[ccp4bb] iNEXT-Discovery 2021 webinars

[Anastassis Perrakis]

Dear all,

I do hope that after a full week for recovery from the CCP4 “study weekend”, 
you might want to consider joining our iNEXT-Discovery webinar series! We have 
lined up a series of new talks on X-ray tomography, cryo-EM, NMR, and more!

To see the full program and also register (even if you registered for the 2020 
series, you need to register again!) at :

https://zoom.us/webinar/register/4016104403708/WN_Ofbx1MrNS-OAK95yMbtmXA

We hope to see many of you there, and wanted to also remind you that you are 
more than welcome to apply for access for X-rays (SAXS, MX, fragment and 
ligands screens, tomography), cryo-EM (single particles, tomography, FIB, etc), 
NMR (solution, solid, fragment screens, etc, and macromolecular biophysics 
(SPR, ITC, MST, FP, Stopped Flow, etc) at any time through the iNEXT-Discovery 
web site:

https://inext-discovery.eu/network/inext-d/home

Best regards,

Tassos











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[ccp4bb] iNEXT-Discovery

[Anastassis Perrakis]

Dear all,

I would like to share with you an iNEXT and iNEXT-Discovery video for 
Structural Biology and the importance of research infrastructures funding, 
which we think is suitable for explaining things to the wider scientific 
community and to the public with a specific interest to science:

https://www.youtube.com/watch?v=T7waoQUfwNs

We want to also remind you all that all iNEXT-Discovery facilities are 
(remotely) accessible even during the lockdowns! We will be waiting for your 
proposals for access to X-ray crystallography, SAXS, cryo-EM SP and tomography, 
CLEM, solution and solid state-NMR, and many more at:

https://inext-discovery.eu/network/inext-d/home

Best regards and best wishes for the holiday season,

Tassos

Prof. Dr. Anastassis Perrakis
Group leader, Dept. of Biochemistry, Netherlands Cancer Institute
Professor of Macromolecular Structures, University of Utrecht
Oncode Institute Investigator
Coordinator iNEXT-Discovery H2020











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Re: [ccp4bb] Finding partial occupancy monomer by MR ?

[Anastassis Perrakis]

Dear Phil,

What is the space group? Could it be that the 4th monomer makes also a trimer 
by symmetry, in an alternative SG?

A.


> On 10 Dec 2020, at 14:49, Phil Jeffrey  wrote:
> 
> Preamble:
> I have an interesting crystal form with 3 monomers (~400aa) at full occupancy 
> and apparently one at much reduced occupancy.  It was built recently from 
> Se-SAD and was in moderately good condition: Rfree=32% for trimer, 2.6 Å.  In 
> recent refinement cycles it became obvious that there was a 4th monomer in a 
> region of weaker/choppy 2Fo-Fc and Fo-Fc density that corresponded to a 
> "confusing" set of low-occupancy SeMet sites found by SHELXD and Phaser-EP.  
> The experimental map was bad in that region and was probably flattened during 
> density modification anyway, in retrospect.
> 
> Question:
> Phaser failed to find the 4th monomer after trivially finding the other 3 
> with a recent version of the monomer.  I'm wondering if there's a way to 
> indicate "this one is partial occupancy" to Phaser, or if there's a way to 
> improve the odds of success beyond just lowering the expected % homology.  Or 
> if anyone has had success with other programs.  This is perhaps a rare edge 
> case but I naively expected Phaser to work.
> 
> In the end I used the weak SeMet sites to locate the monomer and the 
> occupancy appears to be around 0.32 in refinement.
> 
> Cheers,
> Phil Jeffrey
> Princeton
> 
> 
> 
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Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Anastassis Perrakis]

Dear Boaz,

The archimboldo model gives Rot z-score: 8.1, Trans Z-score 13.8

Not sure this matters, as it lacks a few loops that even good old arp/warp can 
fill up in ten minutes ;-)

A.

On Dec 4, 2020, at 0:40, Boaz Shaanan 
mailto:bshaa...@bgu.ac.il>> wrote:

Just curious, how does the result of the Phaser run  with the Alphafold model 
compare with a Phaser run using the Arcimboldo phased model as a probe?
Boaz

Boaz Shaanan, Ph.D.
Department of Life Sciences
Ben Gurion University of the Negev
Beer Sheva
Israel

On Dec 4, 2020 00:32, Anastassis Perrakis 
mailto:a.perra...@nki.nl>> wrote:
AlphaFold - or similar ideas that will surface up sooner or later - will beyond 
doubt have major impact. The accuracy it demonstrated compared to others is 
excellent.

“Our” target (T1068) that was not solvable by MR with the homologous search 
structure or a homology model (it was phased with Archimboldo, rather easily), 
is easily solvable with the AlphaFold model as a search model. In PHASER I get 
Rotation Z-score 17.9, translation Z-score 26.0, using defaults.


imho what remains to be seen is:

a. how and when will a prediction server be available?
b. even if training needs computing that will surely unaccessible to most, will 
there be code that can be installed in a “reasonable” number of GPUs and how 
fast will it be?
c. how do model quality metrics (that do not compared with the known answer) 
correlate with the expected RMSD? AlphaFold, no matter how impressive, still 
gets things wrong.
c. will the AI efforts now gear to ligand (fragment?) prediction with similarly 
impressive performance?

Exciting times.

A.




On 3 Dec 2020, at 21:55, Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk<mailto:488a26d62010-dmarc-requ...@jiscmail.ac.uk>>
 wrote:

Hello. A quick look suggests that a lot of the test structures were solved by 
phaser or molrep, suggesting it is a very welcome improvement on homology 
modelling. It would be interesting to know how it performs with structures of 
new or uncertain fold, if there are any left these days. Without resorting to 
jokes about artificial intelligence, I couldn't make that out from the CASP14 
website or the many excellent articles that have appeared. Best wishes, Jon 
Cooper.


Sent from ProtonMail mobile



 Original Message 
On 3 Dec 2020, 11:17, Isabel Garcia-Saez < 
isabel.gar...@ibs.fr<mailto:isabel.gar...@ibs.fr>> wrote:

Dear all,

Just commenting that after the stunning performance of AlphaFold that uses AI 
from Google maybe some of us we could dedicate ourselves to the noble art of 
gardening, baking, doing Chinese Calligraphy, enjoying the clouds pass or 
everything together (just in case I have already prepared my subscription to 
Netflix).

https://www.nature.com/articles/d41586-020-03348-4

Well, I suppose that we still have the structures of complexes (at the moment). 
I am wondering how the labs will have access to this technology in the future 
(would it be for free coming from the company DeepMind - Google?). It seems 
that they have already published some code. Well, exciting times.

Cheers,

Isabel


Isabel Garcia-Saez PhD
Institut de Biologie Structurale
Viral Infection and Cancer Group (VIC)-Cell Division Team
71, Avenue des Martyrs
CS 10090
38044 Grenoble Cedex 9
France
Tel.: 00 33 (0) 457 42 86 15
e-mail: isabel.gar...@ibs.fr<mailto:isabel.gar...@ibs.fr>
FAX: 00 33 (0) 476 50 18 90
http://www.ibs.fr/




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

[Anastassis Perrakis]

btw, if anyone has any leverage to the people making the CASP#14 pages, having 
info in acronyms (e.g. GDT) accessible by a simple “mouse over” instead of 
re-directing to the explanation page would be handy.

In any case, the Casp web-pages in general, leave quite a bit to be desired for 
the average user - they seem more like an "API for humans” and less concerned 
about modern design principles, to put it mildly.

Tassos

On Dec 4, 2020, at 8:53, Joana Pereira 
mailto:joana.pere...@tuebingen.mpg.de>> wrote:

Hi everybody,

As one of the persons playing with the CASP14 data before all news came out, I 
can answer some of the questions raised in this thread.

- "Does anyone know how AlphaFold performs on sequences with little 
conservation?"
One of the things we looked at was how the accuracy of the models was dependent 
on the Neff (number of effective sequences, relates to how deep alignments are 
for that sequence and, thus, to the number of homologs and the conservation of 
the sequence). What we could see is that, basically, in CASP14 it does not 
anymore and that (near-)singleton sequences could be modeled with a pretty good 
accuracy.

- "It would be interesting to know how it performs with structures of new or 
uncertain fold."
It does pretty well! Similarly to the Neff relationship, we also see a 
basically flat line at a GDT of 70-80 at any level of target difficulty. Of 
course the accuracy is slightly higher for easy targets (those for which there 
are templates in the PDB), but to have a GDT of around 70 in Free-Modelling, 
hard targets, is quite impressive.

- "I don't think they have all the side chain placement so perfect as to be 
able to predict the fold and how a compound or another protein binds"
Yap, sidechains remain the poorest modeled parts. Still, those modeled by 
AlphaFold were the closest to the "reality" of the target...

- "I'm curious how well AlphaFold would do on an Intrinsically Disordered 
Protein (IDP)"
Oh yes, that is a super good point and I have been thinking about it too. Maybe 
one should start throwing some IDPs into CASP too :) There's the CAID 
experiment but, on its current state, AlphaFold would not be possible to test.

Best wishes
Joana

---
Dr. Joana Pereira
Postdoctoral Researcher
Department of Protein Evolution

Max Planck Institute for Developmental Biology
Max-Planck-Ring 5
72076 Tübingen
GERMANY


On 03.12.20 23:46, Reza Khayat wrote:
​Does anyone know how AlphaFold performs on sequences with little conservation? 
Virus and phage proteins are like this. Their structures are homologous, but 
sequence identity can be less than 10%.

Reza

Reza Khayat, PhD
Associate Professor
City College of New York
Department of Chemistry and Biochemistry
New York, NY 10031

From: CCP4 bulletin board <mailto:CCP4BB@JISCMAIL.AC.UK> 
on behalf of Anastassis Perrakis <mailto:a.perra...@nki.nl>
Sent: Thursday, December 3, 2020 5:31 PM
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [EXTERNAL] Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

AlphaFold - or similar ideas that will surface up sooner or later - will beyond 
doubt have major impact. The accuracy it demonstrated compared to others is 
excellent.

“Our” target (T1068) that was not solvable by MR with the homologous search 
structure or a homology model (it was phased with Archimboldo, rather easily), 
is easily solvable with the AlphaFold model as a search model. In PHASER I get 
Rotation Z-score 17.9, translation Z-score 26.0, using defaults.


imho what remains to be seen is:

a. how and when will a prediction server be available?
b. even if training needs computing that will surely unaccessible to most, will 
there be code that can be installed in a “reasonable” number of GPUs and how 
fast will it be?
c. how do model quality metrics (that do not compared with the known answer) 
correlate with the expected RMSD? AlphaFold, no matter how impressive, still 
gets things wrong.
c. will the AI efforts now gear to ligand (fragment?) prediction with similarly 
impressive performance?

Exciting times.

A.




On 3 Dec 2020, at 21:55, Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk<mailto:488a26d62010-dmarc-requ...@jiscmail.ac.uk>>
 wrote:

Hello. A quick look suggests that a lot of the test structures were solved by 
phaser or molrep, suggesting it is a very welcome improvement on homology 
modelling. It would be interesting to know how it performs with structures of 
new or uncertain fold, if there are any left these days. Without resorting to 
jokes about artificial intelligence, I couldn't make that out from the CASP14 
website or the many excellent articles that have appeared. Best wishes, Jon 
Cooper.


Sent from ProtonMail mobile



 Original Message 
On 3 Dec 2020, 11:17, Isabel Garcia-Saez < 
isabel.gar...@ibs.fr<mai

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

[Anastassis Perrakis]

AlphaFold - or similar ideas that will surface up sooner or later - will beyond 
doubt have major impact. The accuracy it demonstrated compared to others is 
excellent.

“Our” target (T1068) that was not solvable by MR with the homologous search 
structure or a homology model (it was phased with Archimboldo, rather easily), 
is easily solvable with the AlphaFold model as a search model. In PHASER I get 
Rotation Z-score 17.9, translation Z-score 26.0, using defaults.


imho what remains to be seen is:

a. how and when will a prediction server be available?
b. even if training needs computing that will surely unaccessible to most, will 
there be code that can be installed in a “reasonable” number of GPUs and how 
fast will it be?
c. how do model quality metrics (that do not compared with the known answer) 
correlate with the expected RMSD? AlphaFold, no matter how impressive, still 
gets things wrong.
c. will the AI efforts now gear to ligand (fragment?) prediction with similarly 
impressive performance?

Exciting times.

A.




On 3 Dec 2020, at 21:55, Jon Cooper 
<488a26d62010-dmarc-requ...@jiscmail.ac.uk>
 wrote:

Hello. A quick look suggests that a lot of the test structures were solved by 
phaser or molrep, suggesting it is a very welcome improvement on homology 
modelling. It would be interesting to know how it performs with structures of 
new or uncertain fold, if there are any left these days. Without resorting to 
jokes about artificial intelligence, I couldn't make that out from the CASP14 
website or the many excellent articles that have appeared. Best wishes, Jon 
Cooper.


Sent from ProtonMail mobile



 Original Message 
On 3 Dec 2020, 11:17, Isabel Garcia-Saez < 
isabel.gar...@ibs.fr> wrote:

Dear all,

Just commenting that after the stunning performance of AlphaFold that uses AI 
from Google maybe some of us we could dedicate ourselves to the noble art of 
gardening, baking, doing Chinese Calligraphy, enjoying the clouds pass or 
everything together (just in case I have already prepared my subscription to 
Netflix).

https://www.nature.com/articles/d41586-020-03348-4

Well, I suppose that we still have the structures of complexes (at the moment). 
I am wondering how the labs will have access to this technology in the future 
(would it be for free coming from the company DeepMind - Google?). It seems 
that they have already published some code. Well, exciting times.

Cheers,

Isabel


Isabel Garcia-Saez PhD
Institut de Biologie Structurale
Viral Infection and Cancer Group (VIC)-Cell Division Team
71, Avenue des Martyrs
CS 10090
38044 Grenoble Cedex 9
France
Tel.: 00 33 (0) 457 42 86 15
e-mail: isabel.gar...@ibs.fr
FAX: 00 33 (0) 476 50 18 90
http://www.ibs.fr/




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[ccp4bb] What can iNEXT-Discovery do for us?

[Anastassis Perrakis]

Dear all,

We are happy to announce a webinar series highlighting some of the experimental 
possibilities offered by iNEXT-Discovery facilities.

iNEXT-Discovery is an H2020 program, that allows scientists from all over 
Europe (and outside Europe, to some extent) to access state of the art 
facilities for Structural Biology (X-rays, cryo-EM, NMR, Biophysics). Our 
offers extend beyond the use of machines, to hands-on expertise and (in many 
cases) help in designing experiments and interpreting data and of course remote 
services! Access to iNEXT-Discovery is free (often including samples shipment 
or visitor expenses) and is on the basis of (rapid) independent peer-review, 
for projects with a translational research component; the offer extends to 
industry partners under specific rules.

These webinars will be short presentations (20 minutes) presented by experts in 
the various facilities, and will not be in-depth science talks, but mostly 
geared towards user awareness for the technical and scientific capabilities we 
can offer!

We will offer one webinar every Monday at 16:00 CET, until Xmas, and then a few 
more!

We will cover topics like cryo-EM single particles and tomography, X-ray 
tomography, fragment screening by X-ray crystallography, in-cell NMR, and more. 
Every presentation will be followed by a live discussion.

A more detailed program is at: 
https://inext-discovery.eu/events/what-can-inext-discovery-do-for-us/

Registration is open at: 
https://zoom.us/webinar/register/WN_ZPGVwe7xR9GIyqESRS-dxA

Best regards on behalf of the iNEXT-Discovery team,

A. Perrakis

Prof. Dr. Anastassis Perrakis, PhD
Group leader, Dept. of Biochemistry, Netherlands Cancer Institute
Professor of Macromolecular Structures, University of Utrecht
Oncode Institute Investigator
Coordinator iNEXT-Discovery H2020











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[ccp4bb] iNEXT-Discovery: Access to facilities for structural biology research

[Anastassis Perrakis]

Dear all,

While being in turbulent times, iNEXT-Discovery, the EC Horizon-2020 project 
offering access to facilities for structural biology (EM, NMR, X-rays and 
macromolecular biophysics), has officially started its operations. 
iNEXT-Discovery encourages applications for structural biology projects for 
‘translational research projects’ in the areas of biomedicine, biotechnology, 
biomaterials and in the food sector, in particular from non-experts and for 
researchers from academia and industry.

We are now happy to announce to the wider research community our new website 
with details about the project and where applications for access to the 
participating facilities can be submitted:

https://inext-discovery.eu

Regardless of current corona-measures external researchers are more than 
welcome to apply for access and we expect reviewing of incoming proposals to 
proceed as normal. However, please keep in mind that most facilities are 
currently not operational, and it is uncertain when they will open again to 
deliver access and to receive visitors. We will not be able to give a detailed 
overview about the status of all facilities, as the situation is changing 
almost daily.

Having accepted applications in our access pipelines will help both our 
scheduling and yours. Our facilities can arrange experiments for approved user 
projects to be executed immediately after local policies allow.

Please note that applications addressing SARS-CoV-2 or Covid-19 and mentioning 
this in the title will be funneled towards rapid review. Operational facilities 
will prioritize such projects if they can be accommodated.

On behalf of the iNEXT-Discovery team,

Hans Wienk (Project Manager) - email 
h.wienk.AT.nki.nl<http://h.wienk.AT.nki.nl> for questions about access
Anastassis Perrakis (Coordinator)








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Re: [ccp4bb] Macromolecular Crystallography workshop in South America 2020

[Anastassis Perrakis]

Dear all,

As this discussion is receiving so much deserving attention, I should say that 
being an organiser of crystallography events in the past and in the future, we 
always strive to *intentionally* achieve a good gender balance of speakers. 
Sometimes we did even well, albeit often not well enough.

I also admit that this is not the case in our CSHL Macromolecular 
Crystallography, https://meetings.cshl.edu/courses.aspx?course=C-CRYS=20 , 
where we have much less women than we would like to as lecturers (while for 
students we typically have more than 50% women).

As I am embarrassed to admit that we might have missed brilliant female 
teachers in the past, If there are any female volunteers for teaching specific 
subjects during the CSHL course, I would really appreciate an Inbox message for 
what you would like to teach, so we can discuss these with the co-organisers 
(at least there now we are super-happy to have Janet after 30 years of male 
domination).

Thanks in advance,

Tassos

>
> El 4 feb. 2020, a la(s) 22:02, Edward Snell
> mailto:esn...@hwi.buffalo.edu>>
>  escribió:

>
> ​It is great that this workshop is occurring but I couldn't help but notice
> that there seem to be a lot of male speakers and tutors. I was wondering if
> it might be appropriate to add some female role models. There are some
> great candidates?

>
>
> 
> From: CCP4 bulletin board
> mailto:CCP4BB@JISCMAIL.AC.UK>>
>  on behalf of
> Alejandro Buschiazzo 
> mailto:ale...@pasteur.edu.uy>>
> Sent: Tuesday, February 4, 2020 7:30 PM
> To: 
> CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Macromolecular Crystallography workshop in South America
> 2020

> Dear colleagues,
>
> We are pleased to announce the 8th South American Macromolecular
> Crystallography School:

> Macromolecular Crystallography School 2020
> "Structural Biology to enhance high impact research in health and disease”
>
> To be held at the Institut Pasteur de Montevideo (Uruguay) - September 9-19,
> 2020

> http://pasteur.uy/novedades/mx2020/
>
> The application deadline is July 9, 2020. For further inquiries :
> mx2...@pasteur.edu.uy

>
> Main Topics:
>
> •   data processing;
>
> •   phasing and structure determination;
>
> •   model refinement and validation;
>
> •   introduction to crystallography + cryo-electron microscopy
> integration

> Confirmed speakers and tutors (so far... a few more will join the crew):
>
> Alejandro Buschiazzo (Institut Pasteur de Montevideo, Uruguay)
> Paul Emsley (Laboratory of Molecular Biology MRC, Cambridge, UK)
> Rafael Junqueira Borges (Instituto de Biociências UNESP, Botucatu, Brazil)
> Ronan Keegan (STFC Rutherford Appleton Lab - CCP4, Didcot, UK)
> Eugene Krissinel (STFC Rutherford Appleton Lab - CCP4, Didcot, UK)
> Joāo Muniz (Instituto de Fisica de São Carlos, Brazil)
> Garib Murshudov (Laboratory of Molecular Biology MRC, Cambridge, UK)
> Colin Palmer (STFC Rutherford Appleton Lab - CCP-EM, Didcot, UK)
> James Parkhurst (Diamond Light Source, Didcot, UK)
> Randy Read (University of Cambridge, UK)
> Kyle Stevenson (STFC Rutherford Appleton Lab - CCP4, Didcot, UK)
> Clemens Vonrhein (Global Phasing Ltd, Cambridge, UK)
>
> Please find the application form and further contact information at
> http://pasteur.uy/novedades/mx2020/
 (this www site will be updated
> regularly, so stay tuned!)
>
> This Workshop is supported by the Collaborative Computational Project Nº4
> (CCP4, UK) & Science and Technology Facilities Council (UK); the Centro de
> Biologia Estructural del Mercosur (CeBEM); and the Programa Iberoamericano
> de Ciencia y Tecnologia para el Desarrollo (CYTED) through de MICROBES
> consortium.

> Organizers:
> Alejandro Buschiazzo, PhD. Institut Pasteur de Montevideo, Uruguay
> Kyle Stevenson, DPhil. CCP4, STFC Rutherford Appleton Laboratory, United
> Kingdom
 Richard Garratt, PhD. Instituto de Fisica de Sao Carlos, USP,
> Brazil
> Applicants:
> 25 students will be selected, prioritizing advanced PhD, postdocs and young
> researchers. The Course will provide financial support covering
> registration fees, and for the case of those students coming from abroad,
> all local expenses (lodging, per diem and local transportation). Look in
> the www site for details on application procedures.




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[ccp4bb] PhD / PostDoc positions @NKI Amsterdam

[Anastassis Perrakis]

Dear all,

We are looking for enthusiastic, motivated PhD students and Post-Docsto join 
the Division of Biochemistry in the Netherlands Cancer Institute 
(https://www.nki.nl) in the groups of Tassos Perrakis and Titia Sixma. If you 
have a keen interest in the general field of integrative structural cell 
biology, and previous experience in cryo-EM, X-ray crystallography, cell 
biology, biochemistry, or biophysics … well, read on!

The project in the laboratory of Titia Sixma 
(https://www.nki.nl/divisions/biochemistry/sixma-t-group/ ) is in the area of 
DNA repair.

The projects in Anastassis (Tassos) Perrakis group 
(https://www.nki.nl/divisions/biochemistry/perrakis-a-group/) are in the 
general field of controlling the progress of cell division and microtubule 
interacting proteins, in lysolipids signaling focusing on Autotaxin and its 
interactions, and together with Robbie Joosten in  PDB-REDO .

The Netherlands Cancer Institute is an international center of excellence with 
a high standard of biological research and outstanding research facilities, and 
has an interactive atmosphere. It is located in Amsterdam, with all its 
cultural amenities, close to the Schiphol airport.

The two groups share a common research infrastructure in the Department of 
Biochemistry, and have an interest in structural studies coupled to functional 
analysis. Facilities include the ability to produce and purify proteins in 
bacterial, insect, and mammalian cells, and a well-equipped macromolecular 
biophysics laboratory that are sites in the Instruct Centre NL 
(https://instruct-eric.eu/centre/protein-facility---nki/<http://eric.eu/centre/protein-facility---nki/>).
 Equipment for structural analysis is spearheaded by a brand-new cryo-EM (Jeol 
F2 200kV with a K2 detector) and high-throughput crystallisation robotics; 
these local facilities are coupled with regular access to Krios cryo-EM at 
NeCEN (https://www.necen.nl , https://instruct-eric.eu/centre/necen---lu/) and 
to the ESRF, SLS and DLS synchrotrons, and access possibilities for NMR 
experiments 
(https://instruct-eric.eu/centre/bijvoet-center---uu/<http://eric.eu/centre/bijvoet-center---uu/>).
 Dedicated computing facilities include privileged access to both CPU and GPU 
clusters. Both groups participate in various European and National 
collaborative projects and are members of the Oncode Institute 
(https://www.oncode.nl).

We are looking flexibly for either highly motivated PhD students (strong 
master-level placements in reputable laboratories and familiarity with some of 
the methods we outline would be strong plus) or post-docs (experience in 
cryo-EM would be highly desirable for most positions; candidates with a strong 
background and track record in X-ray crystallography, protein purification, 
cell biology, would be taken seriously under considerations depending on the 
project). For the PDB-REDO project a background in bioinformatics and some 
experience in computerprogramming would be necessary. Applicants should write 
an email, stating their specific interests and attaching a CV and names of 
three references to t.sixma.AT.nki.nl<http://t.sixma.AT.nki.nl> or 
a.perrakis.AT.nki.nl<http://a.perrakis.AT.nki.nl>; feel free to also make 
informal inquiries.

We expect applications to be sent in by Monday 13th January and all positions 
are available asap. PhD candidate positions are for four years; post-doc 
positions can be for 1-4 years depending on qualifications and experience; the 
CAO salary levels apply according to the NKI collective agreement.


