Re: [ccp4bb] pointless error-summary

[Phil Evans]

Do note that all the Pointless 1.5.x versions had a serious bug and should not 
be used
Phil

On 19 Nov 2011, at 09:49, Rajesh kumar wrote:

> Thanks to Harry Powell, Phil Evans, De-Feng Li for suggestions and special 
> thanks to Charles Ballard looking in to my data.
> 
> The summary is 
> 
> Harry Powell and Phil Evans-Pointless  needs update as newer version is up to 
> version 1.6.8 -  the latest copy is at 
> ftp://ftp.mrc-lmb.cam.ac.uk/pub/pre/pointless-1.6.8.linux
> 
> De-Feng Li- In the log file, Number of batches (1) show that the dataset used 
> in Pointless have been scaled. In fact, the input dataset should be not 
> scaled. You can try it again.
> 
> Charles Ballard-I can reproduce the failure using pointless 1.4.10 (ccp4 
> 6.1.13).  It, however, runs successfully with pointless 1.5.22 (ccp4-6.2.0). 
> 1.4.10 is throwing a lot of memory errors, so it is probably a memory 
> problem. I would suggest using a more up-to-date pointless.  You can get that 
> either from CCP4, or just pointless itself from 
> ftp://mrc-lmb.cam.ac.uk/pub/pre. 
> ps - you will probably get more from the unmerged data
> 
> Thanks
> Rajesh
> 
> Date: Tue, 15 Nov 2011 09:17:34 -0500
> From: ccp4...@hotmail.com
> Subject: [ccp4bb] pointless error
> To: CCP4BB@JISCMAIL.AC.UK
> 
> Dear all,
> 
> mtzdump shows my space group as 'C 2 2 21' (number 20)
> pointless gives error "Space group is not in library". part of the log file 
> is following.
> 
> I appreciate all the help.
> 
> Thanks
> Raj
> 
> 
> **
> **
> * POINTLESS  *
> *   1.4.5*
> **
> *   Determine Laue group from unmerged intensities   *
> * Phil Evans MRC LMB, Cambridge  *
> * Uses cctbx routines by Ralf Grosse-Kunstleve et al.*
> **
> **
> 
> Maximum resolution in file output_7-21.mtz:2.740
> Columns for I, sigI: IMEAN  SIGIMEAN
> 
>  Spacegroup information obtained from library file: 
>  Logical Name: SYMINFO   Filename: 
> /opt/programs/ccp4/ccp4-6.1.13/lib/data/syminfo.lib
> 
> 
> ===
> 
> >*> Summary of test data read in:
>Resolution range accepted:47.922.74
> 
>Number of reflections  = 18008
>Number of observations = 18008
>Number of parts= 18008
>Number of batches  = 1
>Number of datasets = 1
>   Project:   Crystal: unknown  Dataset: 
> unknown210711:15:24:58
>  Run number:   1 consists of batches  1 to  1
>Average unit cell:  110.66   112.08   120.7890.0090.0090.00  
> Unit cell (from HKLIN file) used to derive lattice symmetry with tolerance   
> 2.0 degrees
>  110.66 112.08 120.78  90.00  90.00  90.00
> 
> Tolerance (and delta) is the maximum deviation from the
>  expected angle between two-fold axes in the lattice group
> 
> Lattice point group: P 4 2 2
> Reindexing or changing symmetry
> Reindex operator from input cell to lattice cell: [-1/2h-1/2k,-1/2h+1/2k,-l]
> 
>h'   = ( h k l ) (-0.5-0.5   0 )
> (-0.5 0.5   0 )
> (   0   0  -1 )
> 
> 
> Lattice unit cell after reindexing: deviation 0.73 degrees
>   78.75  78.75 120.78  90.00  90.00  90.73
> 
> 
> Number of reflections  = 10213
> Number of observations = 18008
> Average multiplicity = 1.8
> 
> Resolution range in list:  47.92 ->   2.74
>
> Intensity normalisation: B-factor =  -13.3
> 
> Resolution range reset to47.92 to 3.00
>using I/sigmaI cutoff6.0
> 
>4 pairs rejected for E^2 too large
>
> 
> Overall CC for  6256 unrelated pairs:   0.014  N= 6256
> 
> Estimated expectation value of true correlation coefficient E(CC) =  0.973
> 
> Estimated sd(CC) = 1.110 / Sqrt(N)
> 
>   Number of reflections omitted from ice rings:  613
>
> Estimated E(CC) of true correlation coefficient from identity =  0.973
> 
>  Failed to find spacegroup in SYMINFO! 
> 
>  Space group is not in library 
> Symop:  X,Y,Z
> Symop:  Y,X,-Z
> Symop:  -Y,-X,-Z
> Symop:  -X,-Y,Z
> Space group is not in library
> 
> 
> 
> 
> --
> 
> FATAL ERROR message: 
> Space group is not in library
> --
> 
> 
> 
> 

Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

[Eleanor Dodson]

Just a plea for less molecular replacement.

