[ccp4bb] FW: [ccp4bb] Assistant Professor in Structural Biology, University of Nebraska-Lincoln

[Phoebe A. Rice]

Another point of confusion worth pointing out:


  *   Many US medical-school-affiliated faculty positions are “12 months” but 
you may be expected (and heavily pressured) to recover well over 50% of your 
salary from your grants.  The fine print varies widely as to what you will be 
paid if you fail to recover enough salary from grants.  Fortunately for me, my 
institution pays the full salary anyway but that is not true everywhere – some 
institutions are totally “soft money” (100% of that salary they offered you has 
to come from your own research grants).



  *   Many US “arts and sciences” schools (that is, not affiliated with a 
medical school) offer 9 months salary, BUT they will probably never ask you to 
recover more than that that 3 months’ worth of “additional” summer salary from 
your grants, which means you can do more science with your grant money.



  *   Be sure to ask about the fine print as soon as they offer you the job!  
It isn’t obvious – for example, here Biochemistry is affiliated with the med 
school and is on a 12-month plan, but Chemistry is not affiliated with the med 
school and is on a 9-month plan.  Some universities have two biochemistry 
departments, one on each plan.  Don’t assume logic.

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


From: CCP4 bulletin board  on behalf of Hekstra, Doeke 
Romke 
Date: Sunday, October 15, 2023 at 2:45 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Assistant Professor in Structural Biology, University of 
Nebraska-Lincoln
Hi everyone,

When I was looking for faculty positions, I completely misunderstood this 
language too. I didn’t apply to some places because of this odd “9-month” 
language, only to realize I was getting a 9-month salary after all!

Mark is, of course, right that this practice is nearly universal, but it would 
help to reconsider our collective terminology or better explain this in job 
postings.

Best, Doeke

=

Doeke Hekstra
Assistant Professor of Molecular & Cellular Biology, and of Applied Physics 
(SEAS),
Director of Undergraduate Studies, Chemical and Physical Biology
Center for Systems Biology, Harvard University
52 Oxford Street, NW311
Cambridge, MA 02138
Office:617-496-4740
Admin:   617-495-5651 (Lin Song)



From: CCP4 bulletin board  On Behalf Of Mark Wilson
Sent: Sunday, October 15, 2023 12:05 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Assistant Professor in Structural Biology, University of 
Nebraska-Lincoln

Hi Gerlind,
To clarify, the position is tenure-leading, where tenure dossiers are typically 
submitted in the 5th year of employment.  If tenure is granted, then the 
position is a continuous appointment.  The 9-month language in the ad refers to 
the academic year-typically faculty can either choose to take their salary over 
either 9 or 12 months.   Faculty can also raise additional funds for salary 
over the summer via grants.  This is common (nearly universal) in the US 
system. Importantly, this is _not_ simply a 9-month contract position, which 
would not make sense for an Assistant Professor.

Best regards,
Mark

Mark A. Wilson (he/him)
Professor
Department of Biochemistry/Redox Biology Center
University of Nebraska
N118 Beadle Center
1901 Vine St.
Lincoln, NE 68588

From: Gerlind Sulzenbacher 
mailto:gerlind.sulzenbac...@univ-amu.fr>>
Date: Sunday, October 15, 2023 at 10:15 AM
To: Mark Wilson mailto:mwilso...@unl.edu>>, 
CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] Assistant Professor in Structural Biology, University of 
Nebraska-Lincoln
Non-NU Email

Dear Mark,

thank you for posting this job offer, probably very interesting for people 
currently unemployed.

Personally, I feel that working conditions and job offers in the scientific 
field have deteriorated considerably in recent years.
I've always been shocked by one-year job offers involving international 
movements.
Now we seem to be down at job offers for 9 months.

>From a human point of view, I find this extremely worrying.
Sorry to pollute the mailing list with my personal opinions.

All the best.
Gerlind


On 15/10/2023 16:46, Mark Wilson wrote:
Dear Colleagues,

The Department of Biochemistry at the University of Nebraska-Lincoln (UNL) 
invites applications for a tenure-track nine-month (academic year) faculty 
position at the rank of Assistant Professor. Areas of particular interest 
include but are not limited to time-resolved approaches to understanding 
macromolecular function, multiscale imaging, and integrated computational and 
experimental approaches to structural biology. Researchers at UNL have 
collaborations with national user facilities pioneering time-resolved X-ray 
diffraction techniques and have established a state-of-the-art cryo-EM facility 
housing a Thermo 

Re: [ccp4bb] the structures of Nucleic acid

[Phoebe A. Rice]

For complex RNA structures,

  *   Molecular replacement can stumble
  *   If made by simple T7 transcription methods (the most common way), the RNA 
would have EVERY U replaced with 5-bromo-U
  *   Adding bromine to the 5 position of U could affect the structure (it 
would be replacing -H not -CH3)

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


From: CCP4 bulletin board  on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
Date: Monday, September 18, 2023 at 4:44 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] the structures of Nucleic acid
I am afraid most scientists will use the most straightforward technique!
If SAD is available the PHOSPHATE backbone of DNA will provide sufficient 
signal to allow SAD to work, and you get an unambiguous answer to whether it is 
A-DNA or B or Z...
MR will usually work of course as well
Eleanor


On Mon, 18 Sept 2023 at 09:18, Natesh Ramanathan 
mailto:nat...@iisertvm.ac.in>> wrote:
Dear Fu Xingke,

 Depends on what Nucleic Acid you are talking of.  If it is RNA, you 
can expect some sequence to tertiary structure correspondence so you might be 
able to try more MR as compared to DNA.   DNA may have double helical 
architecture but less sequence to tertiary structure correspondence, and hence 
DNA is less likely to have a 3D structure like RNA specific structure for a 
sequence.

 SAD has become a straight forward method to avoid all these problems 
to get ab-initio structure.  So many go for it directly.

Hope that helps.
Best wishes,
Natesh

On Mon, 18 Sept 2023 at 13:36, fuxingke 
mailto:fuxingke0...@163.com>> wrote:
Dear Colleagues,
 Reacently, I find the structures of Nucleic acid are solved by 
single-wavelength anomalous diffraction(SAD). So, why molecular replacement 
(MR) not?

Regards



Best wishes,
Fu Xingke
Institute of Physics CAS



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--
--
"Live Simply and do Serious Things .. "
- Dorothy Mary Crowfoot Hodgkin OM, FRS

"In Science truth always wins"
- Max Ferdinand Perutz OM FRS
--
Dr. Ramanathan Natesh
Associate Professor,
School of Biology and Center for High-Performance Computing (CHPC),
Founding and Current President of Cryo Electron Microscopy and 3 Dimensional 
Image Processing Society of India (CEM3DIPSI),
Indian Institute of Science Education and Research Thiruvananthapuram 
(IISER-TVM),
Maruthamala P.O., Vithura,
Thiruvananthapuram,  695551, Kerala, India

nat...@iisertvm.ac.in<mailto:nat...@iisertvm.ac.in>
http://faculty.iisertvm.ac.in/natesh

Researcher ID: http://www.researcherid.com/rid/C-4488-2008
ORCID: http://orcid.org/-0002-1145-5962
Vidwan-ID : 94134: http://iisertvm.irins.org/profile/94134
PUBLONS: https://publons.com/author/1520837/ramanathan-natesh#profile

Office Ph. 0091- 471-2778087



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Re: [ccp4bb] Future Diffraction Methods

[Phoebe A. Rice]

Excellent idea.

From: CCP4 bulletin board  on behalf of Debanu Das 

Date: Saturday, December 24, 2022 at 5:57 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Future Diffraction Methods
Consider merging with the Annual ACA meeting. The ACA meeting can also benefit 
from more X-ray diffraction methods rigor and training and it will help to 
elevate the continuity and history of the ACA and its previous and current 
member participation and contributions in the field.
Best,
Debanu

On Tue, Dec 20, 2022 at 6:40 PM Orville, Allen (DLSLtd,RAL,LSCI) 
<66b6f959eb28-dmarc-requ...@jiscmail.ac.uk>
 wrote:
Hi,

Thanks James, for organising our last GRC meeting.

In the past few years, I’ve also attended one or more Annual BioXFEL 
International Conferences (open, but ending?) and Ringberg Workshop on Science 
with FELs (by invitation only). Both were excellent meetings held in the 
winter, complemented the GRC in diffraction methods, and benefited from 
relatively small attendance with very focused discussions.  I will also add 
that dynamic structural biology is a rapidly growing area that can include 
simultaneous functional, spectroscopic, and structural data collection from the 
same samples, and using synchrotron, XFEL, and/or electron sources. Moreover, 
many of the other life-science centric GRC’s also include significant 
structural data in their frontier programs and discussions (e.g. metals in 
biology, hemes and tetrapyrroles, enzymes, etc.). We are fundamental to 
biochemistry R… a broad community indeed.

AMO
~ ~ ~ ~ ~
Allen M. Orville, Ph.D.
Wellcome Investigator and Royal Society Wolfson Fellow
Principal Scientist, XFEL Hub at Diamond Light Source
Harwell Science and Innovation Campus
Didcot, Oxfordshire
OX11 0DE
United Kingdom

Phone: +44 (0) 1235 567505
Mobile: +44 (0) 7471 026061
email: allen.orvi...@diamond.ac.uk

AMO site: https://www.diamond.ac.uk/Instruments/Mx/XFEL-Hub/Staff/Orville.html
XFEL-Hub site: https://www.diamond.ac.uk/Instruments/Mx/XFEL-Hub.html





From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Frank von Delft 
mailto:frank.vonde...@cmd.ox.ac.uk>>
Date: Monday, 19 December 2022 at 09:22
To: CCP4BB@JISCMAIL.AC.UK 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: Re: [ccp4bb] Future Diffraction Methods
Shoot... that sucks.

Yes, we do need something!

"Structural Biology Methods"?


More interesting question:  does it need the GRC to host?  What about
reimagining the CCP4 study weekend - the question keeps coming up there
anyway.

That's one for CCP4 WG1 to discuss - they're meeting straight after New
Year, so maybe James, you and Ivo should hop on a zoom and kick the idea
around.

Frank



On 16/12/2022 22:10, James Holton wrote:
> I want to thank everyone who attended the 2022 Gordon Research
> Conference and Gordon Research Seminar on Diffraction Methods in
> Structural Biology, as well as all those who contributed to these
> great gatherings in the past.  It was an outstanding meeting if I do
> say so myself. Not just because it had been so long without in-person
> interaction, not just because we had zero covid cases (which I see as
> no small feat of Mind over Virus), but because of this amazing
> community. It is rare in this world to have such a strong spirit of
> collaboration, camaraderie and openness in undertakings as high-impact
> as this. Surmounting the barriers to atomic-detail imaging of
> biological systems has never been more exciting and more relevant.  I
> am proud to be a part of it, and honored to have served as Chair.
>
> It is therefore with heavy heart that I report to this community that
> I was the last Chair of the Diffraction Methods GRC.
>
> The GRC Conference Evaluation Committee
> (https://www.grc.org/about/conference-evaluation-committee/) voted
> this year to discontinue the Diffraction Methods GRC and GRS. This
> ends a 46-year tradition that I feel played a vital, and vibrant role
> in the work of the people who answer questions on this BB. The reason
> given was insufficient attendance.  All other metrics, such as
> evaluation surveys and demographics were very strong. I have tried to
> appeal, but I'm told the vote was unanimous and final. I understand
> that like so many conference organizing bodies the GRC is having to
> make tough financial decisions. I must say I disagree with this one,
> but it was not my decision to make.
>
> Many of the past and elected Chairs have been gathering and discussing
> how to replace the Diffraction Methods GRC/GRS going forward. Many
> great ideas, advice and perspectives have been provided, but that is a
> select group. I feel it is now time to open up this discussion to the
> broader community of structural methods developers and practitioners.
> There are some important questions to ask:
>
> * How do we define this community?
> Yes, many of us do cryoEM 

[ccp4bb] postdoctoral position

[Phoebe A. Rice]

[Text  Description automatically generated]
Postdoctoral Position in the Rice Lab at the University of Chicago.

Mechanism, Engineering, and Structural Biology of Large Serine Integrases
A postdoctoral position is available immediately the laboratory of Dr. Phoebe 
Rice.


Large serine integrases (LSIs; derived from lysogenic phages) mediate precise 
site-specific DNA recombination (integration, excision, or inversion), leaving 
not a single nick in the DNA backbone.  Control of LSI directionality is 
uniquely simple.  The integrase itself catalyzes phage insertion, but not the 
reverse (excision) reaction. Excision requires a 2nd protein called a 
recombination directionality factor (RDF), that binds its cognate integrase and 
alters its conformation in an unknown manner that somehow inhibits integration 
and activates excision. LSI-RDF pairs can catalyze the insertion of large 
payloads without relying on host DNA repair (unlike CRISPR-Cas-based systems) 
and can scarlessly and controllably reverse those insertions (unlike any other 
system).


This project seeks to (1) understand, through cryo electron microscopy and 
biochemistry, how an RDF interacts with its cognate integrase to control the 
reaction direction; (2) through bioinformatics and protein engineering, 
eliminate the current bottleneck in identifying cognate RDFs for individual 
LSIs and (3) enhance the usefulness of RDFs by engineering new control 
mechanisms into them.  The project is funded by a new large, collaborative NSF 
+ UKRI/BBSRC award to the Rice (https://voices.uchicago.edu/phoebericelab/) and 
Olorunniji (Olorunniji 
lab<https://cm-uat.ljmu.ac.uk/about-us/staff-profiles/faculty-of-science/pharmacy-and-biomolecular-sciences/femi-olorunniji>)
 labs.



Successful applicants will have a Ph.D. or MD/PhD in the broadly defined fields 
of Biochemistry, Biophysics, and/or Molecular Biology.  Experience with the 
tools of structural biology and / or nucleic acid biochemistry is preferred.  
Applicants should be eager to learn new skills and systems, and should have 
effective communication, teamworking and problem-solving skills.



The postdoctoral fellow will be part of the vibrant local structural biology, 
protein engineering and microbiology communities, with access to our 
state-of-the-art electron microscopy facilities 
https://voices.uchicago.edu/advancedem/.  Mentorship will be tailored to the 
fellow’s goals and can include, for instance, participation in the University’s 
MyChoice career development program and guided mentorship of diverse 
undergraduate students.


To apply, email Dr. Rice at pr...@uchicago.edu and include a cover letter 
explaining relevant work experience and interest in this position, a CV and the 
contact information of 2-3 references.

Compensation in the Biological Sciences Division follows the NIH NRSA Stipend 
scale. Additional information on benefits and being a postdoc in the University 
of Chicago Biological Sciences Division can be found at 
https://bsdpostdoc.uchicago.edu/.

The University of Chicago is an Affirmative Action/Equal 
Opportunity/Disabled/Veterans Employer and does not discriminate on the basis 
of race, color, religion, sex, sexual orientation, gender identity, national or 
ethnic origin, age, status as an individual with a disability, protected 
veteran status, genetic information, or other protected classes under the law. 
For additional information please see the University's Notice of 
Nondiscrimination at: 
www.uchicago.edu/non-discrimination<http://www.uchicago.edu/non-discrimination> 
. Job seekers in need of a reasonable accommodation to complete the application 
process should call +1 773-834-6693 or email hmcgu...@bsd.uchicago.edu with 
their request.





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Re: [ccp4bb] RNA's tertiary structure prediction ways with high accuracy

[Phoebe A. Rice]

You might try this:

https://seq2fun.dcmb.med.umich.edu/DeepFoldRNA/

I’m recommending it with the caveat that I’ve tested it on exactly 1 
unpublished structure, and it did a really impressive but far from perfect job.


From: CCP4 bulletin board  on behalf of Abhilasha Thakur 

Date: Thursday, September 8, 2022 at 3:02 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] RNA's tertiary structure prediction ways with high accuracy
Hello everyone..
Greetings of the day!!

I am working on RNA and it's structure prediction.
I have a RNA sequence which is about less than 160 nucleotide sequence, I don't 
have it's structural information. Please suggest me what kind of software can 
be used which can provide me structure with High accuracy and if there is 
python script for predicting structure please provide me if possible, or there 
is others methods for predicting the structure of RNA please suggest that also.

Thankyou



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Re: [ccp4bb] Lower b-factors with increasing T

[Phoebe A. Rice]

Apologies for the stupid post about freezing conditions last night – my brain 
was clearly very low on glucose when I read that list of temperatures!

From: CCP4 bulletin board  on behalf of Phoebe A. Rice 

Date: Wednesday, September 7, 2022 at 7:49 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Lower b-factors with increasing T
I guess the big question is what is the question that you’re trying to address 
from those numbers?   I’d be nervous about making conclusions about trends in B 
factors from just 1 data set per temperature.  As you probably know, the B 
factors will reflect static differences in atomic position across asymmetric 
units as well as thermal motion, and it can be difficult to control variables 
such as exactly how fast a crystal freezes or how much trauma it experiences in 
its journey from sitting happily in a drop to the frozen state.

From: CCP4 bulletin board  on behalf of Matt McLeod 

Date: Wednesday, September 7, 2022 at 1:57 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Lower b-factors with increasing T
Hi everyone,

I have a series of datasets at 253K (~2.0A), 273K (2.0A), 293K (2.0A), 313K 
(2.2A) and I am curious as to the details in determining B-factors.

I have treated these datasets more-or-less identically for comparison's sake.  
I used DIALS to index, integrate, and scale the data.  I scaled the data to a 
~0.6 CC1/2 cutoff.

After fully refining the datasets, there is an odd trend with respect to 
temperature (from what has been previously published) and I assume that this is 
because of "behind-the-scenes" computation rather than a biophysical 
observation.  The B-factors slightly decrease from 252-293K, and then 
significantly drop at 313K.  The maps look pretty well identical across the 
datasets.

253K - 53.8 A^2
273K - 48.4 A^2
293K - 45.5 A^2
313K - 18.6 A^2

I compared the wilson intensity plots from DIALS scaling for 273K and 313K and 
they are very comparable.

I am looking for suggestions as to where to look at how these b-factors are 
selected or how to validate that these B-factor are or are not accurate.  Also, 
any relevant literature would be welcomed.  From what I have read, there is a 
general trend that as T increase, the atoms have more thermal energy which 
raises the b-factors and this trend is universal when comparing datasets from 
different temperatures.

Thank you and happy to supply more information if that is helpful,
Matt



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Re: [ccp4bb] Lower b-factors with increasing T

[Phoebe A. Rice]

I guess the big question is what is the question that you’re trying to address 
from those numbers?   I’d be nervous about making conclusions about trends in B 
factors from just 1 data set per temperature.  As you probably know, the B 
factors will reflect static differences in atomic position across asymmetric 
units as well as thermal motion, and it can be difficult to control variables 
such as exactly how fast a crystal freezes or how much trauma it experiences in 
its journey from sitting happily in a drop to the frozen state.

From: CCP4 bulletin board  on behalf of Matt McLeod 

Date: Wednesday, September 7, 2022 at 1:57 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Lower b-factors with increasing T
Hi everyone,

I have a series of datasets at 253K (~2.0A), 273K (2.0A), 293K (2.0A), 313K 
(2.2A) and I am curious as to the details in determining B-factors.

I have treated these datasets more-or-less identically for comparison's sake.  
I used DIALS to index, integrate, and scale the data.  I scaled the data to a 
~0.6 CC1/2 cutoff.

After fully refining the datasets, there is an odd trend with respect to 
temperature (from what has been previously published) and I assume that this is 
because of "behind-the-scenes" computation rather than a biophysical 
observation.  The B-factors slightly decrease from 252-293K, and then 
significantly drop at 313K.  The maps look pretty well identical across the 
datasets.

253K - 53.8 A^2
273K - 48.4 A^2
293K - 45.5 A^2
313K - 18.6 A^2

I compared the wilson intensity plots from DIALS scaling for 273K and 313K and 
they are very comparable.

I am looking for suggestions as to where to look at how these b-factors are 
selected or how to validate that these B-factor are or are not accurate.  Also, 
any relevant literature would be welcomed.  From what I have read, there is a 
general trend that as T increase, the atoms have more thermal energy which 
raises the b-factors and this trend is universal when comparing datasets from 
different temperatures.

Thank you and happy to supply more information if that is helpful,
Matt



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Re: [ccp4bb] problems in Rfree-flags when refining with REFMAC and processing with STARANISO

[Phoebe A. Rice]

Why why why doesn’t REFMAC just write a new column – something like FOBS_mod?  
Shouldn’t it be a general rule that if you change something, you give it a 
different name?

From: CCP4 bulletin board  on behalf of Randy John Read 

Date: Wednesday, July 13, 2022 at 5:22 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] problems in Rfree-flags when refining with REFMAC and 
processing with STARANISO
Also, this might be a good time for the regular reminder *never* to use the MTZ 
output by Refmac as input for anything else, because the FOBS coming out of 
Refmac is not the same as the FOBS going in. It might well have had various 
corrections applied, including a correction for twinning. I think the newer 
ccp4i2 and CCP4Cloud interfaces might make it harder for you to do that than 
the old ccp4i did.

Best wishes,

Randy Read

> On 13 Jul 2022, at 10:35, Clemens Vonrhein  wrote:
>
> Dear Vera,
>
> On Wed, Jul 13, 2022 at 10:13:28AM +0200, Vera Pfanzagl wrote:
>> You write that I should make sure REFMAC reads in the test-set from
>> the staraniso.mtz and does not replace it with a new one.  This
>> would only be the case if it generates a new test set when I
>> initially import the reflection file, right? How can I check that
>> retrospectively with my history-riddled file?
>
> The REFMAC program/binary itself will obviously read an MTZ file
> exactly as-is when run via
>
>  refmac5 xyzin your.pdb hklin your.mtz xyzout refmac.pdb hklout refmac.mtz <  ... some commands ...
>  e
>
> But "running REFMAC" can mean very different things when this is done
> via one of the available interfaces: there might be various
> preparation steps involved before the actual REFMAC binary is called.
>
> Best thing: run e.g.
>
>  mtzdmp input.mtz  -n 100 > tmp.1
>  mtzdmp output.mtz -n 100 > tmp.2
>
> and compare those two text files (same number of reflections reported,
> same completeness for test-set flags etc).
>
>> PS: the links in [2] are not accurate anymore, the interface of
>> CCP4i2 export alone already looks completely different and does not
>> seem to want external processing files.
>
> Probably something for wwPDB/CCP4/REFMAC to look into and to provide
> an update.
>
> Cheers
>
> Clemens
>
> 
>
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-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: +44 1223 336500
The Keith Peters Building   Fax: +44 1223 336827
Hills Road   E-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.  
www-structmed.cimr.cam.ac.uk



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Re: [ccp4bb] Maps on mobile phones.

