[ccp4bb] Hi prep column from GE

[Michael Colaneri]

We have a hi prep SP column from GE.  We try to load ~14 mg protein but all
goes to FT.  We lowered the pH and changed the buffer but no luck.  We would
appreciate all suggestions.  Mike Colaneri

[ccp4bb] 14th Annual Structural Biology Symposium, March 27th, 2009

[Mark A. White]

Dear Colleague: 

You and your colleagues are cordially invited to join us for the 14th
Annual Structural Biology Symposium to be held at the University of
Texas Medical Branch at Galveston on March 27th, 2009. The meeting is
organized by the Sealy Center for Structural Biology & Molecular
Biophysics and co-sponsored by the Keck Center for Computational &
Structural Biology. In previous years, over 300 scientists from the U.S.
and abroad, have participated in the annual event. The goal of the
symposia is to bring together leaders in all areas of structural biology
to present current topics. Following hurricane Ike, this year’s
Symposium has been scaled back to a single day event and will include
the following speakers: 



   Keynote Speaker: 

  James R. Williamson

The Scripps Research Institute 




  Michael Rosen, UT Southwestern, HHMI

Yizhi Jane Tao, Rice University

Marty Scholtz,Texas A&M 

 Marc Morais, UTMB at Galveston

 C. S. Raman, UT Health Science Center Houston










Poster sessions and social events, included in the program, provide
ample opportunity for formal and informal discussions with the
speakers.Awards will be given to graduate students and post-docs for
best poster presentations. To insure maximum participation, the costs of
attending the symposium have been kept to a minimum. Reasonably priced
rooms have been reserved at the historic beachfront Hotel Galvez on
Galveston Island for symposium participants and meals are provided
on-site during the Symposium.


Please visit our symposium web-site at
http://www.scsb.utmb.edu/symposium/ or contact Angelina Johnson, Phone:
(409) 772-8083, Fax: (409) 772-6334 or email at: ajohn...@utmb.edu, for
on-line registration and abstract submission. Registration for the
Symposium will open January 1, 2009 and we encourage you and your
colleagues to go on-line and register. We are looking forward to seeing
you on March 27th, 2009.

On behalf of the Organizing Committee:

Sincerely yours,


Robert O. Fox, Ph.D. 
Symposium Chair 
Deputy Director, 
  Sealy Center for Structural Biology and Molecular Biophysics 
Professor,
  Department of Biochemistry and Molecular Biology 

Vincent J. Hilser, Ph.D.
Symposium Co-Chair
Director,
  Sealy Center for Structural Biology and Molecular Biophysics
Professor, 
  Department of Biochemistry and Molecular Biology







Yours sincerely,

Mark A. White, Ph.D.
Assistant Professor, Dept. Biochemistry and Molecular Biology, 
Manager, Sealy Center for Structural Biology and Molecular Biophysics
X-ray Crystallography Laboratory,
Basic Science Building, Room 6.660 C
University of Texas Medical Branch
Galveston, TX 77555-0647
Tel. (409) 747-4747
Cell. (409) 539-9138
Fax. (409) 747-4745
mailto://wh...@xray.utmb.edu
http://xray.utmb.edu
http://xray.utmb.edu/~white

Re: [ccp4bb] Off topic: Crystal degredation with age

[Engin Ozkan]

I have to second that.
I recently had crystals that will only grow after seeding and will live 
for exactly five days. On the sixth day, the same drop will have tracks 
of dissolved crystals left in every drop: they almost look like tire 
tracks. The crystals frozen on the fifth day diffract to 2.2 A, and I 
phased with these crystals using Se-Met, so they were definitely good on 
the fifth day.  In this case, the structure did show why the crystals 
were so unstable, a whole domain (40% of the protein) was swerving 
several degrees without much crystal contacts holding it down. Actually, 
there was almost no crystal contacts in one dimension: these crystals 
should not have formed!


So aging may be good until a certain day, and then it can turn really 
badly after that.


Engin

Edward A. Berry wrote:

For a counterexample, from Iwata's group,
Horsefield et al. Succinate: quinone oxidoreductase Acta. 
Cryst.(2003). D59, 600-602:


"It proved critical to freeze the crystals within 72 h of 
crystallization set-up. Crystals that were frozen after this time 
limit showed no diffraction. This alteration in properties was 
apparent by the change in colour from deep orange to pale yellow that 
was observed in crystals more than four weeks old (Fig. 1c). The deep 
orange colour of the crystals is attributed to the presence of haem b 
within the protein. The loss or breakdown of haem, demonstrated by the 
change of colour in the crystals, could lead to structural instability 
and consequently loss of diffraction. "


I've heard the Fe-S clusters of E. coli SDH are oxygen-sensitive in 
the isolated enzyme.
The mitochondrial counterpart is more stable, we've collected data 
from crystals 1-2 years old,
and seen "conversion" (new crystals grow while old crystals dissolve) 
to a new space group

with better diffraction (that was a within few months after setup).