Anastassis Perrakis and Titia K. Sixma







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Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Problem with structural alignment

[Anastassis Perrakis]

Dear Herman et al,

On Dec 16, 2019, at 17:52, Schreuder, Herman /DE 
mailto:herman.schreu...@sanofi.com>> wrote:

Dear Ishan,

A structural alignment should be independent of the software used.
However, the residues being selected for the alignment can make a big 
difference and different programs have different selection criteria.
There are two cases:

  1.  Different proteins. Here one needs to decide what the equivalent residues 
are. Usually this is based on a sequence alignment, but once a crude 
superposition has been obtained, the equivalent residue pairs can be optimized.

Theseus can do that well. https://theobald.brandeis.edu/theseus/


  1.  The same protein undergoing a conformation change (e.g. loop or domain 
movement). Here one has to decide what the rigid regions are and where the 
moving loop or linker is. By looking at the coarse superposition with coot or 
any other graphics program, one quickly sees what would be the best borders.

In this case I would be inclined to use the RAPIDO server.

http://webapps.embl-hamburg.de/rapido/

I like algorithms making these choice ;-) I am lazy!

Best regards,

Tassos



  1.

If it is really critical for your hypothesis, you will have to define manually 
the equivalent pairs.

Best,
Herman


Von: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
Im Auftrag von Eleanor Dodson
Gesendet: Montag, 16. Dezember 2019 11:53
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] Re: [ccp4bb] Problem with structural alignment


EXTERNAL : Real sender is 
owner-ccp...@jiscmail.ac.uk

Well CCP4mg does this very nicely and it is easy to check the results - all 
displayed on the screen so you can pinpoint outliers..
Eleanor

On Mon, 16 Dec 2019 at 06:38, Ishan Rathore 
mailto:ishanrathor...@gmail.com>> wrote:
Hi,

I am trying to compare multiple homologous structures of a protein, where I am 
analysing the active site residues and the bound substrate/peptide. I have used 
multiple methods for alignment in coot and pymol. Every method gives a slightly 
different orientation in the active site. Based on the analysis I am trying to 
propose a hypothesis for the catalytic mechanism of the protein. But, I am a 
bit wary of getting biased with the alignment if that supports my hypothesis.

What are the parameters that have to be considered for a reliable alignment?
What are the other Softwares available for alignment?



Thanks and regards
Ishan



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Re: [ccp4bb] Unusual dataset with high Rmerge and extremely low b-factors?

[Anastassis Perrakis]

Dear Michael,

Exactly because the B factor is only 8, the intensity as function of resolution 
is droping really slowly, and thus the I/sigI 4.7 at low resolution drops so 
slowly to 1.0.

I would almost bet you have a low solvent content.

The data are fine imo.

Best,

Tassos

On Nov 4, 2019, at 0:18, Michael Jarva 
mailto:jarv...@wehi.edu.au>> wrote:

Hi CCP4BB,

I have some unusual crystal diffraction data I'd like to get your input on.

Almost a year ago I shot some small rods sticking out of a loop, so basically 
no liquid around them - using the microfocus MX2 beamline at the australian 
synchrotron, collected on an EIGER 16M detector.

The crystals diffracted weakly and was seemingly not viable at first glance 
because of high Rmerge/Rpims. See the aimless summary at the bottom of this 
post. This seemed to stem from a low spot intensity at low resolutions 
(I/sd(I)=4.6), but since the CC1/2 was fine I went with it anyway.

Here I also noted an unusually low Mosaicity, 0.05, and Wilson B-factors, 8.02 
Å^2.

Density maps looked great and the build refined easily enough (R/Rfree 
0.1939/0.2259) with a mean B-factor of 19.85, which according to phenix is 
lower than any other structure deposited in that resolution bin. Furthermore, 
the molprobity score is 0.83, and overall real-space correlation CC is 0.855.

So my question is, can I feel comfortable depositing this?

best regards
Michael

Chosen Solution:space group P 1 21 1
Unit cell:44.93   41.90   45.83  90.00  115.57   90.00
Number of batches in file:   1659
The data do not appear to be twinned, from the L-test
Overall InnerShell OuterShell

Low resolution limit   41.34 41.34  2.49

High resolution limit   2.40  8.98  2.40


Rmerge  (within I+/I-) 0.231 0.084 0.782

Rmerge  (all I+ and I-)0.266 0.099 0.983

Rmeas (within I+/I-)   0.323 0.118 1.091

Rmeas (all I+ & I-)0.317 0.118 1.167

Rpim (within I+/I-)0.225 0.084 0.759

Rpim (all I+ & I-) 0.171 0.063 0.623

Rmerge in top intensity bin0.079- -

Total number of observations   19901   362  2067

Total number unique 6054   126   611

Mean((I)/sd(I))  2.7   4.6   1.0

Mn(I) half-set correlation CC(1/2) 0.958 0.991 0.570

Completeness98.6  98.2  97.3

Multiplicity 3.3   2.9   3.4

Mean(Chi^2) 0.48  0.33  0.50


Anomalous completeness  81.7  92.2  75.1

Anomalous multiplicity   1.5   1.8   1.9

DelAnom correlation between half-sets -0.003 0.041 0.045

Mid-Slope of Anom Normal Probability   0.704   - -


The anomalous signal appears to be weak so anomalous flag was left OFF


Estimates of resolution limits: overall

   from half-dataset correlation CC(1/2) >  0.30: limit =  2.40A  == maximum 
resolution

   from Mn(I/sd) >  1.50: limit =  2.67A

   from Mn(I/sd) >  2.00: limit =  2.87A


Estimates of resolution limits in reciprocal lattice directions:

  Along0.96 a* - 0.28 c*

   from half-dataset correlation CC(1/2) >  0.30: limit =  2.40A  == maximum 
resolution

   from Mn(I/sd) >  1.50: limit =  2.40A  == maximum 
resolution

  Along k axis

   from half-dataset correlation CC(1/2) >  0.30: limit =  2.40A  == maximum 
resolution

   from Mn(I/sd) >  1.50: limit =  2.86A

  Along   -0.17 a* + 0.99 c*

   from half-dataset correlation CC(1/2) >  0.30: limit =  2.40A  == maximum 
resolution

   from Mn(I/sd) >  1.50: limit =  2.98A


Anisotropic deltaB (i.e. range of principal components), A^2:  8.62


Average unit cell:44.93   41.90   45.83   90.00  115.57   90.00

Space group: P 1 21 1

Average mosaicity:   0.05


Minimum and maximum SD correction factors: Fulls   1.27   1.28 Partials   0.00  
 0.00





Michael Jarva, PhD
ACRF Chemical Biology Division
The Walter and Eliza Hall Institute of Medical Research
1G Royal Parade
Parkville Victoria 3052
Australia
Phone: +61 3 9345 2493
Email: jarv...@wehi.edu.au | Web: 
http://www.wehi.edu.au/
The ACRF Chemical Biology Division is supported by the
Australian Cancer Research Foundation


___

The information in this email is confidential and intended solely for the 
addressee.
You must not disclose, forward, print or use it without the permission of the 
sender.

The Walter and Eliza Hall Institute acknowledges the Wurundjeri people of the 
Kulin
Nation as the traditional 

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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Re: [ccp4bb] not solely pdb issue: need someone to officially settle the pdb dispute

[Anastassis Perrakis]

If the structure has been deposited in the PDB and thus is public ally 
available, B (or F, G, Φ, Ξ, Δ, Α or whoever else) has every right to use it in 
a publication.

“A” should follow the advice of Frank and do a happy dance for the usefulness 
of the work, or if not feeling like dancing she/he could follow my advice that 
will be offered in Greek: «ξυδάκι».

Sent from my iPhone

On 21 Aug 2019, at 11:13, Flemming Goery 
mailto:flemming_go...@hotmail.com>> wrote:

Dear all:
A has sought a job in the lab of B. B invited A for a interview with a PPT oral 
presentation, as requested B has sent the PPT on the structural biology 
research of XXX to B by e-mail, and presented in front of A and his 
postdoctoral researcher.

After interview, B requested all research documents (including detailed 
reports) on XXX to be sent by A to B by e-mail, A sent, including 2 sets of pdb 
for the same structure, one set with solvent, one without. A told B all 
intellectual property of the Documents and the research belonged to A, based on 
the regulation of A's institute.

B sought a referee from A's institute, to someone A did not agree. It seems the 
referee told B one set of PDB has been deposited (the one without solvent)

Then B did not give the offer to A. A joined Institute D, without independent 
funding for the writing (in fact, no salary to support this writing, and no fee 
for publication of this work).

Several years later, A found B's paper, i.e., the concerned paper published in 
Journal C. In the paper, B has used the information from deposited PDB for 9 
times (already a significant paprt of the paper, not to say the message from 
the other Documents sent to B by A). In the paper, it write something like, 
'based on our work on the structure of  (folowed by 4 letter pdb code)', which 
implied the structure was solved by the authors of the paper, rather than by A.

A contacted Journal C, Journal C contacted B, B claimed the deposited PDB was a 
public domain knowldge. Journal C took the action to add the reference to the 
deposited pdb in the paper.

As mentioned, the paper has mentioned and used the message from the deposited 
pdb 9 times, and in the paper the reference mark was not added to the first 
occurence of the mentioning of the deposited pdb, but added (only once for the 
9 occurences of depositation code) to a paragraph where it can be concluded 
that the authors have used the undeposited pdb with the solvent. In another 
words, although reference to the deposited pdb was added by a correction, from 
where the reference mark was added, it cannot show they have refered to the 
cited pdb, not to say the undeposited pdb with solvent which they used based on 
the paragraph information.

A's concern was that: A cannot exclude the possibility that the research in the 
paper other the part related to PDB, were fabricated, thus A request paper 
retraction as the major clain.

If cannot retratcted, A request to be the correspondence author (sometimes 
requets co-first author, sometimes request both co-first author and 
co-correspondence author), as without A's work (the PPT presentation, 2 sets of 
pdb, all documents), the work in the concerned paper cannot be done. A regard 
as having contributed to the initiation of the paper, thus A prefer to be add 
as a co-correspondence author if appropriate.

First, can the paper deserve a retraction, and second, can B deserve a 
co-author?

Flemming




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Re: [ccp4bb] need someone officially settle a pdb dispute for a publication

[Anastassis Perrakis]

Something is unclear to me in the original question. What does “has used his 
pdb for a publication” mean? Somebody used an entry already in the PDB? 
Somebody used a “.pdb” coordinates file for publication (without “.mtz”)? What 
was and is the relationship between A and B?

In any case, assuming that A and B are not in talking terms (have you tried 
through a mediator?), it is the director or designated ombudsperson of the 
institute of A, that should review the case internally, and officially contact 
the corresponding person of the institute of B. I can’t see what the journal 
has to do with it, without a settlement between institutes. I also do not 
consider a direct contact if A to the director of B appropriate. There should 
be procedures for these cases.

A.

Sent from my iPhone

On 21 Aug 2019, at 10:12, Mark J van Raaij 
mailto:mjvanra...@cnb.csic.es>> wrote:

Dear Flemming,

As I understand it (I may be wrong), the final responsible institutions are 
those where the authors work. But as you say, they sometimes don't even reply - 
or they just may be very slow because they want to be really sure before 
committing to any answer.

But the journal has a responsibility also, to retract the paper if there is a 
serious suspicion the data were not obtained ethically. Of course, it may be 
difficult to prove ownership of a pdb file, if both authors claim ownership 
there is not really a way the journal can decide who is right. In my opinion, 
the journal should officially contact the institutions where the authors work 
to try and resolve this. The institutions may take the journal more seriously 
than a single researcher.

A generally respected institution that may advise on authorship disputes is 
COPE, Committee on Publication Ethics: https://publicationethics.org/
May also take a while though...
They have a database with anonymised examples of previously resolved disputes 
that may be helpful - you may find a similar situation on which they have 
"ruled". These are of course not legal rulings, but are considered by their 
members (most respectable journals) as a strong guideline.
This case may have similarities:
https://publicationethics.org/case/claim-stolen-data-and-demand-retractions

Best of luck,

Mark

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


On 20 Aug 2019, at 17:45, Flemming Goery 
mailto:flemming_go...@hotmail.com>> wrote:

Dear All,

A and B belong to 2 different institutes. A claimed B has used his pdb for a 
publication in Journal C. Journal C did not give the retraction, but permit 
complain related to the journal publication author issue, with the prerequisite 
journal C did not have the authority on authorship dispute. Then A has e-mailed 
to the institute head of B with academic misconduct by B as claim, the 
institute head of B did not give reply.

In this situation, can A have the journal  authorship  dispute settled by a 
neutral reviewer (Journal C view: you (A) need to reach out to the institutions 
that have authority to adjudicate on such matters, as investigation and 
adjudication on authorship claims falls outside the remit of journal editors. 
)? Who are qualified as the neutral reviewer so that the review decision can be 
submitted to Journal C?

If you believe you are qualified, or you know somebody or some organization 
qualified, please let me know and I will introduce the issue to you by separate 
e-mail (it is best not disseminated, am I right?)

Best regards.

Flemming



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Re: [ccp4bb] challenges in structural biology

[Anastassis Perrakis]

I would wonder more if the biological questions you can *ask* with a (crystal) 
structure are sufficiently relevant to justify the resources. 

Sent from my iPhone

> On 15 Jul 2019, at 22:08, Tim Grüne  wrote:
> 
> Dear James,
> 
> 10) are the biological questions that you can answer with a (crystal) 
> structure sufficiently relevant to justify the resources?
> 
> Best,
> Tim
> 
> 
> 
> Am 15.07.2019 21:44, schrieb Holton, James M:
>> Hello folks,
>> I have the distinct honor of chairing the next Gordon Research
>> Conference on Diffraction Methods in Structural Biology (July 26-31
>> 2020).  This meeting will focus on the biggest challenges currently
>> faced by structural biologists, and I mean actual real-world
>> challenges.  As much as possible, these challenges will take the form of
>> friendly competitions with defined parameters, data, a scoring system,
>> and "winners", to be established along with other unpublished results
>> only at the meeting, as is tradition at GRCs.
>> But what are the principle challenges in biological structure
>> determination today?  I of course have my own ideas, but I feel like I'm
>> forgetting something.  Obvious choices are:
>> 1) getting crystals to diffract better
>> 2) building models into low-resolution maps (after failing at #1)
>> 3) telling if a ligand is really there or not
>> 4) the phase problem (dealing with weak signal, twinning and
>> pseudotranslation)
>> 5) what does "resolution" really mean?
>> 6) why are macromolecular R factors so much higher than small-molecule ones?
>> 7) what is the best way to process serial crystallography data?
>> 8) how should one deal with non-isomorphism in multi-crystal methods?
>> 9) what is the "structure" of something that won't sit still?
>> What am I missing?  Is industry facing different problems than
>> academics?  Are there specific challenges facing electron-based
>> techniques?  If so, could the combined strength of all the world's
>> methods developers solve them?  I'm interested in hearing the voice of
>> this community.  On or off-list is fine.
>> -James Holton
>> MAD Scientist
>> 
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> 
> -- 
> --
> Tim Gruene
> Head of the Centre for X-ray Structure Analysis
> Faculty of Chemistry
> University of Vienna
> 
> Phone: +43-1-4277-70202
> 
> GPG Key ID = A46BEE1A
> 
> 
> 
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[ccp4bb] CCP4mg trouble making movies

[Anastassis Perrakis]

Dear all,

I am having some trouble with ccp4mg 2.10.10 and making movies on a Mac running 
OSX 10.13.6

I am trying now for a test a single-scene movie of 2sec “rock”. The movie 
previews  fine.
When I “make movie” the scene directory and the frames are created, and I get a 
window telling me:
“For movie test / Converting frames to scene movie / and merging scene movies” 
However, nothing is happening - no files and my CPU is blissfully idle.

Any hints/clues?

Thanks,

Tassos



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Re: [ccp4bb] ORCID being mandatory for PDB depositions

[Anastassis Perrakis]

I am wondering, are there any arguments that would suggest that ORCID is not in 
line with GDPR requirements? You are disclosing your name etc to the PDB 
anyway, does it matter if its through ORCID or not? 

Tassos


> On Apr 9, 2019, at 14:04, V F  wrote:
> 
> Dear all,
> Did anyone observe that oneDep from EBI made ORCID mandatory for
> deposition? What am I supposed to do if my collaborators do not want
> to create ORCID? (especially with GDPR I do not want to create ORCID)
> Just posting here so that some one will respond? my mails are going to
> /dev/null?
> 
> Many thanks,
> VF
> 
> 
> 
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[ccp4bb] X-Ray Methods in Structural Biology @CSHL

[Anastassis Perrakis]

Dear all,

The June 15th deadline for applications to the CSHL X-ray Methods in Structural 
Biology Course to be held later this year, October 15 through October 30, 2019 
is rapidly approaching.

The official course announcement is here:

https://meetings.cshl.edu/courses.aspx?course=C-CRYS=19

so please pass this on to folks who might be interested and who would benefit. 
The course is designed for researchers who are either new to, or who wish to 
increase their in-depth knowledge of, macromolecular crystallography. 

This immersive course is an outstanding place to learn both the theoretical and 
practical aspects of Macromolecular Crystallography because of the extensive 
lectures from world-renowned teachers, which are combined with hands-on 
experiments. This 2019 course will be lead by Jim Pflugrath (Rigaku, ret.), 
Tassos Perrakis (NKI), Paul Adams (LBL), Janet Newman (CSIRO) and an All-Star 
cast of lecturers. Attendees can expect to participate in a course that is 
unparalleled in the world, with experts each devoting several days to teaching 
the fundamentals, theory and practical considerations of crystallographic 
structure solution.

We expect to have the participants crystallize several proteins and determine 
their structures all in about two weeks.Students may also work on their own 
projects, but not exclusively. They will also become well-versed in the theory 
of X-diffraction and crystal structure determination while having lots of fun, 
but not much sleep. There will be a trip to the state-of-the-art beamlines at 
NSLS-II to collect data.

The course is limited to 16 participants due to the very hands-on nature of the 
experiments and the intimate seminar room and laboratory settings.  Please 
check the above web link for more details. In particular, please note the 
information about fellowships, scholarships, and stipends that are available. 
It is often possible for applicants to receive some level of support to help 
offset the course fee.

This course is supported with funds provided by the National Institute of 
General Medical Sciences for which we are extremely grateful. Also there are 
stipends available from the Leona M. and Harry B. Helmsley Charitable Trust and 
the Howard Hughes Medical Institute to help offset the cost of tuition. 

If anyone has any questions, please send me e-mail, I will be happy to answer 
any queries.

Thanks for spreading the word, Janet, Tassos, Paul, and Jim


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Re: [ccp4bb] coiled-coil "degree"

[Anastassis Perrakis]

Dear Jorge,

The “coiled coil” has a formal definition - not all interacting helices are 
coiled coils. I would refrain from expressions like "more or less 
"coiled-coiled"one to another”. I have found the program “socket” very useful 
for analysing such structures. 
http://coiledcoils.chm.bris.ac.uk/socket/server.html

I must say that I really do not understand completely the actual question, but 
a word of caution: The ‘asymmetric unit’ not has little to do with the 
‘functional unit’ of the protein but furthermore, what we define as AU 
especially in the case of multiple copies of the molecule in the AU, is a 
personal choice. Sometimes, its worth thinking what we call “asymmetric unit 
contents” and what is a “crystallographic contact” in cases of NCS. I am not 
sure this applies here as I cannot completely visualise your problem at hand, 
but keep it in mind.

Best regards,

Tassos

> On Aug 28, 2018, at 15:11, Andrew Lovering  wrote:
> 
> Dear Jorge
> 
> This may not be exactly what you require, but there is a nice example of 
> analysis in the shift of a coiled coil register herein:
> 
> Elife. 2018 Jun 5;7. pii: e34815. doi: 10.7554/eLife.34815.
> Asymmetric activation mechanism of a homodimeric red light-regulated 
> photoreceptor.
> Best,
> Andy
> 
> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
> jiu...@gmail.com
> Sent: 28 August 2018 14:04
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] coiled-coil "degree"
> 
> Dear all,
> 
> 
> I am working currently on a structure that, nicely, presents two 
> different orientations between its domains when one compares monomers of the 
> tetramer in the asymmetric unit.
> 
> I notice, in this nature gift, that a helix, probably central (in its 
> role) for the relation (orientation) between the two domains, assumes 
> different relation to other one (helix, that belongs to one of the domains) 
> such that they are (significantly) more or less "coiled-coiled"one to 
> another, once the domains are in the "close" or "open" conformation. We have 
> already analyzed the hydrogen bonds and salt bridges that are disrupted (or 
> formed) due to the different (domain and helix) conformations.
> 
> I wonder whether there is a metric (easy to evaluate) to characterize how 
> much the two helix are "around each other" (id est, how much "coiled-coiled" 
> they are) and, preferably, a software to calculate this metric.
> 
> Thank you,
> 
> 
> Jorge
> 
> State University of Ponta Grossa
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1




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[ccp4bb] Deadline for the EM and the X-ray bundles of iNEXT extended to 24 April (midnight)

[Anastassis Perrakis]

Dear all,

The deadline for the EM and the X-ray bundles of iNEXT is now extended until 24 
April (midnight, for all timezones …).

For more information http://www.inext-eu.org/inext-em-and-x-ray-bundles/ and to 
submit a proposal https://www.structuralbiology.eu/submit-proposal/?t=inext

Best regards,

the INEXT team

[ccp4bb] Reminder: iNEXT Bundles

[Anastassis Perrakis]

Dear all,

The single deadline for the iNEXT bundles for EM and X-ray projects “from 
purified molecule to high resolution data” is this Friday, 20th April.

Dont miss it.

https://www.structuralbiology.eu/submit-proposal/?t=inext

Best regards,

On behalf of the iNEXT team, Tassos

[ccp4bb] iNEXT EM and X-ray bundles!

[Anastassis Perrakis]

Dear all,

iNEXT, a European program to support access to research infrastructures for 
Structural Biology, is now welcoming applications for the “EM and X-ray 
bundles", to help people carry a project all the way from the starting sample 
to the initial structure determination, by EM or X-rays.

To apply for either bundle you need exciting translational research projects, 
for which you have the macromolecule or complex you want to study in purified 
soluble form, and you want to engage in structural studies. In a short 
application form (less than one thousand words overall), you will need to 
describe the background of the project in your lab and current results, the 
general scientific background, and the value for translational research (short 
term but also long term value in any short of biomedical or biotechnological 
application). No prior experience on either method is required.

For the EM bundle, projects will start with an initial characterisation for 
optimal stability conditions, molecular weight in solution, and negative stain 
EM (if you send enough sample, we can also try to crystallise it if you wish - 
just in case!). At this stage you will only need to send the sample to us. 
Projects that look most promising after that first step, will proceed to 
optimising the cryo-conditions in one of the iNEXT EM facilities, with your 
participation. Finally, projects that do best in cryo-optimisation, will 
proceed to collect data in one of the high-end microscopes available to iNEXT.

For the X-ray bundle, projects will start with an initial characterisation for 
optimal stability conditions, molecular weight in solution, and crystallisation 
trials. Projects that either yield crystals, or promising precipitates, will 
proceed to extensive crystallisation trials. For all projects that will yield 
crystals diffraction data will be collected in one of the fully automated X-ray 
facilities that collaborate in iNEXT. You will only need to send the sample to 
us and in most cases physical presence will not be required.

It should be emphasised, that no re-application will be needed at any step and 
iNEXT will cover all experimental expenses, and contribute to travel costs, 
when needed.

There is a single deadline on Friday 20th April 2018.

You can Submit a Proposal by clicking 
here or go to the 
iNEXT web site: http://www.inext-eu.org and follow the “Submit Proposal” link.

Please note, that given the iNEXT resources availability, this is likely the 
only call for these “bundle” access. These will serve as pilots for likely 
requesting future funding from the EU, if such an opportunity becomes available.

We note that researchers from all EU and associated countries can apply, but 
also international labs are eligible, albeit in a more limited capacity.

Please forward this email beyond this bulletin list, especially to colleagues 
that might be less familiar with structural biology. Consider asking this 
colleague of yous that wanted to do a structural biology project and you never 
had time or the resources to try hers or his exciting idea! Preliminary results 
from this bundle access, or a structure, can be the basis for your next grant 
application!

Please also keep in mind that iNEXT remains open to all kinds of Structural 
Audit, Enhanced Support, and High-End data collection, for projects that 
involve X-ray Crystallography, SAXS, solution and solid state NMR, EM single 
particles, EM tomography or CLEM, biophysics for looking at macromolecular 
interactions, advanced light imaging, and ligand and fragment screening! All 
these access modalities are listed on our web site!

Best regards and looking forward receiving your project,

The iNEXT coordination and management team and all iNEXT partners

[ccp4bb] Access to iNEXT!

[Anastassis Perrakis]

Dear all,

iNEXT has been offering access to X-rays, electrons, 
magnets, and photons for a bit over than two years.

iNEXT has pioneered the so-called “Structural Audit” access, where any user can 
mail-in their sample for initial characterisation by structural biology methods.