[Phoebe A. Rice]

Oo that looks handy!

And it reminds me to ask the community: if you wanted to do outreach to high 
schoolers and get them to, say, look at a DNA structure, is there a PyMol-like 
program that works on tablets?  I see PyMol for ipad has been discontinued.

  Thanks,
Phoebe


From: CCP4 bulletin board  on behalf of Paul Emsley 

Reply-To: Paul Emsley 
Date: Sunday, March 27, 2022 at 11:11 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] Maps on mobile phones.



On 27/03/2022 00:17, Jon Cooper wrote:
Hello, I have been trying to put together a thing for viewing small blocks of 
CCP4 electron density maps with a mobile web browser. If anyone is interested, 
the current state of it is here:

http://ic50.org/jbctest14.html



Have you seen/heard about uglymol? The name is an insult but the project is 
interesting.

https://github.com/uglymol/uglymol



Sorry, the link is not https yet, but nothing gets uploaded to the server! It 
seems to work OK on Android and iPhone and the maps (note: only maps; it 
doesn't do MTZ's, sorry) look similar when viewed in Coot (taken as the gold 
standard ;-),

For the record, I've never much liked the contouring of Coot - too many close 
lines and tiny triangles. I've wanted to change it for a long time, but it's 
never been the most important thing to fix.


but I have a few questions about the contouring algorithm that I have used. It 
is "surfacenets.js" from here:

https://github.com/mikolalysenko/isosurface

and a paper describing it is here:

https://www.merl.com/publications/docs/TR99-24.pdf

Unfortunately, my maths is not good enough to tell if it matters if you give it 
fractional coordinates, rather than orthogonal. I simply give it the electron 
density values on the CCP4 map grid coordinates, which will be on 
non-orthogonal axes for unit cells with non-90 degree angles. It seems to give 
qualitatively similar results to Coot in these cases, so I am cautiously 
optimistic, but not sure.


uglymol makes the transformation that you need - so does CootVR for that matter

https://github.com/hamishtodd1/hamishtodd1.github.io/tree/master/cvr

Here's Chris Hassall playing with it:

https://www.youtube.com/watch?v=-wfopgdN8o4

Just to be clear, this is running inside Firefox.

(It looks like he discovered a contouring bug when he zooms out)



Another thing is that the results of the contouring are sent out in groups of 3 
points which are the vertices of triangles forming the surface. Hence, I 
orthogonalize them and get three.js to draw them as just that - triangles. My 
worry is that, since the triangles all have edges in common, nearly all of the 
contour lines (except the ones at the edges of the map box) get drawn twice, or 
at least are sent to three.js twice for drawing, which doesn't seem terribly 
efficient?!

Coot used to remove double drawing. The large speed up in contouring in the 0.9 
series is a result of removing that test and just drawing the lines twice.


Is there a nicer way of doing this? I think it might be better to have 
FRODO-style contouring just on the 2D sections of the map, rather than having 
lots of diagonal lines?

I think so too, especially as a larger Shannon sampling factor is now not much 
of an issue.


Anyway, my 5 YO phone takes about 3 seconds to step from one  residue to the 
next, so it seems not too bad, although not ideal!



FWIW, Kevin Cowtan recently advertised a position to "develop next generation 
web based molecular graphics software" - sounds related.



Finally, its been asked before, but is there a nice way in CCP4i2 to output 
maps that cover the coordinates of the structure, rather than the asymmetric 
unit? Saving maps in Coot gives the asymmetric unit, too, although using Export 
Map Fragment seems the best option. I know about doing this in the old gui with 
mapmask, or using phenix, so just wondering if I've missed a way of doing this 
in i2, etc? I know that suitably extended CCP4 maps are available from the PDBe 
EBI.


I just use a script that runs mapmask - using a GUI seems like an overhead.



Paul.







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Re: [ccp4bb] protein DNA complex structure and extension of DNA structure

[Phoebe A. Rice]

I would try the tools on this site - http://web.x3dna.org/index.php/protein


From: CCP4 bulletin board  on behalf of "Srivastava, 
Dhiraj" 
Reply-To: "Srivastava, Dhiraj" 
Date: Monday, December 27, 2021 at 8:32 PM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] protein DNA complex structure and extension of DNA structure

Hi
I solved the structure of protein DNA complex where the DNA molecule is 
bent. I want to extend the DNA computationally so I can model two DNA binding 
proteins together on same DNA molecule. is there a way by which I can extend 
the DNA in both direction? its easy to do it with ideal DNA molecule but I 
don't know how to do it with bent DNA.

Thank you
Dhiraj



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Re: [ccp4bb] Can twinning be seen in the diffraction pattern?

[Phoebe A. Rice]

A side point, worth considering depending on your x-ray source: many years ago 
we threw out several crystals in a row for having split spots before we finally 
realized that the BEAM was split, not the crystals.  Oops.

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


From: CCP4 bulletin board  on behalf of Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
Reply-To: Eleanor Dodson 
Date: Wednesday, March 17, 2021 at 5:20 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] Can twinning be seen in the diffraction pattern?

Yes Ana, I agree although some non-merihedrals where the accident of cell 
dimensions mean many spots can overlap but not quite exactly can seem a bit 
smeary and "multiple.
Meridral twins do not usually look multiple - they are usually only revealed by 
the 2nd moment and other stats..
Eleanor

On Wed, 17 Mar 2021 at 10:13, Ana Luísa Moreira de Carvalho 
mailto:a...@fct.unl.pt>> wrote:
Just a short note on this: I often see colleagues using the word “twinning" 
when referring to a crystal that is actually multiple (not single).

I think much confusion arises from this. For me, a twin crystal is the one that 
looks single under the microscope and only intensity statistics reveal that the 
diffraction comes from more than one crystal.

If a crystal looks multiple, i do not call it a twin. Am i being too meticulous 
on this?
Thanks!


On 16 Mar 2021, at 13:31, Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk<mailto:176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>>
 wrote:

You usually detect twinning most reliably from the intensity statistics - 
CCP4I2 and Xtriage report those..
Eleanor

On Tue, 16 Mar 2021 at 07:31, Marina Gárdonyi 
mailto:marina@pharmazie.uni-marburg.de>>
 wrote:
Dear all,

thanks to all who helped me solving the question. You sent me a lot of
comments and information I have not taken into account.
After reading all the answers, I have come to the conclusion that the
spots that are very close to each other come from the long cell axis
(57-57-160) and that twinning can probably not be seen in my case. I
should have mentioned that the diffraction images came from an
in-house x-ray machine, recorded with a 0.5 degree rotation range.

Thank you all again!

Kind regards,
Marina

--
Marina Gárdonyi

PhD Student, Research Group Professor Dr. Klebe

Department of Pharmaceutical Chemistry

Philipps-University Marburg

Marbacher Weg 6, 35032 Marburg, Germany

Phone: +49 6421 28 21392

E-Mail: 
marina@pharmazie.uni-marburg.de<mailto:marina@pharmazie.uni-marburg.de>

http://www.agklebe.de/



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Re: [ccp4bb] Open position - data management in biophysics

[Phoebe A. Rice]

Worth discussion although may be impossible to fix.
My 2 cents:
Standard contracts here seem to be for 1 year, but I've always worked on the 
assumption that the contract will be renewed unless post-doc and PI have 
"irreconcilable differences" or there is an unforeseen funding shortfall. 
Then again, I have seen people at other institutions tell post-docs that they 
will automatically be "out" after just 1 year if they fail to find their own 
fellowships, which seems rather exploitative to me - one can do absolutely 
everything right and still not get a fellowship.

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

On 1/20/21, 3:45 PM, "CCP4 bulletin board on behalf of Gerlind Sulzenbacher" 
 wrote:

Dear Markus,

thank you for opening this discussion.

I'd like to add that in some countries, like France, where I work, this 
goes often along with with 12 months contracts.
Imagine moving continent, eventually with family (yes, PostDocs happen 
to have a family) just for a 12 months contract, under the conditions 
you mentioned.
Sad, as you said, ... and I am quite sure that it has not always been 
like that.
I wish all members of the BB a good mood,
Best,
gerlind


On 20/01/2021 21:48, Markus Heckmann wrote:
> Dear PI s, and senior scientists' involved in recruitment,
>
> Why do so many (especially postdoc) positions these days indicate:
>
>> Readiness for high workload
>> able to work independently but also effectively and collaboratively with 
other lab member
>> Candidates should have a documented publication record in peer-reviewed 
journals, able to work both independently and as an effective team member.
> Do the candidates need to subtly understand that they need to work on
> weekends or holidays? And what does it mean by independently and
> collaboratively at the same time. Or is this a template from HR
> departments.
>
> Was it always like this in science world or we too need to work like
> amazon warehouse workers (you can google it and see the pain)?
>
> Saddened...
>
> Mark
> (not trying to point out any single PI/person but overall it is the
> same words repeated...)
>
>
>
>> We are opening a new position for an upcoming European project.
>>
>> *We are looking for an expert in scientific programming with experience 
in
>> scientific data processing for a European project focused on Standards 
for
>> Data Archival and Exploitation. *
>>
>> Job description:
>>
>> We offer attractive work connected to development of data management
>> infrastructure for biophysical data in the frame of an international
>> project at the Institute of Biotechnology in the centre of excellence
>> Biocev. The main responsibility lies in definition of data standards and
>> models for biophysical data, development of algorithms, design of user
>> interface, and realization of a pilot database of biophysical data. The
>> person is expected to actively participate in multilateral international
>> negotiations, to drive the tasks fulfillment in collaboration with the
>> local international partners, and to present the results.
>>
>
>> Dear all,
>>
>> Two postdoctoral positions are available in the laboratory of Dr. 
Pengxiang
>> Huang, Assistant Professor and CPRIT scholar in cancer research in the
>> Department of Molecular and Cellular Biology at Baylor College of 
Medicine.
>> With the long-standing interest in sterol lipids, the Huang lab 
investigates
>> the poorly understood mechanisms involved in Hedgehog (Hh) and Wnt signal
>> transduction, two related pathways that play critical roles in 
development,
>> regeneration and cancer. We utilize a combination of biochemistry, cell,
>> chemical and structural biology approaches, including both X-ray
>> crystallography and Cryo-EM. Our recent work identified cholesterol as 
the
>> endogenous ligand for Smoothened, the key signal transduer and 
oncoprotein
>> in the Hh pathway (Cell. 166:1176-87). We also characterized the 
structural
>> and oncogenic basis of Smoothened activation, demonstrating for the first
>> time the active conformation of a class F GPCR (Cell. 174:312-24). The
>> highly interdisciplinary and collaborative environment of our group will
>> thus provide unique career development opportunities for future 
postdoctoral
>

Re: [ccp4bb] B-factors very high

[Phoebe A. Rice]

Dear Silvia,
  While high Bs can be a sign that parts of your model are incorrect (and 
therefore some careful manual rebuilding may be required), for things like 
poorly-ordered surface loops, they can simply be truth.  
A point worth remembering for newbies: your goal is to produce the model that 
best reflects the data, which seems obvious, but that means don't try to model 
what you can't see (e.g. seriously disordered surface loops) and don't worry if 
bits where the density was marginal have high Bs.
 Best,
 Phoebe


On 1/4/21, 8:39 AM, "CCP4 bulletin board on behalf of Silvia Napolitano" 
 wrote:

Dear CCP4 Community,
I am currently working on the structure of a monomeric protein of 23KDa. 
The protein is generally quite "loopy" and I think I am mid-way refinement. 
At the moment I am struggling, among other things, with an extremely high 
B-factor and I'm not sure how to lower it.
Do you have any suggestions on how I can proceed to obtain a lower average 
B-factor? (I am sorry if the question is rather naive, but I have worked very 
little on X-ray structures so far)
I attached a screenshot of the polygon obtained from the last refinement 
(the geometry now should be a bit improved, I am running a new job at the 
moment). I will be happy to provide further files if needed.
Many thanks in advance for your precious help!
I wish you a nice start to the new year.
Best
Silvia




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Re: [ccp4bb] [ccp4bb] Up-to-date 3D stereo solution

[Phoebe A. Rice]

Ah yes, fond memories of that mirrored contraption!  
There were also wooden contraptions to strap on one's head with image-merging 
mirrors in them.  We had fun lining up equal-height friends and merging them. 
But both of those did have the downside that getting engrossed in electron 
density while keeping one's head properly positioned caused back issues even in 
my student days.  

On 12/13/20, 6:38 AM, "CCP4 bulletin board on behalf of Goldman, Adrian" 
 wrote:

the “best” I’ve found is stereo glasses and display side-by-side stereo. 

When I was a graduate student in the Steitz lab a WHILE ago, there was a 
frame built that fit over the Evans and Sutherland Ps2 and PS390 monitors (I 
said a while ago :)) that carried two mirrors at 45 degrees at your eye width 
apart, and another two at 45 degrees towards the edge of the screen.The 
whole device was counterweighted and could be lowered into place or pulled up 
for non-stereo work.  You put your nose on the centre of the two mirrors.

For side by side stereo, it can’t be beat.  Immersive, no eye-strain.  I 
have debated having something similar made…

Adrian

> On 12 Dec 2020, at 17:07, Hughes, Jonathan 
 wrote:
> 
> just cross your eyes.
> jon
> 
> -Ursprüngliche Nachricht-
> Von: CCP4 bulletin board  Im Auftrag von Jan 
Stransky
> Gesendet: Samstag, 12. Dezember 2020 12:11
> An: CCP4BB@JISCMAIL.AC.UK
> Betreff: [ccp4bb] Up-to-date 3D stereo solution
> 
> Dear all,
> 
> I am wondering, if anyone has experience with implementing stereo 3D 
setup with currently available hardware. NVIDIA 3D vision seems to be pretty 
much dead.
> 
> The primary usage would be model building in Coot, but also potentially 
Pymol analysis.
> 
> The biggest issue is an access to controls. We played a bit with PS VR, 
VR solutions prsented last year at CCP4 weekend seem to be still far from 
usable.
> 
> Did anyone try AR?
> 
> Best regards,
> 
> Jan
> 
> 
> 
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Re: [ccp4bb] taking information from a deposited structure

[Phoebe A. Rice]

Each PDB entry now has a DOI which should make referencing that structure 
easier in your own publication.

From: CCP4 bulletin board  on behalf of Firdous Tarique 

Reply-To: Firdous Tarique 
Date: Wednesday, September 9, 2020 at 1:35 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] taking information from a deposited structure

Hi

Thanks a lot for your feedback.

Best

Kahkashan

On Wed, Sep 9, 2020 at 11:58 AM Randy Read 
mailto:rj...@cam.ac.uk>> wrote:
It’s perfectly normal and accepted to use a released entry from the PDB, even 
if there’s no underlying publication.  That is true of many of the structures 
from the structural genomics initiatives, for instance.  As you say, you should 
cite the PDB entry ID and it would be nice also to name the authors of the 
entry.

Best wishes,

Randy Read

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research Tel: +44 1223 336500
The Keith Peters Building   Fax: +44 1223 336827
Hills Road   E-mail: 
rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.  
www-structmed.cimr.cam.ac.uk

> On 9 Sep 2020, at 06:41, Firdous Tarique 
> mailto:kahkashantari...@gmail.com>> wrote:
>
> Dear CCP4 community members.
>
> I have solved  a crystal structure of a protein and am now trying to get a 
> structure with a ligand bound to it, but so far unsuccessful. A homologous 
> structure with the same ligand is present in the RCSB PDB (un published). Is 
> it permissible to fetch structural information from that unpublished data and 
> use it for docking and simulation studies with my protein? Will it be 
> copyright infringement or it is just a normal thing.  I will be mentioning in 
> my manuscript that I have used this unpublished structure (deposited by this 
> author) for these studies.
>
> Best
>
> Kahkashan
>
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[ccp4bb] Faculty position

[Phoebe A. Rice]

Biochemistry and Biophysics Faculty at The University of Chicago

The Department of Biochemistry and Molecular Biology at the University of 
Chicago invites

applications for a faculty position, preferably at the assistant professor 
rank. We seek scientists

studying macromolecular dynamics and structure, mechanisms of molecular 
function and

related areas. Appropriate topics include proteins, nucleic acids and their 
assemblies;

membrane proteins; cellular processes in prokaryotes and eukaryotes (for 
example,

proteostasis, condensates and immunology); biomolecular design; chemical 
biology, and

computational analysis, among others. Resources include cryoEM and cryoET 
capabilities (Titan

Krios/K3). The University is affiliated with Argonne National Laboratory and 
the Marine

Biological Laboratory. Rank and tenure status will be commensurate with 
qualifications.

Prior to the start of employment, qualified applicants must have a doctoral 
degree or

equivalent. To be considered, those interested must apply through the 
University of Chicago’s

Academic Recruitment job board, which uses Interfolio to accept applications. 
To apply for a

tenure-track assistant professorship, please visit 
http://apply.interfolio.com/78680 and/or to

apply for a tenured associate or full professorship please visit

http://apply.interfolio.com/78694. Qualifications, required materials, and 
procedures for

applying, which differ for the two postings, are described at these links. 
Review of applications

will begin 15 Oct 2020 (we encourage application as soon as possible 
thereafter) and end when

the position is filled.



Equal Employment Opportunity Statement

We seek a diverse pool of applicants who wish to join an academic community 
that places the

highest value on rigorous inquiry and encourages diverse perspectives, 
experiences, groups of

individuals, and ideas to inform and stimulate intellectual challenge, 
engagement, and

exchange. The University’s Statements on Diversity are

at https://provost.uchicago.edu/statements-diversity.

The University of Chicago is an Affirmative Action/Equal 
Opportunity/Disabled/Veterans

Employer and does not discriminate on the basis of race, color, religion, sex, 
sexual orientation,

gender identity, national or ethnic origin, age, status as an individual with a 
disability, protected

veteran status, genetic information, or other protected classes under the law. 
For additional

information please see the University's Notice of Nondiscrimination.

Job seekers in need of a reasonable accommodation to complete the application 
process should

call 773-702-1032 or email equalopportun...@uchicago.edu with their request.





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Re: [ccp4bb] Paired refinement proves data quality goes beyond the spatial limits of the detector

[Phoebe A. Rice]

This advance brings deep new meaning to crystallographic data having both a 
real and an imaginary component!

On 3/31/20, 11:36 PM, "CCP4 bulletin board on behalf of Petr Kolenko" 
 wrote:

Dear colleagues,
We, the developers of a program for paired refinement, have found a 
remarkable feature that should be shared with the community. The fact that data 
beyond the arbitrary cutoff may cause an improvement of electron density and 
make your models better is generally accepted. We found now that the data 
beyond the dimensions of the detector are still useful, should be used in 
refinement, deposited in your structure factor file and if possible, made 
publicly available in data repositories as well! This leads us to a general 
recommendation, please deposit your raw data including regions beyond the 
detector edge or better the corner.  Share the maximum available and be FAIR.
Stay safe, best regards,
Petr



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Re: [ccp4bb] CCP4BB Digest - 27 Feb 2020 to 28 Feb 2020 (#2020-61)

[Phoebe A. Rice]

While we're at it, it would also be a great improvement if the PDB would list 
nominal resolution in all 3 directions for structures with significantly 
anisotropic data.  I'm never 100% sure what to do in those cases.
 Thanks, 
 Phoebe

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

On 3/1/20, 5:19 PM, "CCP4 bulletin board on behalf of dusan turk" 
 wrote:

Hi again,

First, I wish to thank everyone for their responses. I hope that no one 
minds that I include them in my response letter to the Editor ?

The idea of citing the Karplus and Diedrichs paper in Science has been 
essentially consumed already in our first response letter, which accompanied 
the submission of the revised manuscript,  and did not work. (Instead of the 
Science citation from 2012 we used a quote from the paper suggested below by 
Karplus and Diederichs, Curr Opin Struct Biol. 2015 Oct; 34: 60–68. )

As Phoebe mentioned, it would be good to use the momentum. My intention of 
writing to the bulletin board was not only to get help with argumentation, but 
also to raise the issue of how to address resolution cutoffs in 
crystallographic data in publications  to avoid such situations in general. I 
also think that the issue is more complicated than the last shell criterion 
with I/Isig > xx, Rmerge < yy, or cc1/2 > zz, or ...

1. Number of reflections in a shell effects the numbers significantly. The 
larger numbers of the shells there are the larger the numbers will be.  The 
number of reflections in a shell depends also on the highest resolution and 
unit cell size. Hence we have a dependance of potential criteria on several 
parameters. 

2. Refinement and data processing programs use different numbers of shells 
and even different ways of calculating shells. REFMAC typically uses 20 equal 
volume sliced shells), PHENIX is more complicated, as far as I understand 
(shell number may depend on the number of TEST set reflections in individual 
shell, shells can be defined according to equal  slicing volume,  some kind of 
log dependency or even linearly according to real space resolution), in MAIN 
there are  20 shells by default, but one can choose any of the mentioned 
slicing rules.

I suggest that we use this discussion to shape up guidelines that can later 
proposed for consideration to the IUCr committee for macromolecules.  I prefer 
soft as opposed to strict borders.  In the end, the structures do not speak for 
themselves, but are a mean to support one or more biologically relevant 
conclusions.

???

best wishes,
dusan turk


> On 29 Feb 2020, at 01:00, CCP4BB automatic digest system 
 wrote:
> 
> 
> Date:Fri, 28 Feb 2020 16:03:22 +
> From:"Phoebe A. Rice" 
> Subject: Re: What resolution - X-ray diffraction round this time
> 
> Can we get some momentum for the "standard table 1" including TWO numbers 
- outer limit used in refinement, and nominal resolution based on some standard 
such as I/sigI =2 (or 3, or whatever the community can agree on)? That would 
hopefully cut down on all the reviewer complaints of overstated resolution.
> 
> ~~~
> Phoebe A. Rice
> Dept. of Biochem & Mol. Biol. and
>  Committee on Microbiology
> https://voices.uchicago.edu/phoebericelab/
> 
> 
> On 2/28/20, 6:56 AM, "CCP4 bulletin board on behalf of Malý Martin" 
 wrote:
> 
>Dear colleagues,
> 
>I agree with all the previous responses, it is a pity to throw away
>useful high-resolution data. The problem of high-resolution cutoff
>estimation is also nicely summarized in another paper by Andrew Karplus
>and Kay Diederichs "Assessing and maximizing data quality in
>macromolecular crystallography"
>https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684713/ . It is suggested
>using CC1/2 for the selection of the cutoff for data processing (not
>I/sigI or R_whatever). Later on, the decision should be validated
>performing the paired refinement protocol.
> 
>Good luck with the argumentation.
>Martin
> 
> 
>On 2/28/20 11:08 AM, LMB wrote:
>> Ask the referee - (apart from the other suggestions here)
>> 
>> ‘How would removing data Improve my model?”
>> 
>> Sent from my iPad
>> 
>>> On 28 Feb 2020, at 08:22, dusan turk  wrote:
>>> 
>>> Hi,
>>> 
>>> Browsing through the recent discussion on EM data resolution cutoff
>>> it occurred to me t

Re: [ccp4bb] What resolution - X-ray diffraction round this time

[Phoebe A. Rice]

Can we get some momentum for the "standard table 1" including TWO numbers - 
outer limit used in refinement, and nominal resolution based on some standard 
such as I/sigI =2 (or 3, or whatever the community can agree on)?  That would 
hopefully cut down on all the reviewer complaints of overstated resolution.