Edward Snell wrote:




Dear All,

I was recently trying to find references on how age may degrade a
crystal, i.e. grow them and use them or preserve them as fresh as
possible. I seem to remember seeing a couple of papers on this but my
memory is fading and I have been unable to locate them. Can anyone jog
my memory or tell me if I'm imagining things?  I've found plenty on the
protein prep etc. but nothing on the crystal.

Thanks,

Eddie.


Edward Snell Ph.D.
Assistant Prof. Department of Structural Biology, SUNY Buffalo,
Hauptman-Woodward Medical Research Institute
700 Ellicott Street, Buffalo, NY 14203-1102
Phone: (716) 898 8631 Fax: (716) 898 8660
Email: esn...@hwi.buffalo.edu  Telepathy: 42.2 GHz
 
Heisenberg was probably here!Crystallization, how quaint!
 

Re: [ccp4bb] Rigid body refinement as last refinement?

[Clemens Vonrhein]

Hi,

On Fri, Feb 06, 2009 at 08:13:29AM -0800, Andy Millston wrote:
> Restrained refinement does a good job at refining most of the
> structure except a small region with poor density. Every time I
> refine it, it puts this region completely out of the map.

Do you mean:

  a) after restrained refinement the 2Fo-Fc map tells me the model
 should be were it was before refinement (and were it stays during
 rigid body refinement)

or

  b) after restrained refinement the 2Fo-Fc map and the model don't
 match up.

If it's a), i.e. 2Fo-Fc map before and after restrained refinement is very
similar but model has been moved away: are you sure there isn't some
very bad contact that pushes your model away? Have you checked e.g in
Coot with all symmetry-equivalents switched on?

If it's b): maybe the initial 2fo-Fc map (after rigid-body refinement)
was heavily biased and showed you an artefact? And the restrained
refinement is actually telling you the truth - your model isn't were
you thought it should be and there is no data/information to put it
there.

> It doesn't
> have much effect on overall geometrical factors as its a very small
> part of a relatively large molecule.
> I am annoyed at the fact that Refmac doesn't seem to have much
> respect for the electron density map while refining this region,

But it does (well: it doesn't look at the density per se - for that
you would need to do real-space refinement): just that something else
(probably one of its geometry terms) it telling REFMAC to do what it
does in the end.

> and all my attempts to keep the residues within the map go in vain.

Remember that the map isn't something fixed (unless you have very good
experimental phases): it's based on the model phases after all.

> The refined residues are still ok according to Fo-Fc map, but
> they're completely out of the 2Fo-Fc map boundaries.

What do you mean with '2Fo-Fc map boundaries'?

> Also, my model and the refined model both look bad on Ramachandran
> plot.

That's ok, we're not refining against Ramchandran plot (or at least we
shouldn't) - so as others have mentioned: this is telling you
something.

> But I think its a unique feature of the molecule, at least thats
> what the density suggests. Also, packing score and quality value
> plot suggest that there is nothing seriously wrong with my model.

A lot of these criteria might not pick up details in small
regions. Furthermore, if you start e.g. from a good initial model
(molecular replacement) then all the purely geometrical quality
indicators will be great, since nothing has changed there after
rigid-body refinement.

> As you've suggested I should try altering the restraints and see how it goes.

Or combine restrained refinement for most of your protein with this
part as a rigid body? I don't know if REFMAC can do that though ...

Or leave that part out for the moment and concentrate on getting
everything else as good as possible: this would give you the best
phases frmo the 'good region' and maybe then the 2Fo-Fc and Fo-Fc map
tell you what's going on in the other part. Sometimes its very
beneficial to remove unclear regions again to not bias your own
expectations of what it should look like.

Cheers

Clemens

-- 

***
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
*
*  Global Phasing Ltd.
*  Sheraton House, Castle Park 
*  Cambridge CB3 0AX, UK
*--
* BUSTER Development Group  (http://www.globalphasing.com)
***

Re: [ccp4bb] Rigid body refinement as last refinement?