If you get a new crystal of a known protein with the  same cell 
dimension as youur old crystal, the most likely scenario is that it has 
the same group, and you really should not try MR - use the previous 
solution as input to do rigid body refinement, and then
 a) the R factor will tell you if this is a reasonable hypothesis (it 
usually is..) and
b) you dont have this awful problem of not being able to compare the 
solutions..


 Eleanor

On 11/20/2011 03:57 PM, Napoleão Valadares wrote:

Thank you all for the replies. Felix Frolow, Dan Leahy, Hans
Brandstetter, Boaz Shaanan and Tim Gruene you really helped a lot.

I think I understand it now, I always thought the "one ring to rule them
all" translated in the crystallography realms to "one origin to rule
them all". That probably means I have a long road in front of me.

I'm still half confused, I definitely need to read more, as much as I
read about symmetry and space groups I never seem to improve or get a
better understanding, but I'll keep trying.

About the same origin:
The pdbs of both Solution-1 and Solution-2 present the same space group
and cell, as observed opening the pdbs as text files or in pymol. When I
open both maps on coot they are not superposed but present the same cell
and origin.

If I open both solutions on pymol they clash. If I generate the symmetry
mates of both solutions none of them are superposed, instead they clash.
But I think they are related as you all pointed, I'll check it out.

Thank you all for your kind answers and your patience with a beginner.
Regards from a sunny Brazil,
Napo


On 11/20/2011 2:58 AM, Felix Frolow wrote:

Napoleao,
It is so called alternative origins play a game with you. You do not
change your structure by shifting 1/2 translation (or even combination
of these translations)
into directions of the main axes of your unit cell. Structure factors
after this operation stay the same, however phases change
systematically, producing however the same
map features.
Would I be a begin crystallographer now, I would read a bit more old
fashioned books
on crystallography such as probably Jensen and Stout…
FF
Dr Felix Frolow
Professor of Structural Biology and Biotechnology
Department of Molecular Microbiology
and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

e-mail: mbfro...@post.tau.ac.il 
Tel: ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608

On Nov 20, 2011, at 07:42 , Napoleão Valadares wrote:


Hello,
I'm observing a very strange phenomena (at least to me, I'm a
beginner). It is related to symmetry (I think).

I got a data set at 1.95A (I/Sigma 3.5, R-Factor and R-meas < 35% in
the last shell) and a partially refined solution with R/Rfree 22/24,
166 aminoacids observed and around 30 solvent molecules. I'll call
this Solution-1. The refinement was smooth, the densities were very
clearly "asking" for the correct missing side chains and the map
looks good.

The space group I'm using is P212121, pointless and XDS agree with
that (but me and pointless both have a long history of being wrong
about space groups). Phenix.xtriage says there's no twinning.

I took Solution-1 and used it as a template in a molecular
replacement in the same space group (P212121) using the same mtz as
the one used to refine the template. I got a different (not
superposed in space) solution (called Solution-2, scores by Phaser
RFZ=24.2 TFZ=33.0 PAK=0 LLG=1413 LLG=1899) that was readily refined
in Phenix to R/Rfree 24/26 without any solvent molecule.

- The solutions are not superposed in space, although they are
near-identical and can be superposed yielding a C-alpha rmd =0.001.
- Both structures present VERY similar density maps. The maps are not
superposed in space, but when you "run the chain" in one map in Coot
and do the same in the other it they the present exactly the same
features. It is impossible to ignore their similarities.
- Both structures and maps present the same origin and unit cell.
- If I add to Solution-2 the equivalent solvent molecules of
Solution-1 (I did this by superposing Solution-1 to Solution-2 then
copy/pasting the solvent molecules), the R/Rfree become 22/24. This
is a clear indication that the solutions are related.
- I can't find any MR solutions using the same template and space
groups P222, P2221 and P21212.

How two different sets of phases can yield maps with the same
features? What is happening, wrong space group? I have a feeling my
lack of experience is the problem.
Thank you.
Regards,
Napo

Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

[Ian Tickle]

On Mon, Nov 21, 2011 at 10:23 AM, Eleanor Dodson  wrote:
> Just a plea for less molecular replacement.
>
> If you get a new crystal of a known protein with the  same cell dimension as
> youur old crystal, the most likely scenario is that it has the same group,
> and you really should not try MR - use the previous solution as input to do
> rigid body refinement, and then
>  a) the R factor will tell you if this is a reasonable hypothesis (it
> usually is..) and
> b) you dont have this awful problem of not being able to compare the
> solutions..
>
>  Eleanor

But sometimes (or actually we find quite often) the crystal after
soaking & freezing is sufficiently non-isomorphous (we sometimes see
up tp 10% changes in cell parameters) that RBR just doesn't work, and
then you have to fall back on MR.  The solution in that case to avoid
problems of generating symmetry-related and/or origin-shifted
molecules is to do a limited MR search, e.g. rotating/translating each
molecule in the a.u. independently by up to 5 deg & 5 Ang. from the
model (which of course has 100% similarity making it a lot easier).
Furthermore, because the number of points sampled is much less one can
afford to do the more accurate full 6-D search, as opposed to the
usual 3-D RF followed by 3-D TF on each RF solution.  So now we do
this routinely (we don't even bother with a preliminary RBR).  I
believe the limited 6-D search can be done with Phaser.