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

On 2/28/20, 6:56 AM, "CCP4 bulletin board on behalf of Malý Martin" 
 wrote:

Dear colleagues,

I agree with all the previous responses, it is a pity to throw away
useful high-resolution data. The problem of high-resolution cutoff
estimation is also nicely summarized in another paper by Andrew Karplus
and Kay Diederichs "Assessing and maximizing data quality in
macromolecular crystallography"
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684713/ . It is suggested
using CC1/2 for the selection of the cutoff for data processing (not
I/sigI or R_whatever). Later on, the decision should be validated
performing the paired refinement protocol.

Good luck with the argumentation.
Martin


On 2/28/20 11:08 AM, LMB wrote:
> Ask the referee - (apart from the other suggestions here)
>
> ‘How would removing data Improve my model?”
>
> Sent from my iPad
>
>> On 28 Feb 2020, at 08:22, dusan turk  wrote:
>>
>> Hi,
>>
>> Browsing through the recent discussion on EM data resolution cutoff
>> it occurred to me that the X-ray diffraction community isn’t that
>> unanimous either.
>>
>> My stand:
>>
>> When the default resolution cutoff provided with the data processing
>> software in electron density map calculation and refinement delivers
>> quality maps noisier than expected and/or too high R-factors I start
>> adjusting the resolution cutoff by lowering the resolution and trying
>> alternative space group. Hence, I allow the data processing programs
>> to suggest where to draw the line (be it CC1/2, I/sigI, R merge, R
>> sym, R p.i.m. and R r.i.m, …) , unless there are problems.
>>
>> Doing so, I came into a dispute with a referee who shaped his request:
>>
>> "It is well accepted that the criteria for resolution cutoff should
>> consider both I/SigI and Rmerge for the outer most shell. For data
>> sets collected at synchrotron sources, the criteria of I/SigI > 5 and
>> Rmerge <50% can be taken as a good practical reference.”
>>
>> So where do we stand? Which are the most objective criteria for
>> resolution cutoff to be used in diffraction data processing? Which
>> number of shells to use when calculating the statistics? Do we have a
>> consensus?
>>
>> best wishes,
>>
>> dusan turk
>>
>>
>>
>> Dr. Dusan Turk, Prof.
>> Head of Structural Biology Group http://stef.ijs.si/
>> Head of Centre for Protein and Structure Production
>> Centre of excellence for Integrated Approaches in Chemistry and
>> Biology of Proteins, Scientific Director
>> http://www.cipkebip.org/
>> e-mail: dusan.t...@ijs.si
>> phone: +386 1 477 3857 Dept. of Biochem.& Mol.& Struct. Biology
>> fax: +386 1 477 3984 Jozef Stefan Institute
>> Jamova 39, 1 000 Ljubljana,Slovenia
>> Skype: dusan.turk (voice over internet: www.skype.com
>>
>> 
>>
>> 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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-

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zpráva nebo její přílohy pouze informativní charakter. Tato zpráva ani její 
přílohy v žádném ohledu Biotechnologický ústav AV ČR, v. v. i. k ničemu 
nezavazují. Text této zprávy nebo jejích příloh není návrhem na uzavření 
smlouvy, ani přijetím případného návrhu na uzavření smlouvy, ani jiným právním 
jednáním směřujícím k uzavření jakékoliv smlouvy a nezakládá předsmluvní 
odpovědnost Biotechnologického ústavu AV ČR, v. v. i.

Disclaimer: If not expressly stated otherwise, this e-mail message 
(including any attached files) is intended purely for info

Re: [ccp4bb] ITC Stoichiometry

[Phoebe A. Rice]

Isn’t that what the experiment should help you figure out?
A could conceivably compete with B-B interactions, leading to A-B heterodimers. 
 A could be so large as to occlude some of its own binding sites on the B 
hexamer, leading to less than 6 As bound per hexamer of B.  Or maybe each A 
binds two different surfaces of B, such that some number (< or = 6) of As could 
bridge two B hexamers … or the other way around … Mother Nature is inventive – 
best to get some experimental numbers!

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


From: CCP4 bulletin board  on behalf of Rezaul Karim 
<1e364c8f16de-dmarc-requ...@jiscmail.ac.uk>
Reply-To: "reza...@yahoo.com" 
Date: Friday, February 21, 2020 at 10:06 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] ITC Stoichiometry

Hi Angshu,
Very interesting experiment. Is the B is in hexameric form in solution by 
itself or it requires A for hexamer formation?

Thanks,
Reza

Md Rezaul Karim
PhD candidate
PhD Program in Integrated Biomedical Sciences
Dept. of Molecular Medicine, Morsani College of Medicine, USF,Tampa
Schonbrunn lab, Moffitt Cancer Center, Tampa
Email: rez...@health.usf.edu, md.ka...@moffitt.org
Phone: (813) 745 4673 ext. 5462

On Fri, Feb 21, 2020 at 9:59 AM, DUMAS Philippe (IGBMC)
 wrote:
Dear  Angshu
The answer to your question requires to define precisely the term of 
"stoichiometry".
*If you consider the hexamer as "the molecule B", then the expected 
stoichiometric ratio is 1/1  (one molecule  A  should bind to 1 hexamer B).
*But if you consider the monomer of B as "the molecule" in the cell, then  the 
expected stoichiometric ratio is 1/6 (one molecule of A  should bind to 6 
monomers of the hexamer B).
Accordingly, you have to define the concentration in the cell as follows: if 
you consider the hexamer as "the molecule B" and, let's suppose you have [B] = 
10 µM of hexamer, then  [B] = 60 µM if you consider  the monomer of B as "the 
molecule".
I hope I answered your question.
Philippe Dumas


De: "Angshu Dutta" 
À: "CCP4BB" 
Envoyé: Vendredi 21 Février 2020 15:24:53
Objet: [ccp4bb] ITC Stoichiometry


Dear all,

Apologies for an off-topic question.

There are two proteins- A(monomer) and B(hexamer). As per reports, one molecule 
of A(monomer) should bind to one molecule of B(hexamer). In order to show the 
interaction between the two proteins through ITC, A is taken in the syringe and 
B is taken in the cell. What kind of stoichiometry values should be expected?

I look forward to your responses.

Many thanks in advance.

Best,

Angshu



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

[Phoebe A. Rice]

While there is some truth to that argument, the problem is that it is harder to 
achieve an international reputation in the first place while being routinely 
overlooked.

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


From: CCP4 bulletin board  on behalf of Andrew Leslie 

Reply-To: Andrew Leslie 
Date: Wednesday, February 5, 2020 at 11:56 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] Macromolecular Crystallography workshop in South America 
2020

Dear All,

  In fairness to the organisers, I would like to point out that 
there is nothing that is “lazy” about organising these workshops. It involves a 
considerable effort both in arranging the course, the venue and especially in 
attracting funds to support the workshop (it is important to note that CCP4 
does not supply all the funds). In addition, it is unfair to single out this 
particular workshop for criticism, as I believe it has long been the case that 
these workshops have not had a good gender balance in terms of the tutors. It 
is also important to realise that the gender imbalance does NOT extend to 
choice of the students, where as far as I am aware the gender balance is always 
very good.

One difficulty the organisers face is that funding will typically depend on 
having tutors with an international reputation in the areas in which they are 
teaching, ideally having been involved in developing the software that is being 
used. Unfortunately, this inevitably leads to gender bias.

While I would agree that this is an issue that is worthy of being raised, and I 
feel sure that this point will be taken on board by future organisers, it is 
also important to realise the practical difficulties that organisers face and 
the considerable effort that is involved in running these workshops.

Regards,

Andrew Leslie


On 5 Feb 2020, at 00:30, Alejandro Buschiazzo 
mailto:ale...@pasteur.edu.uy>> wrote:

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<mailto: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.

The application deadline is July 9, 2020.

Please address further inquiries to: 
mx2...@pasteur.edu.uy<mailto:mx2...@pasteur.edu.uy>

Looking forward to hosting you in Montevideo!


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Re: [ccp4bb] microscope camera

[Phoebe A. Rice]

With a little fiddling and patience, one can just point a cell phone camera 
down the eyepiece.  It doesn’t produce the best pics, but works fine if you 
just want to stick a few in a notebook (or a grant …).

From: CCP4 bulletin board  on behalf of Sergei Strelkov 

Reply-To: Sergei Strelkov 
Date: Thursday, November 28, 2019 at 7:47 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] microscope camera


Moticam 5 has been working well for us for a few years now (installed on a 
Leica binocular)



Prof. Sergei V. Strelkov Laboratory for Biocrystallography Department of 
Pharmaceutical Sciences, KU Leuven O, Campus Gasthuisberg, Herestraat 49 bus 
822, 3000 Leuven, Belgium Phone: +32 16 33 08 45, mobile: +32 486 29 41 32 Lab 
pages: 
http://pharm.kuleuven.be/Biocrystallography


From: CCP4 bulletin board  on behalf of Dean Derbyshire 

Sent: Wednesday, November 27, 2019 11:35
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] microscope camera

Forgive the off topic subject:

Has anyone got any experience with moticam microscope cameras?
We are looking into cheap cameras for record keeping etc and this supplier 
looks good but…

Cheers in advance

Sent from Mail for Windows 10




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Re: [ccp4bb] difficult molecular replacement solution

[Phoebe A. Rice]

I like to try molrep with something missing from the search model so that I can 
see if the resulting map includes density for something that I know should be 
there but the computer doesn’t.  As others have pointed out, it also bolsters 
believability if the result agrees with a self-rotation function and shows nice 
packing.

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


From: CCP4 bulletin board  on behalf of Robert S 
Phillips 
Reply-To: Robert S Phillips 
Date: Wednesday, November 13, 2019 at 8:33 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] difficult molecular replacement solution

I have been working on a protein structure which has been hard to solve by 
molecular replacement.

Unit cell: (60.6, 172.34, 196.42, 90, 90, 90)
Space group: P 21 21 21

The problem is that the homologues have only ~20% identity, and there are 
multiple chains in the asymmetric unit.  The question is how many.  It could be 
4, 5, or 6 chains.

N  solvent   P
 4  0.602   3.090.225
 5  0.502   2.470.388
 6  0.403   2.060.229

I have run PHASER with 4, 5 and 6 chains.  I allowed it to search all possible 
space groups, and P212121 was the best solution.  These are the results.

N  LLG   TFZ
4  104.97.5
5  137.57.7
6  166.28.3

 Am I correct to conclude that there are 6 chains in the asymmetric unit?

Rob

Robert S. Phillips
Professor of Chemistry and of Biochemistry and Molecular Biology
University of Georgia
Athens, GA 30602
Phone: (706) 542-1996
Fax: (706) 542-9454
E-mail: rsphill...@chem.uga.edu
Web:  
http://tryptophan.net<https://pod51004.outlook.com/owa/redir.aspx?C=ccbf42ffea5f48b1bf8e9bb950454bab=http%3a%2f%2ftryptophan.net>



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Re: [ccp4bb] challenges in structural biology

[Phoebe A. Rice]

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

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

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


From: CCP4 bulletin board  on behalf of John R Helliwell 

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

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

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

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

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

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

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

Greetings!
John

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



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

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

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

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

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




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

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

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

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

"If I had a trillion dollars for structural biology"

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

Re: [ccp4bb] challenges in structural biology

[Phoebe A. Rice]

Hi All,
  Agreed! 
  Crystallization methods have improved in some ways, but at least in my 
experience the real energy barrier is usually knowing enough about the quirky 
biochemistry of the particular idiosyncratic complex we happen to be working 
on.  That means that one may need a grant's worth of biochemical plans anyway, 
whether or not "determine structure" is included as an aim (which of course 
won't get funded unless diffracting crystals are in hand, which is probably an 
issue for another day ...).  Some of the "magic bullets" of the past have 
turned out to rely on assumptions that remind one of spherical cow jokes.  Or 
to require a very large up-front investment in finicky microfluidics or other 
technologies that just isn't practical for small labs that try to do 
biochemistry as well as structure. 
  A long-term worry of mine is training of the next generation:  it is quite 
possible to solve structures now just by pushing buttons in software such as 
phenix now, and in many cases those suites do make the best decisions, but they 
deprive learners of understanding what is going on inside the black box.  Often 
I just can't find the relevant tables of statistics, etc to explain to a 
student why an autosol run produced an ugly map - e.g. cross R factor vs. 
resolution for native vs. alleged derivative?  FOM vs. resolution?  Clearly 
labeled statistics before and after whatever density modification happened?  
Even a clear, concise log of what kind(s) of density modification were applied? 
 I get frustrated when other people's students can't tell me exactly what 
they've already tried, but it isn't always their fault.
  Best,
 Phoebe

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

On 7/21/19, 12:55 PM, "CCP4 bulletin board on behalf of Kay Diederichs" 
 wrote:

Hi Artem,

you are certainly correct in that James' points 2-9 would be moot if his 
point 1 were solved. But as long as this is not the case, we resort to work 
with few and/or small and/or badly diffracting and/or non-isomorphous crystals, 
which makes points 2-9 very relevant. 

Maybe the reason why crystallization research is not well funded is that it 
is not expected to yield significant improvements. Personally, I think that 
even huge funding would not result in methods that succeed in crystallizing all 
molecules.

best,
Kay

On Sun, 21 Jul 2019 11:28:14 -0400, Artem Evdokimov 
 wrote:

>Excellent question :)
>
>First of all, thank you for putting this out to the community!
>
>Secondly, I agree with several of us who've written that a single
>conference is not enough to discuss all the possible topics.
>
>Thirdly, in my opinion all the other problems are secondary to the main
>(and only remaining!) problem in crystallography: getting
>diffraction-quality protein crystals reproducibly and quickly
>
>The amount of funding for serious crystallization research seems to be
>close to non-existent. In general methodology funding is hard to get, but
>crystallization seems to me like the absolute underdog of the method pool -
>the true 'red headed stepchild' of the methods development funders.
>
>At risk of repeating myself - the other problems (worthy, significant, and
>urgent as they are!) are subservient to the main issue at hand - namely
>that crystallization remains an unpredictable and artful phenomenon while
>literally all other aspects of structure determination process (the gene to
>structure pipeline, whatever you might call it)have made astronomic leaps
>forward.
>
>Artem
>- Cosmic Cats approve of this message
>
>
>On Mon, Jul 15, 2019 at 3:44 PM Holton, James M <
>270165b9f4cf-dmarc-requ...@jiscmail.ac.uk> wrote:
>
>> 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:
>>

[ccp4bb] Post-doc position

[Phoebe A. Rice]

A post-doctoral position is open in the Rice lab at the University of Chicago 
(https://voices.uchicago.edu/phoebericelab/).  The primary project is to figure 
out, through biochemistry and structural biology, the mechanisms of 
self-loading helicases encoded by medically nasty staphylococcal mobile genetic 
elements.  The lab employs a variety of approaches from cryo-EM and 
crystallography through biochemistry and microbiology, all aimed at 
understanding mobile DNA elements at the molecular level.  A variety of 
additional project opportunities are also available.
The Advanced Electron Microscopy Facility at the University of Chicago 
currently houses a 200 KV Talos F200C microscope with side-entry holders and a 
Vitrobot plunge freezer for cryo screening, a 120 KV Spirit microscope for 
negative-staining, and a 300 KV Tecnai F30 for plastic tomography.
The University has made > 10M dollars in investments to upgrade the facility 
with a state-of-the-art 300 KV Titan Krios microscope equipped with a K3 direct 
detection camera and Volta phase plate, which will be fully installed by the 
end of 2019. Additional upgrades include a Aquilos cryo FIB-SEM Leica, Apreo 
volume scope and Leica EM GP2 cryo-plunge.
The Advanced Photon Source’s fantastic x-ray beamlines are also a short drive 
away.
Top candidates will have a PhD (or be about to receive one) and experience in 
cryo-EM and biochemistry.  To apply, please send your CV, with names and 
contact information for 3 references, to me by e-mail: 
pr...@uchicago.edu<mailto:pr...@uchicago.edu>

Be the one who turns this science fiction figure into reality!
[cid:image001.png@01D5281E.B2A586E0]
The University of Chicago is an Affirmative Action/Equal 
Opportunity/Disabled/Veterans Employer and does not discriminate on the basis 
of race, color, religion, sex, sexual orientation, gender identity, national or 
ethnic origin, age, status as an individual with a disability, protected 
veteran status, genetic information, or other protected classes under the law. 
For additional information please see the University's Notice of 
Nondiscrimination at 
http://www.uchicago.edu/about/non_discrimination_statement/. Job seekers in 
need of a reasonable accommodation to complete the application process should 
call 773-702-0287 or email 
acoppadministra...@uchicago.edu<mailto:acoppadministra...@uchicago.edu> with 
their request.

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




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Re: [ccp4bb] naive question how to add GTP as a residue to a nucleic acid chain and refine!

[Phoebe A. Rice]

At least last fall, the state of the art for getting your T7-transcribed RNA to 
start with a triphosphate in phenix was to add this file (edited for your 
chains, of course) through the gui (if you use the gui).
Thanks to Deepak Koirala and Nigel Moriarty for help in working this out.

  *   Phoebe

refinement.geometry_restraints.edits {
bond {
  action = *add
  atom_selection_1 = chain A and resname GTP and name O3'
  atom_selection_2 = chain A and resid 594 and name P
  symmetry_operation = None
  distance_ideal = 1.6
  sigma = 0.02
  slack = None
}
angle {
  action = *add
  atom_selection_1 = chain A and resname GTP and name C3'
  atom_selection_2 = chain A and resname GTP and name O3'
  atom_selection_3 = chain A and resid 594 and name P
  angle_ideal = 120
  sigma = 1
}
bond {
  action = *add
  atom_selection_1 = chain B and resname GTP and name O3'
  atom_selection_2 = chain B and resid 594 and name P
  symmetry_operation = None
  distance_ideal = 1.6
  sigma = 0.02
  slack = None
}
angle {
  action = *add
  atom_selection_1 = chain B and resname GTP and name C3'
  atom_selection_2 = chain B and resname GTP and name O3'
  atom_selection_3 = chain B and resid 594 and name P
  angle_ideal = 120
  sigma = 1
}
}



From: CCP4 bulletin board  on behalf of Almudena Ponce 
Salvatierra 
Reply-To: Almudena Ponce Salvatierra 
Date: Friday, April 12, 2019 at 10:43 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] naive question how to add GTP as a residue to a nucleic acid 
chain and refine!

Dear all,

I would like to add a GTP residue to a nucleic acid chain. For so I followed 
the following steps in coot: add monomer from library, GTP, changed chain ID 
and residue number accordingly, and then Extensions --> Modelling --> link 2 
atoms. I got a dashed line between the 3'O of the GTP and the phosphate of the 
neighboring nucleotide, and then I used the real space refine tool. Up to here, 
it all looked promising.

In phenix refine I run elbow and readyset and provided cif files, but after 
phenix.refine I find out that the link between GTP and it's neighbor is gone.

How to preserve the bond?

Thank you very much in advance!!

Best,

Almudena



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Re: [ccp4bb] ORCID being mandatory for PDB depositions

[Phoebe A. Rice]

Very good point, and a good argument against the current trend of publications 
in the glossies including everything from mice to omics to structure all in one 
manuscript with one set of authors.  Especially since it is being pointed out 
more vociferously these days (as it should be) that accepting authorship 
implies accepting responsibility.

  *   Phoebe

From: CCP4 bulletin board  on behalf of Mark J van Raaij 

Reply-To: Mark J van Raaij 
Date: Tuesday, April 9, 2019 at 9:32 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] ORCID being mandatory for PDB depositions

There is something to be said for not everyone being an author of the PDB entry 
anyway.
If I were a non structural biologist who for example had made and tested some 
essential knock-out mice for the paper, I might prefer not to be an author of 
the PDB entry in case there was something wrong with it (which I might not 
necessarily be able to judge).
Similarly, as a structural biologist I might prefer not to be on the knock-out 
mice database entry in case there was something wrong or unethical in the way 
the mice were made...

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
http://wwwuser.cnb.csic.es/~mjvanraaij
Section Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/

On 9 Apr 2019, at 14:49, Robbie Joosten 
mailto:robbie_joos...@hotmail.com>> wrote:

Than it is easy enough. You mustn't create an ORCid for others, this could 
indeed get you into GDPR trouble as you are sharing personal data without 
consent.

So that leaves the practical bit. If deposition requires an ORCid and a 
collaborator does not have an ORCid despite your requests, then that 
collaborator cannot be a depositor. Let the record show that this is not your 
fault as your hands are tied here.

HTH,
Robbie

On Apr 9, 2019 14:33, V F 
mailto:veronicapfiorent...@gmail.com>> wrote:
On 09/04/2019, Mark J van Raaij 
mailto:mjvanra...@cnb.csic.es>> wrote:
> Perhaps the poster is referring to the legality of creating an ORCID on
> behalf of the collaborator?

Yes that is what I meant. (English not my first language)!

> That is how I interpreted it - but perhaps I over-interpreted...
>
> Another thing that came to my mind, not everyone has to be an author of the
> PDB structure. Often all the authors of the corresponding paper are also on
> the PDB entry, and there is nothing wrong with that, but if a
> (non-crystallographer?) collaborator really doesn't want an ORCID, he or she
> doesn't have to be an author of the PDB entry.
>
>
> 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
> http://wwwuser.cnb.csic.es/~mjvanraaij
> Section Editor of Acta Crystallographica F, Structural Biology
> Communications
> http://journals.iucr.org/f/
>
>
>> On 9 Apr 2019, at 14:20, Anastassis Perrakis 
>> mailto:a.perra...@nki.nl>> wrote:
>>
>> 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 
>>> mailto:veronicapfiorent...@gmail.com>> 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
>>>
>>> 
>>>
>>> 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
>
>
> 
>
> 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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Re: [ccp4bb] Ramachandran outliers

[Phoebe A. Rice]

In this sort of case, I find that often the Rama-bad residues appear unfixable 
because small distortions in many bond lengths and angles have made the side 
chain appear correctly fit even though the rotamer choice was wrong.