[William G. Scott]

Hi Andy:

Just be very careful of special pleading type arguments.  I really  
think this is trying to tell you something, along with the  
Ramachandran plot.  On a more practical level, the PDB will flag it  
when you try to deposit it.  There is something wrong.


It might be worth making a composite-omit map in CNS, and if you have  
experimental phases, go back and look at the original MIR (or  
whatever) map.  Double-check the space group is right.


Do all that before you change any defaults.

It is very good by the way you are asking for feedback at this stage.   
If it turns out there is a problem, it is much better to catch it  
now.  Also, if you do have to do something non-canonical during the  
refinement, the burden is placed on the paper's authors to justify  
why.  So either way this will be time well spent.


Best of luck.

Bill



On Feb 6, 2009, at 8:13 AM, Andy Millston wrote:


Thank you for your responses.

Restrained refinement does a good job at refining most of the  
structure except a small region with poor density. Every time I  
refine it, it puts this region completely out of the map. It doesn't  
have much effect on overall geometrical factors as its a very small  
part of a relatively large molecule.
I am annoyed at the fact that Refmac doesn't seem to have much  
respect for the electron density map while refining this region, and  
all my attempts to keep the residues within the map go in vain. The  
refined residues are still ok according to Fo-Fc map, but they're  
completely out of the 2Fo-Fc map boundaries. Also, my model and the  
refined model both look bad on Ramachandran plot. But I think its a  
unique feature of the molecule, at least thats what the density  
suggests. Also, packing score and quality value plot suggest that  
there is nothing seriously wrong with my model.


As you've suggested I should try altering the restraints and see how  
it goes.


Thank you.

Andy

Re: [ccp4bb] Rigid body refinement as last refinement?

[Andy Millston]

Thank you for your responses.

Restrained refinement does a good job at refining most of the structure except 
a small region with poor density. Every time I refine it, it puts this region 
completely out of the map. It doesn't have much effect on overall geometrical 
factors as its a very small part of a relatively large molecule. 
I am annoyed at the fact that Refmac doesn't seem to have much respect for the 
electron density map while refining this region, and all my attempts to keep 
the residues within the map go in vain. The refined residues are still ok 
according to Fo-Fc map, but they're completely out of the 2Fo-Fc map 
boundaries. Also, my model and the refined model both look bad on Ramachandran 
plot. But I think its a unique feature of the molecule, at least thats what the 
density suggests. Also, packing score and quality value plot suggest that there 
is nothing seriously wrong with my model.

As you've suggested I should try altering the restraints and see how it goes.

Thank you.

Andy


  

Re: [ccp4bb] Off topic: Crystal degredation with age

[Edward A. Berry]

For a counterexample, from Iwata's group,
Horsefield et al. Succinate: quinone oxidoreductase Acta. Cryst.(2003). D59, 
600-602:

"It proved critical to freeze the crystals within 72 h of crystallization set-up. Crystals that were frozen after this time limit 
showed no diffraction. This alteration in properties was apparent by the change in colour from deep orange to pale yellow that was 
observed in crystals more than four weeks old (Fig. 1c). The deep orange colour of the crystals is attributed to the presence of 
haem b within the protein. The loss or breakdown of haem, demonstrated by the change of colour in the crystals, could lead to 
structural instability and consequently loss of diffraction. "


I've heard the Fe-S clusters of E. coli SDH are oxygen-sensitive in the 
isolated enzyme.
The mitochondrial counterpart is more stable, we've collected data from 
crystals 1-2 years old,
and seen "conversion" (new crystals grow while old crystals dissolve) to a new 
space group
with better diffraction (that was a within few months after setup).


Edward Snell wrote:




Dear All,

I was recently trying to find references on how age may degrade a
crystal, i.e. grow them and use them or preserve them as fresh as
possible. I seem to remember seeing a couple of papers on this but my
memory is fading and I have been unable to locate them. Can anyone jog
my memory or tell me if I'm imagining things?  I've found plenty on the
protein prep etc. but nothing on the crystal.

Thanks,

Eddie.


Edward Snell Ph.D.
Assistant Prof. Department of Structural Biology, SUNY Buffalo,
Hauptman-Woodward Medical Research Institute
700 Ellicott Street, Buffalo, NY 14203-1102
Phone: (716) 898 8631 Fax: (716) 898 8660
Email: esn...@hwi.buffalo.edu  Telepathy: 42.2 GHz
 
Heisenberg was probably here!Crystallization, how quaint!
 