We have very recently revamped the Structural Audit access which now allows you 
to submit a sample for:


  *   Crystallisation, stability and aggregation (Prometheus), oligomerisation 
(SEC-MALS)
  *   SAXS measurements in batch, or with inline SEC.
  *   Initial characterisation for NMR, typically 15N-HSQC
  *   Negative stain EM

You can mix and match these access routes, in any way you want, with as many 
samples per experiment as you wish, in a single application.

We aim for rapid review by our external experts, and you are welcome to Submit 
a Proposal now!

Thank you and stay tuned for more from iNEXT in the near future!

Best regards,

Tassos

PS Our website, in case links do not work: http://www.inext-eu.org

[ccp4bb] Helical bundles vs coiled coils

[Anastassis Perrakis]

Dear all,

I suspect that helical bundles and coiled coils would have different CD 
spectra? Are you aware of a paper that has shown that (or the opposite …).

Would anyone know another experimental method (*) to check this quickly?

Thanks

A.

(*) Other than solving the structure by crystallography or EM; or SAXS (we have 
the SAXS data and they are clear, but “I" need something on top).
“I” Imaginary referee #3

Re: [ccp4bb] Crystal structure of an unknown protein

[Anastassis Perrakis]

I wonder why you assume know there are "about 20 point mutation sites” if "this 
protein is an unknown protein”.
It looks like you are comparing the sequence of a protein you do not know what 
it is to the sequence of a protein you dont really know what it is (1).

I would consider it more likely, it is the E. coli protein and the electron 
density is ambigious so some side chains might have been erronously assigned to 
a false identity.

A.

(1)
Percy: You know, they do say that the Infanta's eyes are more beautiful than 
the famous Stone of Galveston. 
Edmund: Mm! ... What?
Percy: The famous Stone of Galveston, My Lord.
Edmund: And what's that, exactly?
Percy: Well, it's a famous blue stone, and it comes ... from Galveston.
Edmund: I see. And what about it?
Percy: Well, My Lord, the Infanta's eyes are bluer than it, for a start.
Edmund: I see. And have you ever seen this stone?
Percy: (nods) No, not as such, My Lord, but I know a couple of people who have, 
and they say it's very very blue indeed. 
Edmund: And have these people seen the Infanta's eyes?
Percy: No, I shouldn't think so, My Lord.
Edmund: And neither have you, presumably.
Percy: No, My Lord.
Edmund: So, what you're telling me, Percy, is that something you have never 
seen is slightly less blue than something else you have never seen.


>> Dear CCP4bb,
>> In 2014, I collected a high quality data set from a crystal. But I could not 
>> solve the structure of that crystal because this protein is a contaminate.
>> Recently, I used StruBE's Contaminer and fortunately got the solution. 
>> Thanks ContaMiner!!!  This protein is a contaminate protein.
>> However, I found this protein is an unknown protein (about 180 residues) 
>> whose amino acid sequence is not totally same as E.coli. There are about 20 
>> point mutation sites comparing to the E.coli protein. This means this 
>> protein may be from an unknown bacteria.
>> The space group of this crystal is new. There is also a new ligand in this 
>> protein.
>> My question is how could I found the primary structure of this protein and 
>> how to deposit this protein in PDB.
>> Best regards,
>> Jiyong
> 
> 

Re: [ccp4bb] secondary structure prediction

[Anastassis Perrakis]

Dear Zheng,

You might want to try the tools at:

https://ccd.rhpc.nki.nl

Tassos

On Dec 6, 2017, at 15:14, zheng zhou 
> wrote:

Dear CCP4 community,

Sorry for the off-topic question. I am trying to design constructs for
structure studies. It only has a homolog structure in PDB with
sequence identity ~20%. When I blast against PDB sequence, there are
quite a few motif hits (30~40aa, identity 40~50%). Any prediction
tools utilize this information?

Thanks for your advice in advance.

Best,

Zheng

[ccp4bb] Biophysical Characterisation of Macromolecules and Quantification of Biomolecular Interactions

[Anastassis Perrakis]

Dear all,

I would like to remind you that the deadline for applying to this course is 10 
October 2017

https://www.structuralbiology.eu/events/biophysical-characterisation-of-macromolecules-and-quantification-of-biomolecular-interactions/

You can apply at:

https://www.aanmelder.nl/96461/subscribe

… and don’t forget to write a strong motivation letter as the places are 
limited!

Best regards,

Tassos

[ccp4bb] Master Your Proteins, NKI Amsterdam, 20-23 November 2017

[Anastassis Perrakis]

Dear all,

Applications for the Instruct-ERIC course on Biophysical Characterisation of 
Macromolecules and Quantification of Biomolecular Interactions are now open.

The course aims to provide the theoretical background of popular biophysical 
methods that are often used in relation to Structural Biology research, and 
practical experience both in performing experiments and in analysing the 
resulting data. The rationale behind an extended kit of experiments to 
characterise macromolecules and their interactions will be explained. You will 
be shown how to perform and analyse the related experiments in dedicated 
practical sessions.

The course will include theory and practical sessions on thermal shift assays 
(also label free), Surface Plasmon Resonance, sothermal Titration Calorimetry, 
Miscroscale Thermophoresis , Fluoresence Methods, Stopped Flow, and the 
Graphpad/Prism and Kintek analysis software.

Details can be found at:

https://www.structuralbiology.eu/events/biophysical-characterisation-of-macromolecules-and-quantification-of-biomolecular-interactions/
 

 


On the behalf of the organisers,

Tassos

[ccp4bb] Course: Bridging solution methods: from NMR to X-ray scattering and biophysics"

[Anastassis Perrakis]

Dear all,

Announcing in the mailing list for crystallography and in the era of the single 
particles EM resolution revolution, a course for solution methods? 
NMR, SAXS, calorimetry, fluorescent methods and modeling? Seriously?

Well, yes! For many, many reasons, you should be at least curious, and make 
sure you read through - and apply!

Registrations are open for this iNEXT (http://www.inext-eu.org 
) 4-day training course 

September 18-22, 2017, University of Patras, Greece

The course, organized with lectures in the mornings followed by hands-on 
training in the afternoon, will cover the main approaches for the 
characterization of the structure and dynamics of biomolecules in solution. The 
course is aimed primarily at PhD students and young Post-Docs, but more senior 
researchers interested in structural biology are also welcome. The registration 
fee, including accommodation, is 150 Euro; personal fellowships are also 
available.

For applications and further information please visit

http://www.bionmr.upatras.gr/index.php?option=com_content=article=251=220
 


On the behalf of the organizers Georgios Spyroulias and Giacomo Parigi

Tassos Perrakis

[ccp4bb] iNEXT access: NMR Extended Support and more!

[Anastassis Perrakis]

Dear all,

I should perhaps offer sincere apologies for posting the crystallography boards 
with NMR news ;-). 

However, I will not do that, as I sincerely hope you will see that in an era of 
integration of structural methods, such opportunities can offer the chance for 
excellent and exciting scientific endeavours.

iNEXT (http://www.inext-eu.org ) is committed to 
bring structural methods closer to non-experts, by funding access to world 
class infrastructures for Structural Biology. Major tools for achieving that 
goal are the so-called Extended Support modalities. 

We are happy to announce the start for acess to the iNEXT Enhanced Support 
modality for NMR (NMR assignment, structure, dynamics). In this mode, the NMR 
groups in Florence, Frankfurt and Utrecht are involved in guiding and training 
NMR novices. This iNEXT access modality also targets "new users with 
backgrounds other than Structural Biology”, so please feel free to forward that 
also to your biochemistry friends. Facility personnel will be involved to guide 
assignments, titrations, structure calculations, relaxation analyses, etc.

I also want to bring your attention to a few more iNEXT news:

- The Structural Audit, now allows multiple samples in a related project to be 
submitted in a single application for stability assessment and crystallisation 
screening; a basic analysis by SAXS, EM, or NMR is also supported (single click 
in application!) if an appropriate sample is available.

- The Enhanced Support modality "From macromolecular sample to X-ray data 
collection” is now better integrated with Structural Audit; applications can be 
combined, so in case of a successful Audit, you could rapidly proceed to 
further X-ray experiments (crystal optimisation, SAXS and/or crystallography)

- The Enhanced Support modality "Advanced light imaging” to support structural 
studies with in cells measurement of protein interaction kinetics, have also 
been made recently available.

- The Enhanced Support modality "Cryo-EM sample optimisation” for hands-on 
assistance in optimizing a sample for single particle EM is a great chance to 
move your sample towards cryo-EM!

- The Enhanced Support modality "Macromolecular interactions” is your chance 
for access to a Biophysics facility with various instruments and hands-on 
support for performing and analysing experiments to quantify a variety of 
interactions.

- All the High-End modalities (X-rays, NMR, cryo-EM) are always open for access!

So, please, submit your proposal now! 
 … and keep in 
mind that a limited amount of the funding (~10%) can go towards projects that 
are not in an EU-country or associated country.

On behalf of the iNEXT Management,

Best wishes,

Tassos


Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute, 
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] Superpose program in CCP4

[Anastassis Perrakis]

Dear Wenhe,

Besides this advice, have a look at the http://webapps.embl-hamburg.de/rapido/ 
 server.

Sometimes its goodo to re-think of what you want to do, and wonder why its not 
easily doable in software (perhaps because its not the right thing to do …)

A.


> On 30 Oct 2016, at 11:45, Gert Vriend  wrote:
> 
> Dear Wenhe,
> 
> No 3D superpose tool will always align/map all Calphas. If in the one protein 
> the loop turns left, and in the other it turns right, then mapping those 
> loops is meaningless and thus not done by good software. The other problem is 
> that often two proteins that get compared do not even have equally many 
> residues so that there will always be some unaligned/unmapped Calphas left at 
> the end. Look for some articles by Arthur M Lesk on this topic, he has 
> explained protein superposition (problems) very clearly.
> 
> Gert
> 
> Ps, if you want proteins superposed and get different output from what the 
> standard software gives you, just mail me those PDB files and I can see what 
> I can do.
> 
> 
> On 29-10-2016 17:47, WENHE ZHONG wrote:
>> Dear all,
>> 
>> I always use the SUPERPOSE tool in CCP4 to superpose molecules. This time I 
>> want to use the RMSD values of superposed C-alpha atoms to plot a RMSD graph 
>> (instead of using the graph automatically made by the program). However, 
>> there are many atoms missing in the RMSD list.
>> 
>> In the settings I chose “Superpose specific atoms/residues”, checked “Output 
>> all distances to a file”, fit “C-alpha atoms”. The superposed structures 
>> have exactly the same sequence.
>> 
>> My question is: is there any way to get the completed list of RMSD value for 
>> each C-alpha atom? Or is there any other program for this purpose?
>> 
>> Thank you!
>> 
>> Kind regards,
>> Wenhe

Re: [ccp4bb] problem in space group!!

[Anastassis Perrakis]

 On 15 Apr 2015, at 7:55, 高艺娜 gaoy...@cau.edu.cn wrote:
 
 Dear all,
 Recentely I have got a dataset of 3.0A resolution and I have a big problem in 
 choosing space group because  the crystal were found in both P4,P1and P222 
 space groups,here are the figures of self rotation function on P4,P1 and P222 
 performed by MOLREP program for your reference.
 
 The unit cell parameters of P1 space group is a=90,b=90,c=90

I would think these are alpha=beta=gamma=90 … and I suspect (a=b)!=90 and c!=90

Instead of looking self rotation functions, which is always a brave and 
honourable endeavour, I would just run Pointless or phenix.xtriage (on the P1 
processed data) and read carefully the log...

Tassos

 
 Many thanks for any advices..!!!
 
 Best wishes,
 
 ynK
 P222.jpgP4.jpgP1.jpg

Re: [ccp4bb] funding experiences - query

[Anastassis Perrakis]

Dear Bernhard,

I think you need to clarify the first question. “Denied funding’ means to me 
that a committee officially denied the proposal for these reasons, which is 
rare, as typically they are very careful, or at least that is my experience. 
However, its rather common that one of the referees uses any of these as a 
negative point during a review.

Most importantly though, I think that these days almost any grant that is 
purely based on determining structures will be denied by definition. Thus I 
doubt if the question as it is posed now warrants investigation … I think the 
time for ‘crystallographic studies’ has passed and almost all grants that 
include structural work are in the context of a wider scientific framework...

best 

Tassos

 Were you denied funding for a structure study because you did not have yet
 (a) large scale protein expression
 (b) first crystals
 (c) diffraction crystals
 (d) data
 (e) maps
 
 Did you have to 'reverse engineer' an application, i.e. you had the
 structure already (almost) and 
 then write the grant?
 
 Any other peculiarities/comments you received, and what you feel would need 
 improvement/should be addressed.
 
 Again, any comments will be confidential and will contribute to raise
 awareness for the special
 requirements of, and funding for, the significant up-front work necessary
 for crystallographic studies.
 Get it off your soul and make the world a better place.
 
 Thanks and best regards, BR
 -
 Bernhard Rupp
 001 (925) 209-7429
 +43 (676) 571-0536
 b...@ruppweb.org
 hofkristall...@gmail.com
 http://www.ruppweb.org/
 ---
 The road to scientific serfdom is paved with Nature papers
 ---

[ccp4bb] GRC/GRS Diffraction Methods

[Anastassis Perrakis]

Dear all,

I am sorry to send one more post to the already overloaded bb, but please let 
me remind you for the last time that the deadline for applications for the GRC 
and the GRS is approaching, and is only three days from now.

http://www.grc.org/programs.aspx?id=11654

We are all looking forward to welcome you at Bates College in a month from now, 
and we do hope for a few more last minute participations: we need all of you to 
make it as exciting and fun as possible!

Best regards,

Tassos

[ccp4bb] GRC and GRS on Diffraction Methods in Structural Biology 7/27/2014 - 8/1/2014

[Anastassis Perrakis]

Dear all,

As I did give the full promotional message for why these are just outstanding 
meetings to attend, I will only invite you to
look at the latest programs 

GRC (http://www.grc.org/programs.aspx?id=11654)  
GRS (http://www.grc.org/programs.aspx?year=2014program=grs_diff).

and also remind you that the registration is closing in 15 days (June 29).
There are still spots available for selected abstracts to be presented towards 
the end of the meeting, and participant places
are filling up soon (especially after a wave of 15 new registrations only 
yesterday!)

We hope to see you all at Bates college and promise you two exciting meetings 
and a great time!

On behalf of the GRC and GRS organizers,

Tassos

[ccp4bb] GRC Diffraction 2014 Instruct fellowships

[Anastassis Perrakis]

Dear all,

Unfortunately something went wrong in email archiving, and I recall receiving 
more applications from specific people, than what are now in my mailbox. May I 
kindly ask the people that did send an application, to re-send me their PDF 
file by reply to this email?

Sorry again to everybody for the double posting and the rather silly error on 
my side -

Tassos

[ccp4bb] GRC on Diffraction Methods: end July 2014

[Anastassis Perrakis]

Dear all,

We are getting closer and closer to the 2014 edition of my favorite Gordon 
Research Conference 
(https://www.grc.org/programs.aspx?year=2014program=diffrac ) on Diffraction 
Methods in Structural Biology Faster, Smaller, Better: Novel Technologies for 
Diffraction Experiments in Molecular Biology and Drug Discovery , which this 
year will also feature before it starts the Gordon Research Seminars Towards 
Integrative Structural Biology 
(https://www.grc.org/programs.aspx?year=2014program=grs_diff), targeting young 
scientists!

Thanks to INSTRUCT (http://www.structuralbiology.eu) there will be four 
fellowships for young European researchers coming originally from an INSTRUCT 
member country or working in an INSTRUCT member country 
(http://www.structuralbiology.eu/resources/countries). Each fellowship will 
amount to 750 Euro, covering registration for both the GRC and the GRS, and all 
local expenses. It is a condition that you have to attend both the GRS and the 
GRC and submit a poster for both events.

If you want to apply for such a fellowship, please send me an email reply 
(maintain the subject line please), including a single-page PDF file, a very 
short CV, an equally short abstract of your research, and a motivation 
statement explaining why you think this event is crucial for your career. 
Please include the email of your direct supervisor in the end of this letter, 
and a statement that she/he has agreed to cover the travel expenses for you to 
attend, if your fellowship application comes through. 

The deadline for receiving these letters is Wednesday 16 April at 12:CET (yes, 
thats less than a week from now! get writing!).
The letters will be reviewed by an INSTRUCT panel, and the chosen four 
candidates will get a notification by the 8th of May!

And, of course, I do take this opportunity to remind to everyone that the 
(almost) final program is on-line, we have an absolutely amazing group of 
speakers lined up, and the available places are filling up fast! So please 
remember to register on time!

Looking forward to see you all at Bates College for what I trust you can make 
an exciting and memorable meeting!

Best regards,

Tassos

PS If the file is not PDF, if its more than one page, if the characters you use 
are too small to read, etc etc, don't be too surprised if you do not get the 
travel fellowship award!

Department of Biochemistry (B8)
Netherlands Cancer Institute, 
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] Can not see density map when I turn off normalization in PYMOL

[Anastassis Perrakis]

Dear Hongshi -

I cant help but to first suggest that as this is the CCP4bb, the first thing 
you should try is using CCP4mg, if you want to make pretty pictures.
If you just want to look at the map and work with it, you should use Coot.

If still for some reason you want to use Pymol, you should ask the experts of 
Pymol, 
and its best to mail pymol-us...@lists.sourceforge.net

These said, you do not specify what you really want to do. Is the ligand 
already there in the coordinates file?
Then an fo-fc (difference) map should show you nothing, as the ligand is there 
and there should be no difference.
If the ligands is not there in the coordinates file, and you see no density, 
then the ligand is simply not there.

Hope these help,

Tassos


On 19 Feb 2014, at 18:30, hongshi WANG wrote:

 Hello there,
 
  
 I am making a fo-fc map for one ligand using pymol. I strictly followed the 
 pymol wiki protocol (Display CCP4 Maps). Finally, I can get the ligand map 
 using command:
 
  isomesh fo-fc_ligand, omitmap, 3, ligand, carve=2.
 
 However, the problem is the map I got from pymol is smaller than the one I 
 can see in coot at the same contour level (3.0). 
 
 So I gave a second trial based on the assumption that it may be caused by the 
 mis-normalization.  I input the command: “unset normalize_ccp4_maps” to stop 
 PyMOL from normalizing a cpp4 map. After that I loaded my ccp4 map file and 
 tried to do the same things as what I did for the first time. But I could not 
 see any mesh net (density map) shown up. I check the command window.
 
 PyMOLunset normalize_ccp4_maps
 
  Setting: normalize_ccp4_maps set to off.
 
  ObjectMapCCP4: Map Size 134 x 128 x 122
 
  ObjectMapCCP4: Map will not be normalized.
 
  ObjectMapCCP4: Current mean = -0.66 and stdev = 0.074981.
 
  ObjectMap: Map read.  Range: -0.511 to 0.616
 
  Crystal: Unit Cell  200  300  100
 
  Crystal: Alpha Beta Gamma90.000  100.354   90.000
 
  Crystal: RealToFrac Matrix
 
  Crystal:0.0060   -0.0.0011
 
  Crystal:0.0.0045   -0.
 
  Crystal:0.0.0.0053
 
  Crystal: FracToReal Matrix
 
  Crystal:  2000.  -34.5817
 
  Crystal:0.  3000.
 
  Crystal:0.0.  100
 
  Crystal: Unit Cell Volume  6993536.
 
  ExecutiveLoad: E:/ bdligand002.ccp4 loaded as bdligand002, through state 
 1.
 
  PyMOLisomesh fo-fc_ligand, bdligand002, 3, ligand, carve=2
 
  Executive: object fo-fc_ligand created.
 
  Isomesh: created fo-fc_ligand, setting level to 2
 
  ObjectMesh: updating fo-fc_ligand.
 
  
  It seems like no error, but my ligand map, fo-fc_ligand has no density map 
 shown up. I also tried to show the whole mesh at level 2.0 for bdligand002. I 
 still could not see the density map.
 
  
  My pymol is version 1.3 in windows 8 operation system. 
 
  
  
 Any help will be greatly appreciated!
 
  
 Thanks in advance
 
  
 hongshi 
 

[ccp4bb] Gordon Conference on Diffraction Methods in Structural Biology

[Anastassis Perrakis]

Dear all,

The biannual Gordon Research Conference in Structural Biology, accompanied by 
the first Gordon Research Seminar, will take place in the last week of July at 
Bates College, New England, a few hours drive from the IUCr meeting that 
follows in the first week of August.

The theme for the GRC is Faster, Smaller, Better: Novel Technologies for 
Diffraction Experiments in Molecular Biology and Drug Discovery and of the GRS 
(which precedes the GRC and is targeting young scientists giving them an 
opportunity to present their own work to their peers prior to the meeting) 
Towards Integrative Structural Biology.

We have a truly fantastic line of speakers and discussion leaders, including 
John Kuriyan , Randy Read, Ana Gonzales, Ilme Schlichting , Henry Chapman , 
John Spence, Graeme Winter , Aina Cohen, Thomas Schneider , Gwyndaf Evans , Bob 
Fischetti , Flora Meilleur, Janet Newman , John Hunt , Michael Duszenko , Jose 
Antonio Marquez, Paul Adams , Clemens Vonrhein , Airlie McCoy , Brent Nannenga, 
Zbyszek Dauter , Tom Terwilliger , Garib Murshudov , Gerard Kleywegt , Paul 
Emsley, Dmitri Svergun , Peter Zwart , Michael Hammel , Lois Pollack, Elspeth 
Garman, Lisa Keefe , Gary Gililland , Aydnan Achour and Giovanna Scapin.

For more details visit our web site: 
https://www.grc.org/programs.aspx?year=2014program=diffrac

And here is the largely unavoidable motivational speech for anyone interested 
to my highly biased personal view:

I  first went to this meeting in 1998, as a young post-doc, presenting results 
that later led to the 'ARP/wARP' software: in fact, I had changed my slides 
(for the younger audience: slides were pieces of photographic film that were 
typically projected as mirror images of what you really wanted) a month or so 
before the meeting, as we got our very first models auto-built. I thought it 
was the most educational and exciting meeting I have ever been to, and in many 
ways it has shaped my research plan and my career (and my hate for golf). I 
wish I could also say that I never missed any of the subsequent meetings, but 
courtesy of the US Visa authorities and the Greek Army, I actually did a miss a 
couple. However, I still find them as exciting as so many years ago, but for a 
whole different set of scientific reasons: the diffraction methods landscape is 
changing rapidly, new machines and concepts make possible experiments that we 
do not even know what they are going to be! I am honored to be chairing this 
year's edition, and I hope that I will hear from at least one of you, what I 
had heard from a few before: this is the best meeting I have been in my life. 
Science aside, I am glad there is no golf course nearby the Bates College site 
where we are holding this meeting for the last decade, I am equally happy for 
the Great Outdoors site where we do the mid-week excursion, and I am looking 
forwards to the football (sorry: soccer) and basketball games. The Bates 
College boasts an excellent auditorium for the talks, a truly outstanding 
lounge for the poster sessions (that are always accompanied by drinks, an 
observation that might partially explain the tendency to finish well after 
midnight in a very positive spirit) and a somewhat confusingly and an 
unexpectedly good quality restaurant in the very friendly College site. 

A limited amount of (partial) bursaries to young scientists will be available 
for this year - when this becomes definitive we will post the news. I should 
also mention that one session will host eight seminars that will be selected 
from the poster sessions, giving all particpants the opportunity to present 
their own research!

In the meantime, I am looking forward to welcome you all at the meeting and I 
hope you will register ... today!

Best regards,

Tassos

Anastassis (Tassos, Perrakis, Principal Investigator , Staff Member
Department of Biochemistry (B8,
Netherlands Cancer Institute, 
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile , SMS: +31 6 28 597791

Re: [ccp4bb] Sister CCPs

[Anastassis Perrakis]

It is an interesting observation, Tim.

The fact that people do not read tutorials or manuals, is somewhat related to 
the fact
that many questions asked in the ccp4bb could be answered by a more experienced 
colleague,
a supervisor, a manual, a wiki, St Google the revealer, not to mention the 
somewhat extreme
suggestion of an actual text book.

Tassos

PS Many questions can also be answered by coming to the Gordon Conference for 
Diffraction Methods
in Structural Biology this summer!!! A great investment of your time, suitable 
for
both seasoned crystallographers and wannabe stars of the next edition of Cell 
alike !!!

https://www.grc.org/programs.aspx?year=2014program=diffrac

But more on that next week!!!


On 14 Feb 2014, at 19:25, Tim Gruene wrote:

 Hi Robbie,
 
 I think lack of feedback is a serious problem for wikis. I once started
 an XDS tutorial at Kay's XDS-wiki, stating that it was still under
 development and encouraging readers to send me an email if they want to
 see faster progress in the completion of the tutorial. I never received
 a single email, so I did not see the point of spending more time there
 and the tutorial is still pretty incomplete (although Kay also
 contributed to it from time to time).
 
 It may be hard to believe (why?) for newcomers, but crystallographers
 are actually very open to criticism and discussions (cf. ccp4bb ;-).
 