My recipe for fixing that:

  *   Mutate the offending residue to glycine or alanine in coot
  *   Optimize the bond lengths & angles of the backbone
 *   If that doesn’t move the residue into an allowed region, manually fix 
it and re-optimize the bond lengths & angles
  *   Then put the side chain back on and choose the rotamer that best fits the 
density.  You’ll probably fit it to be different than the original one.

My colleagues wrote a rebuild + refine server based on automating, more 
rigorously, that sort of logic:
https://godzilla.uchicago.edu/pages/projects.html
In my own experience at least, you should use the results from their server as 
a buffet of (often quite useful) suggestions for how to rebuild your refmac- or 
phenix- refined model.

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



From: CCP4 bulletin board  on behalf of Tereza Skalova 

Reply-To: Tereza Skalova 
Date: Friday, March 8, 2019 at 3:08 AM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] Ramachandran outliers

Dear all,

I have structure at 3.3A resolution and I have ca. 35 Ramachandran outliers.
Do you have any idea how to reduce the number?
I refine in Refmac, using h-bond based Prosmart restraints based on PDB 
structures (identical molecules with high resolution) and I use NCS, medium 
between AB (protein 1) and loose between CDE (protein 2). I use overall 
B-factor and 8 TLS groups.
Is it possible to optimize Ramachandran plot directly in Refmac?

Thank you

Tereza Skalova







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Re: [ccp4bb] Purchasing slides with diffraction gratings for teaching diffraction

[Phoebe A. Rice]

Slightly off-topic, and I may have recommended these some time ago on the BB 
(sorry), but we find these toy turtles very useful for explaining symmetry, 
particularly screw axes which some students have a really hard time visualizing 
from 2D diagrams:

https://www.amazon.com/Fat-Brain-Toys-FA042-1-Reptangles/dp/B00392NSQ4/ref=sr_1_2?ie=UTF8=1549988668=8-2=reptangles

You can assemble an amazing array of symmetries with these things.

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



From: CCP4 bulletin board  on behalf of Marin van Heel 
<057a89ab08a1-dmarc-requ...@jiscmail.ac.uk>
Reply-To: Marin van Heel 
Date: Tuesday, March 5, 2019 at 3:28 PM
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: Re: [ccp4bb] Purchasing slides with diffraction gratings for teaching 
diffraction

Dear Pietro

I have used many different gratings for the purpose over many years...  Ones 
that I found very rewarding are writeable CDs and DVDs. They often come in a 
pack "protected" by a empty CD / DVD matrix hat is only plastic with grooves 
and no silver (or whatever the shiny recording material is).  Burnig your own 
CDs DVDs has gone somwhat out of fashion so you may have a problem finding 
those transparent ones. But if you have a RED/GREEN laser pointer and empty CD 
/ DVDs all kinds of diffraction experiments are possible. Since DVDs are 
designed for green/blue lasers, the red laser diffraction rapidly goes 
evanescent on a DVD. On the other hand a green or blue laser on a CD matrix 
gives you many diffraction orders on white wall in a dark lecture hall. Nylon 
flags and other woven materials can give excellent diffraction patterns.  Great 
fun! Decades of students at Imperial and other places 
(www.brazil-school.org<http://www.brazil-school.org>) enjoyed those 
crystallography/cryo-EM demos!
My two cents,

Marin

On Tue, Mar 5, 2019 at 6:52 AM Roversi, Pietro (Dr.) 
mailto:pr...@leicester.ac.uk>> wrote:
Dear all,

I'd like to purchase slides with diffraction gratings for educational purposes.

Ideally, to be used with a visible-light laser pointer.

And: with 1D and 2D patterns, of variable repeats - so as to be able to 
illustrate the effect on the spacing in reciprocal space, as it were.

I am looking online but not having much joy.

Can anyone recommend a good provider to purchase from?

Thank you!

Pietro



Pietro Roversi

LISCB Wellcome Trust ISSF Fellow

https://bit.ly/2I4Wm5Z


Leicester Institute of Structural and Chemical Biology
Department of Molecular and Cell Biology, University of Leicester
Henry Wellcome Building
Lancaster Road, Leicester, LE1 7HB
England, United Kingdom

Tel. +44 (0)116 2297237




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Re: [ccp4bb] translational NCS & twinning

[Phoebe A. Rice]

For two projects in the distant past, we dealt with tNCS by initially telling 
lies to the software (1szp and 3pkz).  The tNCS was strong enough that there 
was a clear weak/dark pattern in the diffraction pattern, so for the initial 
molecular replacement we used a data set in the smaller unit cell where only 
the strong spots had been boxed and reduced.  Then we moved that model to the 
proper, full unit cell (weak spots included) data set and finished the 
molecular replacement either by manually pushing the model around according to 
the appropriate vector(s) or just doing more molrep.

If you're desperate, and your weak spots are weak enough, it is worth a try!

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

On 1/10/19, 12:18 PM, "CCP4 bulletin board on behalf of Ethan A Merritt" 
 wrote:

On Thursday, January 10, 2019 2:11:52 AM PST Donghyuk Shin wrote:
> Dear all,
> 
> I am having tough time with my Xtal data sets those seem to be twinned or 
have translational NCS, and it will be greatly appreciated if you can give me 
some advices or comments!
> 
> Data was initially processed with XDS and scaled with aimless without 
specifying certain space group (SG).
> Aimless picked the P 63 2 2 for the best SG, but the xtriage indicates 
there is non-origin peak after patterson analysis. (attached log)
> And, I could not get the proper MR-solution from this data sets.
> 
> Because I read that twinning and tNCS cannot be properly distinguished at 
high SG, I went down to subgroup either P32 or P6 assuming that there is 
twinning which make data set seems to have apparently high SG. (procedure was 
same XDS->aimless but I specified the SG to keep them)
> Then, xtriage still indicates there is non-origin peak as before, but 
found twin laws for the data sets (attached log).
> However, I still could not get the right MR-solution.
> Then, I went even further down to P3 or C2, and xtriage found more twin 
laws which is make sense because of the lower SG. (attached log)

> Again, I could not get the MR-solution.
> For all the MR running above, I assumed that phaser(ccp4 module) 
automatically applied tNCS if they present. or should I have to tick on button 
in the expert parameters?
   ^^^

Hi Donghuk,

This may indeed be a possible source of problems.   I have recent 
experience with a
case where aimless/xtriage/phaser all report a large tNCS component, but 
allowing
phaser to use this information prevents finding a correct MR solution.  I 
can only
obtain the known correct solution by telling phaser explicitly _not_ to use 
tNCS.
In my case the true space group is P1 but the angles are all close to 90., 
causing
most indexing programs to falsely identify it as P2.  I cannot say how or 
why the
tNCS test triggers, but the known correct solution in P1 does not contain 
tNCS.

Ethan


> 
> Then, I went back to the image and processed the datasets with mosflm by 
checking the indexed spots.
> During this step, I played with the threshold for indexing to follow the 
strong spot for get correct SG.
> I am not sure whether this is correct or not, but by putting high 
threshold for indexing (e.g. ~15) I could index the data with C2 which has half 
dimension for a,b axes (116.348,  67.218, 182.861,  90, 90, 90) to the original 
unit cell (232.533, 134.202, 182.67, 90, 90, 90).
> With this, I could put 3 molecules in ASU by MR. During refinement, I 
felt that the R values were not dropping, and I applied twin refinement.
> without twin refinement the R values were (0.39/0.44, work/free), and 
applying twin refinement gave me significantly better values (0.23/0.26).
> Because there were 6 twin operators which may cause this huge R value 
drop, I speculate whether this is true or not.
> 
> Your comments will be greatly helpful! 
> 
> With you all the best,
> Donghyuk
> 
> 
> 
> 
> 
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> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1


-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742



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Re: [ccp4bb] Experimental phasing vs molecular replacement

[Phoebe A. Rice]

A point I like to make to new trainees trying to solve structures:
Model phases from a good model are pretty good, but from a practical viewpoint, 
if your initial model for molecular replacement is that good, the resulting 
structure will probably not tell you much you didn't already know (with 
exceptions of course).
If you only have an existing structure for, say, half of what's in your 
asymmetric unit, SAD phases will be less biased than model phases from 
molecular replacement, even though both may be noisy. 

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

On 12/5/18, 9:14 PM, "CCP4 bulletin board on behalf of James Holton" 
 wrote:

It is true that MAD phasing can give you hyper-accurate phases. This is 
because you are measuring the heavy atom signal in both directions on 
the Harker diagram, allowing the phase to be solved analytically.  The 
phasing signal is noisy, of course, but you can fix that either with a 
bigger heavy atom (more signal) or by doing a lot of averaging (less 
noise). You will probably need more than one crystal.

SAD, on the other hand, can never give you very good phases because the 
phase probability distributions are all bimodal. The technology that 
makes SAD practical is solvent flattening, but as soon as you start 
doing things like solvent flattening you are already imposing a model, 
and every model comes with some amount of bias.  How important that bias 
is depends on the question you are trying to answer.

MIR, like MAD, can get arbitrarily accurate phases, but this and every 
other technique requires a high degree of isomorphism.

In practice, essentially all experimental phasing attempts are really 
trying to get you just over that ever-elusive tipping point of phase 
quality where solvent flattening and model building can take you the 
rest of the way.  So, in the end what you have are model phases, just 
like if you had done MR.  It's sad really how fleeting the involvement 
of experimental phases are in essentially all MAD/SAD structure 
determinations.  Pun intended.

That said, model phases are not so bad.  In fact, in all my experiments 
with fake data the model-phased 2mFo-DFc map always has the best 
correlation to the "true" map.  If you substitute the "true" phases and 
use the 2mFo-DFc coefficients you actually make things worse.  
Counter-intuitive, but true.

-James Holton
MAD Scientist

On 12/5/2018 12:07 AM, 香川 亘 wrote:
> Dear all,
>
> It is my understanding that experimental phasing (e.g. Se-SAD), in 
principle, yields better electron density maps than molecular replacement for 
protein regions with weak electron densities (partially disordered or 
flexible).  I would appreciate if someone could provide comments on whether my 
understanding is correct or not.  If there any good examples or literatures on 
this issue I would be grateful to know about it.
>
> I thank you in advance.
>
> Wataru Kagawa
> 
>
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[ccp4bb] teaching question: graphics for chromebook?

[Phoebe A. Rice]

One for fellow educators:
For years, I’ve given students pymol-heavy homework assignments.  Today I found 
out that at least one has a chromebook and can’t install Pymol.
I gather that these chrome thingies are becoming more and more popular with 
students, so I’m wondering if someone out there already come up with a good 
solution – either an easy way to install pymol, or an alternative 
undergraduate-user-friendly, free macromolecular graphic program that will work 
for chromebook users.
  Thanks,
  Phoebe

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

Re: [ccp4bb] According correct space group assignment...

[Phoebe A. Rice]

Sadly Rafal is right the unit cell dimensions don't make sense - either the 
space group is wrong or the contents have been digested before crystallization.
The size of the overall unit cell is ~21 x 23 x 43.  Given a (DNA-centric but 
close enough) view that a duplex is ~25A wide and there are 3.4A per bp (and 
43A/3.4  gives 12bp) , if this 11mer was a simple symmetric duplex, there could 
only be 2 chains, max, but I222 has 8 asymmetric units.   

This handy tool gives the same answer in a more accurate manner:
http://csb.wfu.edu/tools/vmcalc/vm.html
It says that if the RNA is intact and the space group is P2, there is about 56% 
solvent.  If the space group is really I222, the solvent content would be a 
rather fascinating negative 75%.

   -Phoebe

On 4/20/18, 2:37 PM, "CCP4 bulletin board on behalf of Randy Read" 
 wrote:

I think that starting with a direct methods program like shelxt would be 
fun.  If that doesn’t work, it could be interesting to try to solve it by 
molecular replacement with fragments varying from a tetraplex, a base pair or 
even just single bases.  (Assuming that Phoebe’s concern about twinning does 
not turn out to be correct…)

Best wishes,

Randy Read

-
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical ResearchTel: +44 1223 336500
Wellcome Trust/MRC Building Fax: +44 1223 336827
Hills Road
E-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   
www-structmed.cimr.cam.ac.uk

> On 20 Apr 2018, at 20:09, Tim Gruene  wrote:
> 
> Dear Rafal,
> 
> shelxt does not require the space group, it only needs the Laue group. If 
it 
> finds a decent solution, it'll find also the space group for you.
> 
> Best,
> Tim
> 
> On Friday, April 20, 2018 3:30:36 PM CEST Rafal Dolot wrote:
>> Dear CCP4BB,
>> 
>> I've recently collected data for 11mer build of DNA (9xG, 2xT). XDS, and
>> DIALS gave me similar solution - SG: I2(1)2(1)2(1) or I222, with cell
>> dimension 20.65, 22.96, 43.37, 90, 90, 90, what is too small for this
>> size of the molecule. 11mer is rich in G, so we expect the G-tetraplex
>> formation. Data were collected to almost 1 A, so it should be enough for
>> trials with direct methods/ab initio solution. What I should do first to
>> find correct SG and/or cell parameters?
>> 
>> Best regards,
>> 
>> Rafal
> 
> -- 
> --
> Paul Scherrer Institut
> Tim Gruene
> - persoenlich -
> OSUA/204
> CH-5232 Villigen PSI
> phone: +41 (0)56 310 5297
> 
> GPG Key ID = A46BEE1A

Re: [ccp4bb] According correct space group assignment...

[Phoebe A. Rice]