Re: [ccp4bb] Rigid body refinement as last refinement?

[William G. Scott]

On Feb 6, 2009, at 6:58 AM, Andy Millston wrote:

I am told by a mentor that rigid body refinement should never be the  
last refinement before submission to pdb. Any idea why?


It is counter-productive and he/she doesn't want to get jumped and  
beaten up in the parking lot at the next conference.


Structures in which every round of retrained refinement messes up a  
conformation, is there any alternative to not using rigid body  
refinement?


Find out why the conformation is being "messed up" because that is  
usually telling you something has been fit wrongly.



Andy





Bill

Re: [ccp4bb] Rigid body refinement as last refinement?

[Anastassis Perrakis]

On Feb 6, 2009, at 15:58, Andy Millston wrote:

I am told by a mentor that rigid body refinement should never be the  
last refinement before submission to pdb. Any idea why?


When restrained refinement 'messes up a conformation' it is extremely  
likely it does it for a perfectly sound reason.
This could be that for example that the conformation is wrong, or that  
the restraints used to describe it are wrong.

You should use that as a warning that your structure needs more work.

Structures in which every round of retrained refinement messes up a  
conformation, is there any alternative to not using rigid body  
refinement?


Yes, work more on the refinement. Did you validate your structure  
using eg MolProbity?


A.




Andy




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

Re: [ccp4bb] Rigid body refinement as last refinement?

[Ed Pozharski]

Andy,

you should probably direct this question to the mentor first.  What is
her explanation?

What do you mean by "refinement messes up a conformation"?  Sometimes
with poor quality data refinement might lead to poor Ramachandran map.
You fix it manually, only to see it deteriorating again.  You may try to
play with the weight of geometry restraints.  Since polypeptide backbone
conformation is forced to occupy certain areas of (phi,psi) space
because of steric clashes, one would expect that increasing contribution
of van der Waals repulsion term may help. I heard an opinion (which I
share to some degree) that it is never a good idea to enforce
Ramachandran directly, by setting (phi,psi) restraints, but you may try,
for instance, restraining the hydrogen bonds that stabilize the
secondary structure.  What this means ideologically is that your data is
not of good enough quality, so you incorporate the knowledge of
secondary structure.  Think of it as incorporating additional
experimental data to boost your data/parameter ratio.

If I understand correctly, what you want to do is to fix the model
manually and then just do rigid body to tidy things up a bit.
Alternative could be to fix/restrain the problematic parts of the
structure and do refine the rest.

HTH,

Ed.

On Fri, 2009-02-06 at 06:58 -0800, Andy Millston wrote:
> I am told by a mentor that rigid body refinement should never be the
> last refinement before submission to pdb. Any idea why? Structures in
> which every round of retrained refinement messes up a conformation, is
> there any alternative to not using rigid body refinement?
> 
> Andy
> 
> 
-- 
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
--   / Lao Tse /

[ccp4bb] TLS Create/Edit module error erases project folder

[Michael Jackson]

Hello,
  I was using the TLS Create/Edit module to create a TLS file from scratch. In 
the module I ran it once but wanted to change some parameters.  While I had the 
module interface still open, I selected to delete the TLS project entry just 
made in the list of jobs.  When I did this I did not realize it was going to 
erase the entire project directory I was currently in.  There was a warning 
that it was going to erase a directory but I thought it was some created 
subdirectory in the project folder when I had ran the TLS module intially.  I 
did not have any trace back or log file to recover to send with this message.   
 I am using the ccp4 6.0.2 with the interface 1.4.4.2 versions on a linux intel 
dual core PC using Fedora 9 OS. 





  

[ccp4bb] Rigid body refinement as last refinement?

[Andy Millston]

I am told by a mentor that rigid body refinement should never be the last 
refinement before submission to pdb. Any idea why? Structures in which every 
round of retrained refinement messes up a conformation, is there any 
alternative to not using rigid body refinement?

Andy



  

Re: [ccp4bb] Off topic: Crystal degredation with age

[Brad Bennett]

FWIW, many of the crystals used in "classical" neutron diffraction
experiments were pretty elderly samples by the time data collection was
initiated, partly to allow large crystals to grow ever larger, partly
because of the mandatory deuterium exchange process and partly because the
experiments lasted weeks to months. To be specific, I believe the crystal
that led to the first endothiapepsin neutron structure was 11 years old
before it began D2O-soaking prior to the neutron experiment. It diffracted
pretty well, 2.1A I think...