 Cheers,
 Tim
 
 On 02/14/2014 07:14 PM, Robbie Joosten wrote:
 Limited contributions are a common problem with wikis. This paper
 describes a way to add some incentive to contributing to a wiki:
 http://bioinformatics.oxfordjournals.org/content/29/14/1837.full
 
 Cheers, Robbie
 
 Sent from my Windows Phone  Van: Kay
 Diederichs Verzonden: 14-2-2014 8:21 Aan: CCP4BB@JISCMAIL.AC.UK 
 Onderwerp: Re: [ccp4bb] Sister CCPs
 
 Nat,
 
 that's why I set up the CCP4 wiki at
 http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Main_Page
 ! The idea is that everybody benefits: experienced
 crystallographers/biologists can concentrate on the new and difficult
 questions coming up on the bulletin board, and novices find answers
 to those ever-recurring questions. Everybody can contribute answers,
 or improve existing ones!
 
 But the wiki can only be useful in the long run if there are
 contributors. Why are there (almost) no contributors? It cannot be
 due to technical difficulty; it's very easy to contribute to a wiki.
 One guess is that a posting on a BB is more socially rewarding,
 because the interaction via emails is more immediate.
 
 Re-vitalize the wiki!
 
 Kay
 
 On Thu, 13 Feb 2014 07:21:14 -0800, Nat Echols
 nathaniel.ech...@gmail.com wrote:
 
 One comment (not a complaint) on all this: it seems like the same
 questions get asked over and over again.  If there is a good place
 for a general crystallography FAQ list it is well past time for one
 to be put together - or maybe it just needs to be better
 advertised?  At a minimum, for instance:
 
 - what cryoprotectant should I use? - how do I get big single
 crystals? - how do I improve diffraction? - how can I tell if I've
 solved my structure? - why is my R-free stuck? - is pick random
 statistic suitable for publication?
 
 Some of the other common queries (name my blob!) still need to be
 handled on a case-by-case basis, but it would be much more
 efficient for everyone if the standard answers were collected
 somewhere permanent.
 
 -Nat
 
 
 
 On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov
 eugene.val...@gmail.comwrote:
 
 I absolutely agree with Juergen.
 
 Leaving aside methods developers, who are a completely different
 breed, there is no such thing as a crystallographer sitting in
 a dark room solving structures all day. If there are, these are
 anachronisms destined for evolutionary demise.
 
 More and more cell biologists, immunologists and all other kinds
 of biologists are having a go at doing structural work with their
 molecules of interest themselves without involving the
 professionals. Typically, they learn on the job and they need
 advice with all kinds of things ranging from cloning and protein
 preps through to issues with tetartohedrally-twinned data and
 interpreting their structures.
 
 So, a modern structural biologist is one who is equipped for the
 wet lab and has some idea of how to go about solving structures.
 CCP4BB is a wonderful resource that is great for both the quality
 of the advice offered to those that seek it and for the variety
 of topics that are addressed in the scope of structural biology.
 I have learnt greatly from reading posts from very skilled and
 knowledgeable scientists at this forum and then implemented these
 insights into my own research. I am very grateful for this.
 
 In short, please do not discourage your colleagues, particularly
 very junior ones, from posting to the CCP4BB. Some of the
 questions may appear quaint or irrelevant but it is easy to
 simply ignore topics that are of no 

Re: [ccp4bb] About protein precipitation problem during dialysis

[Anastassis Perrakis]

On 20 Nov 2013, at 21:56, Dhanasekaran Varudharasu wrote:

 Dear Crystallographers,
 
 I dialysed a 30 kDa protein 
 (Recombinant protein which was eluted by 20 mM Tris, 500 mM NaCl, 120 mM 
 imidazole) against water for overnight. But it gets precipitated after 12  
 hours. Can anybody give some suggestion to avoid precipitation.  


... dont dialyse it against water!

A.


 Thanks
 Dhana 
 
 
 
 

Re: [ccp4bb] OT: Who's Afraid of Peer Review?

[Anastassis Perrakis]

I could not resist but comment at the end ... (sorry for cleaning up the thread 
text)

I agree with Roberto that the system is actually not that bad when you think of 
it. Or, it could be much worse.

In my experience, the editors of many journals - professional or academic - try 
very hard, and most referees make an honest effort and are objective.
Of course there are glitches to that system - but, in my experience no matter 
how annoying they are (and oh yes I have been very annoyed), they are 
relatively few.

Should things change in the peer review process? Surely yes. And things are 
changing!!! For example:
- eLife now gets the referees to talk to each other and get a consensus report 
- so one referee cannot say unreasonable things in one review
- EMBO press has many innovations awaiting 
http://www.embo.org/scientific-publications/transparent-process

These said I would had liked to see similar policies implemented to the brand 
new IUCrJ and other IUCr journals, 
and I would have preferred it if the co-editor name in the IUCr journals would 
stay unknown 
(possibly revealed only at publication time, but not before a decision is made).

Best -

Tassos

[ccp4bb] 2014 Gordon Research Conference and Research Seminars on Diffraction Methods in Structural Biology

[Anastassis Perrakis]

Dear all,

In a bit more than a year from now, we will be running the Gordon Research 
Conference on Diffraction Methods in Structural Biology, 
this time preceded for the first time by a two-day Gordon Research Seminar, 
exclusively for young scientists and a few mentors.

http://www.grc.org/programs.aspx?year=2014program=diffrac

http://www.grc.org/programs.aspx?year=2014program=grs_diff

We are in the process of inviting the right people to make the schedule as 
exciting and inviting as possible, hoping we will be
able to attract a diverse, enthusiastic, and motivated crowd!

At this stage, we would like to ask you for ideas for 

1. Specific subjects that are emerging and you would like to see covered, to 
make it worth your time to join us!
2. Specific people that would be excellent speakers, that would make you attend 
the Research Conference!
3. Specific people that would be great mentors, that would make you attend the 
Research Seminars!

Many thanks from the team (but please send all replied to me!)

Tassos Perrakis (GRC chair), Eddie Snell (GRC vice chair), Jeff Headd (GRS 
chair) and Maike Bublitz (GRS assoc. chair)

Re: [ccp4bb] Off-topic: NMR and crystallography

[Anastassis Perrakis]

I would agree with Mark.

It would be also good to state that neither of us is cross-trained, but are 
one-trick dogs  as far as NMR vs X-rays goes.

Still, I think that if you get any protein in good amounts, try to crystallize 
it (there are even good facilities for that these days,
and funding to set up crystallization through Biostruct-X), and if that fails, 
consider NMR. Many good NMR labs try to crystallize
their targets first - if it crystallizes its cheaper, easier and the structure 
is of higher resolution.

Disclaimer: I am not saying NMR is useless - quite the contrary. But, I cant 
see why you would do an NMR structure of something
that crystallizes in a straightforward cheap crystallization screen. I see why 
you might do NMR to answer many different questions.

I will leave the membrane proteins question to experts - but these days we all 
see many membrane proteins coming from X-rays and as
far as NMR goes you will be on the solid state regime and not solution as far 
as I understand: great promise, but again I suspect the
impact to be different than 'structure solution'.

A.

On 9 Jun 2013, at 19:33, Mark van Raaij wrote:

 Well, if you do NMR you avoid the possible bottlenecks of having to obtain 
 well-diffracting crystals, and having to phase the protein (i.e. obtain SeMet 
 protein crystals or suitable heavy atom derivatives; or a suitable MR model).
 But instead, you'll need to prepare labelled protein (15N and/or 13C), which 
 is expensive and for which your protein needs to be able to be expressed in 
 minimal medium, and your protein will need to be very soluble, monodisperse 
 (in general monomeric) and stable in a minimal NMR-compatible buffer for data 
 collections lasting for hours. Assigning all the protons and calculating the 
 final structure can also be months of work, while a high-resolution crystal 
 structure can be finished in days, if the above-mentioned bottle-necks can be 
 overcome.
 
 
 On 9 Jun 2013, at 17:36, Theresa Hsu wrote:
 
 Dear all
 
 A question for the cross-trained members of this forum - for small sized 
 proteins, is NMR better than crystallography in terms of data collection 
 (having crystals in the first place) and data processing? How about membrane 
 proteins?
 
 I would appreciate replies to the board, instead of off-board, to allow for 
 a good discussion.
 
 Thank you.
 
 Theresa

Re: [ccp4bb] delete subject

[Anastassis Perrakis]

Dear all,

Let me start by apologizing for finally making this email longer than I 
intended - I did not have the time to make it shorter.

I must say I am humbled by the amount of positive energy and constructive 
thinking that Bill has. That must explain also how he manages to keep up a 
fantastic resource for the community, what is a largely thankless task with 
little academic reward, but still so helpful. His response did get me thinking, 
as his opinions often do. In principle, in an era of information sharing, why 
don't we indeed solve structures with the collective brain of world 
crystallographers? We could share data, and educate people, and also get better 
structures at the end of the day. As indeed many people are in a small 
institution somewhat off the beaten path - I work in a cancer research 
institute after all and I am the weirdo here - and as indeed I see my knowledge 
getting obsoleted in a steady pace, this idea sounds great!

However, its a bit like 'true' socialism - a grant idea, that we may find it 
will not work too well in practice, at least not under the constraints of human 
nature.

I will keep advocating the greatness of ccp4bb. Its a fantastic resource, which 
is made truly amazing by the many questions that are being asked, and keep 
educating everybody.
But, at the same time, I will keep advocating the usefulness of the typical 
constructions we have in science, whereupon people work in teams. These teams 
are there
to share experience and help each other with overlapping expertise. Why such a 
basic question (and others in the past) need to come out of the 'team'? 
Is there no competence within the team to address it, or is there no correct 
communication? 
In either case, should we as scientists encourage such teams with low-level 
competence? May I remind you that Tom is in Lueven/Belgium,
a large and outstanding University, with at least two very competent (and 
friendly) crystallographers in campus. Does the fact he has to post the ccp4bb 
with a basic question
testify for a complete failure of his supervisor to either help him or get 
other people on site to help him? Should such supervisors be left to guide 
students?

I have nothing against sharing data. I am the fool that submits data at the 
same time as I submit my paper, a practice that is followed by surprisingly a 
few people,
as most people wait a few weeks until the paper is accepted to submit their 
structure (some data mining shows that 1/3 of the PDB entries associated with 
papers
even in journals like Acta D are only deposited to the PDB at least a week 
after the paper submission date!.. no think what this % is in other journals). 
And the mild consequence of this is that somebody picks the structure up, 
panics to be scooped, submits his/her story, and scoops you  while your paper 
is being rejected 
for reasons that are not connected to the structure  (I am not implying foul 
play here, but suggesting a consequence of basic openness).

Are we finally, at the end, with this open and sharing spirit, encouraging 
people to think that crystallography is too trivial? It has once been said in 
this bb, that
'solving a structure is trivial in the same way that climbing mountain Everest 
is trivial: it has been down before, its being done now, and it will be done 
again,
by many well-trained and determined people'. Many people have read and trained 
for this task. If you do not read a couple of books and train before attempting 
the climb, 
and you send an email asking the everestbb 'does anybody know how to open this 
oxygen valve?' you are asking for trouble though 
… and the people that let you attempt the climb without that knowledge, are 
also in the wrong.

The end result of this open and sharing spirit, which downgrades the importance 
of competence in major methodologies like X-ray crystallography, 
was summarized recently is some text I recently got by email from Brussels:
 ... advanced methods for X-ray crystallography and Electron Microscopy is a 
very narrow field that will limit the employability of the graduates
There are the wise words of the referees of a joined grant (with 10 other 
people from Europe) advocating to educate students to get an in-depth PhD-level 
training in crystallography and in EM. 

Maybe that explains my grumpiness. 
Or, as the crystallographer previously known as DVD mailed me in private 
welcome to the club of grumpy old men. 
Maybe I am just being grumpy. 
Or I am justifiably worried.

A.

PS For the record I admire Tom's spirit and courage - he is the kind of guy I 
would hire for a PhD (not that he will ever want to work with me any more). 
I am less impressed by the team and his supervisors, as it stands, and without 
knowing all the details of what might be behind this.

On Mar 28, 2013, at 1:04, William G. Scott wrote:

 Dear Tom et al:
 
 Although arriving too late to participate in the snark-fest, it occurred to 
 me that maybe this is almost exactly how we 

Re: [ccp4bb] process data from two crystals in-house data

[Anastassis Perrakis]

Hi -

You can use Xia2

or, you can use Pointless (Find or Match Laue group) to combine the two 
datasets after Mosflm in one  MTZ with common correct origin etc, 
and then use SCALA to scale them together.

For more info: 
http://ccp4wiki.org/~ccp4wiki/wiki/index.php?title=Data_processing_with_CCP4

Its btw the same procedures (albeit for a different problem) as suggested a 
week ago under the subject Scaling with SCALA high and low resolution data 
sets

A.


On Mar 28, 2013, at 11:08, S. Thiyagarajan wrote:

 Dear all
 I have two data sets, 75 frames each from crystals of the same protein - same 
 cell parameters/space group (P4).
 I could process them seperately each yielding  90% completeness but with  
 poor multiplicity (  2 )
 
 If I merge the data sets using CAD, I loose the data reduction statistics of 
 the combined data set.
 
 I do not have HKL2000. 
 
 Which (free) tool can handle two different diffraction data sets, process 
 them together to give a single final data statistics.
  
 Thanks and regards
 Thiyaga
 
 
 
 S. Thiyagarajan
 Centre of Excellence in Bioinformatics
 School of Biotechnology
 Madurai Kamaraj University
 Madurai - 625021
 Ph: +91-9159224881 (cell)

Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute, 
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] Isothermal titration calorimetry

[Anastassis Perrakis]

It might be worth to consider the question more in detail.

Do you want to study thermodynamics of the interaction, or a KD would do? If 
the former, you need ITC. If the latter, and you want to study things at the 
level of KD only, maybe investing on a plate reader, thermophoresis, or some 
biosensor technology (spr or interferometry based systems) should be 
considered. 

Then, what interactions will you study with the ITC? In general, I would agree 
that the lower sample volume is worth the nano options, but depending on the 
typical systems under study, sometimes the gain on sample quantity is not worth 
the money - while many times its worth it. 

John is if course right that for studying specific systems as the one he 
describes the 200 is great. 

A. 

Sent from my iPhone

On 23 Mar 2013, at 11:00, John Fisher johncfishe...@gmail.com wrote:

 I would recommend the Microcal ITC 200, hands down. Not only is it an amazing 
 instrument with the optional automated sample loader (which is worth every 
 penny), but we were able to do experiments (multiple) using FULL-LENGTH p53 
 binding to a weak cognate protein. I believe this was the first time ITC was 
 ever used with full length p53, as it is so labile and just loves immediately 
 to oligomerize. Sample sizes pay for the instrument.
 Best,
 John
 
 John Fisher, M.D./PhD
 St. Jude Children's Research Hospital
 Department of Oncology
 Department of Structural Biology
 W: 901-595-6193
 C: 901-409-5699
 
 On Mar 23, 2013, at 4:45 AM, Sameh Soror shamd...@googlemail.com wrote:
 
 Dear All,
 
 
 I am sorry for the off topic question. I am going to buy ITC to study 
 protein-protein  protein-ligand interactions
 
 I am comparing microcal, GE and nanoITC, TA instrument..
 any suggestions, recommendations, good experiences or bad experiences. is 
 there a better system.
 
 
 Thank in advance for the help.
 
 
 Regards
 
 
 Sameh
 
 -- 
 Sameh Soror
 
 Postdoc. fellow
 
 

Re: [ccp4bb] first use of synchrotron radiation in PX

[Anastassis Perrakis]

And indeed this experiment was done properly ... in a suit and tie!

http://www.embl-hamburg.de/aboutus/general_information/HH_about/history/HH-holmes.jpg

A.

PS The journal is indeed a bit obscure ... 


On 13 Mar 2013, at 20:22, DUMAS Philippe (UDS) wrote:

 Jean Witz  (now deceased) once told me that the following paper is the first 
 one mentionning data collection on a synchrotron.
 The journal is not really obscure and the paper should easily be found.
 The work was done in Germany, if I remember well.
 
 G. Rosenbaum, K.C. Holmes and J. Witz, Synchrotron radiation as a source for 
 X-ray diffraction, Nature, 230, 434-437 (1971). 
 
 Philippe Dumas

Re: [ccp4bb] how to make protein topology figure based on its structure

[Anastassis Perrakis]

My point was that the second hit in the google list, was the CCP4's own program 
to make topology diagrams.

There was no issue of installing anything - it was an issue of looking if ccp4 
has a program that makes
topology diagrams. If you don't like that program, you could  ask is there 
another program other than ccp4's TopDraw
to make topological diagrams?. Asking the ccp4bb without looking at the ccp4 
list of software
for what you are looking for, is - in my view - sloppy and shows lack of 
effort. Possibly I am too demanding.

I should add, that if you type to google something like topology diagram 
protein structure you get lots
of useful hits, including PDBSUM and Pro-Origami, in the first page.

I do not want to discourage people asking question in the bb, but at the same 
time I think that
some problem solving hierarchy of the order 'google it' - 'ask your lab-mates' 
- 'ask your boss' -
'mail 5,000 people' can save everybody lots of time, can help people to grow, 
and might even make
the bb more fun.

My two cents - and sorry for the long and likely unnecessary and redundant 
email.

A.


 On Sat, Mar 9, 2013 at 1:02 PM, Edward A. Berry ber...@upstate.edu wrote:
 Still, I would second Lisa's question. Your google search gives a lot of 
 false hits, and one doesn't want to download and install a dozen programs 
 (with license forms to fill out in some cases) to see which one works, when 
 Partha can tell us that Pro-origami will do it and provide a link to the 
 description. Seems to be just what I've been looking for. Thanks, Partha!
 
 Anastassis Perrakis wrote:
 
 On 9 Mar 2013, at 12:11, Jon Agirre wrote:
 
 Espript will do. http://espript.ibcp.fr
 
 
 
 Well, if you want a topology diagram, espipt will not do. Brilliant as it may 
 be, it will annotate the topology linearly
 on top of a set of aligned sequences (and more!).
 
 As for a topology diagram, I could not resist pasting your question to google 
 - its a good thing to do before pasting
 the question
 in your email client - it can give you a strange sense of satisfaction and us 
 (or at least me) less Saturday morning fun.
 
 http://lmgtfy.com/?q=%5Bccp4%5D+how+to+make+protein+topology+figure+based+on+its+structure
 
 A.
 
 
 Good luck,
 
 Jon
 
 On 9 Mar 2013 11:52, LISA science...@gmail.com 
 mailto:science...@gmail.com wrote:
 
 Hi all,
 I want to make a topological figure of my protein with a b-sheet and 
 helix based on its crystal structure. Please
 recommend some online sever of software?
 Thank you.
 
 lisa
 
 
 
 

Re: [ccp4bb] how to make protein topology figure based on its structure

[Anastassis Perrakis]

On 9 Mar 2013, at 12:11, Jon Agirre wrote:

 Espript will do. http://espript.ibcp.fr
 
 

Well, if you want a topology diagram, espipt will not do. Brilliant as it may 
be, it will annotate the topology linearly on top of a set of aligned sequences 
(and more!).

As for a topology diagram, I could not resist pasting your question to google - 
its a good thing to do before pasting the question
in your email client - it can give you a strange sense of satisfaction and us 
(or at least me) less Saturday morning fun.

http://lmgtfy.com/?q=%5Bccp4%5D+how+to+make+protein+topology+figure+based+on+its+structure

A.


 Good luck,
 
 Jon
 
 On 9 Mar 2013 11:52, LISA science...@gmail.com wrote:
 Hi all,
 I want to make a topological figure of my protein  with a b-sheet and helix 
 based on its crystal structure. Please recommend some online sever of 
 software? 
 Thank you.
 
 lisa

Re: [ccp4bb] laboratory phage infection [SEC=UNCLASSIFIED]

[Anastassis Perrakis]

We have suffered from that a lot at the NKI a few years back.

What worked for us was a combination of strategies, that were all mentioned 
before but I will repeat:

1. Buy T1 phage-resistant strains, and also use an old incubator (and luckily 
an old building
that was available at the time) to 'evolve' more strains that would be 
resistant.
2. Stop storing bacteria and only store plasmids. Work with bacteria strictly 
within our 'VMT' lab.
3. Perform 'phage' tests (PCR based) in a few (20) places in the lab that are 
labeled on a monthly basis.
Any positive result is red-alter and means to stop working and start cleaning.
4. Buy our 'phage buster', a machine that can bleach a closed room with acid, 
as part of the cleaning procedure
(ideas to lock in the same room as being bleached team members that work 
'dirty' in the lab have been
voiced - but not (yet) implemented)

It has costed us months of trouble, and its a serious matter. I would recommend 
anyone to even close
the lab down for a couple of weeks to clean, destroy all glycerol stocks, clean 
fridges, etc,
rather than try to handle it while 'normal operations' are in place. We have 
tried that and only
when we got really serious and changed the way we work, we are now phage-free 
for a few years
in a row and have minor trouble.

Tassos

On 3 Mar 2013, at 10:09, aidong wrote:

 Dear Opher,
 
 It was also our idea how to get over it since several years ago.  We  
 did it based on a philosophy that any bacteria infected with a phage  
 will not be infected by the same phage again. Therefore, we let our  
 BL21/DE3 strain infected in the lab and when most were died, some  
 cells were survived.  We picked these to make the competent cells for  
 expression constructs.  We believe these actually contained phages.   
 In order to avoid this bomb to blow up, we managed to get a commercial  
 strain (TonA and TonB deletion) purchased from Sigma USA but found it  
 useless.  I wonder how you screened your phage-resistant strains.
 
 In addition, it is our regular practice to use bleach to extensively  
 disinfect all surfaces, including benches, used flasks, instruments  
 and floors. Would virkon work more efficiently in our case?
 
 Sincerely,
 
 Aidong
 
 
 On Mar 3, 2013, at 3:36 AM, Opher Gileadi wrote:
 
 Hi,
 
 We had a massive phage infection at the SGC in 2004; all our washing  
 and sterilization efforts could only solve the problem temporarily. I  
 then recovered a phage-resistant subclone of BL21(DE3), and prepared  
 derivative strains with pRARE2 (the tRNA plasmid in Rosetta2) or other  
 plasmids. We have expressed thousands of protein since and never had a  
 recurrence of phage problems. Although the resistance is to T1 phages,  
 it seems these are the most common lab infections.
 
 Write me if you want the strains.
 
 Opher
 
 Aidong Han, Ph.D
 
 Department of Biomedical Sciences
 School of Life Sciences
 Xiamen University
 3 South Xiangan Road
 Xiangan, Xiamen 361102
 China
 Phone: 0592-218-8172 (O)
   0592-218-8173 (L)
 Web: http://life.xmu.edu.cn/adhanlab/

Re: [ccp4bb] Mac mini advice

[Anastassis Perrakis]

I am of the opinion that the truth lies somewhere in between ...

Here are my two cents based on personal experience ...

For example, I am happy myself using a MacBook Pro, which is sufficient for all 
my activities, and has all software and data that I need.
Thus, I am myself on the 'new' paradigm side, having a machine with mainframe 
levels of storage and computing power (I do not run git,
but time machine in a mac has the bits I need from the git idea - as far as I 
know git that is).

In the department, we have about 20-25 scientists. These people need to 
''maintain and be proficient in many software suites, many more than
a traditional crystallographer (like me in my PhD time for example) would need:
vector design software for cloning, databases for keeping track of clones, 
sequencing viewing software for their clones, 
 interfaces for crystallisation and biophysical equipment, analysis suites like 
Graphpad/Prism, Origin, Kintek (etc etc)  for biophysical experiments ... 
 and lets not forget SAXS software ... Our experience, is that most of 
these people like to use a Windows workstation for these (the choice is free),
others prefer a Mac, thats not my point here. Many of that software also needs 
to maintained by these people...
Also, for a variety of reasons which have to do with IT support restrictions, 
the Windows machines
we have for them are miss-configured with ancient versions of the windows 
operating system, but still Ok for many things, but not for really 
straightforward use of CCP4/Phenix ...

My point here is, that these people are less likely to be keen of the idea to 
also install and run ccp4/coot/phenix/buster in their machines 
(they use pymol/yassara/chimera though locally since they can copy/paste to 
their presentations and papers then). 
So, we find it useful to keep an old fashioned setup running in parallel. Linux 
boxes, hooked to Zalmans or really
big or double LCDs, in a specific room ... People like these for data 
processing, all data of many years back are online, incremental backup is 
running etc.
For historical reasons we even run NFS/NIS there (I agree its not a great 
choice if one would start now).

My conclusion and advice for labs or departments that have more than 5-6 
people, and are doing crystallography but not as their
full-time business is that besides personal PC/Mac, a common room with a few 
relatively powerful machines with nice, big, double,
screens, likely also Stereo, is useful for a few reasons:

1. Easier to make sure everybody is using the same software more or less
2. Same machine to everybody - not the situation that a new student gets a new 
machine at year 0, which is redundant by graduation time at +3 years (...or 
+5,6,7...)
3. Mixing of people in the room and ability for people to look over the 
shoulder of others, the point that my colleague Titia Sixma always favours,
which has indeed proved great for teaching others and learning from others.
4. Centralised real backup, availability of diffraction data on-line with 
less mounts...

For these machines, centralised user account information and 'home' sharing is 
in my view essential, as it allows to blindly choose any of the
machines that is available at a time ... and, being a Mac fun, I think Linmux 
is better suited for that purpose, financially and practically...