How do your twinning statistics look?  It could be that the real space group 
has lower symmetry (and thus more space for your G4 DNA).

~~~
Phoebe A. Rice
Dept. of Biochem & Mol. Biol. and
  Committee on Microbiology
https://voices.uchicago.edu/phoebericelab/
 
 
On 4/20/18, 8:31 AM, "CCP4 bulletin board on behalf of Rafal Dolot" 
<CCP4BB@JISCMAIL.AC.UK on behalf of rdo...@cbmm.lodz.pl> wrote:

Dear CCP4BB,

I've recently collected data for 11mer build of DNA (9xG, 2xT). XDS, and 
DIALS gave me similar solution - SG: I2(1)2(1)2(1) or I222, with cell 
dimension 20.65, 22.96, 43.37, 90, 90, 90, what is too small for this 
size of the molecule. 11mer is rich in G, so we expect the G-tetraplex 
formation. Data were collected to almost 1 A, so it should be enough for 
trials with direct methods/ab initio solution. What I should do first to 
find correct SG and/or cell parameters?

Best regards,

Rafal
-- 
|--|
|Rafal Dolot, Ph.D.|
|  |
|Polish Academy of Sciences|
|Centre of Molecular and Macromolecular Studies|
|Department of Bioorganic Chemistry|
|Macromolecular Crystallography Team   |
|Sienkiewicza 112  |
|90-363 Lodz, Poland   |
|Phone: +48(42)6803215 |
|Cell:  +48 502897781  |
|--|

Re: [ccp4bb] does 12 A diffraction worth optimization

[Phoebe A. Rice]

In addition to the other useful advice offered, I’d also suggest determining if 
the whole complex or just the DNA fits in the asymmetric unit.  Particularly if 
the crystals are hexagonal rods or plates, you might have DNA-only crystals.

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


From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Natalia O 
<natalie.c...@gmail.com>
Reply-To: Natalia O <natalie.c...@gmail.com>
Date: Friday, March 2, 2018 at 7:35 PM
To: "CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] does 12 A diffraction worth optimization

Hello,

I got crystals of protein-nucleic acid complex, rod-shape, reproducible, don’t 
visibly get damaged upon freezing; however they gave diffraction only to about 
12 A. I tried several crystals. My question is whether such crystals worth 
optimization. Clearly a 4A diffracting crystal could potentially be optimized 
to 3 – 2.5A, but if the diffraction that I am getting now is 12A it could 
suggest that the system is so flexible that getting to 3A with this crystal 
form is not possible at all. I just wonder if there is any statistics or a rule 
of thumb about what initial diffraction worth optimization?

Thank you!
-Natalia

Re: [ccp4bb] secondary structure prediction

[Phoebe A. Rice]

I'm fond of RaptorX


++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago

pr...@uchicago.edu<mailto:pr...@uchicago.edu>
https://voices.uchicago.edu/phoebericelab/


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Missoury Sophia 
[sophia.misso...@u-psud.fr]
Sent: Wednesday, December 06, 2017 9:20 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] secondary structure prediction

Modeller


Sophia Missoury





De: "Smith Liu" <smith_liu...@163.com>
À: CCP4BB@JISCMAIL.AC.UK
Envoyé: Mercredi 6 Décembre 2017 16:04:12
Objet: Re: [ccp4bb] secondary structure prediction

Rosetta



<https://maas.mail.163.com/dashi-web-extend/html/proSignature.html?iconUrl=https://nos.netease.com/mail-online/qiyelogo/defaultAvatar.png=Smith%20Liu=smith_liu123%40163.com=1=%5B%22%E9%82%AE%E7%AE%B1%EF%BC%9Asmith_liu123%40163.com%22%5D>
[https://nos.netease.com/mail-online/qiyelogo/defaultAvatar.png]
Smith Liu

邮箱：smith_liu...@163.com


签名由 网易邮箱大师<https://mail.163.com/dashi/dlpro.html?from=mail88> 定制


在2017年12月06日 22:14，zheng zhou<mailto:zhengzho...@gmail.com> 写道：
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

Re: [ccp4bb] Electron density in PDBe

[Phoebe A. Rice]

I agree that density looks dubious, the B-factor distribution among DNA atoms 
is odd (sudden shifts from blue to red) and the little wwPDB bar charts are on 
the red side.  

But what about the density for the protein?  
The structure is of a hexameric helicase with with dsDNA going through the 
hole, so the space group being nearly-R3 or R32 doesn't shock me: one would 
expect the DNA to subtly break the symmetry. 

Could it simply be very poorly ordered DNA going making a pseudo-continuous 
helix going through the crystal? Or maybe the symmetry isn't broken, and DNA 
backbone is in 3 different rotational states even if there's rough density for 
the planes of the bases?

++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago

pr...@uchicago.edu
https://voices.uchicago.edu/phoebericelab/



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Anastassis 
Perrakis [a.perra...@nki.nl]
Sent: Thursday, November 30, 2017 12:56 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Electron density in PDBe

Interesting structure:

a=b=c=α=β=γ=107 in P1
Estimated twinning fraction 0.439 for k,l,h and 0.439 for l,h,k

In other words R32? ;-)

The PDB-REDO map has some interesting features, one strand has bases density at 
.0 sigma, the other not, see attched.

The NCS averaging they used is kind of interesting. It reads correct if I read 
through, but if something is done wrong, I am guessing it might result in 
erronous features.

As a side note, the refereeing in eLife is interesting. The reviewing editor is 
Stephen Bell; an excellent scientist, but not a crystallographer. Referees 
opted to stay anonymous - but one could speculate they are not 
crystallogrpahers either, as they did not raise any crystallographic questions. 
The validation summary, well, it could have raised some red flags … sorry, it 
did raise red flags, or to be exact red bold boxes. Evidently, nobody looked at 
them.

Have fun -

A.



[cid:B25D7E2E-FD64-40A6-9DBD-60DEF50D9249@home]
[cid:5B0AA6A0-834B-46C7-AE56-8C6DFFB2F62D@home]
A.



On 30 Nov 2017, at 19:01, Wim Burmeister 
<wim.burmeis...@ibs.fr<mailto:wim.burmeis...@ibs.fr>> wrote:

Dear all,
I am doing some analysis on pdb entry 5tct.
I realized that the electron density on the PDBe site (obtained from EDS) shows 
much more model bias than the one obtained from the pdb file and the cif 
reflection data file using ccp4i (import + refmac5 with 0 cycle positional 
refinement). Using the mtz file from pdb-redo a similar map is obtained.
When I read the publication 5kleywegt et al, 2004) on EDS, I understood that 
essentially the same approach is used (refmac with 0 cycle refinement followed 
by fft map calculation).
Does anybody have an idea where the discrepancy comes from ?
I join a jpg with the electron density of the 3 approaches. The contour level 
is about 3 sigma.
Best,
Wim

ps: I expect that the electron density of the DNA is not really present as the 
entry has a number of flaws.
--

Wim Burmeister
Professeur
Institut de Biologie Structurale (IBS) CIBB
71 avenue des Martyrs

CS 20192
38044 Grenoble Cedex 9, FRANCE
E-mail: wim.burmeis...@ibs.fr<mailto:wim.burmeis...@ibs.fr>
Tel:+33 (0) 457 42 87 41   Fax: +33 (0) 476 20 94 00
website<http://www.ibs.fr/research/research-groups/viral-replication-machines-group-m-jamin/team-03/article/poxvirus-replication-machinery-presentation/>

Re: [ccp4bb] Off topic: denaturing urea gels

[Phoebe A. Rice]

Even though the protein should be denatured by all that urea, we find such gels 
sometimes look nicer if stop the reaction with SDS and protease K, and/or 
phenol extract the remains of the protein before loading the high-urea gel.

++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
pr...@uchicago.edu



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Opher Gileadi 
[opher.gile...@sgc.ox.ac.uk]
Sent: Saturday, September 30, 2017 3:44 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic: denaturing urea gels

In addition to the previous suggestions:

With very small gels, the sample composition and depth (in the well) have a 
strong effect on the resolution.
Rinse the wells with TBE buffer just before loading, as urea from the gel 
diffuses into the well and may prevent the sample from settling at the bottom. 
Minimize the amount of salt in the samples; try to load very small volumes (1-3 
ul), even if this means longer exposures later.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Phoebe A. Rice]

You might also be getting aggregation.
If you do an old-fashioned EMSA ("gel shift") assay, does it hang up in the 
well?


++++++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago

pr...@uchicago.edu<mailto:pr...@uchicago.edu>


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Morgan Milton 
[eilise.mil...@gmail.com]
Sent: Friday, July 21, 2017 9:44 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

Completely agree, you need a higher DNA concentration. We have had luck with 10 
nM DNA.

Also, bubbles have a HUGE impact on how the fluorescent signal is measured. 
Make sure you spin your plates down (assuming you are using them) to remove any 
bubbles. We just had an undergrad read his anisotropy assay and the data looked 
horrible. He then realized he had not spun his plate down, after doing so the 
data was much more consistent.

Are you doing technical replicates? We do at least triplicates per plate.

All the best,
Morgan


Morgan E. Milton, PhD


On Fri, Jul 21, 2017 at 9:48 AM, Opher Gileadi 
<opher.gile...@sgc.ox.ac.uk<mailto:opher.gile...@sgc.ox.ac.uk>> wrote:
FP is the ratio between two fluorescence measurements; if the fluorescence 
signal is too low, you will still get a ratio but it will be essentially noise. 
Try to perform the measurements at 10-50 nM DNA. If your binding affinity is in 
the low nM range, you may have to use other methods to measure KD.

Re: [ccp4bb] Ramachandran oultliers increase upon restrained refinement

[Phoebe A. Rice]

4% outliers at < 3A seems a little high to me.
A trick that often works for me is to truncate the side chains surrounding the 
offending peptide to Ala or Gly, optimize the backbone, and then put the side 
chains back on.  Often that makes it clear that a different rotamer would work 
better.


++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago

pr...@uchicago.edu<mailto:pr...@uchicago.edu>
Mobile DNA III: mobile elements are everywhere!  
http://www.asmscience.org/content/book/10.1128/9781555819217


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Vipul Panchal 
[panchal.vi...@igib.in]
Sent: Sunday, July 02, 2017 12:31 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Ramachandran oultliers increase upon restrained refinement

I do agree with Eleanor. When I was solving structure at 2.16A resolution, 
outlier residues were having stearic clash or required side chain flipping.

At 2.76A resolution, hydrogen bonds would be difficult to visulize.

I found phenix.refine more user friendly. You may too find it useful.

Vipul Panchal,
Ph.D. student
CSIR-IGIB
(M)- 091 7678617949

On 02-Jul-2017 9:14 PM, "Eleanor Dodson" 
<eleanor.dod...@york.ac.uk<mailto:eleanor.dod...@york.ac.uk>> wrote:
Just remember - most Ramachandran outliers are due to errors in the environment 
- eg: maybe some side chains clash hence refinement tries to move things to 
accommodate that bad feature, etc etc etc...
At that resolution it is inevitable you will have some level of 
mis-interpretation ..
4% outliers does not surprise me.

EDSTATS is a useful tool to find things such as pep flips - you can access it 
most simply from CCP4 GUI2 .

But unless the outliers are in a important part of the structure I would 
suggest checking, then living with them.

Eleanor






On 2 July 2017 at 16:31, Rajesh Kumar 
<rsu.iitku...@gmail.com<mailto:rsu.iitku...@gmail.com>> wrote:
Dear Meytal,

PHENIX-REFINE has an option for Ramachandran outlier refine. If you check this 
on, it will do the work. But after this refinement, you must check every 
residues to make sure residues are in the density.

Thank you
Rajesh


 ---x
With regards
Rajesh K. Harijan, Ph.D.
Schramm Laboratory
Albert Einstein College of Medicine
1300 Morris Park Ave., Bronx, NY 10461
Tel: 718.430.2777<tel:(718)%20430-2777>  |  Fax: 
718.430.8565<tel:(718)%20430-8565>



On Sun, Jul 2, 2017 at 7:26 AM, Meytal Galilee 
<meytal.gali...@gmail.com<mailto:meytal.gali...@gmail.com>> wrote:
Dear all,
I am refining a 2.76A structure using refmac5.
I keep fixing the Ramachandran outliers down to 18 (1.9%), however, upon 
restrained refinement, the outliers increase back to 35.
Do you have any suggestions how can I fix my structure?
Thanks,
Meytal Galilee

Re: [ccp4bb] Separating Monomers and Dimers

[Phoebe A. Rice]

Sorry if this is an insulting question, but did you store it in enough glycerol 
to prevent it from freezing?  Proteins don't like freeze/thaw cycles, 
especially slow ones.


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of jai mohan 
[0cab66323371-dmarc-requ...@jiscmail.ac.uk]
Sent: Tuesday, June 27, 2017 7:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Separating Monomers and Dimers

Dear all,
I am working on a Red Fluorescent Protein (His-Tag) molecular weight around 
27kDa. After purification I ran a SDS page, the band at 27kDa confirms the 
monomer. The protein was stored at -20C, a week later again I ran a gel, this 
time I saw another new band between 50-60kDa, it confirms the protein solution 
contains both monomers and dimers. I would like to know, what is the best way 
to separate the monomers and dimers? One of my colleague advice me to go for 
sucrose gradient centrifugation and size exclusion chromatography.
However, I seek all your valuable suggestions and advice.

With best regards
Dr. S.M.Jaimohan

Re: [ccp4bb] Protein or DNA crystals

[Phoebe A. Rice]

This is not at all conclusive, but one depressing thing is that DNA-only 
crystals are often hexagonal (like yours are).  
Can you reproduce them without the protein? 

++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
pr...@uchicago.edu



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Joseph Ho 
[sbddintai...@gmail.com]
Sent: Monday, June 19, 2017 9:20 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein or DNA crystals

Dear all:

I would like to seek your opinion on our crystal hits. We are working
on protein/dsDNA complex. By changing different protein and DNA
(14-22bp) constructs, we recently got some hits from commercial
screens using sitting drop vapor diffusion (very small xtals). The
precipitant is PEG and the picture of crystals are attached. In this
particular condition, it is 30%PEG3350, sodium succinate pH5.5 and
100mM NaCl. The crystal seems floating and sit in the bottom. We do
some test shot from other conditions and it is not salt crystals. The
crystals can suck in izit dye.  I do some google and it seems izit dye
also turns dsDNA crystal into blue. We also do UV/Vis microscope but
no Trp fluorescence (6 Trp in 256 aa). It may due to low Trp.

This is our first time to work on protein/DNA complex crystals and we
are not certain if this is just DNA or protein/DNA crystals. Can you
provide your comments on our hits?

Thank you for your help

Joseph

Re: [ccp4bb] radiation damage-induced phasing (RIP) tutorial

[Phoebe A. Rice]

In the distant past, we did get some additional phasing signal by a simplified 
version of the RIP concept based on the lability of the bromines in a 
5-bromo-dU substituted DNA in an x-ray beam. 
We collected the peak anomalous signal first and fast, and then just kept 
collecting (we may probably to the edge wavelength too).  If my memory serves 
me right, we binned the data into reasonably complete subsets, and then tested 
which initial - decayed gave us the best difference signal (which would be a 
combination of delta f' and delta f at that point).  We could have solved the 
structure without going to such lengths, but it was kind of fun and it did 
improve the phasing a bit.  And heck, the crystal was in the beam, so why not 
milk it for every smidgen of signal it could provide?


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of George Sheldrick 
[gshe...@shelx.uni-ac.gwdg.de]
Sent: Saturday, April 29, 2017 2:14 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] radiation damage-induced phasing (RIP) tutorial

Dear Murpholino,

I must apologize, there was a mistake in may last email. The critical
parameter is, as you correctly pointed out, DSCA not RIPW and it is
necessary to try a range of values for DSCA. For this reason I am CCing
this to CCP4bb.

As you will be discovering, RIP phasing is not easy. We treat it like
SIR, but it is complicated by the presence of both positive and negative
difference peaks caused by radiation damage. Also density modification
is less effective for SIR and RIP than for SAD phasing. In the case of
SAD, just replacing negative density with zero improves the phases, but
this is not true of SIR and RIP, However using the anomalous signal as
well (RIPAS) helps, similar to SIRAS.

Best wishes, George

--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-33021 or -33068
Fax. +49-551-39-22582

Re: [ccp4bb] High Rfree: Phasing issue or partial crystal disorder

[Phoebe A. Rice]

Hi,
  Sometimes automated model building needs more manual intervention than one 
might expect, although it sounds like you've already carefully inspected the 
"good" regions.
  Could you use a model of the N-ter (from a homolog) simply to create a 
solvent envelope, then see if solvent flattening (or more sophisticated modern 
solvent-massaging tricks) improves the density in that region?
Phoebe


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Pravinkumar 
Jagtap [pravinja...@gmail.com]
Sent: Tuesday, April 11, 2017 1:54 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] High Rfree: Phasing issue or partial crystal disorder

Dear All,
I am stuck with this problem for 2 months and hope you could help.

We have a 2.1 A dataset for a 380 amino acid long protein. The space group is 
I4 (single molecule in asymmetric unit, 48% solvent content) and the dataset is 
quite perfect (no obvious pathologies). The protein itself is organised in 2 
lobes (N and C terminal lobes). The sequence identity to nearest homologue 
structure is 17%.

We could  get the phases by SeMet SAD phasing (3A resolution dataset, 5 SeMet 
(excluding N-terminal Met), 3 full occupancy SeMet in C-terminal lobe and 2 
partial occupancy (~0.5 each; present on surface) SeMet in N-terminal lobe). 
Automated  model building (at 2.1 A) yielded nice model for the C-terminal lobe 
(215 residues)  and manually I could build parts (around 80 residues) of 
N-terminal lobe with high confidence. In addition we could also build a ligand 
which is sandwiched between C and N terminal lobe.

However the Rfree is stuck at 0.39 (Rwork 0.33). There is indeed some patchy 
density left at the N terminal lobe but as it is discontinuous, I cannot build 
anything in it (except lots of water molecules). In total I am missing around 
85 residues. These residues are predicted to be present in secondary structure 
(and not flexible).

As I have around 75-80% model built, I would expect that I would have all the 
phases and  should get nice density for the remaining part. But as I dont see 
it, could the rest part be flexible? But again, this is not reflected in the R 
factors (I would then expect low Rfree).

Could it be that I still lack phases (due to partial occupancy of SeMeth in 
N-terminal lobe ) and have to try to get them by heavy metal soaking, or there 
is disorder in the N-terminal lobe? I have also tried solving different 
datasets for same crystal but this has not been useful.

Regards,
Pravin.

Re: [ccp4bb] suggestion on protein crystallization optimization from phase separation

[Phoebe A. Rice]

We had a complex many years ago that could sometimes be convinced to 
crystallize by poking the drops (probably with a hair): if I remember 
correctly, both poking some of the skin into the drop and pulling some of the 
oily goo into more contact with the aqueous phase.  Then again, that complex 
also sometimes crystallized without help, so it might not be the best example 
for your case.

My group has generally found that the most important variable is the ends of 
the DNA, and often when we get the "winner" DNA we get at least crappy 
microcrystals under a variety of conditions.

Hope you get nice fat crystals soon!
 Phoebe


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Johannes Cramer 
[johannes.cra...@gmail.com]
Sent: Friday, April 07, 2017 7:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] suggestion on protein crystallization optimization from 
phase separation

Dear Joseph,

You have probably already done so, but have you tried in- or decreasing your 
protein+DNA concentration?
I had a similar problem with proteins that were concentrated too low. I had to 
increase protein and decrease Ammonium sulfate (precipitant in my case) 
concentration.

Cheers,
Johannes


2017-04-06 17:16 GMT+02:00 Joseph Ho 
>:
Dear all:

I would like to seek your suggestion on protein crystallization from
phase separation.
We recently observed many small round droplets shown in our
protein/DNA crystallization. Condition are 0.8M-1.6M LiSO4; 20mM
MgCl2; pH 5-8;  protein conc. ~15mg/ml). The UV microscope confirms
those are protein-rich phase separation.
We have tried to change conc. of LiSO4 and pH. Still we got different
size and amount of small round droplets. At 20 degree, those droplets
appear within one day and at 4 degree, it takes two-three days.  We
also tried additive and silver bullet screen. So far, we have not
found a condition to have protein crystals. The protein is already
truncated. Several DNA constructs are on-going.
At this point, I would like to seek your advice on the method to
optimize the condition. Based on


PS. Any people have luck with protein crystallization by streaking the
Gelationous protein to new drop as shown in
http://xray.bmc.uu.se/terese/tutorial3.html . Can you please share
your experience with us?

Thanks for your help.

Joseph Ho

Re: [ccp4bb] Ligand electron density

[Phoebe A. Rice]

At long last, a brilliant theoretical framework for observations previously 
dismissed as mere discrepancies!
This being Saturday, can the postulated time-dependent decay of previously 
observed observables also be applied to explain the apparent discrepancy in 
number of socks entering the wash vs. number recovered?
  Phoebe


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Bernhard Rupp 
[hofkristall...@gmail.com]
Sent: Friday, March 31, 2017 4:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Ligand electron density

Hi Fellows,

due to the change of month on Sa I am releasing already today
the first page of an until Sa embargoed reprint on concerns
about theoretical principles of electron density reconstruction.

The paper is available on my dropbox (full paper after Sa):
https://dl.dropboxusercontent.com/u/30662912/Rupp_2017_Phys_Rev_Letters_Electron_Density.pdf

Best wishes, BR
--
Bernhard Rupp
Crystallographiae Vindicis Militum Ordo
http://www.hofkristallamt.org/
b...@hofkristallamt.org
+1 925 209 7429
+43 676 571 0536
--
Many plausible ideas vanish
at the presence of thought
--

Re: [ccp4bb] Positive densities map on selective residues on Coot or Pymol

[Phoebe A. Rice]

Thank you Bernard.

I'd like to add that carving the density shown in a figure (especially without 
explicitly describing the carve radius used) is the moral equivalent of showing 
only 1mm of background above and below the band of interest in a western blot.

 Phoebe


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Bernhard Rupp 
[hofkristall...@gmail.com]
Sent: Tuesday, March 21, 2017 3:29 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Positive densities map on selective residues on Coot or 
Pymol

> and carve=2.0 means the mesh is created within 2.0 Å of the selected atoms.  
> Adjust these values to suit.

I would advise extreme care when employing such presentation features. They are 
useful for display
purposes and only when their use is stated as such, but they also open a venue 
for cognitive biases.
One might want to have a look at how creative use of these ‘blob’ features 
until things ‘suit’ can
betray the true believer…

http://www.ruppweb.org/cvs/br/rupp_2001_NSB_questions_BotA.pdf
https://www.dropbox.com/s/iul2cjbuu4x2o9f/Hanson_2009_retraction_bot_nsmb0709-795.pdf?dl=0

Best, BR

The first principle is that you must not fool yourself and you are the easiest 
person to fool.”
Richard P. Feynman

Re: [ccp4bb] How to refine this needle crystal of membrane complex?

[Phoebe A. Rice]

For historical perspective: 

Molecular structure determination by electron microscopy of unstained 
crystalline specimens
PNT Unwin, R Henderson - Journal of molecular biology, 1975 
http://www.sciencedirect.com/science/article/pii/0022283675902120


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, March 01, 2017 1:50 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How to refine this needle crystal of membrane complex?

There are now quite a few structures solved by ED of 3D crystals, esp by Tamir 
Gonen's group here at Janelia, and a quick count in the pdb yields ~15 
3D-crystal ED structures, some of which are not released. They're getting 
better at it all the time. I guess the news has not spread as much as I 
thought. FYI, last I checked they were looking for good samples to try, 
although you'd have to contact Tamir about that to see whether it's still true, 
or whether they've now got their hands full.

All the best,

Jacob Keller





-Original Message-
From: Tim Gruene [mailto:tim.gru...@psi.ch]
Sent: Wednesday, March 01, 2017 2:22 PM
To: Keller, Jacob 
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] How to refine this needle crystal of membrane complex?

Dear Jacob, dear TO,

(I have wondered what can be 'micro' about diffraction - would you know?)

it would be very exciting to get the structure by electron diffraction - as 
much as I know it would be the first new structure solved from a 3D crystal 
with electron radiation in the PDB. You would have to contact one of the 3-4 
labs that to electron diffraction of protein samples (Abrahams, Gonen, 
Hovmoeller / Zou (possibly), and Yonekura/Toyoshima in alphabetic order). The 
first and the last group are equipped with sensitive hybrid pixel detectors 
that might help.

A good test to get an idea about the resolution you might get from electron 
diffraction is to scratch up as many of your crystals and analyse the powder 
pattern with a strong X-ray source (synchrotron, or very good home source).

Note that when you can see the crystals with a light microscope, they are most 
likely too big for electron diffraction - you might end up seeing a black 
screen with no electron penetrating your samples.

Best regards,
Tim

On Wednesday, March 1, 2017 1:59:44 PM CET Keller, Jacob wrote:
> Although trying to refine the crystals is perhaps the most
> straightforward thing, you might consider trying micro electron