Cheers-
Brad

[ccp4bb] Marie Curie Fellowship

[Demetres D. Leonidas]

Please find attached an advert for a short Marie-Curie fellowship at the 
National Hellenic Research Foundation, Athens, Greece.


We seek to recruit a biology/biochemistry graduate, with not more than 4 
years of research experience at post-graduate or equivalent level, who 
will be trained by research in the Molecular Endocrinology Laboratory of 
the National Hellenic Research Foundation: 
http://www.eie.gr/nhrf/institutes/ibrb/programmes/molendocrinology-en.html


in the context of the EURODESY project:

http://www.eie.gr/nhrf/institutes/iopc/eu-projects/eurodesy/index-en.html

Familiarity with cell culture and molecular cell biology and 
pharmacology methodologies will be considered an asset. The position 
will be available from March 2009. The duration of the fellowship will 
be 6 months. Eligibility criteria and reimbursement rates for Marie 
Curie Fellows are described in the handbook at: 
http://ec.europa.eu/research/fp6/mariecurie-actions/action/stage_en.html


Applications should be e-mailed to Dr. MN Alexis (mnale...@eie.gr) till 
1st of March 2009 and should include a full CV and the names and 
addresses of 1-2 referees.


--
Demetres D. Leonidas, Ph.D.
Senior Researcher
Structural Biology & Chemistry Group
Institute of Organic and Pharmaceutical Chemistry
The National Hellenic Research Foundation
48, Vassileos Constantinou Avenue
Athens 116 35, Greece
==
Tel. +30 210 7273841 (office)
+30 210 7273895 (lab) 
Fax. +30 210 7273831

E-mail: d...@eie.gr
URL: http://athena.eie.gr
==

[ccp4bb] SLS non-PX Beamlines: Call for Proposals will be launched on 8 February

[Stefan Mueller]

Dear SLS users, 



Next call for proposals for the non-PX beamlines of the Swiss Light Source, SLS 
will be launched on the 8th of February, 2009.



Deadline for proposal submission: Sunday, March 15, 2009

Submission: All submissions will be handled by the SLS Digital Users Office 
(DUO) /contact: sl...@psi.ch



Please consult the following website to obtain information about the status of 
the different beamlines and the procedure to set-up the proposal:

http://sls.web.psi.ch/view.php/users/experiments/proposals/opencalls/Other/index.html
 



In case of questions related to the experiments at the non-PX beamlines, please 
contact the responsible beamline scientist: 
http://sls.web.psi.ch/view.php/beamlines/index.html



With kind regards



Stefan Müller

on behalf of the SLS team


-
To unsubscribe from this mailing list, please click the following link:
https://duo.psi.ch/duo/change_mailing.php?CMD=UkVNOjIwNzQ6MTY%3D
-

Re: [ccp4bb] comparison of maps, intensities and other basics

[Peter Schmidtke]

Thank you very much for this clear and long answer, Dale. You gave me
exactly the information I needed. 

Thanks again having taken the time to answer to my questions.

Cheers.