These said, it reminds me that we need to update the OS, buy a few new 
machines, new LCDs ... argh.

Sorry of this lecture was outside the scope of the original thread.

Tassos



On 23 Jan 2013, at 9:54, James Stroud wrote:

 On Jan 22, 2013, at 11:20 PM, Nat Echols wrote:
 The real difficulty is integrating Macs into a
 Linux-centric environment, for example configuring NFS, NIS, etc.
 
 That's because NFS and NIS are antiquities left over from the days of 
 mainframes. Distributed file systems and user information databases are 
 designed for an environment of many workers and few machines, when the 
 typical graphics workstation cost $50,000. These days, we argue whether to 
 spend an extra $200 on a $500 computer. We have moved to a new paradigm: many 
 workers with many more machines, with each machine having 
 essentiallymainframe levels of storage and computing power. In other words, 
 instead of NFS, you should run git.
 
 James

Re: [ccp4bb] protein solubility predictions

[Anastassis Perrakis]

Indeed there are many web tools predicting solubility.

My personal bias is that your brain is the best tool for ideas on how to design 
expression constructs.
(since one question in a multi-domain protein, is which bit is interesting ...!)
Many brains are better than one (talk to your colleagues - even to your 
supervisor ! - for ideas).

A tool we like (since we developed it ...) helps to combine information from 
many other tools and help us make decisions
(and order pcr primers to implement our decisions) is available at:

http://xtal.nki.nl/ccd

Another advice is whatever constructs you try, try them in many vectors - I 
could give another self-plugin here
but I will not. There are many good systems allowing to put the same PCR 
product to many vectors for expression.

A.



 From: Careina Edgooms careinaedgo...@yahoo.com
 Reply-To: Careina Edgooms careinaedgo...@yahoo.com
 Date: Tue, 22 Jan 2013 04:22:44 -0800
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] protein solubility predictions
 
 Dear ccp4
 
 Apologies for the off topic question. I was wondering whether anyone could 
 suggest a good tool or methodology to use to predict protein solubility and 
 ability to fold from the sequence? I am working with a large protein of 
 multiple domains. I would like to work with as close to the full length 
 protein as possible without affecting its solubility and ability to fold 
 correctly. I know there are web based tools where you can upload a sequence 
 and see the predicted solubility but I wonder if there is any good strategy 
 to use to determine how best to construct the truncated protein? ie which 
 parts of the sequence to keep and which to remove so as to maximise 
 solubility. Also I have an eye to crystallising this protein in the future 
 and I wonder if there are any specific things I should look out for with that 
 in mind? I'm sure minimising flexible loops is one such thing.
 All help appreciated
 Best
 Careina

Re: [ccp4bb] Mac mini advice

[Anastassis Perrakis]

We did work with a full blown OSX Server in 2004 - indeed many issues on NFS 
were Ok, but NIS was a problem - or we could not figure it out.
We used it as server for developers, running X-grid, SVN, WebObjects servers 
for a couple of EC networks, but never deployed it fully
as a departmental-level server, due to the NIS issues. For a while it also 
hosted the protein-ccd server. 
We wanted to move to Kerberos if I recall correctly, but since my colleague 
Serge Cohen moved out and our IT are mac-o-phobic I opted to continue with 
Linux.

The Server was decommissioned a few weeks ago, and will likely get a second 
life in Soleil/Ipanema, 
but I think as an electric heat generator - almost as good on it as my old 
8-proc Alpha (2000) ;-)

A.

On 23 Jan 2013, at 15:05, Bosch, Juergen wrote:

 I assume nobody of you is running an actual Osx server ? I mean the upgrade 
 to a full server version of the commonly distributed normal Osx releases ?
 
 I have not done it yet but I do think many of the issues mentioned regarding 
 NFS/NIS could be addressed there. Regarding the missing macpro upgrades I 
 expect to see new machines with thunderbolt connectivity in the next 4 
 months. And I will buy my third macpro then to run it as a true server.
 
 Jürgen 
 
 Sent from my iPad
 
 On Jan 23, 2013, at 5:21, Peter Keller pkel...@globalphasing.com wrote:
 
 On Wed, 2013-01-23 at 01:54 -0700, James Stroud wrote:
 On Jan 22, 2013, at 11:20 PM, Nat Echols wrote:
 The real difficulty is integrating Macs into a
 Linux-centric environment, for example configuring NFS, NIS, etc.
 
 That's because NFS and NIS are antiquities left over from the days of
 mainframes. Distributed file systems and user information databases
 are designed for an environment of many workers and few machines, when
 the typical graphics workstation cost $50,000. These days, we argue
 whether to spend an extra $200 on a $500 computer. We have moved to a
 new paradigm: many workers with many more machines, with each machine
 having essentially mainframe levels of storage and computing power.
 
 Technically there is something in what you say as a pattern for
 day-to-day work (for some people, although not all), but I think that
 describing the debate in terms of modern vs. antiquated is missing the
 point completely. The real difference between local vs. centralised
 storage is to do with responsibility for the hardware and the data that
 it contains.
 
 Local workstation storage is OK for the following kinds of cases:
 
 (i) the data that are stored locally have no value, so it doesn't matter
 if they are lost (either through hardware failure, misbehaving software
 or accidental deletion).
 
 (ii) the user has the expertise and the time to set up and maintain a
 strategy for recovering data that are lost from local disks
 
 (iii) the institution that the user works for allows the user to include
 data on local workstation disks in the institution's regular backup
 operations
 
 When none of these apply, there is a real, contemporary case for using
 something like NFS, where the storage is centrally maintained and backed
 up. The cost of storage has fallen of course, but what that means is
 that the real questions now are about the value of the data. In some
 fields, you could store your entire career's data on a few USB memory
 sticks, but I doubt that many people would want to do that without
 having made other copies somewhere else, and the same applies to local
 workstation storage too :-).
 
 There are other considerations in favour of connecting a workstation to
 networked services: if you use more than one machine it can be an
 incredible pain to be constantly moving data around from one to the
 other, and to keep track of what the authoritative versions are. Having
 independent, local user id's and passwords on every workstation can also
 cause difficulties. I could go on
 
 In other words, instead of NFS, you should run git.
 
 This is simply not an option for many crystallographers, who do not have
 a background in software development or data management. Advocating and
 supporting git (or indeed any content/version management system) for
 those kind of users is a losing battle: they see it as an unnecessary
 complication to their daily work, and will avoid using it as far as they
 can.
 
 Regards,
 Peter.
 
 -- 
 Peter Keller Tel.: +44 (0)1223 353033
 Global Phasing Ltd., Fax.: +44 (0)1223 366889
 Sheraton House,
 Castle Park,
 Cambridge CB3 0AX
 United Kingdom

[ccp4bb] Post-doc positions @Amsterdam

[Anastassis Perrakis]

Post-doctoral positions are available in the Netherlands Cancer Institute in 
the groups of Anastassis (Tassos) Perrakis and Titia Sixma (http://xtal.nki.nl).
 
The Netherlands Cancer Institute (http://www.nki.nl) is a center of excellence 
with a high standard of biological research and an interactive international 
atmosphere. It is located in Amsterdam, with all its cultural amenities, close 
to the Schiphol airport.
 
The groups are closely associated and have an interest in structural studies 
coupled to functional analysis, combined with activities in method development 
for structural biology. Our department also hosts the Protein Facility with 
equipment that includes high throughput crystallization and crystal 
visualization robotics, an X-ray facility, Biacore surface plasmon resonance, 
isothermal titration calorimetry (ITC), static light scattering (MALLS), 
versatile systems for fluorescence based equilibrium and transient state 
kinetics analysis (a versatile plate reader and stopped-flow),  an Orbitrap 
mass spec, and operational facilities for E.coli, insect cells and mammalian 
cells based protein expression. 
 
The project in the laboratory of Titia Sixma is focused at the interface of  
ubiquitin (de)conjugation and DNA repair. The projects in Tassos Perrakis group 
are about Autotaxin and/or proteins that regulate mitotic progression.
 
We are looking for enthusiastic researchers with experience in molecular 
biology and/or biochemistry and/or protein crystallography. Applicants should 
write an e-mail with CV and names of three references to t.si...@nki.nl or 
a.perra...@nki.nl
 

Tassos  Titia
 

Re: [ccp4bb] a challenge

[Anastassis Perrakis]

 I think the real challenge (and one that makes for an excellent 
 macromolecular crystallographer) is how well one can interpret a map with 
 poor phases. 

Let me disagree ... An excellent macromolecular crystallographer, is one that 
given some crystals can derive the best strategy to collect data,
process the data optimally, derive phases using all available information, 
build a model and refine it in such a way that it best explains both data
and geometrical expectations, and do these as efficiently as possible.

Efficiency may suggest using one automated suite or another - or indeed may 
best be achieved by manual labor - be it in the map or in data
collection strategy or refinement or another step: and here I am ignoring the 
art of transforming hair-needle-crystalline-like-dingbits to a diffracting 
crystal.

One that can interpret a map with poor phases can be either a genius in 3d 
orientation - or a not necessarily too intelligent nor experienced but 
determined student 
that can drink and breathe this map for a few weeks in a row until a solution 
is in place. Neither would make an excellent macromolecular crystallographer by 
necessity.

Tassos

Re: [ccp4bb] vitrification vs freezing

[Anastassis Perrakis]

talking semantics, kruos (Κρυος), means just cold, not icy cold. 
Cold in Greece is not nearly icy. Unlike the Netherlands ... it only gets cold 
when its really icy ;-)


Tassos


On 15 Nov 2012, at 19:45, Ethan Merritt wrote:

 From Greek kruos, icy cold

Re: [ccp4bb] PNAS on fraud

[Anastassis Perrakis]

Just to add in the controversy, with a somewhat related issue:

Current crystallographic ethic presumes that a structure is deposited just 
before
the submission of the paper. In a survey we did, we found that while
in one journal only 2% of structures are deposited after the paper submission 
date,
on another thats 5%, on another one that is 29% and in yet another one close to 
50%.

The journals are Nature, Science, ActaD and Proteins in order of decreasing IF.
Is there any correlation?

To get some guesses first, Robbie can send the answer tomorrow at around noon 
(as I will be unavailable travelling ...)

Tassos

On 18 Oct 2012, at 21:13, Bernhard Rupp (Hofkristallrat a.D.) wrote:

 Randy Read just pointed out to me that in their case-controlled analysis
 paper
 http://journals.iucr.org/d/issues/2009/02/00/ba5130/index.html
 
 when considering lower resolution and other factors, the vanity journals
 seem to come out 
 no worse than the rest. 
 
 In any case I suspect any retractions are underrepresented in those journals
 because they fight it harder ;-)
 
 Best, BR
 
 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ethan
 Merritt
 Sent: Thursday, October 18, 2012 11:11 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] PNAS on fraud
 
 
 Fang et al. claim that R^2 = 0.0866, which means that CC = 0.29.
 While a correlation coefficient of less than 0.3 is not a complete lack of
 correlation, it's still rather weak.
 
 The highly significant must be taken in a purely statistical sense.
 That is, it doesn't mean the measures are highly correlated, it means the
 evidence for non-zero correlation is very strong.
 
   Ethan

Re: [ccp4bb] PNAS on fraud

[Anastassis Perrakis]

On 18 Oct 2012, at 21:30, Bosch, Juergen wrote:

 Tassos,
 
 just to clarify what you are saying in the Journal with 2% deposition after 
 submission, 98% have been deposited prior to submission (the way it should 
 be). Is that what you are saying or am I reading that wrong ?

Yes, that is what I am saying! 2% is good, 50% is bad.

(btw, the 'worse' is close to 70% - any guesses?)

A.


 Or are you saying only 2% of structures are deposited in that journal ?
 
 Jürgen
 
 On Oct 18, 2012, at 3:24 PM, Anastassis Perrakis wrote:
 
 Just to add in the controversy, with a somewhat related issue:
 
 Current crystallographic ethic presumes that a structure is deposited just 
 before
 the submission of the paper. In a survey we did, we found that while
 in one journal only 2% of structures are deposited after the paper 
 submission date,
 on another thats 5%, on another one that is 29% and in yet another one close 
 to 50%.
 
 The journals are Nature, Science, ActaD and Proteins in order of decreasing 
 IF.
 Is there any correlation?
 
 To get some guesses first, Robbie can send the answer tomorrow at around 
 noon 
 (as I will be unavailable travelling ...)
 
 Tassos
 
 On 18 Oct 2012, at 21:13, Bernhard Rupp (Hofkristallrat a.D.) wrote:
 
 Randy Read just pointed out to me that in their case-controlled analysis
 paper
 http://journals.iucr.org/d/issues/2009/02/00/ba5130/index.html
 
 when considering lower resolution and other factors, the vanity journals
 seem to come out 
 no worse than the rest. 
 
 In any case I suspect any retractions are underrepresented in those journals
 because they fight it harder ;-)
 
 Best, BR
 
 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ethan
 Merritt
 Sent: Thursday, October 18, 2012 11:11 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] PNAS on fraud
 
 
 Fang et al. claim that R^2 = 0.0866, which means that CC = 0.29.
 While a correlation coefficient of less than 0.3 is not a complete lack of
 correlation, it's still rather weak.
 
 The highly significant must be taken in a purely statistical sense.
 That is, it doesn't mean the measures are highly correlated, it means the
 evidence for non-zero correlation is very strong.
 
 Ethan
 
 ..
 Jürgen Bosch
 Johns Hopkins University
 Bloomberg School of Public Health
 Department of Biochemistry  Molecular Biology
 Johns Hopkins Malaria Research Institute
 615 North Wolfe Street, W8708
 Baltimore, MD 21205
 Office: +1-410-614-4742
 Lab:  +1-410-614-4894
 Fax:  +1-410-955-2926
 http://lupo.jhsph.edu
 
 
 
 

Re: [ccp4bb] Do my SAXS data agree with the crystal structure?

[Anastassis Perrakis]

If you main point is dimer vs monomer, Guinier plots is in fact all you would 
need, in my opinion according to what I have read so far...

A.

On 17 Jun 2012, at 13:01, David Briggs wrote:

 Dear Xun,
 
 Regarding your monomer vs dimer, theoretical vs observed crysol plots
 - yes - they are significantly different.
 
 If you focus at the very lowest q part of the curve - the deviation
 there in your monomer plots indicate that there is a significant size
 difference between your PX monomer and your SAXS data - the PX dimer
 is a much better fit at low q.
 
 This should be enough to demonstrate to a reviewer that the dimer you
 see in PX is also present in solution.
 
 Other experiments that could support this are SEC-MALLS or perhaps AUC.
 
 HTH,
 
 Dave
 
 David C. Briggs PhD
 Father, Structural Biologist and Sceptic
 
 University of Manchester E-mail:
 david.c.bri...@manchester.ac.uk
 
 Webs : http://flavors.me/xtaldave
 Twitter: @xtaldave
 Skype: DocDCB
 
 
 
 On 17 June 2012 06:11, Xun Lu xlun...@gmail.com wrote:
 Drs.Caldwell, Briggs, and Gupta,
 
Thank you very much for the advices.   I regret that I didn't show any
 figure in the earlier post.  Here I've attached a figure showing the data
 quality and some fittings.
Data look OK, right? This question may sound silly, but I just want to
 make sure.
As I said in the earlier post, I tried Crysol.  I used the crystal
 structure (dimer+DNA) as the model, and the fitting was OK, right?  In fact,
 I also tried monomer+DNA as the model (I simply deleted one monomer from the
 PDB file).  This kind of comparison may be meaningless, but I was just
 curious.  I am wondering how people judge whether the fit is good or not.
 
 
 Another question, I tried to generate an envelope from SAXS data using
 Gasbor and Dammin (people say Dammin is better at protein-DNA complex,
 although it still uses the same bead for both DNA and protein?).  The
 generated envelope was nothing like my crystal structure.  As people have
 pointed out, protein and DNA scatter differently.  SANS is the way to go.
  So I should give up on modeling SAXS data?  I've almost given up, because
 anyways I have the crystal structure, and SAXS is only a small part of this
 paper.
 
 
 
 Thanks,
 
 Xun
 
 
 
 On Sat, Jun 16, 2012 at 6:36 PM, Kushol Gupta kushol.gu...@gmail.com
 wrote:
 
 Two cents -
 
 
 
 A good deal of caution must be exercised when working with composite
 particles such as a protein-DNA complex in SAXS because of the contrast
 problem.  Simply, protein and DNA scatter differently in x-rays, with a bias
 towards the DNA component.  As a result, experimental Rgs could be slightly
 deflated versus what their true values would be at infinite contrast.  Mass
 estimation by I(0) analysis with a protein standard of known mass and
 concentration is not really valid because the contrast terms are different.
 Because the particle is heterogeneous in composition and distribution, shape
 reconstruction from SAXS alone, which assumes homogeneity, can also be
 misleading (although in practice it is still reasonably instructive).  It is
 for these reasons that SANS and the contrast variation approach can be
 extremely useful.
 
 
 
 With those caveats, the strategy you describe - comparison of experimental
 and theoretical profiles from an experimental structure using CRYSOL or FoxS
 is definitely the best way to go in the case of a protein-DNA complex with
 SAXS alone.  Showing comparisons of the experimental with the calculated
 should make the point.  Test other possible models inferred from lattice
 packing to further your point (if applicable).
 
 
 
 Regarding populations of monomer and dimer -
 
 
 
 · it is generally good to constrain your interpretation of
 scattering data with other orthogonal solution measures which demonstrates
 the homogeneity of your complex in comparable experimental conditions, such
 as sedimentation velocity or gel filtration.
 
 
 
 · Have some determination of affinity of the complex in the same
 solution conditions (including temperature!).  This will allow you to argue
 that your sample concentrations are well in excess of any monomer-dimer
 association behavior (eg, mixtures!).  Scattering of mixtures can undermine
 your ability to accurately assess the structural properties of your complex.
 
 
 
 · Collect a concentration series and extrapolate to infinite
 dilution, if possible, to ensure elimination of the S(q) term from your
 data.  Interparticle interactions can be an issue with complexes containing
 DNA if the buffers aren’t quite right. (I’ve seen this a lot)
 
 
 
 Lastly, remember that the scattering profile represents the solution
 average of the particle, not just a single snapshot.  Some discrepancies
 like those you note should be expected.
 
 
 
 Hope that helps,
 
 
 
 Kushol
 
 
 
 Kushol 

Re: [ccp4bb] very informative - Trends in Data Fabrication

[Anastassis Perrakis]

 I still believe Prof. Dr. Hofkristallrat außer Dienst, is written as 
 Bernhard - unless you are referring to some other guy with a  french name 
 Bernard. And the book indeed is a bible of xtallography.
Jürgen

ausser Dienst ... now I get it ... my German is a lot worse than just spelling 
names wrong ;-)
(and sorry for the 'ss' - no clue where Escet is in my keyboard)

and indeed the book is great - maybe get the publication year and count PDB 
structures before and after it ... We are in year 3 Anno Rupp ... (and no 
worries Bernhard about the errata ... the Bible likely contains many more ... 
each chapter is contradicting every other ... at least you are consistent!)

Best -

A.

Re: [ccp4bb] very informative - Trends in Data Fabrication

[Anastassis Perrakis]

Reading the paper from Dr. Hofkristallrat a.D. and the editorial in ActaF, I 
must say that besides the rather reasonable demand for journals to include 
crystallography experts as referees, Table 1 would have fooled me as referee. 
A validation report of the VTF style might not had helped either in refereeing 
- in this case. Alarm bells could had rung possibly if the PDB was re-refining 
all submitted structures and look for 'too good to be true' improvements (sorry 
Robbie ... we are not there yet to improve things SO much!). Saving the images 
in a repository would had been equally unlikely to have helped (they would had 
submitted some data ... unless these were systematically validated and 
cross-matched to the CRYST data cards no alarm bells either - even if running 
PDB_REDO in all submissions appears a tad unrealistic, re-processing all images 
and matching them to CRYST records seems more troublesome at the present 
moment).

A thing that could had helped, would had been if our biology colleagues who 
want a structure for their story would had valued more the structural 
contribution by scrutinising the data (a corresponding author must scrutinise 
all data before accepting responsibility - and not when questioned throw the 
hands up waving 'it was not me ...'). Maybe ourselves as a community could also 
help by making our colleagues aware that crystallographic work is a tad more 
than 'and the author in the middle of the paper just contributed a structure' 
and explain them that if they want to be using structures for their 
publications they should be always prepared to engage in close and real 
collaborations where both sides accept responsibility for the data of each 
other, as it happens in many fruitful collaborations between biologists and 
crystallographers (such as these I had the privilege to engage with 
collaborators that criticised my data, as I did theirs ...).

regards to all -

Tassos

(and please, no 1st April joke with fraud cases !)

Re: [ccp4bb] Help! weird thing

[Anastassis Perrakis]

Hi -

I agree with Garib that its likely a pseudo-translation issue.
I also agree with that the advice he gives is correct, but ...
... since I am evidently less smart to follow all these steps,
I like to use phenix.xtriage that will  tell me if there is pseudo- 
translation or not,
and will give a p-value for that being significant. Its at the end of  
the text output.


I am not sure if Phaser deals these days with pseudo-translation - I  
guess Randy can tell us.
If not, there is a very simple trick to make Phaser work with pseudo- 
translation,
but since I threw the ball to Randy's court and he told me the trick a  
few years ago,

I will let him explain only if needed ;-)

Best,

Tassos

On Mar 11, 2012, at 12:55, Garib N Murshudov wrote:


Hi

Could you please check:
1) If there is psedotranslation. It could be done by using sfcheck,  
molrep, ctruncate or calculating patterson map and displaying using  
coot at 8-10 sigma level (it is my favourite method for analysis of  
pseudo translations), whole unit cell ( a bit bigger than whole unit  
cell). Then if you see large no origin peak (very likely along one  
of the axis, could be a). If yes then you have several options:  
using phaser - 1) reduce cell, find solution in smaller cell and  
then expand; 2) use molrep to solve. When there are two copies  
related with pseudo translation molrep can give you solution; 3) as  
far as I am aware latest version of phaser works with pseudo  
translation. If you have pseudtranslation you should be aware that  
even if you solve the structure starting R factors could be 70-80%.  
Then you may want to do 40 cycles of rigid body and 40-100 cycles of  
ljelly body
2) Check your space group in pdb and mtz file. They may not be  
consistent.


I hope it helps.

Garib

On 11 Mar 2012, at 07:33, xiaoyazi2008 wrote:


Hi All,

I have an interesting thing to share.
2.3A dataset with good quality, P21
Partial model is available (~60% of the target protein).
It seems that there are 4 copies in the ASU (Matthews_coef 2.6,  
53%solvent)
Molecular replacement gave two copies of the model (Z scores are  
R6.2, T6.2, R6.8, T13.4). The solution is very clear. It could not  
locate the rest two copies.


However, a quick refmac5 refinement gave a very high R factor.
The funny part is the symmetry operation in Coot.
As shown in the JPEG figure, it looks like there should be another  
two copies (based on strong fo-fc green map), which locate in the  
empty space between models found by Phaser.


Why is that Phaser could not find the remaining two copies even  
there are strong fo-fc density?

Any suggestions...


Thanks a lot!

Zhihong
weird thing.jpg


Garib N Murshudov
Structural Studies Division
MRC Laboratory of Molecular Biology
Hills Road
Cambridge
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk
Web http://www.mrc-lmb.cam.ac.uk





P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] Envelope Phasing.

[Anastassis Perrakis]

Hi David -

I think you have four hurdles to overcome:

1. How good is a SAXS envelope
2. How will you place it in the right place
3. How will you extend from ~15-20 A to around 4 A
4. How will you extend from 4 A to beyond

Steps 2 and 4 might be the easiest - albeit far from trivial. 
Point 1 can be argued...

However, I have not seen anyone cracking 3 in a convincing manner...
(in the absence of serious NCS)

I hope the next emails prove me wrong though!

A.

On 12 Mar 2012, at 21:10, David Briggs wrote:

 Hi CCP4bb,
 
 I would like to ask about envelope phasing - specifically with SAXS data.
 
 There are papers (1) and tutorials (2) describing how this might be
 done, but I have also found comments on the ccp4bb, such as this one
 (http://www.proteincrystallography.org/ccp4bb/message11690.html) which
 are somewhat less optimistic.
 
 I get the impression from my reading around that SAXS envelope phasing
 is somewhat difficult to do unless you have some NCS you can use to
 help the phase extension process. Does anybody have any
 opinions/evidence/examples/anecdotes/tips about how SAXS envelope
 phasing can be done successfully?
 
 Cheers,
 
 Dave
 
 (1) - eg - http://scripts.iucr.org/cgi-bin/paper?dz5081
 (2) - eg - 
 http://www.phaser.cimr.cam.ac.uk/index.php/Using_Electron_Density_as_a_Model
 
 
 David C. Briggs PhD
 Father, Structural Biologist and Sceptic
 
 University of Manchester E-mail:
 david.c.bri...@manchester.ac.uk
 
 Webs : http://flavors.me/xtaldave
 Twitter: @xtaldave
 Skype: DocDCB
 

Re: [ccp4bb] Introducing an ELN

[Anastassis Perrakis]

I think that all these points are interesting and valid.