Cheers

-- Ian

Re: [ccp4bb] Strange spots

[David Goldstone]

Dear All,

I posted some odd diffraction late last year consisting of Bragg 
diffraction spots with a diffuse ring or halo. Along with Richard 
Welberry at ANU we have now published an explanation for this diffuse 
scattering. For those that are interested the reference is:-


Acta Cryst. (2011). B67, 516-524  [ doi:10.1107/S0108768111037542 ]

Diffuse scattering resulting from macromolecular frustration
T. R. Welberry, A. P. Heerdegen, D. C. Goldstone and I. A. Taylor

Kind Regards

David


--
David Goldstone, PhD
National Institute for Medical Research
Molecular Structure
The Ridgeway
Mill Hill
London NW7 1AA

Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

[Boaz Shaanan]

Or use Kevin's csymmatch which does wonders on scrambled oligomers that (nearly 
always, at least in my hands) come out of Phaser in cases like those mentioned 
by Ian and which do need MR.

  Boaz


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Ian Tickle 
[ianj...@gmail.com]
Sent: Monday, November 21, 2011 1:02 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

On Mon, Nov 21, 2011 at 10:23 AM, Eleanor Dodson  wrote:
> Just a plea for less molecular replacement.
>
> If you get a new crystal of a known protein with the  same cell dimension as
> youur old crystal, the most likely scenario is that it has the same group,
> and you really should not try MR - use the previous solution as input to do
> rigid body refinement, and then
>  a) the R factor will tell you if this is a reasonable hypothesis (it
> usually is..) and
> b) you dont have this awful problem of not being able to compare the
> solutions..
>
>  Eleanor

But sometimes (or actually we find quite often) the crystal after
soaking & freezing is sufficiently non-isomorphous (we sometimes see
up tp 10% changes in cell parameters) that RBR just doesn't work, and
then you have to fall back on MR.  The solution in that case to avoid
problems of generating symmetry-related and/or origin-shifted
molecules is to do a limited MR search, e.g. rotating/translating each
molecule in the a.u. independently by up to 5 deg & 5 Ang. from the
model (which of course has 100% similarity making it a lot easier).
Furthermore, because the number of points sampled is much less one can
afford to do the more accurate full 6-D search, as opposed to the
usual 3-D RF followed by 3-D TF on each RF solution.  So now we do
this routinely (we don't even bother with a preliminary RBR).  I
believe the limited 6-D search can be done with Phaser.

Cheers

-- Ian

Re: [ccp4bb] how to change DNA bases in pdb to match the target DNA sequence

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Wei,

you could use coot "Calculate->Model/Fit/Refine->Simple Mutate" also for
nucleic acids.
It may be worth running geometry idealisation in coot or refmac5 before
using the model in MR.

Tim

On 11/19/2011 09:44 PM, Wei Shi wrote:
> Hi all,
> I want to change a piece of DNA in pdb file to match the target DNA
> sequence in order to be used as a search model in molecular replacement.
> Does anyone happen to know how I could do this? Thank you so much!
> 
> Best,
> Wei
> 

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

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.10 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFOyjTzUxlJ7aRr7hoRApYSAJ4xf8R1kjy+sl16WlQRGuOZuW4kbQCbBwB7
hY5x91qUynn4xFxrYHsN/kY=
=HaRr
-END PGP SIGNATURE-

Re: [ccp4bb] help with the structures

[Eleanor Dodson]

I think you are proving yet again that refinement at 3.3A is not easy.
Indeed there are probably multiple conformations for parts of the 
structure and that may well be why your data is at low resolution and 
anisotropic.  Maybe this is the best you can do..


I think I would make sure the apo structure is as good as it can be, 
then fit that to the 3.3A data set, and only use that 3.3A data to 
deduce whatever features differ from the APO structure.


 Eleanor
On 11/19/2011 12:09 PM, Rajesh kumar wrote:


Dear All,



We have an anisotropic dataset of 3.3 A  and it was solved (not by me) with 
P6522 with R/freeR
29.1/37.3.