Peter


On Thu, 5 Feb 2009 14:51:15 -0800, Dale Tronrud 
wrote:
> A map file stores a density value for each point on a grid.  The
> units and nature of that item is not defined in the format of the map.
> A map can store any number of things.  The actual values are defined
> by the process that created the map file.
> 
>For electron density maps you will find that some contain values
> measured in e/A^3, others contain values that are normalized Z scores
> (The standard deviation of the variation about the mean is set to
> 1.0), or just a bunch of numbers with arbitrary and mysterious units.
> 
>One tends to use e/A^3 when trying to relate the map to expected
> electron density or to compare one map to another.  A normalized map
> is useful if you are interested in the frequency that a density value
> of that magnitude appears in the map. (Is this value common or rare?)
> One uses arbitrary values if one has an attachment to honesty.
> 
>Calculating an electron density map in units of e/A^3 is not an
> easy task.  The diffracted intensities are not measured, themselves,
> in "real" units.  Their magnitude only has meaning as intensities
> relative to the other intensities in the same dataset.   For the map
> to be expressed in units of e/A^3 the diffraction intensities must
> be expressed in units of e/Unit Cell (at least that is the convention).
> This is a hard problem and many papers have been written on the topic.
> 
>If you have a well refined and complete model for the contents of
> the crystal you can use the calculated diffraction pattern as a template
> to scale the observed intensities and calculate maps in e/A^3, but
> this is an approximation as no model is complete or completely correct.
> 
>The other big issue is that we cannot measure the one reflection
> that defines the average of the electron density in the crystal.  It
> happens to always hit the beamstop.  Because of this problem our maps
> usually have an average value of zero, which is of course wrong.  Even
> when the density values are expressed in e/A^3 the intention is that
> each value in the map must have a number added to it to achieve the
> true value at that point.  At least it's the same number everywhere
> in the map, although we don't know its value.
> 
>Because of these issues and uncertainties, when maps are compared
> they are usually compared using a correlation coefficient.  The
> correlation coefficient is relatively unaffected by these scaling
> problems and will usually give the same answer when given any of
> the kinds of maps I described.
> 
>If you want a more detailed comparison of electron density values
> you really have to get into the details of each of the datasets and
> scaling that was applied to ensure that your results are meaningful.
> 
>Estimating the error bars of an electron density map is another
> enormous problem. As you would expect, it depends critically on the
> origin of the map.  The error analysis of a map calculated from MAD
> phasing is quite different than that of a map calculated using a
> refined model as a reference.
> 
>One complication is that the error level is not necessarily the
> same everywhere in the map. In addition the errors at different
> regions of the map are not independent.  The correlation of deviations
> at different regions of the map are likely more important to any
> analysis then any simple overall error bar.
> 
>However, if you insist on an error level, my best guess would be
> to identify the regions of bulk solvent and calculate the rms deviation
> from the mean there.  Since these regions should be flat, and deviations
> from the mean must be due to something that does not represent election
> density. We might as well call it "error".
> 
> Dale Tronrud
> 
> 
> Peter Schmidtke wrote:
>> Dear CCP4BB List Members,
>> 
>> first of all I am not a crystallographer, but I would like to get some
>> things clear, things I did not find in "Crystallography Made Crystal
>> Clear"
>> and on the internet for now. 
>> 
>> I am trying to read electron density maps in the EZD format. These maps
>> contain scaled values of electron density and size and shape of the unit
>> cell. How can I convert the values of intensities (what is the unit of
>> these values?) to the probabilities you can see in coot for example
(1.03
>> electron / A^3), 
>> Once I have achieved this conversion, can I compare densities of
>> different
>> maps of different proteins? If not directly, is there a way to do so?
>> 
>> Last, is there a way to know the experimental error made on intensity
>> values of a map?
>> 
>> Thanks in advance.
>> 
>>

-- 

Peter Schmidtke

--
PhD Student at the Molecular Modeling and Bioinformatics Group
Dep. Physical Chemistry
Faculty of P

Re: [ccp4bb] PHIDM AND FOMDM from DM

[Clemens Vonrhein]

Dear Xie,

you need to use the right columns for calculating maps. From your
description it seems you could do the following in FFT:

  a) map after density modification:
  
   F1=Fobs PHI=PHIDM W=FOMDM

 or (DM should write out these columns as well):

   F1=FDM PHI=PHIDM

 See: http://www.ccp4.ac.uk/dist/html/dm.html#labout
  
  b) map before density modification (I assume you have some
 f.o.m. attached to your phicalc)

   F1=Fobs PHI=phicalc W=FOMcalc

Doing

  F1=Fobs PHI=phicalc W=FOMDM

combines the wrong bits of information - and probably just shows you
the map before density modification (since the phases haven't
changed).

A map in the end is calculated with an amplitude and a phase. The
f.o.m. is a way to generate that amplitude by doing (Fobs*fom) - which
should give you the same thing (the amplitude of the centroid).

Usually all density modification program (or refinement programs for
that matter) will write out specific map coefficients to calculate
maps with. So in the same way REFMAC will give you FWT/PHWT for a map
you get FDM/PHIDM from DM.

If you always use these map coefficients (usually described in the
documentation) you'll be doing the right thing and won't get confused
with mixing up different amplitudes and phases (adding f.o.m. for good
measure).

I can happily live without the need to use a FOM-column in any step
during structure solution or refinement, which avoids a very common
mistake of using a weight on an already weighted amplitude (F1=FDM
PHI=PHIDM W=FOMDM ... brrr!). The columns in an output MTZ file are
nearly always very well documented in the program writeup.

Hope that simple explanation helps ...