On Jan 25, 2012, at 10:37, Chris Morris wrote:


Tassos reports:


1. None of the twenty test-users was satisfied with any of the two
solutions - and each was annoyed for a different reason.


This suggests that the choice of ELN is not the most difficult part  
of the adoption process. Maybe the test users at the NKI were  
annoyed by the idea of using an ELN at all.




That would surely apply to some users. Some were actually very keen,  
and thats why they signed up for it.


In my experience, the hardest part is ensuring that it provides  
benefits to the people who have to enter the data, and provides them  
early. The fact that it will make information retrieval easier in  
three years is not enough.


I suggest focussing on electronic support for housekeeping: booking  
time on an instrument, finding the files the instrument created,  
ordering oligos, recording when you use the last of a reagent.  
Scientists work very independently in most respects, but they do  
have certain obligations that flow from sharing the lab space. You  
can make use of these to encourage compliance with the ELN. If you  
do, then most of the science will get recorded in passing.


I think that this was exactly one of the problems. The ELNs we tested  
had no option for booking instruments, no way to find files from  
instruments let alone read them (it would support only TIF, JPEG, Doc,  
XLS, PDF), and would not do stock keeping: all these are thought to be  
out of the ELN scope. And that makes an ELN inherently less useful.


Lack of instrument support is another issue: a machine that would  
allow us to import real chromatograms to ELN would be cool - alas, the  
solution that was suggested to us is to save as PDF or XLS and  
reload ...! (it took 3 weeks to come back with this great plan!)


For the rest I have nothing much to say, I basically agree.


A.

I suggest also ensuring that it includes electronic tools that  
actually help. Two examples from PiMS are primer design, and  
automatically uploading and interpreting results from the Caliper GX  
instrument.


It must allow round trips with spreadsheets, i.e. dump ELN data as a  
spreadsheet, edit it, upload it again. Despite their substantial  
disadvantages, some scientists will not give them up. It should also  
allow crossreferencing with paper note books. Some will continue to  
use a lab notebook. When they discover that the ELN serves as a  
searchable index to it, they will warm to the ELN.


I suggest aiming for no paper at your lab progress meetings within  
say 12 months. When you reach that point, everything important is in  
the ELN. Before then, the ELN is not giving real value.


You will need someone who is keen on the introduction of the ELN, to  
customise it, provide first line user support, and act as a single  
point of contact with the supplier. This might be a scientist or an  
IT person. I have also seen this done well by a technician, Delphine  
Chesnel when she was at the EMBL Hamburg. If you can't find such a  
champion, then introduction will not be successful.


Some of the problem here is an own goal by the community:  
scientists are trained to use paper during their degrees, so ELNs  
are a controversial change of practice. One person who, unusually,  
began with an ELN told me how inconvenient it is now she works in a  
paper-based lab.


PepTalk 2012 had a workshop on this topic. The recording and notes  
are here:

   
http://www.structuralbiology.eu/support/forums/networks/pims/why-dont-scientists-use-limselns

regards,
Chris

Chris Morris
chris.mor...@stfc.ac.uk
Tel: +44 (0)1925 603689  Fax: +44 (0)1925 603634
Mobile: 07921-717915
Skype: chrishgmorris
http://pims.structuralbiology.eu/
http://www.citeulike.org/blog/chrishmorris
Daresbury Lab,  Daresbury,  Warrington,  UK,  WA4 4AD




P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] R/Rfree problem

[Anastassis Perrakis]

Any crystal can be worked at as P1. 

What you know is that your crystal is not p212121

It can be 

P21212
P21221
P22121
P1121
P1211
P2111
P112
P121
P211
P1

And then there is twinning ...

Suggestions:

Use Pointless or Phenix.xtriage, feeding the unmerged p1 data to decide. 

Use the Zanuda server with the P212121 data to see if any sub-space group is 
best. 

Use a molecular replacement program with the option to test all space groups. 

A. 



Sent from my iPad

On 19 Jan 2012, at 13:06, 조기준 c-a...@korea.ac.kr wrote:

 Dear all,
  
 I have a data, and it has R/Rfree problem.
 The data could be processed as P212121 and P1.
 The resolution was 2.9, and unit cell parameters were a=73.527, b=90.035, 
 c=237.980, α=β=μ=90 and a=73.709, b=90.099, c=238.172, α=89.939, β=89.945, 
 μ=89.993 for P212121 and P1, respectively.
 R/Rfree was 22.3/29.4 for P1 after several refinements.
 However, I couldn’t decrease R/Rfree below 30.0/37.6 for P212121.
 There were almost no differences on monomer structure and crystal packing 
 between P212121 and P1 after MR, and the electron densities followed trace of 
 peptide chain well.
 Can anybody suggest me about this problem?
  
 Thank you.
 Ki Joon Cho
  
  

Re: [ccp4bb] MAD

[Anastassis Perrakis]

A, yes, inventor's names. Anyone reading who is less than 40 and knows what MTZ 
stands for?

;-)

My favorite technique remains SADDAM - a side product of Gerard's War On Error, 
that never did catch-up with the masses - experimentally or as an acronym.

A.

On 19 Jan 2012, at 21:51, Petr Leiman wrote:

 It would be so much more convenient to call these techniques (MAD, SAD, etc.) 
 by their inventor's name. This would simplify things immensely simultaneously 
 eliminating CCP4BB MADisagreements. 
 
 Although in our days of copyrights wars, the journals and perhaps conferences 
 where these methods were presented for the first time would insist on using 
 their names as part of the method's name...
 
 Petr
 
 
 On Jan 19, 2012, at 7:42 PM, Ethan Merritt wrote:
 
 On Thursday, 19 January 2012, Ian Tickle wrote:
 So what does this have to do with the MAD acronym?  I think it stemmed
 from a visit by Wayne Hendrickson to Birkbeck in London some time
 around 1990: he was invited by Tom Blundell to give a lecture on his
 MAD experiments.  At that time Wayne called it multi-wavelength
 anomalous dispersion.  Tom pointed out that this was really a misnomer
 for the reasons I've elucidated above.  Wayne liked the MAD acronym
 and wanted to keep it so he needed a replacement term starting with D
 and diffraction was the obvious choice, and if you look at the
 literature from then on Wayne at least consistently called it
 multi-wavelength anomalous diffraction.
 
 Ian:
 
 The change-over from dispersion to diffraction in MAD protein 
 crystallography happened a couple of years earlier, at least with regard 
 to work being done at SSRL.  I think the last paper using the term 
 dispersion was the 1988 Lamprey hemoglobin paper.  The next two papers, 
 one a collaboration  with Wayne's group and the other a collaboration
 with Hans Freeman's group, used the term diffraction.
 
 WA Hendrickson, JL Smith, RP Phizackerley, EA Merritt. 
 Crystallographic structure-analysis of lamprey hemoglobin from 
 anomalous dispersion of synchrotron radiation.
 PROTEINS-STRUCTURE FUNCTION AND GENETICS, 4(2):77–88, 1988.
 
 JM Guss, EA Merritt, RP Phizackerley, B Hedman, M Murata, 
 KO Hodgson, HC Freeman. 
 Phase determination by multiple-wavelength X-ray-diffraction - 
 crystal-structure of a basic blue copper protein from cucumbers. 
 SCIENCE, 241(4867):806–811, AUG 12 1988.
 
 WA Hendrickson, A Pahler, JL Smith, Y Satow, EA Merritt, RP Phizackerley. 
 Crystal structure of core streptavidin determined from multiwavelength 
 anomalous diffraction of synchrotron radiation. 
 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF
 AMERICA, 86(7):2190–2194, APR 1989.
 
 On the other hand, David and Lilo Templeton continued to use the term 
 anomalous dispersion for at least another decade, describing their 
 diffraction experiments exploring polarization effects and other
 characteristics of near-edge X-ray scattering by elements all over the
 periodic table.
 
  Ethan
 
 
 Cheers
 
 -- Ian
 
 On 18 January 2012 18:23, Phil Jeffrey pjeff...@princeton.edu wrote:
 Can I be dogmatic about this ?
 
 Multiwavelength anomalous diffraction from Hendrickson (1991) Science Vol.
 254 no. 5028 pp. 51-58
 
 Multiwavelength anomalous diffraction (MAD) from the CCP4 proceedings
 http://www.ccp4.ac.uk/courses/proceedings/1997/j_smith/main.html
 
 Multi-wavelength anomalous-diffraction (MAD) from Terwilliger Acta Cryst.
 (1994). D50, 11-16
 
 etc.
 
 
 I don't see where the problem lies:
 
 a SAD experiment is a single wavelength experiment where you are using the
 anomalous/dispersive signals for phasing
 
 a MAD experiment is a multiple wavelength version of SAD.  Hopefully one
 picks an appropriate range of wavelengths for whatever complex case one 
 has.
 
 One can have SAD and MAD datasets that exploit anomalous/dispersive signals
 from multiple difference sources.  This after all is one of the things that
 SHARP is particularly good at accommodating.
 
 If you're not using the anomalous/dispersive signals for phasing, you're
 collecting native data.  After all C,N,O,S etc all have a small anomalous
 signal at all wavelengths, and metalloproteins usually have even larger
 signals so the mere presence of a theoretical d difference does not make 
 it
 a SAD dataset.  ALL datasets contain some anomalous/dispersive signals, 
 most
 of the time way down in the noise.
 
 Phil Jeffrey
 Princeton
 
 
 
 On 1/18/12 12:48 PM, Francis E Reyes wrote:
 
 
 Using the terms 'MAD' and 'SAD' have always been confusing to me when
 considering more complex phasing cases.  What happens if you have 
 intrinsic
 Zn's, collect a 3wvl experiment and then derivatize it with SeMet or a 
 heavy
 atom?  Or the MAD+native scenario (SHARP) ?
 
 Instead of using MAD/SAD nomenclature I favor explicitly stating whether
 dispersive/anomalous/isomorphous differences (and what heavy atoms for 
 each
 ) were used in phasing.   Aren't analyzing the 

Re: [ccp4bb] Off-topic: ELNs

[Anastassis Perrakis]

Hi -

Here at the NKI, we had formed a committee to look at ELN solution two  
years ago.

We had interviewed three vendors, and run two tests with twenty users.

A brief description of the outcome:

1. None of the twenty test-users was satisfied with any of the two  
solutions - and each was annoyed for a different reason.
2. If that would work at all, better be prepared to dedicate one  
person for technical support (unless security considerations
allow you to have cloud-based usage or remote hosting) and another  
person for scientific support (making templates for
experiments - else in a year you would have 37 templates for how to  
run a gel filtration column).


I would add, that for the whole thing to work at all you need an IT  
department that are really good and collaborative,

and especially for cloud-based solutions you need a fast network.

My personal conclusion - and not that of the NKI which continues to be  
dedicated to implementing an ELN solution may
I clarify - was that I have wasted enough time, and I quit from the  
committee before Christmas.


And, with no offense as well to my friends in the PiMS project,  
besides spending time and resources on that between 2004-2008,
I also do not think that this is a solution for small lab like mine -  
admittedly it works well for bigger and better organized labs.


Sorry for the negative vibes.

A.


On Jan 16, 2012, at 22:28, Seiji Sugiman-Marangos wrote:


Hi, off-topic question regarding electronic laboratory notebooks. Our
lab is planning on moving from paper to digital record keeping and I
was wondering which of the available ELN platforms are being used by
the ccp4 community.

We are primarily a crystallography lab but we would also need some
versatility in the platform as some of our lab members are more  
focused

on biochemistry.

Any suggestions or comments would be greatly appreciated!

Thanks,
Seiji


P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] Reference for Resolution Cutoffs

[Anastassis Perrakis]

Also

Nat Struct Biol. 1997 Apr;4(4):269-75.
Improved R-factors for diffraction data analysis in macromolecular 
crystallography.
Diederichs K, Karplus PA.

But none of these are in any way 'discrediting' Rmerge, they are just proposing 
more statistically sound alternatives. That is not the same ...

A.


On 6 Dec 2011, at 21:44, Ed Pozharski wrote:

 On Tue, 2011-12-06 at 13:43 -0600, Jacob Keller wrote:
 The question is: is there a reference in which Rmerge has been
 thoroughly, clearly, and authoritatively discredited as a data
 evaluation metric in the favor of Rmeas, Rpim, etc., and if so, what
 is that reference?
 
 
 Aren't these sufficient?  
 
 Manfred Weiss  Rolf Hilgenfeld, On the use of the merging R factor as
 a quality indicator for X-ray data, J.Appl.Cryst. 30, 203-205 (1997)
 
 Manfred Weiss, Global Indicators of X-ray data quality J.Appl.Cryst.
 34, 130-135 (2001)
 
 -- 
 Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

Re: [ccp4bb] Archiving Images for PDB Depositions

[Anastassis Perrakis]

Dear all,

Apologies for a lengthy email in a lengthy chain of emails.

I think Jacob did here a good job refocusing the question. I will try  
to answer it in a rather simplistic manner,
but from the view point of somebody who might only have relatively  
little time in the field, but has enjoyed the
privilege of seeing it both from the developer and from the user  
perspective, as well as from environments
as the synchrotron service-oriented sites, as well as from a cancer  
hospital. I will only claim my weight=1 obviously,
but I want to emphasize that where you stand influences your  
perspective.


let me first present the background that shapes my views.

you can skip this

When we started with ARP/wARP (two decades for Victor and getting  
pretty close for myself!), we (like others) hardly
had the benefit of large datasets. We had some friends that gladly  
donated their data to us to play with,
and we have assembled enough data to aid our primitive efforts back  
then. The same holds true for many.


At some point, around 2002, we started XtalDepot with Serge Cohen: the  
idea was to systematically collect phased data,
moving one step away from  HKL F/SigF to include either HLA/B/C/D or  
the search model for the molecular replacement solution.
Despite several calls, that archive only acquired around hundred  
structures, and yesterday morning was taken off-line
as it was not useful any more and was not visited by anyone any more.  
Very likely, our effort was redundant because of the JCSG
dataset, which has been used by many and many people who are grateful  
for it (I guess the 'almost every talk' of Frank refers to me,

I have never used the JCSG set).

Lately, I am involved to the PDB_REDO project, who was pioneered by  
Gert Vriend and Robbie Joosten (who is now in my lab).
Thanks to Gerard K. EDS clean-up and subsequent effort of both Robbie  
and Garib who made gadzillions of fixes to refmac,
now we can not only make maps of PDB entries, but also refine them -  
all but less than 100 structures. That has costed a significant part of
the last four-five years of Robbie's life (and has received limited  
appreciation from editors of 'important' journals and from referees of  
our grants).


/you can skip this

These experiences are what shapes my view, and my train of thought  
goes like this:


The PDB collected F/sigF, and to be able to really use them to get  
maps first, to re-refine later, and re-build now, has received rather
limited attention. It starts to have impact to some fields, mostly to  
modeling efforts and unlike referee nr.3 I strongly believe it

has a great potential for impact.

My team collected also phases, so did JCSG in a more successful and  
consistent scale,
and that effort has been used indeed by developers to deliver better  
benchmarking
of many software (to my knowledge it has escaped my attention if  
anyone used JCSG data directly for eg by learning techniques,
but I apologize if I have missed that). This benchmarking of software,  
based on 'real' maps for a rather limited set of data,

hundreds and not tens of thousands, was important enough anyway.

That leads me to conclude that archiving images is a good idea on a  
voluntary basis. Somebody who needs it should convince the funding  
bodies
to make the money available, and then take the effort to make the  
infrastructure available. I would predict then 100-200 datasets would  
be collected,
and that would really really help developers to make these important  
new algorithms and software we all need. Thats a modest investment,
that can teach us a lot. One of the SG groups can make this effort and  
most of us would support it, myself included.


Would such data help more than the developers? I doubt it. Is it  
important to make such a resource available to developers? Absolutely?
What is the size of the resource needed? Limited to a few hundreds of  
datasets, that can be curated and stored on a modest budget.


Talking about archiving in a PDB-scale might be fantastic in  
principle, but it would require time and resources to a scale that  
would not clearly stand the

cost-benefit trial, especially at times of austerity.

In contrast, a systematic effort of our community to deposit DNA in  
existing databanks like AddGene.com, and annotate PDB entries with  
such deposition
numbers, would be cheap, efficient, and could have far-reaching  
implications for many people that could really get easily the DNA to  
start studying
structures in the database. That would surely lead to new science,  
because people interested enough in these structures to claim the DNA  
and
'redo' the project would add new science. One can imagine even SG  
centers offering such a service 'please redo structure X for this and  
that reason',
for a fee that would represent the real costs, that must be low given  
the investment already existing in experience and technology over  
there - a subset

of targets could be on a 

Re: [ccp4bb] Archiving Images for PDB Depositions

[Anastassis Perrakis]

To avoid misunderstandings, since I received a couple of emails already:

 Is it important to make such a resource available to developers?  
Absolutely?


? was a typo. I meant Absolutely!
 I think such data are essential for development of better processing  
software, and I find the development of better

processing software of paramount importance!

we can of course now have a referendum to decide in the best curse  
of action! :-(


Curse was not a typo.
I am Greek. Today, thinking of referendums, I see many curses of  
action, and limited courses of action.


A.



A.

PS Rob, you are of course right about sequencing costs, but I was  
only trying to paint the bigger picture...




On Oct 31, 2011, at 18:00, Frank von Delft wrote:

Loathe being forced to do things?  You mean, like being forced to  
use

programs developed by others at no cost to yourself?

I'm in a bit of a time-warp here - how exactly do users think our
current suite of software got to be as astonishingly good as it  
is?  10
years ago people (non-developers) were saying exactly the same  
things -
yet almost every talk on phasing and auto-building that I've heard  
ends

up acknowledging the JCSG datasets.

Must have been a waste of time then, I suppose.

phx.




On 31/10/2011 16:29, Adrian Goldman wrote:
I have no problem with this idea as an opt-in. However I loathe  
being forced to do things - for my own good or anyone else's. But  
unless I read the tenor of this discussion completely wrongly, opt- 
in is precisely what is not being proposed.


Adrian Goldman

Sent from my iPhone

On 31 Oct 2011, at 18:02, Jacob Kellerj-kell...@fsm.northwestern.edu 
  wrote:



Dear Crystallographers,

I am sending this to try to start a thread which addresses only the
specific issue of whether to archive, at least as a start, images
corresponding to PDB-deposited structures. I believe there could  
be a

real consensus about the low cost and usefulness of this degree of
archiving, but the discussion keeps swinging around to all levels  
of
archiving, obfuscating who's for what and for what reason. What  
about

this level, alone? All of the accompanying info is already entered
into the PDB, so there would be no additional costs on that score.
There could just be a simple link, added to the download files
pulldown, which could say go to image archive, or something along
those lines. Images would be pre-zipped, maybe even tarred, and  
people

could just download from there. What's so bad?

The benefits are that sometimes there are structures in which
resolution cutoffs might be unreasonable, or perhaps there is some
potential radiation damage in the later frames that might be
deleterious to interpretations, or perhaps there are ugly  
features in

the images which are invisible or obscure in the statistics.

In any case, it seems to me that this step would be pretty  
painless,
as it is merely an extension of the current system--just add a  
link to

the pulldown menu!

Best Regards,

Jacob Keller

--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***



P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28  
597791







P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] To archive or not to archive, that's the question!

[Anastassis Perrakis]

Dear all,

The discussion about keeping primary data, and what level of data can  
be considered 'primary', has - rather unsurprisingly - come up also in  
areas other than structural biology.
An example is next generation sequencing. A full-dataset is a few tera  
bytes, but post-processing reduces it to sub-Gb size. However, the  
post-processed data, as in our case,
have suffered the inadequacy of computational reduction ... At least  
out institute has decided to create double back-up of the primary data  
in triplicate. For that reason our facility bought
three -80 freezers, one on site at the basement, on at the top floor,  
and one off-site, and they keep the DNA to be sequenced. A sequencing  
run is already sub-1k$ and it will not become
more expensive. So, if its important, do it again. Its cheaper and its  
better.


At first sight, that does not apply to MX. Or does it?

So, maybe the question is not To archive or not to archive but What  
to archive.


(similarly, it never crossed my mind if I should be or not be - I  
always wondered what to be)


A.


On Oct 30, 2011, at 11:59, Kay Diederichs wrote:


Am 20:59, schrieb Jrh:
...

So:-  Universities are now establishing their own institutional
repositories, driven largely by Open Access demands of funders. For
these to host raw datasets that underpin publications is a reasonable
role in my view and indeed they already have this category in the
University of Manchester eScholar system, for example.  I am set to
explore locally here whether they would accommodate all our Lab's raw
Xray images datasets per annum that underpin our published crystal
structures.

It would be helpful if readers of this CCP4bb could kindly also
explore with their own universities if they have such an
institutional repository and if raw data sets could be accommodated.
Please do email me off list with this information if you prefer but
within the CCP4bb is also good.



Dear John,

I'm pretty sure that there exists no consistent policy to provide an  
institutional repository for deposition of scientific data at  
German universities or Max-Planck institutes or Helmholtz  
institutions, at least I never heard of something like this. More  
specifically, our University of Konstanz certainly does not have the  
infrastructure to provide this.


I don't think that Germany is the only country which is the  
exception to any rule of availability of institutional  
repository . Rather, I'm almost amazed that British and American  
institutions seem to support this.


Thus I suggest to not focus exclusively on official institutional  
repositories, but to explore alternatives: distributed filestores  
like Google's BigTable, Bittorrent or others might be just as  
suitable - check out http://en.wikipedia.org/wiki/Distributed_data_store 
. I guess that any crystallographic lab could easily sacrifice/ 
donate a TB of storage for the purposes of this project in 2011 (and  
maybe 2 TB in 2012, 3 in 2013, ...), but clearly the level of work  
to set this up should be kept as low as possible (a bittorrent  
daemon seems simple enough).


Just my 2 cents,

Kay






P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] should the final model be refined against full datset

[Anastassis Perrakis]

 
 
 For structures with a small number of reflections, the statistical noise 
 in the 5% sets can be very significant indeed. We have seen differences 
 between Rfree values obtained from different sets reaching up to 4%. 

This is very intriguing indeed!
Is there something specific in these structures that Rfree differences depending
on the set used reach 4%? NCS? Or the 5% set having less than ~1000-1500 
reflections?

It would be indeed very interesting if there was a correlation there!

A.

 
 Ideally, and instead of PDBSET+REFMAC we should have been using simulated 
 annealing (without positional refinement), but moving continuously between 
 the CNS-XPLOR and CCP4 was too much for my laziness.
 
 All the best,
 Nicholas
 
 
 -- 
 
 
  Dr Nicholas M. Glykos, Department of Molecular Biology
 and Genetics, Democritus University of Thrace, University Campus,
  Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office) +302551030620,
Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/

Re: [ccp4bb] is codon optimization worth it?

[Anastassis Perrakis]

Well, codon optimization is not really trouble, it's money. The money are worth 
it usually anyway, since the optimized genes are easy to clone if you make many 
constructs out of one gene, as you better do anyway ... Compared with 
downstream expenses, optimized genes are these days almost always worth the 
trouble...

A. 

Sent from my iPad

On 30 Sep 2011, at 19:02, Segelke, Brent W. segel...@llnl.gov wrote:

 To me, the key question would seem to be, if I can’t win them all, how many 
 more do I win if I go to the trouble?
 
  
 
 Brent
 
  
 
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Keys
 Sent: Friday, September 30, 2011 8:29 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] is codon optimization worth it?
 
  
 
 We codon optimised a poorly expressed gene from neisseria meningitides based 
 on a codon usage table derived from the Welch (etal, 2009) paper below. The 
 optimisation is specifically for overexpression in BL21 (DE3). The optimised 
 gene increased protein expression by at least a factor of 10, and changed 
 (somewhat reduced) the degradation pattern we observed. Unfortunately it 
 didn't do anything to improve the folding (ie. we ended up with lots of 
 half-folded, semi-soluble protein).
 
 With other neisserial derived proteins we have had an almost undetectable 
 effect.
 
 You can't win 'em all.
 
 Cheers,
 Tim
 
 Design Parameters to Control Synthetic Gene Expression in Escherichia coli
 Welch et al, PlosONE 2009
 
 
 
 
 Medizinische Hochschule Hannover
 Zelluläre Chemie, OE 4330
 Zentrum Biochemie
 Carl-Neubergstr. 1
 30625 Hannover
 
 
 
 -Original Message-
 From: CCP4 bulletin board on behalf of Patrick Loll
 Sent: Fri 30.09.2011 16:49
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] is codon optimization worth it?
 
 Has anyone encountered a case in which a construct with the native sequence 
 expressed poorly (or not at all?) in Rosetta(DE3), but the corresponding 
 construct with a codon-optimized sequence expressed well? (The gene in 
 question is from cerevesiae)
 Thanks,
 Pat
 
 ---
 Patrick J. Loll, Ph. D. 
 Professor of Biochemistry  Molecular Biology
 Director, Biochemistry Graduate Program
 Drexel University College of Medicine
 Room 10-102 New College Building
 245 N. 15th St., Mailstop 497
 Philadelphia, PA  19102-1192  USA
 
 (215) 762-7706
 pat.l...@drexelmed.edu
 

Re: [ccp4bb] ARP/wARP install on 6.2.0 RHEL 6?