I got the corrected
mtz file by plugging in the .HKL (P6122) file to anisotropy diffraction server 
at
2.04 A. I reindexed this p6122 to p6522 and extended the resolution and refined
(refmac) the structure to R/freeR
36.40/38.50. With aotoncs option, fixing all Ramachnadran and rotamer
outliers I got it 30/32. When I added waters and it went down to 27.5/31.2. At
this point I recognized that my new .mtz file from anisotropy server has
different R flag than the earlier one (3.3A) so I copied the R flag and did 
refinemnt  to get R/Rfree 0.2682/0.3247. When I looked at
the refined structure I found  more outliers
than I fixed in earlier round. I did fix all the outliers and without NCS and
waters it gives R/Rfree 0.2906/0.3325. At all the stages I look at outliers at
molprobity server which suggested structure is 10th percentile and after
refinement more outliers comes back. At stage-1 map looked far better so was
happy that anisotropy correction has worked for me (this was my first time
handling this type of dataset) but further refinement didn’t make it look any
better.I use both refmac and autoBuster for refinement. 
http://www.flickr.com/photos/rajesh_ccp4/sets/72157628048657095/
This protein is an human enzyme and a bacterial homologue
which has 38% identity has been used to solve the  Apo structure (2.7 A, 
pC2221, R/freeR
23.03/27.96, molprobity is around 50th percentile). I looked in to this I try 
to fix all the outliers and try to improve
molprobity score but it just refused to improve as after refinement I get more
outliers. This Apo structure was used to solve the mutant structure at
3.3 A.  I believe that both structure could
have better R/freeR and excellent molprobity scores than what they have now. I 
am not able to recognise
if there is any problem in Apo structure and if errors have come to mutant so
both of them refuse to improve.


I wondered if there is any model bias (I don’t know if it’s
the case but nothing was coming to my mind) so thought using ARP/wARP classic
to build model from existing model but it complained that "The wilson plot
is very bad and ARp/wARP is very unlikely to run in a sensible way. Please
check your data" .  
http://www.flickr.com/photos/rajesh_ccp4/sets/72157628048687955/



At this point I dont know how to systematically dissect this problem. I know 
there could be wrong in several places but with my only '2-3 structure 
experience' I am not able to identify the regions to look for
error but I think something is not right. I really appreciate if you give me
some suggestions/ideas/directions/tips so that I could recognize problem and
improve structure and learn some more.
I appreciate your valuable time.
Regards,Rajesh  

Re: [ccp4bb] adxv

[Rajesh kumar]

Dear Mark,

 $ locate XKeysymDB - didnt come with any thing suggests probably openmotif lib 
is not installed.
I linux server has Fedora and I am using latest version of Adxv so details on 
the sbgrid suggest its same problem.

So how would I fix this.  

Thanks for your time.

Regards
Rajesh


> Date: Sun, 20 Nov 2011 23:18:32 +
> Subject: Re: [ccp4bb] adxv
> From: mark.x.bro...@gmail.com
> To: ccp4...@hotmail.com
> CC: CCP4BB@jiscmail.ac.uk
> 
> Dear Rajesh,
>   Are you using the Openmotif library? If so, do you
> have an XKeysymDB file installed?
> In as shell, issue:
> $ locate XKeysymDB
> You may need to symlink it to /usr/X11R6/lib/X11/XKeysymDB if it is
> elsewhere [1].
> I suspect you're not the only one to have seen this [2].
> 
> I hope this helps.
> 
> Mark
> [1] 
> ftp://ftp.parallelgeo.com/SPW_Products/Linux/Current_Release/ReadMe_for_recent_Linux_distributions.txt
> [2] http://sbgrid.org/news/newsletters/2009/06 (search for the string "adxv")
> 
> 
> On 20 November 2011 19:45, Rajesh kumar  wrote:
> >
> > Dear Tim,
> > Thanks. Your suggestion of adding to PATH  works and its not completely 
> > functional may be due to the Ximg/ssh or some thing to do with display.
> > All three windows opened but couldn't open any image as it didn't display 
> > in the list of autoload window. Pattern shows *.0.
> >
> > $adxv pin8_1_180.img
> >
> > (standard_in) 1: parse error
> > (standard_in) 1: parse error
> > (standard_in) 1: parse error
> > beam_center x = pixels , mm
> > beam_center y = pixels , mm
> > distance = mm
> > overload = counts
> > pixelsize = 0.172 mm
> > wavelength = Angstroem
> > Adxv Version 1.9.4-beta
> > Copyright (C) 1994-2007 by Andrew Arvai, Area Detector Systems Corporation
> > Using 24-bit TrueColor visual
> > Warning: translation table syntax error: Unknown keysym name:  osfActivate
> > Warning: ... found while parsing ':osfActivate:
> > ManagerParentActivate()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: translation table syntax error: Unknown keysym name:  osfBeginLine
> > Warning: ... found while parsing ':osfBeginLine:   
> > ManagerGadgetTraverseHome()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: translation table syntax error: Unknown keysym name:  osfHelp
> > Warning: ... found while parsing ':osfHelp:
> > ManagerGadgetHelp()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: translation table syntax error: Unknown keysym name:  osfActivate
> > Warning: ... found while parsing ':osfActivate:
> > ManagerParentActivate()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: translation table syntax error: Unknown keysym name:  osfActivate
> > Warning: ... found while parsing ':osfActivate:
> > PrimitiveParentActivate()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: translation table syntax error: Unknown keysym name:  osfHelp
> > Warning: ... found while parsing ':osfHelp:Help()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: translation table syntax error: Unknown keysym name:  osfActivate
> > Warning: ... found while parsing ':osfActivate:
> > PrimitiveParentActivate()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: translation table syntax error: Unknown keysym name:  osfCancel
> > Warning: ... found while parsing ':osfCancel:  MenuEscape()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: translation table syntax error: Unknown keysym name:  osfSelect
> > Warning: ... found while parsing ':osfSelect:  ArmAndActivate()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: translation table syntax error: Unknown keysym name:  osfActivate
> > Warning: ... found while parsing ':osfActivate:
> > PrimitiveParentActivate()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: Locale not supported for XmbTextListToTextProperty
> > Warning: Cannot convert XmString to compound text
> > Warning: translation table syntax error: Unknown keysym name:  
> > osfPrimaryPaste
> > Warning: ... found while parsing ':m osfPrimaryPaste:cut-primary()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: translation table syntax error: Unknown keysym name:  osfActivate
> > Warning: ... found while parsing ':osfActivate:
> > PrimitiveParentActivate()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: translation table syntax error: Unknown keysym name:  osfActivate
> > Warning: ... found while parsing ':osfActivate:
> > PrimitiveParentActivate()'
> > Warning: String to TranslationTable conversion encountered errors
> > Warning: translation table syntax error: Unknown key