> diffraction (micro-ED) with the existing samples—the technique is
> working increasingly better, and would work well with your tiny
> crystals. Look at Tamir Gonen’s recent papers for more info.

> All the best,
>
> Jacob Keller
>
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
> Sent: Wednesday, March 01, 2017 3:53 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] How to refine this needle crystal of membrane complex?
>
> Dear all,
> I am trying to obtain crystals of membrane complex. Now, I have
> obtained the complex with appropriate stoichiometry after a series of
> purification in DDM, then I get extremely thin needles (as shown in
> figure) in  some conditions such as 28% MPD, 0.1 M NaAc pH 4.5 or 45%
> MPD,0.1 M Bis-Tris pH 6.5, 0.2 M CaCl2. However, there are not any
> improvements through changing the concentration of precipitants/salts
> or adding the additives/detergents to the protein.

> I would be very happy if anybody could give me some advice about this
> crystal refinement.

> With best regards.
>
>
> [cid:image001.png@01D2926A.28CB0C70]

--
--
Paul Scherrer Institut
Tim Gruene
- persoenlich -
OFLC/104
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A

Re: [ccp4bb] Bad density for chains

[Phoebe A. Rice]

Are you sure there really are 4 chains?  50% solvent may be average, but we've 
had crystals with closer to 80%.



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Pooja Kesari 
[pkesar...@gmail.com]
Sent: Tuesday, January 24, 2017 10:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Bad density for chains

Dear All,

I have a 2.6 A resolution structure having four chains in an asymmetric unit.
The chain A and B have density for almost all residues however we don't have 
proper residue density in chain C and D.What can be tried to build chain C and 
D ?



Many Thanks
Pooja

Re: [ccp4bb] CCP4BB Digest - 13 Jan 2017 to 14 Jan 2017 (#2017-15)

[Phoebe A. Rice]

Excellent point.  I've heard apocryphal stories of someone obtaining beautiful 
salt crystals (magnesium ammonium phosphate) by trying to crystallize an ATPase 
with ADP and Mg++ using (NH4)2SO4 as a precipitant.  Wait long enough, and the 
ADP becomes ATP plus AMP, the ATPase takes the 3rd phosphate off, and then the 
salt crystals grow.  

Yours sounds like a much less depressing "accident"!


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phil Evans 
[p...@mrc-lmb.cam.ac.uk]
Sent: Thursday, January 19, 2017 4:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] CCP4BB Digest - 13 Jan 2017 to 14 Jan 2017 (#2017-15)

Remember that 2xADP can disproportionate into ATP + AMP

> On 19 Jan 2017, at 20:54, Panneerselvam, Saravanan 
>  wrote:
>
> Dear All,
> Sorry for the little bit off topic. Is there a possibility for covalent
> bond formation between beta phosphate of ADP and acetate molecule both
> are coordinated by divalent metal ions? I am working on a Kinase
> structure  which was crystallized with ADP and in presence of 1M sodium
> acetate. We observed additional density around ADP that fits perfectly
> like a gamma phosphate , mimicking like ATP bound state, surrounded and
> coordinated by two metal ions(resolution is 1.4A). There is a change in
> space group (from I212121 to P212121 ) and further important
> conformation changes are observed around ATP binding pocket and distant
> region. This is the only xtal we obtained in this space group, and all
> other xtals(measured 10 xtals)  from the same plate belong to I212121.
>
>
>
> Thanks your help and time!
>
> Saravanan
> - Original Message -
> From: "CCP4BB automatic digest system" 
> To: CCP4BB@JISCMAIL.AC.UK
> Sent: Sunday, 15 January, 2017 01:01:37
> Subject: CCP4BB Digest - 13 Jan 2017 to 14 Jan 2017 (#2017-15)
>
> There are 2 messages totaling 45 lines in this issue.
>
> Topics of the day:
>
>   1. Phenix (2)
>
> --
>
> Date:Sat, 14 Jan 2017 20:58:09 +
> From:D Bonsor 
> Subject: Phenix
>
> Is the phenix website down? Anyone know when it will be back up?
>
> --
>
> Date:Sat, 14 Jan 2017 14:45:06 -0800
> From:Pavel Afonine 
> Subject: Re: Phenix
>
> See notice on Phenix mailing list that answers your question.
>
> On Sat, Jan 14, 2017 at 12:58 PM, D Bonsor 
> wrote:
>
>> Is the phenix website down? Anyone know when it will be back up?
>>
>
> --
>
> End of CCP4BB Digest - 13 Jan 2017 to 14 Jan 2017 (#2017-15)
> 

Re: [ccp4bb] Calculation of RSRZ Score in PDB Validation Reports

[Phoebe A. Rice]

I found that one can get RSRZ to go way down by loosening the geometry 
restraints.  The result is a crappy structure and I don't recommend doing that, 
but it does get all the atoms crammed into some sort of density.

RSRZ, in my most humble of opinions, seems like one of those statistics that is 
far more useful in theory than reality.   Particularly for medium-resolution 
structures, the fit of each entire side chain to the density is likely to be 
imperfect because the density is imperfect, especially toward the tips of those 
side chains.

Then again, it can be a good flag for bits of the structure worth a second look 
in rebuilding.


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Matthew 
Bratkowski [mab...@cornell.edu]
Sent: Tuesday, November 22, 2016 10:12 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Calculation of RSRZ Score in PDB Validation Reports

Hello all,

I was wondering if anyone knew how the RSRZ score was calculated in the protein 
data bank validation reports and how useful of a metric this actually is for 
structure validation?  I am trying to improve this score on a structure that I 
am working on, but I'm not really sure where to begin.  From my understanding, 
the score is based on the number of RSRZ outliers with a score >2.  In my case, 
I have several residues with scores between 2 and 4, but at least by eye, fit 
to the electron density does not look that bad.  Hence, I can't justify 
deleting them to try to improve the score.  If the score is just based on 
percent of outlier residues, then for instance wouldn't a structure with say 20 
residues modeled with no corresponding electron density have the same score as 
a structure with 20 residues with RSRZ values of say 2.5?


I was also wondering how the resolution of the structure relates to the score?  
Glancing through several pdb validation reports, I noticed some structure with 
low resolution (3.5 A or lower) with relatively high scores, while others with 
high resolution (2 A or higher) getting low scores.  It is reasonable to assume 
that a structure of lower than 3.5 A would be missing several side chains and 
may also have some ambiguous main chain electron density, which should in 
theory increase the RSRZ score.  While of course every structure is different 
and the quality of it due to the rigor of the person building the model, I was 
wondering if there were any general trends related to resolution and RSRZ score.

Thanks,
Matt

Re: [ccp4bb] Two SGs in one droplet?

[Phoebe A. Rice]

Back in my grad school days, we had crystals (of gamma delta resolvase large 
fragment) like that: at high protein concentrations, we'd get P6422 with 
1/asymmetric unit, and at lower concentrations, C2221 with 3/asymmetric unit 
(with an imperfect ncs 2fold 60 degrees from the xtal fold, and very similar 
overall packing) that diffracted better.  Sometimes the latter would grow off 
the sides of the former.
 Phoebe

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Roger Rowlett 
[rrowl...@colgate.edu]
Sent: Friday, October 28, 2016 9:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Two SGs in one droplet?

I've seen this kind of thing before.

Case 1: two crystal forms in the same droplet, C2 and C222. If you looked 
closely, you could tell them apart and I was pretty good at getting a high 
percentage of the desired space group by looking at the crystal forms. The C2 
form diffracted better, so I fished for that one.

Case 2: A mixture of crystals, C2 and C2 with the long axis doubled in length, 
caused by asymmetric ligand binding. In the "double-size" C2 crystal form, 
ligands bound to only 10 of the 12 chains in the double-size ASU, which no 
longer conformed to two adjacent "normal-size" C2 ASUs. In the "normal" size C2 
form, all 6 protein chains in the ASU bound ligand.

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 10/28/2016 9:13 AM, Sam Tang wrote:
Dear all

Sorry for going a bit off-topic in this thread.
May I seek your advice as on whether you have experienced that crystals being 
obtained from the same droplet, looking alike under microscope (rod shape) and 
in fact growing possibly from a same nuclei, give two space groups after 
indexing?

I recently obtain crystals for a protein (co-crystallized with a nucleic acid 
ligand) and collected two datasets from synchrotron. Although these two 
crystals are from the same drop, the SG and unit cell dimensions are very 
different:

Xtal1: C121 (156 60 105 90 111 90) (L-test, Pointless shows that there is no 
twinning), ~2.5 Angstrom
Xtal2: P1 (53 60 79 106 105 98), ~3 Angstorm

Would it be possible that the ligand changes the SG of the crystal so that only 
one of the forms contains the ligand?

Any advice is appreciated and thanks a lot in advance for your input.

Regards

Sam Tang
Biochemistry Programme, School of Life Sciences, CUHK

Re: [ccp4bb] Antw: Re: [ccp4bb] High B factor

[Phoebe A. Rice]

Interesting way to look at it.  But those loop residues are really in the 
crystal with an occupancy of 1, so wouldn't letting the B factor fly give a 
clearer description of what's in the crystal?  Especially as many people know 
to color the structure by B factors to get a feel for which bits are wiggly, 
but they'll never think to color it by occupancy.


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Matthias Barone 
[bar...@fmp-berlin.de]
Sent: Friday, October 14, 2016 8:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Antw: Re: [ccp4bb] High B factor

Picking up the mail of Pavel, Phenix refines occupancies..
If you expect the loops to be disordered, did you try to lower the occupancy of 
these residues, following Ethan Merritt statement that "general uncertainty 
[...] is represented better by occupancy <1 rather than an arbitrary large B 
factor" (To B or not to B, doi:10.1107/S0907444911028320).
If this attempt does not bring the B-factors down, it will surely make the 
model more accurate, as the atom coordinates of the loops may not be correct at 
all, no?

matthias


>>> Pavel Afonine  14.10.16 9.36 Uhr >>>


If you are still worry about your Bfactor, you could try TLS,

Or NCS, but SA with MLHL might be better.

(A joke).

Re: [ccp4bb] Interesting DNA contamination

[Phoebe A. Rice]

That an entire 500bp would be protected from DNase seems very very strange - 
but very interesting if true!

Could that band on the agarose gel be something else?  Protein will stain a bit 
with ethidium as well as with coomassie.  Did you add SDS before running the 
agarose gel or is it native (in which case the size of an intact protein-DNA 
complex is not directly related to the mobility, as others have noted)?  How do 
the 280 and 260nm peaks in your UV spectrum compare to those expected for your 
protein (before adding ATP, and possibly expecting 1 ATP / molecule to come 
along for the ride during purification)?

Good luck,
  Phoebe Rice


++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

pr...@uchicago.edumailto:pr...@uchicago.edu

Re: [ccp4bb] Protein precipitation

[Phoebe A. Rice]

For one of our more easily-degraded proteins, we added a mix of protease 
inhibitors (those expensive tablets you can buy) and also put a drop of EDTA 
into each tube in the fraction collector so that any contaminating 
metal-dependent proteases would be knocked out as soon as the protein came off 
the metal affinity column.  That seemed to help.
Also, if you can get your protein to stick to the next column in whatever 
buffer it comes off the IMAC column in, there's no need to dialyze or 
concentration between columns - the faster you get it clean, the better.
Good luck!
 Phoebe Rice


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Michel, Max 
[m.mic...@fz-juelich.de]
Sent: Tuesday, May 19, 2015 1:32 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein precipitation

Hi Manjula,

I had a similar problem: pH, salt, additives and protease inhibitors (I tried 
them all) didn't work. The protein degraded after lysis of bacteria within 20 
min at 24°C to 50 %. I had luck after i cooled down all buffers and materials 
to less than 4 °C or used them icecold ... (1L bottle buffer stirred for 3-4h 
in icewaterbath). I performed IMAC, IEX and SEC to get rid of the degradation. 
Finally the Protein was stable for at least one month.
You have to take care about temperature induced pH changes. Some buffers change 
their pH up to 0.5 (like Tris) when stirring from 24 to 2 °C. This can lead to 
precipitation if you are working close to the pI of your protein.

Maybe you can try to characterize the protease to find a suitable protease 
inhibitor. Make some massspectrometry with your degraded sample. An other 
option is to test via westernblot. If you have some C- oder N-terminal 
monoclonal well characterized antibody.

Greets Max

Von: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] im Auftrag von Manjula Ramu 
[manjula@gmail.com]
Gesendet: Dienstag, 19. Mai 2015 08:05
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Protein precipitation

Thanks all for the suggestions.
@ Pius,
I used bacterial expression system.Yes I centrifuged precipitated protein 
sample and found more amount in soluble form itself, however within a day 
protein gets degraded. If I have to further purify the protein I have to 
concentrate the IMAC elutes and concentrate using filters. In this step protein 
degradation was more and filter used to block and could not complete the process
.
Do you use batch method of elution or gradient???
Also do you add PMSF in the elution buffer???

Yes Nikhil I did batch elution and included PMSF in all the buffers. and I 
tried using 500mM NaCl too but no change was observed.


Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.commailto:manjula@gmail.com
Mobile no:+91-9538553356
http://www.nimhans.kar.nic.in/
https://www.nimhans.kar.nic.in/

On Tue, May 19, 2015 at 11:14 AM, PULSARSTRIAN 
bhanu.hydpri...@gmail.commailto:bhanu.hydpri...@gmail.com wrote:
Try increasing NaCl to 500 mM and glycerol to 10%..And try reduce bME to 1mM 
for IMAC using Ni-NTA. If your protein requires any additional co-factors (like 
Mg, Ca or Zn) add them as chloride salts upto 1 to 5 mM.

On Tue, May 19, 2015 at 10:19 AM, Manjula Ramu 
manjula@gmail.commailto:manjula@gmail.com wrote:
Hi all,

I am purifying a basic protein with pI 7.8. I used 50mM tris 8.8, 300 mm NaCl, 
5% glycerol,  0.1mM PMSFand 5mM beta ME  in the buffer and performed affinity 
purification. Eluted with 200mM imidazole.  While elution I could see slight 
turbid in eluted protein (I get pure protein, single band on SDS-PAGE). 
However, when I centrifuged I couldn't see any pellet. After some time(a day) 
protein got degraded. In other method I tried cation exchange purification of 
lysate with MES buffer pH 6.5. Here also I saw slight precipitation and later 
degradation.

After this again I tried with MES buffer pH 5.5 and PIPES buffer pH 6.0, here 
also same problem I faced.

Please suggest me a method where I can get stable protein.

Thanks and Regards,
Manjula R
Research Scholar
Department of Biophysics
National Institute of Mental Health and Neurosciences
Bengaluru-29, Karnataka
INDIA
E-mail: manjula@gmail.commailto:manjula@gmail.com
Mobile no:+91-9538553356
http://www.nimhans.kar.nic.in/
https://www.nimhans.kar.nic.in/



--
B4U





Forschungszentrum Juelich GmbH
52425 Juelich
Sitz der Gesellschaft: Juelich
Eingetragen im Handelsregister des Amtsgerichts Dueren Nr. HR B 3498
Vorsitzender des Aufsichtsrats: MinDir Dr. Karl Eugen Huthmacher
Geschaeftsfuehrung: Prof. Dr.-Ing. Wolfgang Marquardt (Vorsitzender),
Karsten Beneke (stellv

Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!!!!!

[Phoebe A. Rice]

Dear Misbha,
  Before you throw stones at other peoples' work:
- have YOU tried to refine a structure at 3.9A, particularly with older 
software?
- there are many structures in the database that are related to lambda 
repressor.  Have you checked how the structure in question compares to those 
determined at higher resolution?  Is the overall fold the same?  Is the 
sequence register the same?  Remember that it is unreasonable to expect that 
all the side chain rotamers will be correct at 3.9A.
- When you looked at the actual electron density, what, exactly, did you see 
that suggested the structure may not be correct?
- The structure factors were deposited, so it should be easy for you to try 
re-refining it yourself with newer software, and see if it makes much 
difference to the final model.
- If you've gone through all those steps, and are still convinced that this 
structure is problematic, please do let the community know!


++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

pr...@uchicago.edumailto:pr...@uchicago.edu

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Misbah ud Din 
Ahmad [misba.ah...@gmail.com]
Sent: Thursday, April 23, 2015 11:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!

Dear crystallographers,

The PDB entry
http://www.rcsb.org/pdb/explore.do?structureId=3BDN
has 16.5% Ramachandran outliers. When I opened this PDB file in coot and 
checked for Ramachandran outliers, the results are:
In preffered region: 58.04%
In allowed regions: 19.78%
Outliers: 22.17%  !

With an R-free of 37.4% at 3.9 A resolution, could you please tell me how 
reliable this structure of Lambda repressor bound to DNA is?


Thanks
Misbha

Re: [ccp4bb] Picking water molecules at 4A structure.

[Phoebe A. Rice]

At 4A, I wouldn't unless I had an exceptionally good reason to.
There will always be some blobs, due to random noise and fourier ripples as 
well as due to an imperfect model.  Unless a blob makes nice H-bonds to 
something else that is nicely ordered, I wouldn't model at water into it. If 
you can't see nice density for side chains then you probably aren't really 
seeing density for waters either.


++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

pr...@uchicago.edumailto:pr...@uchicago.edu

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sudipta 
Bhattacharyya [sudiptabhattacharyya.iit...@gmail.com]
Sent: Monday, April 13, 2015 1:14 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Picking water molecules at 4A structure.

Dear community,

Recently we have been able to solve a crystal structure of a DNA/protein 
complex at 4A resolution. After almost the final cycles of model building and 
refinement (with R/Rfree of ~ 22/27) we could see some small water like 
densities...all throughout the complex. Now my query is, whether one should 
pick water molecules at this low resolutions or it is totally unscientific to 
do so?

Many thanks in advance...!!!

My best regards,
Sudipta.

Re: [ccp4bb] Thymine methyl in coot

[Phoebe A. Rice]

To those in the same spot, the fix was to first run this:

http://kinemage.biochem.duke.edu/software/remediator.php

And then remove all the H's and let your favorite refinement software put them 
back:
grep -v  H 52remediated.pdb  52remediated_noH.pdb

Sorry for bothering those of you who are already more up-to-date!


++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

pr...@uchicago.edumailto:pr...@uchicago.edu

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice 
[pr...@uchicago.edu]
Sent: Thursday, February 19, 2015 8:03 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Thymine methyl in coot

When trying to adjust the chi angles of a dT in coot 0.8.1, the methyl and 
its H's (which I called C5M to make phenix happy) rotate with the sugar, 
producing a rather base bizarre geometry.

Re: [ccp4bb] Can nucleoprotein complex inhibit EtBr binding?

[Phoebe A. Rice]

Yes it can.  Ethidium stains DNA by intercalating between base pairs, so if 
most of your DNA is tightly bound by protein the ethidium may not be able to 
wiggle in there.  We've seen that happen with Mu transposase - DNA complexes.  
If that's the problem, soak your gel in SDS plus EtBr and you should see the 
DNA.

Unfortunately, if your protein and DNA are aggregating in the wells, there is a 
possibility of the staining process knocking them loose.  In that case, you'll 
need to play with the conditions to get nice, soluble complexes that enter the 
gel (tweak the [salt], try adding a little non-denaturing detergent, change the 
pH, speak to it kindly but forcefully ...).  Some DNA-binding proteins are not 
soluble at high concentration and low salt, so you may need to lower the 
concentration of everything in your assay, which may require switching to a 
more sensitive stain (or 32P).

Also, because the mobility in a native gel is determined by the charge: mass 
ratio and the shape, it is impossible to predict exactly where the complex 
should run (i.e. your DNA-only markers are meaningless except for marking the 
free-DNA band).

Good luck!


++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

pr...@uchicago.edumailto:pr...@uchicago.edu

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sudipta 
Bhattacharyya [sudiptabhattacharyya.iit...@gmail.com]
Sent: Friday, February 13, 2015 3:50 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Can nucleoprotein complex inhibit EtBr binding?

Dear Community,

Sorry for this off topic question. I am dealing with a non specific DNA binding 
protein. In a non radio-labelled EMSA DNA:protein titration experiment when I 
EtBr stained the 5% native acrylamide gel, I could see the DNA control (101 
bps) but as the amount of my protein increases I saw the free DNA band getting 
thinner and thinner but I could not see any band shift and probably all the 
complex stuck at wells. But, even if I assume the whole DNA length  is occupied 
by my protein (I determined the ratio to be 1:15 ish) the molecular weight 
should not reach the molecular weight of the highest DNA marker band (20kbps = 
13,200 kDa). Then why I could not see a retarded band in the gel? Or is it 
forming a EtBr resistant nucleoprotein filament? Could you guys please give me 
an idea/suggestions?

Many thanks in advance...!!!

My best regards,
Sudipta.

[ccp4bb] Thymine methyl in coot

[Phoebe A. Rice]

When trying to adjust the chi angles of a dT in coot 0.8.1, the methyl and 
its H's (which I called C5M to make phenix happy) rotate with the sugar, 
producing a rather base bizarre geometry.

Re: [ccp4bb] Pseudo-translation problem

[Phoebe A. Rice]

In terms of detecting pseudo translational symmetry, it sounds like both 
crystals have it, but the relationship between the two complexes is closer to 
perfect in the 2nd crystal.  Looking at that problematic density, it does 
indeed suggest that the lattice is slightly messed up.  For instance, you could 
have a sort of twinning-ish problem.  Say, if your molecules usually sit in a 
row like such: Mol1 - x Angstroms - Mol2 - 0.9x Angstroms - Mol1 - etc. but 
some rows of the crystal pack: Mol1 - 0.9x Angstroms - Mol2 - x Angstroms - 
Mol1 etc, the Mol1s will superimpose but the Mol2s won't.  Or could it be that 
sometimes the protein binds 1bp over from where you wanted it to?  Do you have 
any halogens on the DNA that you could use to make sure the DNA sequence 
register is always the same (i.e. if you put in 1 bromo-dU, do you get 1 
anomalous peak or two)?  Since you don't have a problem with the density 
derived from your first data set, you might get lucky just by cranking through 
many different crystals of the 2nd complex.  I might try micro seeding as well 
in hopes of somehow tweaking the crystal growth pattern.
As a side point, a Patterson peak that is 18% of the origin may not be huge, 
but it seems unusually large to me.  I suspect there's a broad grey area where 
you need some additional analysis to decide what is really noteworthy pseudo 
translational symmetry, such as noticeable patterns of dark vs. light spots.   
As a control, I looked up the validation report now posted for one of my 
lab's old structures that had pseudo translational symmetry clearly visible in 
the diffraction pattern (and later in the coordinates) (1szp).  Xtriage found a 
19%-of-origin peak for that, and decided no signifiant pseudo translation.
   Phoebe

++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Minyun Zhou 
[minyunz...@gmail.com]
Sent: Wednesday, July 30, 2014 9:58 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Pseudo-translation problem

Dear all,

I am trying to determine the structure of a protein-DNA complex.  I collected 
several datasets of the same protein in complex with two dsDNAs.  Two DNAs have 
the same sequence, but one DNA contain a single central mispair, which is 
cleaved by the enzyme leaving an abasic site, while the other contains a 
non-cleavable mispair. Two datasets have almost the same space group and cell 
parameter, however, the later one has been detected with pseudo-translation 
while the other not.

The details of two datasets are as follows:

Dataset 1.  Protein with abasic site containing dsDNA:
C2221: a=106, b=111, c=182.2, α=β=γ=90
Resolution: 2.45 A
Xtriage summary: The largest off-origin peak in the Patterson function is 
18.57% of the height of the origin peak. No significant pseudotranslation is 
detected.

Dataset 2. Protein with non-cleavable lesion containing dsDNA:
C2221: a=106.1, b=111.1, c=183, α=β=γ=90
Resolution: 3.0 A
Xtriage summary: The analysis of the Patterson function reveals a significant 
off-origin peak that is 60.75 % of the origin peak, indicating pseudo 
translational symmetry.

I first determined the structure using the dataset 1 by MR. There are two 
protein-DNA complexes in one asymmetric unit, and two molecules are similar 
except the conformation of a small loop at the active site. The final model has 
the Rwork/Rfree of 19.3%/23.0%.  Then I use this refined structure as model to 
solve the second structure with dataset 2.  The final Rwork/Rfree is 
24.3%/28.9%.  The DNA density looks okay while the protein part is problematic. 
 After refinement, although the main chain of the protein is fitted well into 
the density, there is still a lot of unidentified continuous positive density 
next to the polypeptide chain, especially near the region involved in the 
crystal packing. I attached a snapshot below, which shows one of these positive 
densities lies along a helix. Since there is no other polymer in the reservoir 
solution that can fit, the additional density seems to indicate another 
slightly shifted conformation of the protein itself. My question is whether 