[Anastassis Perrakis]

My two cents: I am installing arp/warp on my Mac since ages and it works ;-)

The only thing you need to make sure is that you grab ownership of the 
/usr/local directory to you as a user, if you installed ccp4 from dmg. If you 
do this by eg

sudo chown -R me.mygroup $CCP4

Then install.sh should work for arp/warp

Package type installation for arp/warp is also possible technically, and it can 
be made available in the next release if it is agreed between developers that 
from now we will support system-specific installations and not only an 
all-in-one package as till now. 

A. 

Sent from my iPad

On 28 Jul 2011, at 10:06, Saul Hazledine s.hazled...@embl-hamburg.de wrote:

 Hello,
  My reply is in the text below:
 
 On Jul 28, 2011, at 6:00 AM, ccp4 wrote:
 
 A plea from West Australia too.
 I was sitting with someone yesterday who was trying to install it on a Mac
 , and finding it a nightmare. 
 
 We're working on improving this. I believe the main issue is that CCP4 has 
 become more user friendly and installs from a DMG, while the ARP/waRP 7.1 
 install is still showing its Unix command line roots. 
 
 He finally got it set up as a local installation, whereupon it promtly
 failed. 
 
 We use Macs a lot here with few problems  (and various versions of CCP4) so 
 my first suspicion is that this might be an install problem.
 
 The message said See refmac-last.log but that told us nothing, and indeed
 refmac seemed to have worked..
 
 
 Would it be possible to send me the install.log that is created in the 
 ARP/wARP install directory? Also, the refmac-last.log that will be created in 
 the directory where ARP/wARP was working?
 
 Its probably best if the remaining communication is done by direct email.
 
 I'm sorry for the trouble that you are having.  I hope we can fix the problem 
 and prevent it happening to others in future.
 
 Saul Hazledine

Re: [ccp4bb] Peptide Crystallization

[Anastassis Perrakis]

NMR ... synthesize with a few labeled aa according to taste of local  
NMR guru.
If they cant do a15-aa peptide in a day, let the bb know, it will be  
entertaining ;-)


A.

On May 25, 2011, at 14:04, Buz Barstow wrote:


Dear all,

I am considering trying to crystallize a small peptide (around 15  
amino acids). The peptide is soluble in neutral water or buffer (pH  
7.0) until at least 10 mM (13 mg/mL), and adopts a turn conformation  
when bound to Zn.


What are your thoughts on attempting this?

If you think that it is worthwhile, what crystallization conditions  
would you try? I am thinking of a sparse matrix screen using the  
Hampton Crystal Screen 1 and 2 kits, using hanging drop  
crystallization in Hampton Vdx trays.


Thanks! and all the best,

---Buz


P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] how to remove part of data with bad signal to noise ratio

[Anastassis Perrakis]

I think you are just confused. The solvent flattening is just a step to make 
your map clearer. You do not carry the modified phases from solvent flattening 
to refinement (and I sincerely hope you don't refine against the solvent 
flattened amplitudes but against the original data!)

Herman's observation is right, i think. One of the reasons that high solvent 
content will give better Rfree is also that this wY you have more reflections 
than usual per atom: considering two asymmetric units with the same volume, 
they have the same number of reflections at a given resolution, but if one has 
higher solvent it has less atoms so you refine better. 

A. 

Sent from my iPhone

On 24 May 2011, at 11:18, Clement Angkawidjaja 
clem...@bio.mls.eng.osaka-u.ac.jp wrote:

 But you have to do solvent flattening (density modification), which people 
 often (unintentionally?) skip for structures solved with molecular 
 replacement. Please correct me if I am wrong.
 
 Clement
 
 On May 24, 2011, at 6:01 PM, herman.schreu...@sanofi-aventis.com wrote:
 
 This is not my experience. Provided the solvent is featureless, I find
 that a high solvent contents leads to a lower Rfree due to a kind of
 solvent flattening effect. Of course, if a significant part of the
 molecule(s) is/are disordered, this will lead to a degradation of the
 Rfree. 
 
 My 2 cents,
 Herman

Re: [ccp4bb] problem with pdb its symmetry molecule

[Anastassis Perrakis]

Yes, its surely possible. If there are no clashes start thinking if this dimer 
is the physiological molecule in solution. 

Check probability that this dimer is stable in solution in Pisa - ebiFold

If so, check first with size exclusion columns if it behaves like dimer in 
solution, preferably use a MALLS setup if available. Ssaxs can confirm it's 
shape. AUC is handy for kD. 

And if its a dimer then of course mutate the interface and assay ... Have fun!

Tassos


Sent from my iPhone

On 26 Apr 2011, at 09:37, Ramanuj Banerjee ramanuj.baner...@saha.ac.in wrote:

 Dear All, I have solved a protein structure (experimentally phased) with 1 
 molecule in the asymmetric unit at 2.22 A (high resolution). The present R 
 factor is .22 and R free .27 with Ramachandran favoured 98% and R and R free 
 are decreasing with refinement.The problem is: when the pdb is opened in 
 pymol and symmetry mates generated, the upper part of the molecule shows to 
 be intertwined with the symmetry molecule (attached .jpg), but there are no 
 clashes in between the two.The electron density is so very fine that no 
 alternative choices of chain flow are available.All the processes starting 
 from phasing and refinement have been done in Phenix.Is such a thing possible 
 ?
 a copy.jpg

Re: [ccp4bb] flex-wARP error

[Anastassis Perrakis]

Hi -

Yes, your suspicion is correct, and I think that bug has been silently  
fixed in the latest distribution.


I am pretty sure Victor has now a job-dependent filename, if not we  
will try and fix it now as we prepare
a new release. In that case, pleas email Gerrit telling him details  
about your platform, and he
can send you a new executable hmain and instructions for how to fix  
this till then.


best,

Tassos


On Mar 24, 2011, at 16:56, Matthew Franklin wrote:


Hi all -

I'm finding that Arp/Warp is crashing when I try to run a job.  I  
was working on a challenging molecular replacement job where I  
though Arp tracing the fragmentary density could do a better job  
than I could by hand (this turned out to be true, by the way).  I  
was testing different parameters, and running a few flex-warp jobs  
in parallel.  They would crash at different cycles of the building  
and refinement, but always at the same point in the cycle.  Looking  
at the error messages in the log files led to this one, in the last  
HmainPept log file to be written:


 Density interpolation: points  10539450
0.00 CPU2 Elapsed  Superposition
  11394 rotations have been done, total   46821
0.00 CPU1 Elapsed  Rotations

   2009 flipped peptides are taken

end_tag_cputime
=
data send to stderr:
forrtl: severe (64): input conversion error, unit 15, file /Users/ 
matt_franklin/bin/arp_warp_7.1/fort.15

Image  PCRoutineLineSource
hmain  00106F9F  Unknown   Unknown  Unknown
hmain  001063CB  Unknown   Unknown  Unknown
hmain  000CCD30  Unknown   Unknown  Unknown
hmain  0009217B  Unknown   Unknown  Unknown
hmain  0009192A  Unknown   Unknown  Unknown
hmain  000B35C8  Unknown   Unknown  Unknown
hmain  000B0C79  Unknown   Unknown  Unknown
hmain  00012A35  Unknown   Unknown  Unknown
real11.60
user11.51
sys  0.08

I think what's happening here is that I had multiple jobs running  
simultaneously, and they were all trying to read and write to the  
same fort.15 file.  As you can see, this file is in Arp/Warp's main  
directory, not in any of the working directories.  At some random  
point, one job would step on the other's read or write, and I'd get  
a crash.


Can someone confirm my guess?  Is it possible to fix this bug so  
that multiple flex-wARP jobs can be run in parallel?  I'm running  
Arp/Warp Expert System (i.e. flex-wARP) from ccp4i, and I don't  
think this is happening with other Arp/Warp modes.


(I'll be happy to provide more logfiles, input files, etc.)

Thanks,
Matt

--
Matthew Franklin, Ph. D.
Senior Research Scientist
New York Structural Biology Center
89 Convent Avenue, New York, NY 10027
(646) 275-7165


P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] while on the subject of stereo

[Anastassis Perrakis]

To add my two cents, I am not at all a fun of stereo for routine use,  
but I appreciate it if I have to look at a long ligand bent 90 deg in  
the middle to bury in my protein, and all that at 3.0-3.5 A  
resolution ... So, I must have both types of brain damage that Jan  
refers to. No surprises here I guess.


A.

On Mar 23, 2011, at 3:16, Phoebe Rice wrote:


My 2 cents worth on the stereo-dependent:

1) They have carpal tunnel syndrome that makes it painful to keep  
the molecule in motion while rebuilding it (NOTE: enough constant  
mouse-wiggling and you will get carpal tunnel problems if you don't  
have them yet!)


2) They work on big, low-resolution structures where you need to see  
a bigger-picture view.  I've had people tell me that can fit 3-3.5A  
maps just fine without stereo, but having viewed their work, I beg  
to differ.


 Phoebe

 Original message 

Date: Tue, 1 Mar 2011 22:30:54 +
From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Jan  
Löwe j...@mrc-lmb.cam.ac.uk)

Subject: Re: [ccp4bb] while on the subject of stereo
To: CCP4BB@JISCMAIL.AC.UK

Ah! The question of to stereo or not to stereo! There has to be a
scientific reason why this question is more popular than asking for  
what
Linux distro is more fashionable this spring or why an Rmerge of  
0.90 in

the outermost shell is good for you and your structure.

I am offering my two (conflicting) theories (and apologies that both
seem to imply some problem):

A) people who do use stereo have a problem with their brain because  
they

cannot produce three dimensional vision from depth cues alone.

B) people who do not use stereo have a problem with their brain  
because
they cannot see properly in three dimensions and rely on depth cues  
alone.


I personally prefer people with A) when I am their passenger in a car
since they do not need to rotate by 90° to see how far the braking
lights of the car in front are away :-)

jan



On 01/03/2011 21:35, Jim Pflugrath wrote:

I will offer my view.

I hate stereo glasses and hate stereo in general.

One should be able to see 3D from the depth-cueing and by keeping  
the view
in motion.  For fitting, I like to flip the view by 90 degrees.  I  
know I am
going to move in displayX and displayY, but never in displayZ.  I  
then
rotate the view around the vertical axis so thatn the old displayZ  
becomes

displayX.

Furthermore, I don't waste too much time fitting.  I know the  
software can
fit the map better than me, so I let it do its job.  I only need  
to get the
coordinates within the radius of convergence of the refinement  
program.  I
also know that 9 times out of 10, the displayed electron density  
is probably
suspect, so I believe in stereochemistry more than I believe in  
the map.


The main trick is to realize that as a human being, you really are  
not that
good at fitting the map or that it is unnecessary to waste your  
time since
the software is really so much better than you.  Refinement is  
quick enough
that you can try various hypotheses as in:  If I move this here,  
then
refinement will do the trick and Well, that didn't work, so I  
will move

that over there and see if refinement will do the trick.

As for stereo figures, you should be able to convey what you want  
to say
from a good figure with depth-cueing, shadows, etc.  Don't ever  
use stereo
glasses in a public seminar.  Maybe my opinion will change with  
better

stereo technology.

OK, I know quite a lot of people will disagree with me. :)

Jim

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf  
Of David

Roberts
Sent: Tuesday, March 01, 2011 10:29 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] while on the subject of stereo

Hi again,

I'd like to ask a question about the pedagogy of stereo.  That is,  
using

stereo with students in the classroom.

Do you all find that, after setting up these elaborate stereo  
devices,

students really use the stereo or do they tend not to?

I am a huge fan of stereo - and frankly here we have quite a few  
options for
doing stereo - from the active Nvidia systems that people have  
recently been

discussing to passive zalmans. ...

As I mentioned, I like stereo a lot, but really projecting on a  
nice bright
lcd monitor also has it's advantages, and with the ease of moving  
things
using the mouse (or whatever device you use), the overall need for  
stereo
seems to be decreasing.  I don't know - I just wonder what peoples  
views are
out there for the actual need for stereo.  It's incredibly cool  
- and I
think is a very powerful way to show things - but I'm wondering if  
we focus

too much on it because it's cool and not because it's pedagogically
necessary.

Just wondering, no worries.  Thanks

Dave


P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,

Re: [ccp4bb] Solubilization refolding percentage efficiency

[Anastassis Perrakis]

How:

1. measure total protein before refolding (eg by absorbance at 280)
2. Purify refolded fraction by gel filtration in FPLC
3. measure total protein after refolding
4. calculate efficiency ...

Why?

I don't see the reason to know the efficiency. If after refolding I  
would get enough in a defined molecular species,
soluble in a non-denaturing buffer, I would be happy. 'Enough' depends  
on what you want to do with it.
Some refolding protocols have efficiencies of just 2-5%, but this is  
enough ...


A.


On Feb 10, 2011, at 10:45, megha goyal wrote:


Hello,

Can anyone let me know how to calculate solubilisation and refolding  
efficiency. We perform solubilisation and then refolding and check  
by HPLC if refolding is completed or not [single peak on HPLC]. how  
do i determine % efficiency of refolding. kindly guide me.


regards,


megha


P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] First images of proteins and viruses caught with an X-ray laser

[Anastassis Perrakis]

Anyway, I thought that was a cool idea, but like so many other cool  
things, it had to be cut from the Nature paper.  Admittedly, the  
problem has not actually been solved yet.  This is why we used  
REFMAC in TWIN mode.


Is that a hint on the:

a. wisdom of the editor
b. wisdom of 'the third referee'
c. wisdom of the dogma 'five years of eight eight lifes in 2000 words'
d. All of the above

;-)

A.

Re: [ccp4bb] Problem of Refinement and density map

[Anastassis Perrakis]

Although there might be space group and twining trouble from the sound  
of it, running ARP/wARP starting from your molrep solution, at that  
resolution should really do whatever can be done and reduce R/RFree  
while building the vast majority of the model. If Rfree stays high,  
start looking at sg possibilities and twining.


A.

PS In general I would suggest to download and install arp/warp but a  
quick try for new users can always be through the web interface

http://cluster.embl-hamburg.de/ARPwARP/remote-http.html


On Feb 7, 2011, at 11:49, Md. Munan Shaik wrote:


Dear all,
I have a question regarding the refinement and density map.

My protein is 261 amino acids long and crystalize very nicely with  
very high resolution . There is no multiple spot in the image and  
diffraction looks amazing. I collected a dataset at 1.38 resolution  
and the space group is P43, overall Rmerg is 0.02 (most likely the  
space group is P4122, but then the solvent content is less than 16%  
for one molecule in the assymmetric unit, that are unlikely), so I  
processed the image in P4 (36% solvent) and run molecular  
replacement with a model that have 42 sequence identity. I got a  
solution in P43 that are good enough in some part but in the map  
there are many gap at even lower sigma level. I tried to refine and  
Rfree stacked at 36 along with Rwork, which is 33.


I also checked the degradation patteren of the protein in the  
crystal and it looks not degraded and full length protein crystalize.


Is there anybody can suggest me anything that I can try?



Regards,

Md. Munan Shaik
PhD Student
Department of Biotehnology
School of Bioscience and Biotechnology
via G. Colombo 03
Padova 35131, Italy
Mobile: 00393275671896
E-mail: munanbt2...@yahoo.com
 munan.sh...@unipd.it





P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] Merging data to increase multiplicity

[Anastassis Perrakis]

... but, back to the main point, my advice would be to only limit the  
mosaicity, to get better completeness by avoiding overlaps.
Its not ideal, in the sense that you would be over-estimating the  
partial fraction of most partial reflections, and thus systematically  
underestimating intensities.

(I hope I got my overs and unders right here ...)

But these errors would not matter much for refinement purposes, where  
you would rather have a slightly systematically wrong estimate

for all data, rather than not have the 15% of the data at all.

Or at least thats what I thought back in '99 refining MutS ... where I  
did refine a lot with both datasets and liked the 'fixed mosaicity'  
one better.


A.


On Jan 28, 2011, at 13:26, José Trincão wrote:

Ah, yes, I was missing that. The statistics will be wrong. But in  
principle I will get an mtz with better data, because I am  
integrating more observations which would have been rejected by  
being missed at low resolution if the mosaicity was set too low or  
being rejected by overlaps at high resolution if the mosaicity is  
increased.
So the question is - can I use this data for refinement? Or should I  
stick with the best of the datasets (the one with the highest  
completeness and multiplicity)?


Thanks!

Jose

On Jan 28, 2011, at 28/1/11 - 11:59, Ian Tickle wrote:

Jose - you're missing the fact that the same dataset processed in
different ways are not statistically independent datasets!  Increasing
the multiplicity for independent data reduces the uncertainty because
the calculation of the SU assumes statistical independence.

Cheers

-- Ian

On Fri, Jan 28, 2011 at 11:46 AM, José Trincão  
trin...@dq.fct.unl.pt wrote:

Hello all,
I have been trying to squeeze the most out of a bad data set (P1,  
anisotropic, crystals not reproducible). I had very incomplete data  
due to high mosaicity and lots of overlaps. The completeness was  
about 80% overall to ~3A. Yesterday I noticed that I could process  
the data much better fixing the mosaicity to 0.5 in imosflm. I got  
about 95% complete up to 2.5A but with a multiplicity of 1.7. I  
tried to integrate the same data fixing the mosaicity at different  
values ranging from 0.2 to 0.6 and saw the trend in completeness,  
Rmerge and multiplicity.
Now, is there any reason why I should not just merge all these  
together and feed them to scala in order to increase multiplicity?

Am I missing something?

Thanks for any comments!

Jose


José Trincão, PhD   CQFB@FCT-UNL
2829-516 Caparica, Portugal

It's very hard to make predictions... especially about the future  
- Niels Bohr




José Trincão, PhD   CQFB@FCT-UNL
2829-516 Caparica, Portugal

It's very hard to make predictions... especially about the future  
- Niels Bohr







José Trincão, PhD   CQFB@FCT-UNL
2829-516 Caparica, Portugal

It's very hard to make predictions... especially about the future  
- Niels Bohr


P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

[ccp4bb] Post-doctoral positions at the NKI /Amsterdam to work on Autotaxin

[Anastassis Perrakis]

Dear all,

Post-doctoral positions are available in the Netherlands Cancer  
Institute in the group of Tassos Perrakis  (http://xtal.nki.nl).


Our group, together with that of Wouter Moolenaar and of Huib Ovaa,  
have a long-standing interest and lively collaboration in the function  
and structure of Autotaxin (ATX), a human enzyme that produces the  
well-established lipid mediator LPA.  ATX has a very promising  
potential as a drug target. Together also with our collaborators at  
Pfizer and at Kentucky (Andrew Morris) we have recently published the  
structure of Autotaxin alone and in complex with our favorite  
inhibitor, as well as data that argue for an interaction of ATX with  
integrins.


Structure: http://www.nature.com/nsmb/journal/vaop/ncurrent/full/nsmb.1980.html
Crystals (good fun!): 
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2935246/?tool=pubmed

Our chemistry collaborators (Ovaa group at the NKI) have also recently  
published data on potent inhibitors that act in vivo, as well as an  
activity-based probe.


Best Inhibitor: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2867685/?tool=pubmed
Inhibitor design: http://pubs.acs.org/doi/abs/10.1021/jm1005012
Activity based probe: 
http://onlinelibrary.wiley.com/doi/10.1002/cbic.201000349/abstract;jsessionid=D15B330C3E290F27688458F751F7FC11.d03t02

We are looking for highly motivated post-doctoral scientists to work  
on projects related to the function of Autotaxin, aiming to understand  
how Autotaxin activity regulates the production of LPA and how we can  
design more potent inhibitors and activity based probes. The project  
includes  protein and mutant production, crystallographic studies and/ 
or cell biology analysis, and is funded by the KWF (Netherlands Cancer  
Fund) and the NWO (Netherlands Science Organization).


The Netherlands Cancer Institute (http://www.nki.nl) is a center of  
excellence with a high standard of biological research and an  
interactive international atmosphere. It is located in Amsterdam, with  
all its cultural amenities, close to Schiphol airport. You will join a  
lively team, closely associated with that of Titia Sixma in the  
Department of Biochemistry and next to the NKI Protein facility.


http://xtal.nki.nl/
http://proteinfacility.nki.nl/

The research teams at B8 are closely associated and have an interest  
in structural studies coupled to functional analysis, combined with  
activities in method development for structural biology. Our  
department also hosts the newly established Protein Facility with  
equipment that includes high throughput crystallization and automated  
crystal visualization robotics both for nano-drop and Fluidigm micro- 
fluidics experiments, an X-ray facility (with Micro-star Ultra source  
from Bruker and a MarDTB), Biacore surface plasmon resonance,  
isothermal titration calorimetry (ITC), static light scattering  
(MALLS) , Thermofluor, an automated protein expression testing  
platform, a mass-spectrometry unit, as well as facilities to express  
protein in E.coli, insect and mammalian cells.


We are looking for enthusiastic researchers, which could have  
experience in protein crystallography and/or molecular biology and/or  
biochemistry and/or cell biology. Applicants should send an e-mail  
including a formal CV, a very short motivation letter for why they  
want to work on this project, and include the names of three referees,  
and send it to me.


Tassos

Re: [ccp4bb] brute force MR

[Anastassis Perrakis]

If the two molecules are related by translational symmetry, at least  
Phaser is not happy.
Since in the second rotation function it removes contribution from the  
first orientation,
if the orientation/rotations of the two molecules in identical, it  
will fail to see the

second rotation applies.

Thus if you have pure translation symmetry, which must be manifested  
with a very high peak

in the native Patterson map, you can either:

1. RTFM of Phaser and supply a list with the first solution and the an  
identical rotation, and ask

it to find the second translation
2. Apply the second translation by hand from the native Patterson (as  
you more or less did empirically)
3. 'Fool' Phaser by telling it you look for two different molecules,  
first the one and then the other.


Tassos

On Dec 7, 2010, at 22:38, Arnon Lavie wrote:


Hi there:

The situation: We are facing difficult molecular replacement: we  
believe
we have two molecules in the ASU, but phaser/molrep find only one.  
Using

the electron density calculated using this single molecule, we have
manually placed  the 2nd molecule, albeit not good enough for rigid  
body

refinement.

Our strategy: We are looking for a program to do a 3 dimensional  
search

around the current position of the 2nd molecule. Maybe one that
calculates R-factor at the different positions, to allow to identify  
the

correct one. ...

Does anyone know of such a program, or an alternative approach?

Thanks.

Arnon

--
***
Arnon Lavie, Professor
Dept. of Biochemistry and Molecular Genetics
University of Illinois at Chicago
900 S. Ashland Ave.
Molecular Biology Research Building, Room 1108 (M/C 669)
Chicago, IL 60607
U.S.A.
 Tel:(312) 355-5029
 Fax:(312) 355-4535
 E-mail: la...@uic.edu
 http://www.uic.edu/labs/lavie/
***


P please don't print this e-mail unless you really need to
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Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] diffcult MR case

[Anastassis Perrakis]

Dear Xiuwen,

First, I cant see how the space group is C2221 with a beta of 92.3

Maybe you need to clear the symmetry confusion before getting  
additional pseudo-symmetry suggestions.


A.



On Nov 29, 2010, at 14:16, xiuwen zhang wrote:


Dear colleages,

Currently I met problem in a MR case and hope somebody could  
advise me.


The crystals belong to sg C2221 with the cell parameters a=59A,  
b=302A, c=97A, beta=92.3. The searching model shares 43% identities  
(300aa) with target protein. When searching in PHASER program, only  
one clear solution could be found with high Z-score: RZF=9.8 and  
TZF=24.2. However, though the density is interpretative, the  
solution coordinate could not fit in the density (Rfactor high to  
54%). Moreover, as suggested by matthews_coff, there should be at  
least 2 molecules in one ASU. But I could not find the other  
molecules in the ASU in this case (though the other solution are  
defined with high Z-score RZF=7.8 and TZF=59, this solution has 115  
clashes with the first one). I also find that this solution  
coordinate occupied some layers and most of the unit cell are lack  
of density. As Phenix.xtrage suggested the data is not twinned, I  
was totally confused by this situation.


I will appreciate if you could share your opinions and  
experience on similar case with me.


Best Regards,
Xiuwen


P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] Citations in supplementary material

[Anastassis Perrakis]

Dear John,

I did not have the IUCr journals specifically in mind while making  
these remarks.
Quite the contrary, I think due to you and other colleagues and  
friends, they are
run competently and to the benefit of the community and of science at  
large.


If I may though offer an opinion about the peer review system in IUCr  
journals,
I personally find the concept that the authors know the identity of  
the managing
editor, wrong. I am sure that in the majority of cases its not a  
problem,
but often a managing editor can hesitate to communicate a negative  
referee report
to e.g. an old colleague or good friend, whose manuscript she/he is  
handling,

even when one of the referees is negative.

I much prefer the system of e.g. Proteins (where I act as a managing  
editor),
where authors never learn the identity of the managing editor far more  
comfortable
(there is a few people that I would rather prefer if they don't know  
for sure that I rejected their paper),

and the current system of PNAS were you learn the identity of the
editor only if your paper is accepted and after is published, far  
superior (you make friends but not enemies ...)


I would not mind to had seen IUCr journals adopting a similar system,  
I think it would improve even further

their good reputation.

A.