Re: [ccp4bb] improve protein-DNA complex crystal

[zq deng]

my DNA  contains a 9-1-9 palindromic sequence,i tried different blunt end
DNA,and got two kind of crystal  with different lenght DNA.both don't
diffract.


2011/11/21 Michael Murphy 

> Deng, could you tell us a bit more about the DNA that you used? I think
> you may want to try to optimize the DNA that you are using. Maybe try
> either a couple base pair longer/shorter piece of DNA? or try switching
> from blunt ended DNA to a one or two base pair overhang piece of DNA? (very
> often it is contacts between the ends of the pieces of DNA that drive
> crystallization)
>
> On Mon, Nov 21, 2011 at 2:36 AM, dengzq1987  wrote:
>
>> **
>> Hi all,
>>
>> I got a protein-DNA complex crystal,by running SDS-PAGE and nativePAGE,i
>> prove that the crystal contain both
>> protein and DNA.BUT it does not diffract. does anyone have suggestion to
>> improve the diffraction ability ?
>>
>>
>> Best wishes,
>>
>> deng
>>
>
>

[ccp4bb] Protein-DNA complex crystallization

[umar farook]

Dear All,

I have been trying to crystallize protein DNA complex, but all the time i
end up with DNA crystals. Even i changed the length of DNA many times but
still no complex, DNA only crystallizes! Does anybody has idea, why do DNA
crystallize by itself ? My protein behaves very nicely, Dynamic Light
Scattering always shows nice values implies homogenous but once i tried to
ran acidic native page but it shows little bit aggregated. The protein is
highly hydrophilic and soluble, and has only three cysteines, is it there
any possibility of aggregation due to cysteine, when overexpressed in E.*
coli* ? and one more thing i mixed protein and DNA together and ran agarose
gel to see any gel shift, indeed there is a binding, but when i take the
same thing to set drops, only DNA crystals. Kindly suggest me, what could
be done.


Thanks & Regards,
Umar Farook.S

Re: [ccp4bb] Crystallization plates - 24 well

[Irene Margiolaki]

Greiner Bio One is an alternative with distributors in various 
different countries worldwide

http://www.greinerbioone.com/en/france/articles/catalogue/article-groups/11_5/


On Fri, 18 Nov 2011 12:40:56 -0500, Poorva Dharkar wrote:

Hi,

I need to buy 24-well crystallization plates. I want to know the
review of crystallization plates other than Hampton research &
Molecular Dimensions (cheaper & still good to work with).

Has anyone used & liked the ones from Jena biosciences?

Thank you,

Poorva Dharkar

National Cancer Institute

Building 37 Room 2128

37 Convent Drive Bethesda, MD 20892-4255


--
Irene Margiolaki
Lecturer, Department of Biology, Section of Genetics, Cell Biology and 
Development, University of Patras, GR-26500, Patras, Greece

Tel: +302610997408
Web site: 
http://www.biology.upatras.gr/index.php?option=com_content&view=category&layout=blog&id=83&Itemid=99

&
Visiting Scientist, European Synchrotron Radiation Facility (ESRF), 
Grenoble, France

Re: [ccp4bb] Protein-DNA complex crystallization

[Francis E Reyes]

Curious, how did you assess that your crystals only have DNA?