Cheers

Clemens


On Thu, Feb 05, 2009 at 09:43:02PM -0800, Xie Jiabao wrote:
> Dear all,
> 
> I am using the density modification tool in ccp4 to generate improved phases 
> for/from my model. I find that the electron density map I generate using 
> Fobs, and density modified phases (PHIDM) are not the same as that generated 
> using Fobs, phicalc (original calculated phases) and FOMDM (new improved 
> figure of merit post dm). Can someone please explain to me why this is so?
> 
> Thanks in advance,
> Xie
> 
> 
> 
>   
-- 

***
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
*
*  Global Phasing Ltd.
*  Sheraton House, Castle Park 
*  Cambridge CB3 0AX, UK
*--
* BUSTER Development Group  (http://www.globalphasing.com)
***

Re: [ccp4bb] Off topic: Crystal degredation with age

[Prof. Joel L. Sussman]

As written in
Jovine, L., Djordjevic, S. & Rhodes, D. (2000). “The crystal  
structure of yeast phenylalanine tRNA at 2.0 A resolution: cleavage by  
Mg(2+) in 15-year old crystals” J Mol Biol 301, 401-414.


"Furthermore, the possibility of a Mg2+-catalysed cleavage of the  
phosphodiester bond between H2U16 and H2U17 was also inferred from the  
electron density map of the orthorhombic form of the tRNAPhe crystals  
[Sussman et al 1978].


n.b.  this chain break was seen 31 years ago, also, in fresh crystals.

Sussman, J.L., Holbrook, S.R., Warrant, R.W., Church, G.M. & Kim, S.- 
H. (1978). Crystal structure of yeast phenylalanine transfer RNA. I.  
crystallographic refinement J Mol Biol 123, 607-630.


see Fig 9c: "Residues 16 and 17. Note the discontinuity between  
phosphate 17 and ribose 17. This region is one of the weakest electron  
density regions."


-
Prof. Joel L. Sussmanjoel.suss...@weizmann.ac.il
Pickman Prof. of Structural Biology  +972 (8) 934 4531 - tel
Department of Structural Biology +972 (8) 934 4159 - fax
Weizmann Institute of Sciencewww.weizmann.ac.il/~joel
Rehovot 76100 ISRAEL www.weizmann.ac.il/ISPC

Proteopedia, www.proteopedia.org  (because life has more than 2D)
-

On 5 Feb 2009, at 21:48, William G. Scott wrote:


Some things improve with age. Here is one of my favorite stories:



http://tinyurl.com/oldtrna


The crystal structure of yeast phenylalanine tRNA at 2.0 Å  
resolution: cleavage by Mg2+ in 15-year old crystals


Luca Jovine,  Snezana Djordjevica and Daniela Rhodes

We have re-determined the crystal structure of yeast tRNAPhe to 2.0  
Å resolution using 15 year old crystals. The accuracy of the new  
structure, due both to higher resolution data and formerly  
unavailable refinement methods, consolidates the previous structural  
information, but also reveals novel details. In particular, the  
water structure around the tightly bound Mg2+ is now clearly  
resolved, and hence provides more accurate information on the  
geometry of the magnesium-binding sites and the role of water  
molecules in coordinating the metal ions to the tRNA. We have  
assigned a total of ten magnesium ions and identified a partly  
conserved geometry for high-affinity Mg2+ binding. In the electron  
density map there is also clear density for a spermine molecule  
binding in the major groove of the TΨC arm and also contacting a  
symmetry-related tRNA molecule. Interestingly, we have also found  
that two specific regions of the tRNA in the crystals are partially  
cleaved. The sites of hydrolysis are within the D and anticodon  
loops in the vicinity of Mg2+.








On Feb 5, 2009, at 11:11 AM, Edward Snell wrote:





Dear All,

I was recently trying to find references on how age may degrade a
crystal, i.e. grow them and use them or preserve them as fresh as
possible. I seem to remember seeing a couple of papers on this but my
memory is fading and I have been unable to locate them. Can anyone  
jog
my memory or tell me if I'm imagining things?  I've found plenty on  
the

protein prep etc. but nothing on the crystal.

Thanks,

Eddie.


Edward Snell Ph.D.
Assistant Prof. Department of Structural Biology, SUNY Buffalo,
Hauptman-Woodward Medical Research Institute
700 Ellicott Street, Buffalo, NY 14203-1102
Phone: (716) 898 8631 Fax: (716) 898 8660
Email: esn...@hwi.buffalo.edu  Telepathy: 42.2 GHz

Heisenberg was probably here!Crystallization, how quaint!