this seemingly “dual conformation” is caused by pseudo-translation and 
indicates the solution I found is incorrect. And how can I eliminate the effect 
of pseudo-translation to get the correct structure?

Thanks for your help!

Best regards,

Minyun

Re: [ccp4bb] coot problems to decrease R FREE

[Phoebe A. Rice]

If you are doing real-space refinement of the entire model against a map in 
coot, isn't it also important to make sure that the Rfree reflections were NOT 
used in calculating that map?  Depending on how the map was made, that info can 
be hidden in the fine print.

++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Robbie Joosten 
[robbie_joos...@hotmail.com]
Sent: Monday, April 21, 2014 4:40 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] coot problems to decrease R FREE

Dear Peter,

 I'm a novice of coot and ccp4. Now I'm doing refinement using both refmac5
 and coot.Here are some problems I'm facing. Really hope you can give me
 some suggestions.

 1、THE RESOLUTION OF THE DATA IS 2.5 angstrom. After first refinement of
 refmac5 I got R factor which is 0.26 and R FREE which is 0.31. My question
is
 what the final R factor and R FREE should be after several rounds of
 refinement by refmac5 and coot.
As low as it can reasonably be. Which is of course a lousy target but the
purpose of model refinement is to make it as good as possible, not to
achieve a certain R-free. That said, at this resolution, the PDB average is
about 25% for R-free.

 2、At which map level（e/A3 or rmsd）should I refine the data by coot?
You refine against the whole map, but for viewing purposes you should change
the map contour level during building. For the majority of the map you
should be able to see stuff well above an rmsd of 1.0 for the 2mFo-DFc map.
At the difficult bits you may need to go below 1.0, but no so low that you
can fool yourself. The proper height for the difference map depends as a bit
on the situation. I usually just turn it down until obvious noise peaks
(small negative peaks in the solvent) appear. That typically ends up
somewhere between 3 and 4 rmsd.

 3、Can you give me some tips and strategies about how to use coot to
 decrease R free? now I just use some basic tricks such as fit density and
 Ramachandran plot to refine the data.
This is what I do for your kind of resolution: set the weight for refinement
in COOT to 50 or 40 (the default is 60) and switch on torsion restraints and
possibly Ramachandran restraints. Then I go through the entire structure
residue by residue (space bar) refining windows of three residues (with the
't' key) and fixing everything that I can (that includes obvious solvent
molecules). Symmetry should always be switched on and NCS ghosts (if
available too). When that is done you can focus on the remaining difficult
bits by looking at the difference map peaks in COOT and at validation
reports from WHAT_CHECK, and MolProbity.

So now for the bit where I plug my own stuff: you can try PDB_REDO
(http://xtal.nki.nl/PDB_REDO for the server, there is also a stand-alone
version) to take a lot of work out of your hands. It optimises your
refinement in Refmac and, rebuilds side-chain and tries to find peptide
flips that improve you Ramachandran plot and fit with the maps. You also get
a lot of validation information that may help you with further rebuilding of
your model.

Cheers,
Robbie


 Best regards,


 Peter Chen

[ccp4bb] off-topic: bug busting

[Phoebe A. Rice]

Some time ago, there was a nice discussion of cost-effective, wimpy 
protein-friendly ways to break open E. coli.  We're thinking about replacing an 
aging sonicator.  If people have a favorite gizmo, could they repeat that 
advice?
thank you,
  Phoebe Rice


++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp

Re: [ccp4bb] A question on protein microheterogenity for crystalization

[Phoebe A. Rice]

It might be that the protein is innocent and the FPLC is guilty. If  FPLC pump 
needs maintenance and is stuttering a bit when running at high pressure, the 
salt gradient won't be as smooth as you'd like.  If you have an ionic strength 
detector, that trace will tell you.  Otherwise, see if other people's proteins 
are also looking a bit iffy under similar circumstances.
  Phoebe

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Katherine Sippel 
[katherine.sip...@gmail.com]
Sent: Sunday, December 15, 2013 11:53 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] A question on protein microheterogenity for crystalization

Hi Acoot,

There are a lot of great suggestions here already. I've also run into this 
phenomenon in couple of cases. The first was a binding protein in mixed bound 
and unbound forms. The second was a case of heterogeneous post-translational 
modification.  In both cases I could only get crystals from purified peaks and 
not pooled protein. If protein is precious I'd second Mark's suggestion to try 
screening pooled protein while you scale up your prep.

Cheers,
Katherine


On Sat, Dec 14, 2013 at 6:16 AM, Acoot Brett 
acootbr...@yahoo.commailto:acootbr...@yahoo.com wrote:
Dear All,

When I purified my protein by ion exchange chromatography for crystallization, 
there were several peaks containing the target protein as analyzed by SDS-PAGE. 
All these peaks have the same MW as determined by gel filtration coupled MALLS.

For crystallization purpose, can I merge the corresponding ion exchange 
chromatography peaks together? Otherwise the protein yield will be too low. And 
how to explain the heterogeneity by ion exchange chromatography in this 
situation?

I am looking forward  to getting a reply from you.

Acoot



--
Nil illegitimo carborundum - Didactylos

Re: [ccp4bb] switching space group

[Phoebe A. Rice]

If you're only changing the 2-fold axis along c to a 2-fold screw axis, you 
don't need to go back to the raw image files and reprocess them!   Just tweak 
the header of the .sca file and carry on (and take notes on what you did).


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Vikrant Upadhyay 
[vikrant192...@gmail.com]
Sent: Wednesday, November 06, 2013 5:13 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] switching space group

Hi Maggie,

you can re-process your data using XDS and can provide the desired space group 
in the XDS.INP file. It won't take much time.

Vikrant

Vikrant Upadhyay
Postdoctoral associate
Dr. Crina Nimigean's lab, A-1050
Department of Anesthesiology
Weill Cornell Medical College
525 East 68th Street
New York, NY 10065

On Nov 6, 2013, at 1:13 PM, MAGGIE 
dongmeij...@gmail.commailto:dongmeij...@gmail.com wrote:

Hi,

I have a structure which should have space group P212121, but it has been 
processed to P21212.  It can not be solved and refined.  Right now I do not 
have HKL2000, but I need change the space group to P212121.  Is there a way for 
me to do this using CCP4?

Thank you,

Maggie

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

[Phoebe A. Rice]

Another annoying syndrome is people who download your coordinates, don't read 
the paper, and then assume any insights are their own brand new ideas.
A biophysicist once said to me at a meeting I hope you aren't offended that 
we've found the DNA bend in your structure is flexible.  The TITLE of our 
paper about that structure was Flexible DNA bending ...
Then again, at least somebody did look at the coordinates ;)
  Phoebe Rice

PS - I recently participated in a e-life review and was quite impressed by the 
process.  I think it cleared up several bits of confusion and pointed out where 
the authors needed to explain things a bit better in order for a broad audience 
to be able to follow it.

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Debasish 
Chattopadhyay [debas...@uab.edu]
Sent: Thursday, October 10, 2013 9:20 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] OT: Who's Afraid of Peer Review?

My editorial suggestion:
My suspicion is that many structural papers are not read beyond the author 
list and title, if at all should be corrected as follows:
My suspicion is that many papers are not read beyond the author list and title, 
if at all.


Debasish

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frances 
C. Bernstein
Sent: Thursday, October 10, 2013 6:21 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] OT: Who's Afraid of Peer Review?

To bolster Adrian's argument about people not reading papers:

I periodically identify PDB entries that have not been released becuase they 
are on hold until publication.  But the paper was publshed months earlier.  
This means that nobody has read the paper, then tried to look at the 
coordinates, and then asked the PDB to release them.

My suspicion is that many structural papers are not read beyond the author list 
and title, if at all.

[I am not faulting the PDB in not identifying that the structure should be 
released.  Typically the author list has changed and the title is completely 
different from what was submitted to the PDB.
And there are fewer of these so I suspect that the journals are getting more 
reliable about communicating with the PDB.  And it looks like the PDB is better 
at finding these.]

Frances

=
Bernstein + Sons
*   *   Information Systems Consultants
5 Brewster Lane, Bellport, NY 11713-2803
*   * ***
 *Frances C. Bernstein
   *   ***  f...@bernstein-plus-sons.com
  *** *
   *   *** 1-631-286-1339FAX: 1-631-286-1999
=

On Thu, 10 Oct 2013, Adrian Goldman wrote:

 ?then the issue is to reduce the number of papers people publish: this is the 
 central problem in the system: nobody reads them, nobody cites them, etc etc. 
  There are papers out there - quite a number - that have no cites, meaning 
 that even the authors weren't interested in them.  A long time ago, when I 
 was at Yale, Fred Richards said that people should be judged on their 10 best 
 papers, and that was all you should be asked to put into a grant or whatever.

 If we (the funding agencies, governments etc etc) did this, the number of 
 papers would go down, there would be less rubbish to review, less money to be 
 made by Elsevier and the open-access journals, less money wasted on the whole 
 process - and even the current peer review system would work better because 
 we would have more time to spend on properly reviewing that little that 
 remained.

 My personal contention is that anyone who is publishing more than 10 papers a 
 year isn't reading and understanding their own work - and yet there are 
 many senior authors that have published 300+ papers in 10-15 years.

   Adrian


 On 10 Oct 2013, at 09:11, Miguel Ortiz Lombard?a 
 miguel.ortiz-lombar...@afmb.univ-mrs.fr wrote:

 Ciao Roberto,

 I'm sure the current research system works better in some fields than
 in others. It depends on a number of factors, perhaps the more
 important of them the amount of publications produced. Or it may be
 as we say in
 Spain: everybody talks about the party according to how much fun is
 having :-)

 Agreed that peer-reviewing is a continuous, endless process. But can
 we afford relying on the cleverness of the next generation to carry
 out our present work and mend our present problems? That's why I
 tried to make the distinction between peer-reviewing and really
 existing peer-reviewing. In some fields the latter may get closer to
 the former, sure. You assume that papers are read beyond their title,
 abstract and conclusions, that they are read critically and
 understood, that when flaws or reproducibility problems are found
 these are reported, that those reports are ever widely registered by
 the community. All that happens, fortunately

Re: [ccp4bb] over-expression strategies

[Phoebe A. Rice]

Something I recently learned from a friend: try changing the 4th base in the 
coding sequence to G (e.g. ATGGxx...) and the ribosome will like it better.  We 
tried it recently on a frustrating construct and it worked like a charm.


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Fernandez, Elias 
J [efern...@utk.edu]
Sent: Friday, September 13, 2013 7:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] over-expression strategies


Yes, the gene's been codon-optimized. The predicted mRNA structure appears 
stronger in the optimized version than the native, where it is high too. And, 
expression levels are unchanged. Elias


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Elias Fernandez 
[efern...@utk.edu]
Sent: Thursday, September 12, 2013 10:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] over-expression strategies

Dear CCP4ers,
We’ve been struggling with little (nearly none) expression of our protein, in 
both E coli and with in vitro transcription/translation methods. It appears 
that our mRNA has high 2’ structure with a low dG (theoretically ~760kcal/mol). 
If this is indeed the source of our problem, are there any potential strategies 
to either disrupt the mRNA structure chemically (in E coli or in vitro) or with 
 thermophile expression systems for expression at higher temperatures?
Regards,
Elias

[ccp4bb] FW: Departmental License for Pymol

[Phoebe A. Rice]

This may get us a more advanced version than the one embedded in phenix?


From: Erin Adams [ejad...@uchicago.edu]
Sent: Wednesday, September 11, 2013 9:32 AM
To: Shohei Koide; Herbert C Friedmann; Andrzej Joachimiak; Godfrey Getz; James 
Shapiro; Ronald S. Rock; Robert Haselkorn; Geoffrey L. Greene; Sean Crosson; 
Nancy Schwartz; Joe Piccirilli; Tao Pan; Keith Moffat; David R. Kovar; Tobin R. 
Sosnick; Stephen C. Meredith M.D., Ph.D.; Phoebe A. Rice; Francisco Bezanilla; 
Marvin W. Makinen; Stephen Kent; Donald F. Steiner Md.; Benoit Roux; D Allan 
Drummond; Glyn Dawson; Erin Adams; Robert Keenan; Theodore L. Steck; Tony 
Kossiakoff; Eduardo Perozo
Subject: Departmental License for Pymol

Dear colleagues,

Tobin has generously agreed to fund another three year departmental licensing 
for Pymol.  The information is below, please distribute this to your labs.

Thanks,

Erin


Begin forwarded message:

From: Schrodinger Licensing 
licens...@schrodinger.commailto:licens...@schrodinger.com
Subject: PyMOL Invoice #13348
Date: September 10, 2013 5:24:01 PM CDT
To: ejad...@uchicago.edumailto:ejad...@uchicago.edu


Dear Erin J. Adams,

Thank you for your recent purchase of PyMOL.
Here is the information you need for downloading the software:

Invoice number: 13348
Download  documentation URL: http://pymol.org/dsc
USER: 16sep10
PASSWORD: xoeklc4y

The following links should be helpful to you while using the software:

PyMOL-Users mailing list. Please consider joining the community mailing
list if you haven't done so already.
http://lists.sourceforge.net/lists/listinfo/pymol-users

For technical support issues, please email:
licens...@schrodinger.com

The community-maintained Wiki documentation server:
http://pymolwiki.org

Additional PyMOL-related links:
http://pymol.sourceforge.net/links.html

We hope you enjoy PyMOL.

Sincerely,

Schrodinger PyMOL licensing team


Schrodinger Licensing | licens...@schrodinger.com
http://www.schrodinger.com | 503-299-1150 | 503-299-4532 (fax)
101 SW Main St. Suite 1300, Portland OR 97204



Erin J. Adams Ph.D.
Associate Professor
Department of Biochemistry and Molecular Biology
University of Chicago
929 E. 57th Street
Chicago, IL  60637
office:  CIS W236
lab:  CIS W229
website: http://ejadamslab.bsd.uchicago.eduhttp://ejadamslab.bsd.uchicago.edu/
Office phone: 773-834-9816
Lab phone: 773-834-0660
Department Fax: 773-702-0439

[ccp4bb] Please Delete: FW: Departmental License for Pymol

[Phoebe A. Rice]

MANY APOLOGIES the wrong address got auto-filled when I tried to forward this 
to my lab.
Please delete.


++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice 
[pr...@uchicago.edu]
Sent: Wednesday, September 11, 2013 9:50 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] FW: Departmental License for Pymol

This may get us a more advanced version than the one embedded in phenix?


From: Erin Adams [ejad...@uchicago.edu]
Sent: Wednesday, September 11, 2013 9:32 AM
To: Shohei Koide; Herbert C Friedmann; Andrzej Joachimiak; Godfrey Getz; James 
Shapiro; Ronald S. Rock; Robert Haselkorn; Geoffrey L. Greene; Sean Crosson; 
Nancy Schwartz; Joe Piccirilli; Tao Pan; Keith Moffat; David R. Kovar; Tobin R. 
Sosnick; Stephen C. Meredith M.D., Ph.D.; Phoebe A. Rice; Francisco Bezanilla; 
Marvin W. Makinen; Stephen Kent; Donald F. Steiner Md.; Benoit Roux; D Allan 
Drummond; Glyn Dawson; Erin Adams; Robert Keenan; Theodore L. Steck; Tony 
Kossiakoff; Eduardo Perozo
Subject: Departmental License for Pymol

Dear colleagues,

Tobin has generously agreed to fund another three year departmental licensing 
for Pymol.  The information is below, please distribute this to your labs.

Thanks,

Erin


Begin forwarded message:

From: Schrodinger Licensing 
licens...@schrodinger.commailto:licens...@schrodinger.com
Subject: PyMOL Invoice #13348
Date: September 10, 2013 5:24:01 PM CDT
To: ejad...@uchicago.edumailto:ejad...@uchicago.edu


Dear Erin J. Adams,

Thank you for your recent purchase of PyMOL.
Here is the information you need for downloading the software:

Invoice number: 13348
Download  documentation URL: http://pymol.org/dsc
USER: 16sep10
PASSWORD: xoeklc4y

The following links should be helpful to you while using the software:

PyMOL-Users mailing list. Please consider joining the community mailing
list if you haven't done so already.
http://lists.sourceforge.net/lists/listinfo/pymol-users

For technical support issues, please email:
licens...@schrodinger.com

The community-maintained Wiki documentation server:
http://pymolwiki.org

Additional PyMOL-related links:
http://pymol.sourceforge.net/links.html

We hope you enjoy PyMOL.

Sincerely,

Schrodinger PyMOL licensing team


Schrodinger Licensing | licens...@schrodinger.com
http://www.schrodinger.com | 503-299-1150 | 503-299-4532 (fax)
101 SW Main St. Suite 1300, Portland OR 97204



Erin J. Adams Ph.D.
Associate Professor
Department of Biochemistry and Molecular Biology
University of Chicago
929 E. 57th Street
Chicago, IL  60637
office:  CIS W236
lab:  CIS W229
website: http://ejadamslab.bsd.uchicago.eduhttp://ejadamslab.bsd.uchicago.edu/
Office phone: 773-834-9816
Lab phone: 773-834-0660
Department Fax: 773-702-0439

Re: [ccp4bb] structural search for homologs in pdb?

[Phoebe A. Rice]

Hi Gloria,
  I've gotten useful results from these two threading / prediction servers: 
Raptorx and Phyre (for low homology, I found the original version works better 
than phyre2).
 Phoebe

++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of 
moham...@strubi.ox.ac.uk [moham...@strubi.ox.ac.uk]
Sent: Thursday, August 22, 2013 11:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] structural search for homologs in pdb?

Dear Gloria,

If you are searching with a protein sequence, you could also try FFAS 
(ffas.burnham.org/‎).  This will perform profile-profile alignments to do fold 
recognition.  Works quite well for low homology cases in particular.

HTH and best wishes,

Mohammad

Re: [ccp4bb] Low 280 absorbance imidazole?

[Phoebe A. Rice]

Hi Bernhard,
  If your cheap imidazole works fine aside from the absorption problem, there's 
always my favorite stone-age detection method: pencil a numbered grid onto a 
piece of filter paper, spot the fractions on it, and stain with coomassie.  
It'll tell you which fractions to load on a gel, and it goes easy on the budget 
as well.
 Phoebe


++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Bernhard Rupp 
[hofkristall...@gmail.com]
Sent: Wednesday, August 21, 2013 9:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Low 280 absorbance imidazole?

Hi Fellows,

could someone please point me towards the source of a known high purity 
imidazole
with low absorbance at 280 nm? I am facing the problem of detecting a low 
absorption protein
in high imidazole background after IMAC gradient elution. In the UV spectra of 
the
2 imidazoles I checked there is some contaminant that absorbs at 280…

Thx, BR


Bernhard Rupp
Marie Curie Incoming International Fellow
Innsbruck Medical University
Schöpfstrasse 41
A 6020 Innsbruck – Austria
+43 (676) 571-0536
bernhard.r...@i-med.ac.atmailto:bernhard.r...@i-med.ac.at

Dept. of Forensic Crystallography
k.-k. Hofkristallamt
Vista, CA 92084
001 (925) 209-7429
b...@ruppweb.orgmailto:b...@ruppweb.org
b...@hofkristallamt.orgmailto:b...@hofkristallamt.org
http://www.ruppweb.org/
---

Re: [ccp4bb] Twin or underestimation of symmetry

[Phoebe A. Rice]

Check the pseudo-precession pics (0kl plane, etc).  A rhombohedral crystal that 
is indexed as P6xx may have seemingly-bizarre systematic absences that could 
trick one into thinking its P63xx.  

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Tim Gruene 
[t...@shelx.uni-ac.gwdg.de]
Sent: Tuesday, July 30, 2013 7:26 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Twin or underestimation of symmetry

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Jeffrey,

how complete is you model, i.e. what is the ratio between  the number
of atoms in the PDB file (without solvent/ ligands) and the expected
number of atoms as calculated from the sequence?
If a substantial part of the molecule is disordered you may not be
able to get a much better model.

What is the R-value and Rfree? If you have placed many water atoms you
may have hovered out the difference density allowing you to further
improve your model.

Regards,
Tim

On 07/29/2013 07:37 PM, Jeffrey D Brodin wrote:
 Hi everyone,

 I have a dataset that's been giving me some trouble and wanted to
 get your ideas on the best way to proceed. The data extend to ~2.6
 Å and scale integrate/scale well in P622. According to Pointless,
 the symmetry is either P622 or P6322, however, neither Molrep norm
 Phaser finds molecular replacement solutions in these space groups.
 If I run Phaser and let it check all other space groups it finds a
 solution in P6122, but I can only refine it to an Rfree value of
 ~35%. The data also scales well in P3 and P6 and I get molecular
 replacement solutions in space groups of either P31 or P61.
 However, the Refinement again stalls at an Rfree value of ~35%. I
 tried adding twin refinement in Refmac and it seems to be helping;
 The R/Rfree values dropped to 20.9/27.3 and the map appears much
 better compared to the previous refinements. The refined twin
 fractions are 0.26, 0.24, 0.24 and 0.25. Am I doing something wrong
 with the refinement or is this a legitimate way to treat the data.
 Thank you in advance for your help.


- --
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.14 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFR97DoUxlJ7aRr7hoRAsfrAKCIEYpOkrVfm/VrkIn+Jn9NZ16swACguGLe
G1ZAwYDXmvyx/jK0iE7VjZo=
=q8Ki
-END PGP SIGNATURE-

Re: [ccp4bb] TLS refinement refmac

[Phoebe A. Rice]

EEK, it seems to me that anything called simply FP should be unadulterated 
FP.  If the software modifies a column in some way, it should give it a new 
label, shouldn't it?


++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Eleanor Dodson 
[eleanor.dod...@york.ac.uk]
Sent: Wednesday, July 17, 2013 6:05 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] TLS refinement refmac

Oh dear - you should always start refinement against the original processed 
data - but others have told you that already..

The TLS files will not be appropriate for the rescaled FPs - it is surprising 
that the Rfactor actually goes down though!
Or are you doing several cycles of refinement in thesecond pass too - in that 
case one would hope the Rfactor would continue to fall.
Eleanor




On 17 July 2013 11:00, Guenter Fritz 
guenter.fr...@uni-konstanz.demailto:guenter.fr...@uni-konstanz.de wrote:
Am 17.07.2013 11:59, schrieb Guenter Fritz:
Hi Stefan and Gottfried,
thanks a lot for the answers. This is the point. Wouldn't it make more sense to 
add an extra column that contains the changed Fs?
Best, Guenter

Hi,

it is strongly advised to use the original mtz e.g. scala.mtz as the refmac 
input mtz in all refmac runs,
as this contains the original Fs - Refmac applies some aniso corrections  to 
the Fs and puts them into the output.mtz.

so the output Fs are not the same as in the input F - therefore one should use 
the scala.mtz

cheers
Stefan


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Guenter Fritz
Gesendet: Mittwoch, 17. Juli 2013 10:39
An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] TLS refinement refmac


Dear all,

one gets different R values, if you re-read in the mtz written out by refmac 
after TLS refinement. I think this issue had been a while ago in ccp4bb, but I 
can't find the right track.

Here are the details.

1st run:
If we do TLS + restr. refinement in refmac we get:
InitialFinal
R factor0.3010   0.2170
R free0.3175   0.2695

2nd run:
Now, if we use the same input pdb and the same input tls paramter file, but use 
the mtz written out by the first run, we get:
InitialFinal
R factor0.2274   0.1903
R free0.2482   0.2540


Apparently in the mtz file written out by the 1st refmac must contain
some information that is re-read in the 2nd run. But one just defines
FP, SIGFP and Rfree flags. Do FPs change in the output mtz after TLS
refinement??

Any help to clarify this is appreciated.

Thanks, Guenter

[ccp4bb] atomic coloring for the color blind

[Phoebe A. Rice]

I feel badly that one of my undergrads had trouble telling an O from a C in a 
pymol homework set because he's color blind. (The assignment involved telling 
me why the a GTP analog (GDPCP) wasn't hydrolyzed).
Is there a handy by-atom coloring scheme I can recommend that works for the 
red-green color blind?
  thanks,
  Professor Rice


++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp

Re: [ccp4bb] Strand distorsion and residue disconnectivity in pymol

[Phoebe A. Rice]

In the olden days, when dinosaurs did roam, Ribbons had a nice fix-up feature 
that would gently move the alpha carbon end of the stick so that it met the 
ribbon.  Looked MUCH better than the current wavy ribbon one gets with side 
chain helper, and I haven't been able to find a trick like that in pymol.

So pymol developers, please consider yourselves wheedled ...


++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Jason Vertrees 
[jason.vertr...@schrodinger.com]
Sent: Thursday, May 30, 2013 10:17 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strand distorsion and residue disconnectivity in pymol

Hi Jacob,

Sure, but I believe Donghui wanted to only show sidechains, not any backbone 
atoms, as sticks. Doing this will leave fragments floating in space as the 
original email noted.

Cheers,

-- Jason


On Thu, May 30, 2013 at 10:13 AM, Jacob Keller 
j-kell...@fsm.northwestern.edumailto:j-kell...@fsm.northwestern.edu wrote:

Can't you just show both cartoon (smoothed) and sticks (not smoothed) for the 
given area?

JPK


On Thu, May 30, 2013 at 11:06 AM, Jason Vertrees 