F

On Nov 21, 2011, at 8:59 AM, umar farook wrote:

> Dear All,
> 
> I have been trying to crystallize protein DNA complex, but all the time i end 
> up with DNA crystals. Even i changed the length of DNA many times but still 
> no complex, DNA only crystallizes! Does anybody has idea, why do DNA 
> crystallize by itself ? My protein behaves very nicely, Dynamic Light 
> Scattering always shows nice values implies homogenous but once i tried to 
> ran acidic native page but it shows little bit aggregated. The protein is 
> highly hydrophilic and soluble, and has only three cysteines, is it there any 
> possibility of aggregation due to cysteine, when overexpressed in E.coli ? 
> and one more thing i mixed protein and DNA together and ran agarose gel to 
> see any gel shift, indeed there is a binding, but when i take the same thing 
> to set drops, only DNA crystals. Kindly suggest me, what could be done.
> 
> 
> Thanks & Regards,
> Umar Farook.S



-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

Re: [ccp4bb] adxv

[Edward A. Berry]

yum install lesstif ?

but wouldn't the motif stuff be required for the X-server, i.e. your terminal, not for the 
server running adxv?


Rajesh kumar wrote:

Dear Mark,

$ locate XKeysymDB - didnt come with any thing suggests probably openmotif lib 
is not
installed.
I linux server has Fedora and I am using latest version of Adxv so details on 
the sbgrid
suggest its same problem.

So how would I fix this.

Thanks for your time.

Regards
Rajesh


 > Date: Sun, 20 Nov 2011 23:18:32 +
 > Subject: Re: [ccp4bb] adxv
 > From: mark.x.bro...@gmail.com
 > To: ccp4...@hotmail.com
 > CC: CCP4BB@jiscmail.ac.uk
 >
 > Dear Rajesh,
 > Are you using the Openmotif library? If so, do you
 > have an XKeysymDB file installed?
 > In as shell, issue:
 > $ locate XKeysymDB
 > You may need to symlink it to /usr/X11R6/lib/X11/XKeysymDB if it is
 > elsewhere [1].
 > I suspect you're not the only one to have seen this [2].
 >
 > I hope this helps.
 >
 > Mark
 > [1]
ftp://ftp.parallelgeo.com/SPW_Products/Linux/Current_Release/ReadMe_for_recent_Linux_distributions.txt
 > [2] http://sbgrid.org/news/newsletters/2009/06 (search for the string "adxv")
 >
 >
 > On 20 November 2011 19:45, Rajesh kumar  wrote:
 > >
 > > Dear Tim,
 > > Thanks. Your suggestion of adding to PATH works and its not completely 
functional may
be due to the Ximg/ssh or some thing to do with display.
 > > All three windows opened but couldn't open any image as it didn't display 
in the list
of autoload window. Pattern shows *.0.
 > >
 > > $adxv pin8_1_180.img
 > >
 > > (standard_in) 1: parse error
 > > (standard_in) 1: parse error
 > > (standard_in) 1: parse error
 > > beam_center x = pixels , mm
 > > beam_center y = pixels , mm
 > > distance = mm
 > > overload = counts
 > > pixelsize = 0.172 mm
 > > wavelength = Angstroem
 > > Adxv Version 1.9.4-beta
 > > Copyright (C) 1994-2007 by Andrew Arvai, Area Detector Systems Corporation
 > > Using 24-bit TrueColor visual
 > > Warning: translation table syntax error: Unknown keysym name: osfActivate
 > > Warning: ... found while parsing ':osfActivate: 
ManagerParentActivate()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warning: translation table syntax error: Unknown keysym name: osfBeginLine
 > > Warning: ... found while parsing ':osfBeginLine: 
ManagerGadgetTraverseHome()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warning: translation table syntax error: Unknown keysym name: osfHelp
 > > Warning: ... found while parsing ':osfHelp: ManagerGadgetHelp()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warning: translation table syntax error: Unknown keysym name: osfActivate
 > > Warning: ... found while parsing ':osfActivate: 
ManagerParentActivate()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warning: translation table syntax error: Unknown keysym name: osfActivate
 > > Warning: ... found while parsing ':osfActivate: 
PrimitiveParentActivate()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warning: translation table syntax error: Unknown keysym name: osfHelp
 > > Warning: ... found while parsing ':osfHelp: Help()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warning: translation table syntax error: Unknown keysym name: osfActivate
 > > Warning: ... found while parsing ':osfActivate: 
PrimitiveParentActivate()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warning: translation table syntax error: Unknown keysym name: osfCancel
 > > Warning: ... found while parsing ':osfCancel: MenuEscape()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warning: translation table syntax error: Unknown keysym name: osfSelect
 > > Warning: ... found while parsing ':osfSelect: ArmAndActivate()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warning: translation table syntax error: Unknown keysym name: osfActivate
 > > Warning: ... found while parsing ':osfActivate: 
PrimitiveParentActivate()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warning: Locale not supported for XmbTextListToTextProperty
 > > Warning: Cannot convert XmString to compound text
 > > Warning: translation table syntax error: Unknown keysym name: 
osfPrimaryPaste
 > > Warning: ... found while parsing ':m osfPrimaryPaste:cut-primary()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warning: translation table syntax error: Unknown keysym name: osfActivate
 > > Warning: ... found while parsing ':osfActivate: 
PrimitiveParentActivate()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warning: translation table syntax error: Unknown keysym name: osfActivate
 > > Warning: ... found while parsing ':osfActivate: 
PrimitiveParentActivate()'
 > > Warning: String to TranslationTable conversion encountered errors
 > > Warn