[ccp4bb] Postdoctoral Fellow, Membrane protein structure at the ESRF

[Matthew BOWLER]

Dear CCP4 users,
   We have an opening at the ESRF Macromolecular crystallography group 
(http://www.esrf.fr/UsersAndScience/Experiments/MX) for a Post-Doc to 
work on *the role of membrane proteins in the resistance of 
*/Deinococcus radiodurans/* to extreme conditions (see advert below).  
The ESRF is located in one of the most scientifically stimulating and 
beautiful places in Europe.  *The Macromolecular Crystallography (MX) 
group operates a world leading suite of synchrotron radiation beamlines 
dedicated to the study of biological macromolecules and is also a member 
of the (Partnership for Structural Biology, www.psb-grenoble.eu/) with 
all the laboratory facilities you could need.  Please follow the 
instructions below to apply but feel free to email me with informal 
enquiries.  Many thanks, Matt.



  



 Post-doctoral fellow (m/f) - Ref: 2312
 in the Experiments Division



The ESRF is a multinational research institute, situated in Grenoble, 
France and financed by 19 countries mostly European. It operates a 
powerful synchrotron X-ray source with some 30 beamlines (instruments) 
covering a wide range of scientific research in fields such as biology 
and medicine, chemistry, earth and environmental sciences, materials and 
surface science, and physics. Research at the ESRF is carried out by 
several thousand external users each year from universities, public 
research laboratories and industry, and by the ESRF's own scientists. 
The ESRF employs about 600 staff and is organized as a French /société 
civile/. The working language of the ESRF is English.




/The Experiments Division designs, constructs and runs the ESRF 
experimental stations (= beamlines). Within this division the 
Macromolecular Crystallography group is now seeking to recruit a:/




*Post-doctoral fellow (m/f)*

*THE FUNCTION:*

The subject of your research will be *the role of membrane proteins in 
the resistance of */*Deinococcus radiodurans*/* to extreme conditions*. 
The first line of defence to environmental shock are the surface layer 
(or S-layer) proteins of the outer membrane. These proteins form 
hexagonal crystalline planes covering the outer membrane acting as 
protective layer with proposed roles as diverse as ion traps to that of 
an exoskeleton.  The S-layer has been studied extensively with a wealth 
of biochemical data and its macrostructure has been investigated by 
electron microscopy.  However, molecular details of its assembly and 
response to its environment remain unknown.  The S-layer protein from 
native /D. radiodurans/ membranes has been purified and we are 
proceeding with structural studies. This project will be carried out in 
the state-of-the-art CI Branden building (Partnership for Structural 
Biology, http://www.psb-grenoble.eu/) as well as at the ITQB in Lisbon, 
Portugal.


In parallel, you will also be required to participate in support 
activities for external users on MX beamlines.




*QUALIFICATIONS AND EXPERIENCE:*

You should hold a PhD in Protein Crystallography, Molecular Biology or 
Biochemistry with a strong background in structural biology. Additional 
skills in molecular biology, recombinant protein expression and 
purification and biochemical characterisation of membrane proteins are 
highly desirable.


Only candidates holding a Ph.D. obtained less than 3 years ago are 
eligible for Post-doctoral positions. Post-doctoral fellows are employed 
for a two year-period with a possibility of extension to three years. 
The contract will start as soon as possible.


Contact person: Matthew Bowler (bow...@esrf.fr ), 
Celia Romao (cmro...@itqb.unl.pt ) or Sean 
McSweeney (mcswee...@esrf.fr )




*ADDITIONAL INFORMATION:*

The ESRF shares a site with several other major European scientific 
institutes; it offers an exciting opportunity in an international 
atmosphere. New staff coming from outside the Grenoble area benefit from 
installation allowances to move and settle in the area. An expatriation 
allowance may be payable in accordance with specific regulations. Only 
candidates holding a Ph.D. obtained less than 3 years ago are eligible 
for Post-doctoral positions. /*Post-doctoral fellows are employed for an 
eighteen-month period with a possibility of extension to another six to 
eighteen months */and the contract should start as soon as possible. The 
ESRF is an equal opportunity employer and encourages applications from 
disabled persons.


*If you are interested, please send us a fax (+33 (0)4 76 88 24 60) or 
an e-mail (**recru...@esrf.fr **) with your 
address, and we will provide you with an application form. Or print out 
an application form on the World Wide Web 
http://www.esrf.fr/Jobs/Applying. In addition to the application form, 
you should provide us with a detailed CV and the names of two referees.*




*Ref. 2312 - Deadline for returning application forms: 1 Marc