jason.vertr...@schrodinger.commailto:jason.vertr...@schrodinger.com wrote:
Hi Donghui,

Bernhard is correct: PyMOL flattens out secondary structure to produce more 
aesthetically appealing images. You can disable this for beta sheets and loops 
by typing:

# disable smoothing of sheets

set cartoon_flat_sheets, 0


# disable smoothing of loops

set cartoon_smooth_loops, 0


The cartoon_sidechain_helper setting automatically modulates these settings. If 
for some reason you need to disable cartoon_sidechain_helper you can imitate 
the look with:

hide
show cartoon
show sticks, not (n. C+CA+O+N) extend 1
set cartoon_smooth_loops, 0
set cartoon_flat_sheets, 0

Again as Bernhard noted, smoothing representations adjusts the representations' 
positions in space; therefore, you have the option of being positionally 
correct or aesthetically more pleasing.

Cheers,

-- Jason




On Wed, May 29, 2013 at 10:29 PM, wu donghui 
wdh0...@gmail.commailto:wdh0...@gmail.com wrote:
Dear all,

I found a problem when I use pymol to prepare structure interface. Strand is 
distorted when residue from the strand is connected to the strand by turning on 
side_chain_helper on. However when side_chain_helper is off, the strand turns 
to normal shape but the residue from it is disconnected to the strand. I 
attached the picture for your help. I know there must be some tricks for this. 
Welcome for any input. Thanks a lot.

Best,

Donghui



--
Jason Vertrees, PhD
Director of Core Modeling Products
Schrödinger, Inc.

(e) jason.vertr...@schrodinger.commailto:jason.vertr...@schrodinger.com
(o) +1 (603) 374-7120tel:%2B1%20%28603%29%20374-7120



--
***

Jacob Pearson Keller, PhD

Looger Lab/HHMI Janelia Farms Research Campus

19700 Helix Dr, Ashburn, VA 20147

email: kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org

***



--
Jason Vertrees, PhD
Director of Core Modeling Products
Schrödinger, Inc.

(e) jason.vertr...@schrodinger.commailto:jason.vertr...@schrodinger.com
(o) +1 (603) 374-7120

Re: [ccp4bb] Missing DNA density in Protein DNA complex structure

[Phoebe A. Rice]

Because your protein binds non-specifically, it could be an interesting case of 
static disorder where the DNAs are making a pseudo-continuous helix through the 
crystal regardless of sequence, and 12 bp/turn happens to fit nicely into a 
generic lattice.
How does the density for the individual bases look?
You could test that idea by substituting one T with BrdU.  I suspect you'll see 
multiple partially- occupied anomalous peaks in your asymmetric unit for those 
longer DNAs that pack like 12mers.


Here's a reference where an RNA duplex did a similar thing:

Crystal structures of two plasmid copy control related RNA duplexes: An 18 base 
pair duplex at 1.20 A resolution and a 19 base pair duplex at 1.55 A 
resolution.http://www.ncbi.nlm.nih.gov/pubmed/10555960

Klosterman PS, Shah SA, Steitz TA.

Biochemistry. 1999 Nov 9;38(45):14784-92.

PMID:
10555960


++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Raji 
Edayathumangalam [r...@brandeis.edu]
Sent: Sunday, May 05, 2013 7:39 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Missing DNA density in Protein DNA complex structure

Dear Ashok,

There are many questions underlying your questions. A couple of things to check 
right off the bat:

(1) Do you actually know that your crystal still contains all of the DNA bp 
that you started with? Did you analyze the contents of your crystal by native 
PAGE, mass spec or other methods?

(2) Yes, the number of base pairs do matter, especially if you have 
quasi-helical DNA stacking interactions that facilitate packing along one of 
the unit cell dimensions. For example, 12-bp is a little over a turn in 
contrast to 17-bp, which is a little more than 1.5 turns of a DNA B-form helix.

(3) Are the crystal packing interactions in cases 1, 3, 4 and 5 similar? And, 
is there something unique about the packing in case 2, especially DNA-to-DNA 
packing? Make sure to display symmetry related molecules. That may explain why 
you can accommodate more molecules in the unit cell.

(4) Compare the DNA sequences in cases 1-5 above and see if there is a pattern 
to the type(s) of nucleotides that are bound by protein in each case.

It is hard to say more without knowing what the models look like but if your 
project is to investigate the DNA-protein interactions in more detail, the 
above-mentioned sorts of questions may be a place to start.

Good luck!
Raji








On Sun, May 5, 2013 at 2:21 AM, ASHOK KUMAR Patel 
ashok...@gmail.commailto:ashok...@gmail.com wrote:

Hi all,



I am working on a DNA binding protein (mol wt around 30 kDa), which binds to 
Duplex DNA in a non-specific sequence manner. The structure has been published 
with 12 base pair duplex DNA.



I am trying to understand the DBD protein DNA interaction even more by choosing 
different lengths and sequences. In Co-crystallization I used 16, 18, 20 and 22 
bases palindromic sequence random DNA bases (purchased from IDT), annealed and 
used in crystallization.



I collected some diffraction data on NSLS recently at around 2.1 Å and 2.7 Å. 
But, when I did data processing, model building and refinement. I am getting 
strange results as depicted in the table..


S N


a=


b=


c=


α=


β=


γ=


Space group


No of molecules in asymmetric unit


Length of DNA

Used for crystallization


Duplex DNA found in structure


Resolution


1


38.67


61.43


76.77


90.00


104.17


90.00


P 1 21 1


1


12 base


12 base


2.0 Å


2


86.076


57.099


99.493


90.00


103.90


90.00


P 1 21 1


2


17 base


17 base


3.05  Å


3


37.855


61.668


76.601


90.00


102.24


90.00




P 1 21 1


1


18base


12 base


2.1 Å


4


37.073


61.864


78.242


90.000


100.810


90.000


P 1 21 1


1


20 base


12 base


2.7 Å


5


























20 base


12 base


3.1





My question and concerns are as:

1. How I am getting almost identical Cell parameters with different length of 
DNA (row 3 and 4) to the first row?

2. Why I am getting only 12 base duplex DNA instead of 18mer or 20 mer I used 
in crystallization.

3. Is anything has to do with ODD and EVEN duplex DNA. When odd 17 base duplex 
was used, it has 17 bases in the structure, while in all EVEN case of 18, 20 or 
20, only 12 bases in the structure.

4. The complex having odd DNA length 17 has 2 molecules in ASU while all other 
has 1.



Why only 12 mer DNA density in the complex? Why I am missing 6 or 8 bases in 
the density? How can we explain the missing DNA in the structure?



I will appreciate any kind of explanation and suggestions.



Thanks

Ashok

--
Ashok kumar patel
Department of Biophysics
Johns Hopkins University
Baltimore, MD 21218



--
Raji

Re: [ccp4bb] Puzzling Structure

[Phoebe A. Rice]

Looks like a typo to me: if you change the CRYST space group record from 
P212121 to P21212, as the paper says it is, the packing problem goes away.

++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Michel Fodje 
[michel.fo...@lightsource.ca]
Sent: Friday, April 12, 2013 2:17 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Puzzling Structure

By the way, you will need to show symmetry atoms to see the problem.

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Michel Fodje
Sent: April-12-13 1:14 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Puzzling Structure

Has anyone else noticed a problem with the structure  of the N-terminal
capsid domain of HIV-2  PDB 2wlv.

Load it up to in coot and navigate to residue B118.



/Michel.

Re: [ccp4bb] plate crystal optimization

[Phoebe A. Rice]

Among many possible reasons, the streaking could be caused mechanical stress to 
the thin plates during mounting.  Have you tried those loops that look like 
miniature tennis rackets?

  Phoebe



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Rakesh 
Chatterjee [rakesh.2665...@gmail.com]
Sent: Monday, April 01, 2013 10:01 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] plate crystal optimization

hello everyone,
i have a protein which co-purifies with a ligand (small molecule ~ 810 Dalton). 
i didn't know what kind of ligand is it so i am planing to identify it. 
meanwhile my protein crystallized as plates and while diffracting i found that 
the crystal is showing a good diffraction pattern upto 2.6 Angstroms in home 
source Cu K-alpha. however when the crystal rotates between 90 degrees to 135 
degrees, the spots become streaky. should i try different cryo-MPD/ PEG400 etc? 
to circuvent the problem i did some additive screen but was not of much help. 
any valuable suggestion willbe handfull.

thanx in advance
rakesh

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Phoebe A. Rice]

Hi Zbyszek,
  If the issue is perfect twinning, I agree - good point!
  But you don't want to confuse people who simply have nearly-but-not-quite 
crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of 
newbies read the BB).  We had a case of P31 that was so close to P61 we 
actually solved the molecular replacement problem in P61, then expanded it back 
and re-rigid-bodied it.  We've played similar games with translational 
pseudo-symmetry (ignoring the weak spots at first).  In cases like that it is 
important to properly reprocess the data in the lower symmetry space group (or 
smaller unit cell) because there is real information in those small 
differences.  However, the point about Rfree holds for twinning or rotational 
pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry 
operators, not re-picked in the lower symmetry space group.
   Phoebe

++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago
773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek 
Otwinowski [zbys...@work.swmed.edu]
Sent: Tuesday, March 19, 2013 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

It is a clear-cut case of crystal packing disorder. The tell-tale sign is
that data can be merged in the higher-symmetry lattice, while the number
of molecules in the asymmetric unit (3 in P21) is not divisible by the
higher symmetry factor (2, by going from P21 to P21212).
From my experience, this is more likely a case of order-disorder than
merohedral twinning. The difference between these two is that structure
factors are added for the alternative conformations in the case of
order-disorder, while intensities (structure factors squared) are added in
the case of merohedral twinning.

Now an important comment on how to proceed in the cases where data can be
merged in a higher symmetry, but the structure needs to be solved in a
lower symmetry due to a disorder.

!Such data needs to be merged in the higher symmetry,assigned R-free flag,
and THEN expanded to the lower symmetry. Reprocessing the data in a lower
symmetry is an absolutely wrong procedure and it will artificially reduce
R-free, as the new R-free flags will not follow data symmetry!

Moreover, while this one is likely to be a case of order-disorder, and
these are infrequent, reprocessing the data in a lower symmetry seems to
be frequently abused, essentially in order to reduce R-free. Generally,
when data CAN be merged in a higher symmetry, the only proper procedure in
going to a lower-symmetry structure is by expanding these higher-symmetry
data to a lower symmetry, and not by rescaling and merging the data in a
lower symmetry.

Zbyszek Otwinowski

 Dear all,
 We have solved the problem. Data processing in P1 looks better (six
 molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules
 in
 ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round
 of refinement (without put waters, ligands, etc.).

 Indeed, there were one more molecule in ASU, but the over-merged data in
 an orthorhombic lattice hid the correct solution.

 Thank you very much for all your suggestions, they were very important to
 solve this problem.

 Cheers,

 Andrey

 2013/3/15 Andrey Nascimento andreynascime...@gmail.com

 *Dear all,*

 *I have collected a good quality dataset of a protein with 64% of
 solvent
 in P 2 21 21 space group at 1.7A resolution with good statistical
 parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4;
 Complet.=93%
 Redun.=2.4, the overall values are better than last shell). The
 structure
 solution with molecular replacement goes well, the map quality at the
 protein chain is very good, but in the final of refinement, after
 addition
 of a lot of waters and other solvent molecules, TLS refinement, etc. ...
 the Rfree is a quite high yet, considering this resolution
 (1.77A).(Rfree=
 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower
 symmetry space group (P21), but I got the same problem, and I tried all
 possible space groups for P222, but with other screw axis I can not even
 solve the structure.*

 *A strange thing in the structure are the large solvent channels with a
 lot of electron density positive peaks!? I usually did not see too many
 peaks in the solvent channel like this. This peaks are the only reason
 for
 these high R's in refinement that I can find. But, why are there too
 many
 peaks in the solvent channel???*

 *I put a .pdf file (ccp4bb_maps.pdf) with some more information and map
 figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf*

 *
 *

 *Do someone have an explanation or solution for this?*

 * *

 *Cheers,*

 *Andrey

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Phoebe A. Rice]

oops, I should have expanded my comments to include the sort of funky lattice 
order-disorder Zbyszek so cleverly diagnosed.  Scratch that perfect twinning 
comment in my last message.

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Phoebe A. Rice 
[pr...@uchicago.edu]
Sent: Tuesday, March 19, 2013 10:34 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

Hi Zbyszek,
  If the issue is perfect twinning, I agree - good point!
  But you don't want to confuse people who simply have nearly-but-not-quite 
crystallographic symmetry (OK, I'm being a bit pedagogical here, but a lot of 
newbies read the BB).  We had a case of P31 that was so close to P61 we 
actually solved the molecular replacement problem in P61, then expanded it back 
and re-rigid-bodied it.  We've played similar games with translational 
pseudo-symmetry (ignoring the weak spots at first).  In cases like that it is 
important to properly reprocess the data in the lower symmetry space group (or 
smaller unit cell) because there is real information in those small 
differences.  However, the point about Rfree holds for twinning or rotational 
pseudo-symmetry: the Rfree flags should be expanded by the xtal symmetry 
operators, not re-picked in the lower symmetry space group.
   Phoebe

++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago
773 834 1723; pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/
http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Zbyszek 
Otwinowski [zbys...@work.swmed.edu]
Sent: Tuesday, March 19, 2013 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Strange density in solvent channel and high Rfree

It is a clear-cut case of crystal packing disorder. The tell-tale sign is
that data can be merged in the higher-symmetry lattice, while the number
of molecules in the asymmetric unit (3 in P21) is not divisible by the
higher symmetry factor (2, by going from P21 to P21212).
From my experience, this is more likely a case of order-disorder than
merohedral twinning. The difference between these two is that structure
factors are added for the alternative conformations in the case of
order-disorder, while intensities (structure factors squared) are added in
the case of merohedral twinning.

Now an important comment on how to proceed in the cases where data can be
merged in a higher symmetry, but the structure needs to be solved in a
lower symmetry due to a disorder.

!Such data needs to be merged in the higher symmetry,assigned R-free flag,
and THEN expanded to the lower symmetry. Reprocessing the data in a lower
symmetry is an absolutely wrong procedure and it will artificially reduce
R-free, as the new R-free flags will not follow data symmetry!

Moreover, while this one is likely to be a case of order-disorder, and
these are infrequent, reprocessing the data in a lower symmetry seems to
be frequently abused, essentially in order to reduce R-free. Generally,
when data CAN be merged in a higher symmetry, the only proper procedure in
going to a lower-symmetry structure is by expanding these higher-symmetry
data to a lower symmetry, and not by rescaling and merging the data in a
lower symmetry.

Zbyszek Otwinowski

 Dear all,
 We have solved the problem. Data processing in P1 looks better (six
 molecules in ASU), and Zanuda shows a P 1 21 1 symmetry (three molecules
 in
 ASU), Rfactor/Rfree drops to 0.20978/0.25719 in the first round
 of refinement (without put waters, ligands, etc.).

 Indeed, there were one more molecule in ASU, but the over-merged data in
 an orthorhombic lattice hid the correct solution.

 Thank you very much for all your suggestions, they were very important to
 solve this problem.

 Cheers,

 Andrey

 2013/3/15 Andrey Nascimento andreynascime...@gmail.com

 *Dear all,*

 *I have collected a good quality dataset of a protein with 64% of
 solvent
 in P 2 21 21 space group at 1.7A resolution with good statistical
 parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4;
 Complet.=93%
 Redun.=2.4, the overall values are better than last shell). The
 structure
 solution with molecular replacement goes well, the map quality at the
 protein chain is very good, but in the final of refinement, after
 addition
 of a lot of waters and other solvent molecules, TLS refinement, etc. ...
 the Rfree is a quite high yet, considering this resolution
 (1.77A).(Rfree=
 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower
 symmetry space group (P21), but I got the same problem, and I tried all
 possible space groups for P222, but with other screw axis I can not even
 solve the structure.*

 *A strange thing in the structure are the large solvent channels with a
 lot of electron density positive

Re: [ccp4bb] Strange density in solvent channel and high Rfree

[Phoebe A. Rice]

What happens if you solvent-flatten/flip/massage that map, but tell the 
software the solvent content much lower than what you think it is now?  Maybe 
you'll find another copy of the molecule?




From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Andrey 
Nascimento [andreynascime...@gmail.com]
Sent: Friday, March 15, 2013 1:39 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Strange density in solvent channel and high Rfree

Dear all,
I have collected a good quality dataset of a protein with 64% of solvent in P 2 
21 21 space group at 1.7A resolution with good statistical parameters (values 
for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall 
values are better than last shell). The structure solution with molecular 
replacement goes well, the map quality at the protein chain is very good, but 
in the final of refinement, after addition of a lot of waters and other solvent 
molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering 
this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I 
reprocess the data in a lower symmetry space group (P21), but I got the same 
problem, and I tried all possible space groups for P222, but with other screw 
axis I can not even solve the structure.
A strange thing in the structure are the large solvent channels with a lot of 
electron density positive peaks!? I usually did not see too many peaks in the 
solvent channel like this. This peaks are the only reason for these high R's in 
refinement that I can find. But, why are there too many peaks in the solvent 
channel???
I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures 
in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf

Do someone have an explanation or solution for this?

Cheers,
Andrey

Re: [ccp4bb] Superposing nucleic acids

[Phoebe A. Rice]

As already pointed out, it depends on what you're trying to show / figure out.

But my generic advice would be to go with just C1' because the backbone can 
vary quite a bit even in more-or-less-B-form DNA.

If you feel like getting a little fancier, add the atom on the other end of the 
glycosidic bond (note the atom number is different for pyrimidines vs. 
purines).  The sublime thing about W:C pairing is that it keeps the orientation 
glycosidic bonds constant.



++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Barry Finzel 
[finze...@umn.edu]
Sent: Tuesday, January 22, 2013 9:21 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Superposing nucleic acids

Minimally,  I believe you want to use P and C1'.
(This has been done by others, including Pabo and Pabo  Nekludova, J.Mol. 
Biol., 301:597-694 (2000).

I actually prefer to use all backbone atoms: P O5' C5' C4' O3' C3' C2' C1' O1'


Barry Finzel
Medicinal Chemistry Department
2-160C Weaver-Densford
Tel: (612) 626-5979
Lab: (612) 626-5226
Fax: (612) 624-0139
finze...@umn.edumailto:finze...@umn.edu



On Jan 22, 2013, at 9:00 AM, Alan Cheung wrote:

Dear all - is there a convention for superposing nucleic acids duplexes of 
unrelated sequence?

i.e. which atoms should be superposed : C4' of the sugar, P of the backbone, 
O3'/O5' of the backbone, or some combination thereof?

Alan


--
Alan Cheung
Gene Center
Ludwig-Maximilians-University
Feodor-Lynen-Str. 25
81377 Munich
Germany
Phone:  +49-89-2180-76845
Fax:  +49-89-2180-76999
E-mail: che...@lmb.uni-muenchen.demailto:che...@lmb.uni-muenchen.de

[ccp4bb] off-topic: citations for GST dragging gunk into solution?

[Phoebe A. Rice]

Does anybody have a nice, cite-able reference for the observation that GST tags 
can sometimes drag otherwise-aggregated/unfolding/generally unhappy proteins 
into solution?



We need to politely explain why a His-tagged version of our protein (purified 
in my lab) has activity but a GST-tagged version (purified in a different lab) 
doesn't.



  thanks, Phoebe



++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp

Re: [ccp4bb] Faculty positions at Molecular Biophysics Unit, IISc, Bangalore

[Phoebe A. Rice]

I have no idea about Bangalore, but I know from personal experience that 
already-filled job ads exist and waste everybody's time.  One of the junior 
faculty jobs I intereviewed for in the 90s at a prestigious US medical school 
turned out to be just such a thing - before I went people in the know told me 
which inside post-doc would get the position, faculty turnout to my 
presentation was low, and even during the visit somebody told me they thought 
it would be an inside deal.  And in the end, it was.

I had much better things to do with my time.



++

Phoebe A. Rice
Dept. of Biochemistry  Molecular Biology
The University of Chicago

773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu
http://bmb.bsd.uchicago.edu/Faculty_and_Research/

http://www.rsc.org/shop/books/2008/9780854042722.asp


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Manoj Tiwari 
[diffracti...@gmail.com]
Sent: Monday, October 29, 2012 4:28 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Faculty positions at Molecular Biophysics Unit, IISc, 
Bangalore

Just to let Narayan Viswam know more about HM Krishnamurthy. He did his PhD 
from IISc long long back in 1979 on small molecule crystallography. Then he did 
his postdoc in protein crystallography from two top most Universities of USA. 
He had been mentored by really really great protein crystallographers in USA. 
Everybody will agree that neither the Institute nor the mentors should be 
blamed for such acts. This is individual's character and act.

I am sure that there are many more frustrated people who did not get through 
the prestigious institute at some level of their life. Such comments deserve 
nothing more than the ignorance.

Seems grapes are sour :)

MT



On Tue, Oct 30, 2012 at 1:08 AM, Narayan Viswam 
nvisw...@gmail.commailto:nvisw...@gmail.com wrote:
Sham is very right about the institute.

Narayana Sthanam's illustrious former colleague Krishna Murthy who faked so 
many structures  retracted quite a few papers was an alumnus of the institute.


On Mon, Oct 29, 2012 at 12:21 PM, James Stroud 
xtald...@gmail.commailto:xtald...@gmail.com wrote:
The agism in the advertisement doesn't do the institute much credit. I'm 
inclined to believe Sham, given the Institutes stated policies:

Applicants, preferably below 35 years

James



On Oct 29, 2012, at 11:28 AM, Narayana VL Sthanam wrote:

Sham, who so ever you are, if you have such a long list of complaints, why 
don’t you put your name clearly and complain openly, instead of hiding behind
some anonymous ‘SHAM’ name. What you write about IISc  may be all true or it 
may be reflection of your frustration for not getting a job at IISc!
How do we know? So, grow a ‘spine’, if you have a complaint, say it like a man 
and do not hide behind and bad mouth others anonymously like spoiled child.
You are not only throwing dirt on such a prestigious Indian institution,  and 
also on many decent and capable scientists who do outstanding work and produce 
brilliant graduate students, some of which I was fortunate to have in my lab.

Best
Narayana Sthanam

--
Narayana Sthanam,Ph D
Professor of Structural Biology
244 CBSE 1025 18th Street South
Center for Biophysical Sciences and Engineering
University of Alabama at Birmingham
Birmingham, Al 35294
Phone: 205 934 0119tel:205%20934%200119
URL: 
http://www.opt.uab.edu/narayanalabhttps://mail.ad.uab.edu/owa/redir.aspx?C=80a7a9ef03ea46b58d648b78f13d4272URL=http%3a%2f%2fwww.opt.uab.edu%2fnarayanalab
 “Never let success go to your head, nor failure to your heart”


From: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of U US
Sent: Monday, October 29, 2012 12:03 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Faculty positions at Molecular Biophysics Unit, IISc, 
Bangalore

Dear Friends,

There is no need to apply to this position, we suggest. It is a PREDETERMINED 
SELECTION, i.e. candidate is fixed and this (advertisement, screening, 
selection board, selection and approval) is just the procedure.
It does not matter whether you apply or not. If you apply and called for 
interview, then you have to waste your valuable time as well as huge travel 
money unless some Big Boss is fixing you to the post.
Interestingly Indian Institute of Science recruits and carries faculties and 
trains them in such a way that it has become a epicentre of recruitment scams 
across India and it make rest of Indian Scientists/Faculties in their path of 
scams and CRIME. Students also inherit the character of their boss. They do not 
participate in any form of fair selection in the country. Almost all cases they 
select and load many times inferior candidates even though candidate was not 
seen by anybody or interviewed. Similarly they distribute various national 
awards among themselves and within