Re: [ccp4bb] Protein-DNA complex crystallization

[sxn214]

Sent from my Verizon Wireless Phone

- Reply message -
From: "umar farook" 
Date: Mon, Nov 21, 2011 10:59 am
Subject: [ccp4bb] Protein-DNA complex crystallization
To: 

Dear All,

I have been trying to crystallize protein DNA complex, but all the time i
end up with DNA crystals. Even i changed the length of DNA many times but
still no complex, DNA only crystallizes! Does anybody has idea, why do DNA
crystallize by itself ? My protein behaves very nicely, Dynamic Light
Scattering always shows nice values implies homogenous but once i tried to
ran acidic native page but it shows little bit aggregated. The protein is
highly hydrophilic and soluble, and has only three cysteines, is it there
any possibility of aggregation due to cysteine, when overexpressed in E.*
coli* ? and one more thing i mixed protein and DNA together and ran agarose
gel to see any gel shift, indeed there is a binding, but when i take the
same thing to set drops, only DNA crystals. Kindly suggest me, what could
be done.


Thanks & Regards,
Umar Farook.S

Re: [ccp4bb] Protein-DNA complex crystallization

[Phoebe Rice]

What is Kd?  

Also, in reply to earlier posts: it is sadly common in crystallizing large 
protein-DNA complexes to go through a couple dozen different duplexes and 
several dismally-diffracting crystal forms before finding a good one.

  Phoebe

>From: CCP4 bulletin board  (on behalf of umar farook 
>)
>Subject: [ccp4bb] Protein-DNA complex crystallization  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Dear All,
>   I have been trying to crystallize protein DNA
>   complex, but all the time i end up with DNA
>   crystals. Even i changed the length of DNA many
>   times but still no complex, DNA only crystallizes!
>   Does anybody has idea, why do DNA crystallize by
>   itself ? My protein behaves very nicely, Dynamic
>   Light Scattering always shows nice values implies
>   homogenous but once i tried to ran acidic native
>   page but it shows little bit aggregated. The protein
>   is highly hydrophilic and soluble, and has only
>   three cysteines, is it there any possibility of
>   aggregation due to cysteine, when overexpressed in
>   E.coli ? and one more thing i mixed protein and DNA
>   together and ran agarose gel to see any gel shift,
>   indeed there is a binding, but when i take the same
>   thing to set drops, only DNA crystals. Kindly
>   suggest me, what could be done.
>   Thanks & Regards,
>   Umar Farook.S

Re: [ccp4bb] 1.95A, different phases, maps look the same, R/Rfree 22/24, wrong space group? - RESOLVED

[Napoleão Valadares]

The problem was resolved with your help, and in fact there was no 
problem, except that I was unaware of the alternative origins. I promise 
I'm reading more about it. :$


A side note: I collected another crystal, different crystallization 
condition but same cell parameters, and followed Eleanor Dodson 
suggestion, just did a rigid body and the map looks just like the others.


Thanks you for all the answers, I'm learning a lot by trying to follow 
(and understand) your different suggestions, and by following this list 
in general. It is a good reading.

Best regards,

   Napo



On 11/20/2011 1:57 PM, Napoleão Valadares wrote:
Thank you all for the replies. Felix Frolow, Dan Leahy, Hans 
Brandstetter, Boaz Shaanan and Tim Gruene you really helped a lot.


I think I understand it now, I always thought the "one ring to rule 
them all" translated in the crystallography realms to "one origin to 
rule them all". That probably means I have a long road in front of me.


I'm still half confused, I definitely need to read more, as much as I 
read about symmetry and space groups I never seem to improve or get a 
better understanding, but I'll keep trying.


About the same origin:
The pdbs of both Solution-1 and Solution-2 present the same space 
group and cell, as observed opening the pdbs as text files or in 
pymol. When I open both maps on coot they are not superposed but 
present the same cell and origin.


