of Medicine
635 Barnhill Drive, MS0013C
Indianapolis, IN, 46202
(317)274-4371

> On Sep 19, 2019, at 12:16 PM, Phoebe A. Rice  wrote:
> 
> Is it a complex with DNA?  If so, vary the DNA ends?
>  
> ~~~
> Phoebe A. Rice
> Dept. of Biochem & Mol. Biol. and
>   Committee on Microbiology
> https://voices.uchicago.edu/phoebericelab/ 
> 
>  
>  
> From: CCP4 bulletin board  > on behalf of William Richardson 
>  >
> Reply-To: William Richardson  >
> Date: Thursday, September 19, 2019 at 10:41 AM
> To: "CCP4BB@JISCMAIL.AC.UK " 
> mailto:CCP4BB@JISCMAIL.AC.UK>>
> Subject: [ccp4bb] Optimisation of 2D very thin plates to thick plates
>  
> Dear colleagues, 
> I would like some advice on crystal growth optimisation. I have been trying 
> to crystallise a DNA regulator. I got microcrystals in 1.3M Lithium Sulphate 
> and 50 mM Cacodylate pH = 6. Through a combination of condition refinement 
> and seeding I have attained crystals with a plate-like habit of dimension 
> 50micron, 50micron, <1micron. These have very weak (8Å) anisotropic 
> diffraction at DLS i24 but as they’re so wafer thin there has been no 
> improvement. Any suggestions to make them more 3D would be great. The space 
> group is C2221 
> Any advice?
> Thanks in advance
> Regards,
>  
> Will
>  
>  
> 
>  
> This message and any attachment are intended solely for the addressee
> and may contain confidential information. If you have received this
> message in error, please contact the sender and delete the email and
> attachment. 
>  
> Any views or opinions expressed by the author of this email do not
> necessarily reflect the views of the University of Nottingham. Email
> communications with the University of Nottingham may be monitored 
> where permitted by law.
>  
>  
>  
>  
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 
> 
> To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] Optimisation of 2D very thin plates to thick pl

[Jonathan Cooper]

Adding chymotrypsin to your crystallisations in about 1:100 mass ratio with 
your protein can help. You can quite easily find some papers on this method.  
#yiv8007840479 -- filtered {panose-1:2 4 5 3 5 4 6 3 2 4;}#yiv8007840479 
filtered {font-family:Calibri;panose-1:2 15 5 2 2 2 4 3 2 4;}#yiv8007840479 
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p.yiv8007840479MsoNormal, #yiv8007840479 li.yiv8007840479MsoNormal, 
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[ccp4bb] Are there any proteins capable of crystallizing at a wide range of pH having the same space group?

[Murpholino Peligro]

A quick glance at the entries of hen egg white lysozyme in the PDB show
that it can be crystallized at different pH values, but the space group is
not always the same. I still have to refine the analysis but I was
wondering that maybe there are a few proteins that can crystallize at a
wide range (maybe not that wide) of pH values and still have the same space
group?

To refine the analysis a wee further: By any chance do you know any
proteins overpopulating the PDB (i.e. besides HEWL)?

Lots of thanks as always.

Murphy



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Re: [ccp4bb] Are there any proteins capable of crystallizing at a wide range of pH having the same space group?

[Dale Tronrud]

   My recollection is that gamma-chymotrypsin crystals will persist in
pHs all the way from 3 to 9.  I don't know if the crystals will grow
over that range.

   There are a fair number of phage T4 lysozymes variants in the PDB.  I
don't think this is considered "overpopulation" but a valuable
contribution to humanity.  ;-)

Dale Tronrud

On 9/19/2019 4:03 PM, Murpholino Peligro wrote:
> A quick glance at the entries of hen egg white lysozyme in the PDB show
> that it can be crystallized at different pH values, but the space group
> is not always the same. I still have to refine the analysis but I was
> wondering that maybe there are a few proteins that can crystallize at a
> wide range (maybe not that wide) of pH values and still have the same
> space group?
> 
> To refine the analysis a wee further: By any chance do you know any
> proteins overpopulating the PDB (i.e. besides HEWL)?
> 
> Lots of thanks as always.
> 
> Murphy
> 
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
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> 



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Re: [ccp4bb] Are there any proteins capable of crystallizing at a wide range of pH having the same space group?

[Murpholino Peligro]

Following my last question I was searching for a redundant PDB and found
this paper https://doi.org/10.1016/j.str.2007.07.012. I went to the methods
section and found out that the link to the database of redundant protein
structures is now gone (http://dper.burnham.org/). Anyone knows of a
similar database? Or anyone knows how to make one?

Thanks again for your help.


Murph

El jue., 19 de sep. de 2019 a la(s) 19:04, Dale Tronrud (
de...@daletronrud.com) escribió:

>
>My recollection is that gamma-chymotrypsin crystals will persist in
> pHs all the way from 3 to 9.  I don't know if the crystals will grow
> over that range.
>
>There are a fair number of phage T4 lysozymes variants in the PDB.  I
> don't think this is considered "overpopulation" but a valuable
> contribution to humanity.  ;-)
>
> Dale Tronrud
>
> On 9/19/2019 4:03 PM, Murpholino Peligro wrote:
> > A quick glance at the entries of hen egg white lysozyme in the PDB show
> > that it can be crystallized at different pH values, but the space group
> > is not always the same. I still have to refine the analysis but I was
> > wondering that maybe there are a few proteins that can crystallize at a
> > wide range (maybe not that wide) of pH values and still have the same
> > space group?
> >
> > To refine the analysis a wee further: By any chance do you know any
> > proteins overpopulating the PDB (i.e. besides HEWL)?
> >
> > Lots of thanks as always.
> >
> > Murphy
> >
> >
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
> >
>



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Re: [ccp4bb] Are there any proteins capable of crystallizing at a wide range of pH having the same space group?

[mesters]

As far as lysozyme is concerned, the space group obtained is mainly the 
result of an interplay between the pI of lysozyme on the one hand and 
the pH of the solute and type of salt (or organic small molecule) on the 
other hand (Hofmeister series). The pI is about 9.5 and one should not 
be surprized lysozyme can crystallize in the same space group over a 
wide pH range (4 to 9) Not sure you will find examples in the PDB of 
other protein with a more neutral pI that behave similarly.


Overpopulating the PDB are (to name a few)

Beta-2-microglobulin (1034) 



 *
 o Lysozyme C (1009)
   

 o Carbonic anhydrase 2 (752)
   

 o Endothiapepsin (525)
   

 * Cationic trypsin (445)
   

 * Myoglobin (418)
   


 * Cyclin-dependent kinase 2 (402)
   

 * Prothrombin (380)
   

 * Beta-secretase 1 (377)
   

 * DNA polymerase beta (318)
   


 * Beta-lactamase (552)
   

 * Thermonuclease (290)