On Nov 19, 2010, at 12:32, John R Helliwell wrote:


I don't wish to vear away from Victor's thrust with starting this
thread and I would happily sign the petition you suggest.

But I feel I should respond to the assertions about 'problems of peer
review' at least with respect to Journals of my experience.
Some 'Editor handling of submissions' statistics should help quantify
such matters. These are a matter of public record re my IUCr Journals
submission handling statistics ie therefore not confidential and which
basically are:-
approx 1000 article submissions;
my rejection rate 20%;
appeals against my rejections 0.5%;
As Editor in Chief of Acta Cryst between 1996 to 2005 I received three
appeals (out of approx tens of thousands of submissions through all
Coeditors); I rejected these three. [My judgements were confidential
re the details.]

I can add that for the 2000 referees' reports or so for my article
handling of submissions, that colleagues have kindly supplied to my
Editor requests, problems involve:-
about 1% where the report is 'publish as is' AND without any
commendation given; these are in effect not terribly useful reports to
me as an Editor. Another problem, which is growing, is the number of
declines to my invites to referee (around 10%). Even worse are the no
replies at all from invited referees as time is lost to the authors
who rightly expect as prompt as possible handling.

Re your points I offer replies as follows:-
Let me outline what I think are problems of peer review:

1. 'review by last author name'. Very often the last author is well
known, or a friend, and the reviewers' critical judgement takes a
temporary leave of abesnse.
JRH reply:- Such reports would be easy to spot and are not a problem
in my experience and so resort to double blind review is not necessary
in my experience.

2. 'preferred reviewers'. a double edged sword .. think about it.
JRH reply; these are not so commonly offered suggestions by authors in
fact and where they are one can follow or decide against (see point
1).


3. too much power of decision on editors (professional or academic)
being able to reject papers without peer-review in many journals.
JRH reply;This approach, 'insufficent general interest' is for the
magazines we know and yet still love.

4. Bad refereeing - sometimes I wonder if people read the paper.
JRH reply;Such reports are very few and obvious. The other categories
above are more common (ie 'publish as is' category).

5. Lack of referee expertise: you get papers these days with: a
structure, some biochemistry, some SAXS, some biophysics, and a cell
based assay. Two or three people being
able to pick up all the mistakes is very unlikely.
JRH reply; Papers can be challenging re content and your example here
is a good one. Other chalenging cases are where they include a lot of
maths. That said peer review does its best but can occasionally fail;
this level of failure can be measured by the number of criticism
articles or formal retractions. These are also very few, but it is
true, not zero.

Yours sincerely,
John



On Thu, Nov 18, 2010 at 10:47 AM, Anastassis Perrakis a.perra...@nki.nl 
 wrote:


On Nov 18, 2010, at 11:18, James Stroud wrote:

The future of publishing will be

(1) Publish your own work
(2) Peer review by the entire community

Although I have been remarkably bad at predicting the future, I  
still like

attempting to do so ...!
This will not happen ...! ;-)
To be honest, I am not even sure its a great idea ...
Let me outline what I think are problems of peer review:
1. 'review by last author name'. Very often the last author is well
known, or a friend, and the reviewers' critical judgement

Re: [ccp4bb] Citations in supplementary material

[Anastassis Perrakis]

On Nov 18, 2010, at 11:18, James Stroud wrote:


The future of publishing will be

(1) Publish your own work
(2) Peer review by the entire community


Although I have been remarkably bad at predicting the future, I still  
like attempting to do so ...!

This will not happen ...! ;-)

To be honest, I am not even sure its a great idea ...

Let me outline what I think are problems of peer review:

1. 'review by last author name'. Very often the last author is well  
known, or a friend, and the reviewers' critical judgement takes a  
temporary leave of abesnse.

2. 'preferred reviewers'. a double edged sword .. think about it.
3. too much power of decision on editors (professional or academic)  
being able to reject papers without peer-review in many journals.

4. Bad refereeing - sometimes I wonder if people read the paper.
5. Lack of referee expertise: you get papers these days with: a  
structure, some biochemistry, some SAXS, some biophysics, and a cell  
based assay. Two or three people being

able to pick up all the mistakes is very unlikely.

Having outlines these, I can see ways that all can be amplified if you  
just publish all your work, and anybody can comment on it:

Pairing to the above problems, you just amplify them:

1. Even more tempting to earn brownie points online!
2. you can ask your friends or I can ask your enemies to review
3. the other way around: far too many things out... how to filter ?
4. Lack of 'obligation', or even fear to make yourself look like a  
fool to the editors, will make commenting even more sloppy
5. People that think they are experts dwell on meaningless  
technicalities.


Peer review is like democracy, its the worst publication system we can  
have, except the ones that have been tried or suggested ...


A.



(3) Citation = Link

#3 makes it work.

Give it 25 years. The journals won't be in the position to lobby  
lawmakers to prevent this trend if we make sure the journals die so  
slowly that they don't realize it.


James


On Nov 18, 2010, at 1:14 AM, John R Helliwell wrote:


Dear Jacob,
Your posting reminds me of a Research Information Network  
Conference I

went to in 2006 in London.
Your views coincide with a presenter there, Peter Mika.
His talk can be found at:-
http://www.rin.ac.uk/news/events/data-webs-new-visions-research-data-web
In his talk he referred to:- openacademia.org
Peter Mika and I were on the Closing Panel; he advocated that
refereeing is an imposition on a researcher's
individual freedom and thus he/she should 'publish' their work on
their own website. By contrast, I argued in favour of
Journals and peer review, both with respect to my articles and my
experiences as an Editor of more than one Journal.

I would be happy to continue corresponding on this not least as
publication should be a varied spectrum of options.
Also I feel obliged to say that one cannot apply simply, by rote,
'Learned Society publisher is good', 'commercial publisher is bad';
there are exceptions in both camps. [in effect this was the tone of  
my

last posting.]

Greetings,
John

On Wed, Nov 17, 2010 at 8:13 PM, Jacob Keller
j-kell...@fsm.northwestern.edu wrote:
I guess the practice of being on your best behavior is good in  
terms

of getting the research trimmed into shape, but there is a huge
temptation to fudge things to get published, and to hide unpleasant
artifacts, as can be seen by the many recent (and not so recent)
scandals. Maybe as a lab website things would be more open. Also,
having a comments section always seemed like an excellent idea to  
me,

even for journals as they are, but would be really easy to implement
in a website. I would love to read comments from others in the field
about the papers I read, as sometimes people can help to point out
gaping holes where one might not see them otherwise. It would be  
like

journal club for the whole scientific community.

Jacob

On Wed, Nov 17, 2010 at 2:08 PM, Jrh jrhelliw...@gmail.com wrote:

Dear Jacob
Re journals out of the window:-
Well, like democracy, journals may not be ideal but I believe  
other alternatives such as free for all personal website  
publishing, are worse. So, journals that are community driven  
offer an optimal approach, critically based on specialist peer  
review. That is why our community effort IUCr Journals I believe  
are so important. Open access, where we can sustain it  
financially, also can convey access to the widest readership ie  
that the high impact magazines currently, mainly, command.

All best wishes,
John
Prof John R Helliwell DSc


On 17 Nov 2010, at 18:28, Jacob Keller j-kell...@fsm.northwestern.edu 
 wrote:


Supplementary info seems to me to be a double-edged sword--I  
just read
a Nature article that had 45 pages of supplementary info. This  
means

that you get a lot more for your money, but all of the methods and

Why not have papers be as long as the authors want, now that  
almost

everything is internet-based? It would make the papers much more

Re: [ccp4bb] Citations in supplementary material

[Anastassis Perrakis]

I like the online methods sections with e.g. Nature papers that also  
come with the pdf - they also count for citations, the citations are  
not within the manuscript allowance on numbers, they get peer  
reviewed and they actually leave quite a lot of space for readers to  
understand the experiments and for authors to cite methods properly.  
Easy fix in my mind.


What Poul says about Nature, which is also my understanding, is in  
sharp contrast with the Cell group policy:



Supplemental References
Literature citations from within the Supplemental Information and  
unique to the Supplemental Information should be listed as  
Supplemental References. References that are already included in the  
main body of the paper should not be listed again. Please note that  
as references in Supplemental Information do not contribute towards  
citation measures for those papers, authors are encouraged, when  
possible, to include references in the main body of the paper.


My experience with Elsevier, is that they like to listen to feedback  
and appreciate it.


Nature seems to have fixed the problem with citations in Methods in a  
proper manner (which is largely true for all NPG journals, I think, at  
least NSMB will also do the Online Methods trick!).
So, an important step would be that we as a community, take the steps  
to get everybody to follow that.


So, lets start a petition?
Maybe Victor that started this important thread could champion this  
and see it to an end !!!


A.

Re: [ccp4bb] expression of Cys-rich small protein

[Anastassis Perrakis]

 We are trying to express for structural studies a 257 AA  
eukaryotic intracellular [...]


As you describe about your protein, I guest your protein may  
required disulfide bonds to be folded correctly


As Laurie described her protein is intra-cellular, so it will not need  
disulfide bonds, unless it has some really weird compartmentalization.


To add to the nice tricks about E.coli strains forming disulfide  
bonds, if your institute is well-equipped for cell culture, just  
secret it from HEK293 cells.
Its easier and cheaper than what most people think - all you need is a  
good person to show you around the basic tricks, a hood, and an  
incubator,

which most places have anyway.

A.


On Nov 16, 2010, at 18:22, van dat nguyen wrote:


Hi Laurie,

What E. coli strain did you use? As you describe about your protein,  
I guest your protein may required disulfide bonds to be folded  
correctly. E. coli cytoplasm is a reduced environment, which is not  
suitable to make disulfide bonded proteins.  to solve this problem  
it is recommended to use E. coli strain which a less reduced  
cytoplasm, ex SHuffle® T7 strain (from NEB) or Rosseta Garmi strain,  
or express rich Cysteine proteins in the periplasm of E. coli. using  
those strain with co-expression of Protien Disulfide Isomerase will  
be good to try. However, Recently our group have made a very good  
system in E. coli to expressed disulfide bonded protein in E. coli  
cytoplasm, please have a look at this paper:


http://www.ncbi.nlm.nih.gov/pubmed/20836848

A better system will be published soon.

Best Wishes,

Dat

On Tue, Nov 16, 2010 at 6:13 PM, Laurie Betts laurie.betts0...@gmail.com 
 wrote:

All -

We are trying to express for structural studies a 257 AA eukaryotic  
intracellular (also possibly nuclear) protein (predicted to be  
single domain all-helical) that has 12 Cysteines.  No known metal- 
binding function not that it couldn't happen.  So far (E. coli) it  
expressed solubly as MBP fusion (with an N-terminal region deleted  
predicted disordered) until cleavage of MBP, then it's not soluble,  
including detergents added.  THe MBP fusion is usually soluble  
aggregate so we assume that our part is not folded right.  We have  
so far assumed it needs a lot of reducing agent (5 mM DTT or  
TCEP).Thinking of trying chaperones and insect cells next.


Any experience out there that might help?  Mostly I wonder about all  
the cysteines.  Don't really know if that is the problem.


Laurie Betts



P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] Question on calculation of RMSD

[Anastassis Perrakis]

My favorite tools for doing this are:

http://webapps.embl-hamburg.de/rapido/

and/or

http://www.theseus3d.org/

Reading some of the associated papers will not hurt inchoosing when to  
apply which one!


A

On Nov 14, 2010, at 22:52, E rajakumar wrote:


Dear All
I have two structures of homo-dimeric protein complex with different  
DNA.
I want to calculate RMS deviation between second monomer from these  
two complexes by fixing superposed first monomer.


This I require to know what is the effect of DNA on relative  
orientation of two monomers in the dimer.


Previously I was using MOLEMAN2 to do this calculation.

Please can you suggest me any other program to do this calculation.

Thanking you
Raj


E. Rajakumara
Postdoctoral Fellow  Strcutural Biology Program  Memorial Sloan- 
Kettering Cancer Center  New York-10021  NY  001 212 639 7986 (Lab)   
001 917 674 6266 (Mobile)





P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] High Rmerge with thin frames

[Anastassis Perrakis]

three additional points:

1.

OTOH, if The diffraction is quite weak, one may be limited by  
counting

statistics.  This also cannot be overcome by processing.


As JIm suggests above then, maybe you should look if the 15% Rmerge is  
almost reasonable given the specific I/sigI at low resolution?



2. If there is one thing I do not like in XDS, is that there is no (or  
I have failed to find) statistics of I/sigI and Rmerge as function of  
image.

Have a look at the SCALA output. Maybe some images are bad?

3. making too fine slices of too weak diffraction images ends up with  
either too weak counting statistics or inability to 'lock' the  
refinement.
we did that for one crystal form, collecting 0.1, 0.2, 0.35, 0.5, 0.7,  
1.0 from various crystals (with the same dose per degree, at SLS using  
a PILATUS, mosaicity 0.4-0.6) in an attempt to get better Se signal.  
We miserably failed to get any useful signal at the end, but learned  
that for these very weak diffracting plates (submicron) collecting  
0.5-1.0 degrees was actually giving at the end better data.


A.

Re: [ccp4bb] High Rmerge with thin frames

[Anastassis Perrakis]

On Nov 5, 2010, at 16:57, Ronnie Berntsson wrote:


Dear Tassos,

I'm interested in your third point. Do you have any explanation for  
why 0.5-1 degrees oscillation gave better data? Purely due to the  
fact that the crystals survived longer and thus yielded higher  
redundancy data, or also other parameters?


No, x-ray beam survival had nothing to do with this. The dose was the  
same per degree, so the damage was the same in all, at least on  
principle, and from what I see in practice.


I simply think that the very low partiality of all reflections ends up  
with counts that are just above the noise (even for a well set up  
experiment and an excellent detector). The diffracting volume of these  
crystals in some orientation was really small! 1x40x70 microns, only,  
so the signals were very low. I think that the end integration goes  
wrong because images cannot be well refined with so low-count data. As  
soon as the reflections were stronger, things 'catch up'.


Also do anyone know where the threshold lies for when not to use  
fine phi slicing on the PILATUS? ie, at what level of diffraction  
would one need to increase the exposure (and oscillation in order to  
still get redundant data)?


In general, the slicing of the Pilatus works great for us. My only  
negative experience was really really small crystals.


We'll be in a similar position in the coming weeks with data  
collection using PILATUS detectors, and would like to maximize the  
potential data quality from our weak diffracting crystals. Any input  
on this would be greatly appreciated!


I would aim to be able to see nice spots, to at least 3.5 A: these  
would be enough to 'lock' the orientations, and I would expect either  
XDS or MOSFLM to integrate even very low signals.


A.



Cheers,
Ronnie Berntsson



On Nov 5, 2010, at 16:16, Anastassis Perrakis wrote:


three additional points:

1.

OTOH, if The diffraction is quite weak, one may be limited by  
counting

statistics.  This also cannot be overcome by processing.


As JIm suggests above then, maybe you should look if the 15% Rmerge  
is almost reasonable given the specific I/sigI at low resolution?



2. If there is one thing I do not like in XDS, is that there is no  
(or I have failed to find) statistics of I/sigI and Rmerge as  
function of image.

Have a look at the SCALA output. Maybe some images are bad?

3. making too fine slices of too weak diffraction images ends up  
with either too weak counting statistics or inability to 'lock' the  
refinement.
we did that for one crystal form, collecting 0.1, 0.2, 0.35, 0.5,  
0.7, 1.0 from various crystals (with the same dose per degree, at  
SLS using a PILATUS, mosaicity 0.4-0.6) in an attempt to get better  
Se signal. We miserably failed to get any useful signal at the end,  
but learned that for these very weak diffracting plates (submicron)  
collecting 0.5-1.0 degrees was actually giving at the end better  
data.


A.




P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] High Rmerge with thin frames

[Anastassis Perrakis]

In the Pilatus mode these are open-shutter experiment, where the  
Pilatus integrates over different times
- all these exposure times are slower than the frequency of the  
detector, as far as I understand the setup.


So, the crystal gets the 'full blast' in all cases, and the blast is  
the same for the same rotation, as we did the experiments.


btw, I have the suspicion, that for our system, it was better to do  
'100 sec' in the pilatus, than 100x1 sec and read for 3-4 secs
with a CCD in between, but I have no rigorous proof for that. Its just  
an observation that should be treated with caution.


Tassos

On Nov 5, 2010, at 17:11, Jacob Keller wrote:


3. making too fine slices of too weak diffraction images ends up with
either too weak counting statistics or inability to 'lock' the
refinement.
we did that for one crystal form, collecting 0.1, 0.2, 0.35, 0.5,  
0.7,
1.0 from various crystals (with the same dose per degree, at SLS  
using  a
PILATUS, mosaicity 0.4-0.6) in an attempt to get better Se signal.   
We
miserably failed to get any useful signal at the end, but learned   
that
for these very weak diffracting plates (submicron) collecting   
0.5-1.0

degrees was actually giving at the end better data.



Perhaps the reason for the better data was an instance of the
redundancy-vs-long-exposure dilemma. Given, say, 1deg exposures:  
should one

collect 500x1s exposures or 250x2s exposures? I think this has been
examined, and while it does depend on the details of the parameters,  
it is
often better to collect 250x2s exposures because there is a flat  
rate per

frame noise level in the detector. I am wondering whether you just
increased the number of flat rates in your data sets by increasing  
the

number of frames while keeping the exposure/degree equal?

JPK



P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] DLS

[Anastassis Perrakis]

hear-hear

wise words.

may I add that if you are buying a DLS anyway, maybe consider getting  
a model which can also be connected online to an FPLC to do Static LS  
measurements,
... after all a DLS signal is basically an autocorrelation of the  
static signal over time ... which will tell you about dispersion,
but is incapable of measuring MW, and can only get Rg, unlike static  
LS (MALLS) that will give you absolute MW over your FPLC - which is  
handy.


A.


On Oct 30, 2010, at 20:10, Artem Evdokimov wrote:

I second the question of need: a decent PCR machine capable of  
'thermofluor'-like experiments should cost around $34-$38K, which  
leaves about the same amount of $$ for a decent purification  
machine. One of the questions you have to figure out is: are you  
setting up a high-throughput lab or a regular kind of lab? If you're  
doing HT work then perhaps a plate-based DLS is a good idea,  
although you will undoubtedly need more $$ for the rest of the  
stuff. On the other hand if you're setting up to do 'regular' i.e.  
low-throughput (I prefer the term 'hand crafted', by the way)  
purification then an investment in a Thermofluor machine (be it PCR  
or a multiwell spectrofluorimeter with temperature control) and  
another useful device might be more wise.


Having written this, I would have to add for the sake of honesty  
that my lab has both - but we are in fact straddling the boundary  
between HT and hand-crafted work and we have use both instruments  
daily.


Artem

On Fri, Oct 29, 2010 at 11:02 PM, Mohd Shukuri Mohamad Ali shuk...@biotech.upm.edu.my 
 wrote:

Hi there

Sorry for the off ccp4 topics. I am planning to get a DLS to set up my
crystallography lab. I got a tight budget. Around USD 8 perhaps.  
I'm

quite new to this field.

Thanks in advance for your suggestions

Regards
Shukuri



P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] Fill map/mask with dummy atoms?

[Anastassis Perrakis]

Apologies for not seen the original post, but

$warpbin/arp_warp  EOF
MODE MIRBUILD
FILES CCP4 MAPFIND 2fofc.map XYZOUT1 dummy.pdb
SYMM your_sym
CELL your_cell
RESOLUTION resol
MIRBUILD ATOMS atoms_in_protein MODELS 1 RESN DUM
END
EOF

will do it.
I recommend getting a map in a 0.3 A grid.

A.

and fill up the keywords
On Oct 25, 2010, at 21:16, Pavel Afonine wrote:


Hi Dirk,

may be too late... but (may be) better later than never -:)

Here is the working example of how you can do it. Note, the  
procedure just builds the dummy atoms in spheres with user-defined  
centers and radia. You can specify as many spheres as you wish.  
Dummy atoms clashing with model atoms or other dummy atoms will not  
be added. The procedure doesn't care about map or data (Fobs or  
whatever): it just geometrically adds dummy atoms where requested.  
Also note, it uses a PHENIX command line tool that is not  
specifically designed for this task but simply can do it with  
appropriate set of parameters.

Ok, that was a preambula -:) Now let's do it:

here is where all the example-files:
/net/cci/afonine/public_html/for_Dirk

The command

phenix.grow_density params

will creates this file with dummy atoms: dummies_DA.pdb

which in PyMol looks like this:

http://cci.lbl.gov/~afonine/for_Dirk/da_only.png

or superposed with the model:

http://cci.lbl.gov/~afonine/for_Dirk/da_plus_model.png

Note, the above command requires the data file (remember, this  
command is meant for something else), but if you have just a PDB  
file (it can be empty I guess), and don't have any data file, you  
can fake it just to run this command. To get fake Fobs:


phenix.fmodel model.pdb high_res=3 type=real r_free=0.1 label='F-obs'
mv model.pdb.mtz data.mtz

I guess this is it. Let me know if I can be of any help with this.

Pavel.


On 10/13/10 4:00 AM, Dirk Kostrewa wrote:


 Dear CCP4ers,

is there a program around that allows to fill an input map or mask  
with dummy atoms?


Best regards,

Dirk.

--

***
Dirk Kostrewa
Gene Center Munich, A5.07
Department of Biochemistry
Ludwig-Maximilians-Universität München
Feodor-Lynen-Str. 25
D-81377 Munich
Germany
Phone: +49-89-2180-76845
Fax: +49-89-2180-76999
E-mail:kostr...@genzentrum.lmu.de
WWW:www.genzentrum.lmu.de
***




P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute,
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] R factor R free struck

[Anastassis Perrakis]

A handy way to do what Paul suggests is the Zanuda server at York  
which will try refinement in all lower symmetries ...


From the first email though two things are unclear:

1. How good was the Phaser solution (eg z-score?) ?
2. Did you only try rigid body fitting as you wrote? (I find regid  
body after Phaser frankly useless)


Anyway, what I would do would be to:

a. get the latest version of refmac before doing anything else, and do  
full XYZB refinement, not rigid body, with tight NCS restraints
b. if that does not drop Rfree to say below 45%, submit to Zanuda to  
check symmetry as Poul suggests - do that anyway in fact!
c. if zanuda gives lower symmetry, reprocess your data in lower  
symmetry, run Phaser again, run refmac again, check for twining


Maybe I will go now on preaching the wrong gospel for this BB,
but Buster and/or Phenix are extremely useful at that resolution range.
I must say that Buster sometimes has been really impressive in our  
hands.


A.



On 3 Oct 2010, at 9:09, Poul Nissen wrote:

Try a lower symmetry, e.g. P21 or P1 with one or two octamers in the  
asymmetric unit (well, unit cell for P1), but you already know your  
packing so no problem solving it.
It can sometimes be very subtle and your model refinement will be  
the most sensitive test for the correct space group - so don't be  
misled by seeming perfect scaling in orthorhombic.


Poul
On 03/10/2010, at 04.56, Jack Russel wrote:


Hi all,

I have collected  a data at 2.9 Å and the solved the structure  
using phaser . the space group comes to be P2 21 21. There are 4  
molecules in Assymetric unit and an octamer is generated according  
to the symmetry. But after repeated rounds of rigid body refinement  
with REFMAC5 and model building with coot  the R factor had been  
struck at 40% and R free at 50%.


So my first question is whether my solution after phaser is  
correct. And if it is how can i lower the  R factor and Rfree.


The second question is it possible to have such a large difference  
between R factor and R free.


Thanks in advance

Re: [ccp4bb] Protein melting temperatures

[Anastassis Perrakis]

Hello -

The excellent paper of McCrary, uses differential scanning  
calorimetry, which will give an absolute measure of thermostability.


Using Thermofluor I would be afraid you can only assess the relative  
thermostability of one protein in different conditions.
As your fluorescence reporter would interact differently with exposed  
hydro[hobic patches in different proteins, I would be a bit more careful
in comparing the Thermofluor results between different proteins ... I  
am not aware of anyone correlating differential scanning calorimetrywith
Thermofluor data, but I must admit I have not looked up that  
literature recently.


A.


On 23 Sep 2010, at 18:40, Philippe DUMAS wrote:


Le 23/09/2010 17:28, Raji Edayathumangalam a écrit :

Raji
I suggest having a look to this paper:
McCrary et al. J. Mol. Biol. 264(1996) 784
where you will find an interesting study on protein stability and an
interesting comparison with other proteins.
Philippe Dumas


Hi Folks,

Sorry for the pre-xtallo question; pre-xtallo right now, but hoping  
to

take my protein the xtallo way one of these days!

I am currently performing Thermofluor assays with my protein and the
results show that the Tm is ~45C.  I am looking for some examples of
proteins and their melting temperatures so that I can gauge where my
protein falls in the spectrum of unstable-to-stably folded. For
example, the melting temperature of some forms of lysozyme is 73.8C
(very stable, I suppose).

Just need a sense for whether my protein is considered unstable or
somewhat stable. Please could you share some examples.

Many thanks.
Raji

---
Raji Edayathumangalam
Joint Research Fellow
Harvard Medical School/
Brigham and Women's Hospital
Brandeis University



McCrary-JMB264(1996)784.pdfp_dumas.vcf