If I open both solutions on pymol they clash. If I generate the 
symmetry mates of both solutions none of them are superposed, instead 
they clash. But I think they are related as you all pointed, I'll 
check it out.


Thank you all for your kind answers and your patience with a beginner.
Regards from a sunny Brazil,
  Napo


On 11/20/2011 2:58 AM, Felix Frolow wrote:

Napoleao,
It is so called alternative origins play a game with you. You do not 
change your structure by shifting 1/2 translation (or even 
combination of these translations)
into directions of the main axes of your unit cell. Structure factors 
after this operation stay the same, however phases change 
systematically, producing however the same

map features.
Would I be a begin crystallographer  now, I would read a bit more old 
fashioned books

on crystallography such as probably Jensen and Stout...
FF
Dr Felix Frolow
Professor of Structural Biology and Biotechnology
Department of Molecular Microbiology
and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

e-mail: mbfro...@post.tau.ac.il 
Tel:  ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608

On Nov 20, 2011, at 07:42 , Napoleão Valadares wrote:


Hello,
I'm observing a very strange phenomena (at least to me, I'm a 
beginner). It is related to symmetry (I think).


I got a data set at 1.95A (I/Sigma 3.5, R-Factor and R-meas < 35% in 
the last shell) and a partially refined solution with R/Rfree 22/24, 
166 aminoacids observed and around 30 solvent molecules. I'll call 
this Solution-1. The refinement was smooth, the densities were very 
clearly "asking" for the correct missing side chains and the map 
looks good.


The space group I'm using is P212121, pointless and XDS agree with 
that (but me and pointless both have a long history of being wrong 
about space groups). Phenix.xtriage says there's no twinning.


I took Solution-1 and used it as a template in a molecular 
replacement in the same space group (P212121) using the same mtz as 
the one used to refine the template. I got a different (not 
superposed in space) solution (called Solution-2, scores by Phaser 
RFZ=24.2 TFZ=33.0 PAK=0 LLG=1413 LLG=1899) that was readily refined 
in Phenix to R/Rfree 24/26 without any solvent molecule.


- The solutions are not superposed in space, although they are 
near-identical and can be superposed yielding a C-alpha rmd =0.001.
- Both structures present VERY similar density maps. The maps are 
not superposed in space, but when you "run the chain" in one map in 
Coot and do the same in the other it they the present exactly the 
same features. It is impossible to ignore their similarities.

- Both structures and maps present the same origin and unit cell.
- If I add to Solution-2 the equivalent solvent molecules of 
Solution-1 (I did this by superposing Solution-1 to Solution-2 then 
copy/pasting the solvent molecules), the R/Rfree  become 22/24. This 
is a clear indication that the solutions are related.
- I can't find any MR solutions using the same template and space 
groups P222, P2221 and P21212.


How two different sets of phases can yield maps with the same 
features? What is happening, wrong space group? I have a feeling my 
lack of experience is the problem.

Thank you.
Regards,
 Napo

[ccp4bb] Movements of domains

[Filip Van Petegem]

Dear crystallographers,

I have a general question concerning the comparison of different
 structures.  Suppose you have a crystal structure containing a few
domains.  You also have another structure of the same, but in a different
condition (with a bound ligand, a mutation, or simply a different
crystallization condition,...).  After careful superpositions, you notice
that one of the domains has shifted over a particular distance compared to
the other domains, say  1-1.5 Angstrom.   This is a shift of the entire
domain.  Now how can you know that this is a 'significant' change?  Say the
overall resolution of the structures is lower than the observed distance
(2.5A for example).

Now saying that a 1.5 Angstrom movement of an entire domain is not relevant
at this resolution would seem wrong: we're not talking about some electron
density protruding a bit more in one structure versus another, but all of
the density has moved in a concerted fashion.  So this would seem 'real',
and not due to noise.   I'm not talking about the fact that this movement
was artificially caused by crystal packing or something similar. Just for
whatever the reason (whether packing, pH, ligand binding, ...), you simply
observe the movement.

So the question is: how you can state that a particular movement was
'significantly large' compared to the resolution limit?  In particular,
what is the theoretical framework that allows you to state that some
movement is signifcant? This type of question of course also applies to
other methods such as cryo-EM.  Is a 7A movement of an entire domain
'significant' in a 10A map? If it is, how do we quantify the significance?

If anybody has a great reference or just an individual opinion, I'd like to
hear about it.

Regards,

Filip Van Petegem

-- 
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/

Re: [ccp4bb] Movements of domains

[Steiner, Roberto]

I believe ESCET was designed to answer your kind of question

Best
Roberto

On 21 Nov 2011, at 22:03, "Filip Van Petegem" 
mailto:filip.vanpete...@gmail.com>> wrote:

Dear crystallographers,

I have a general question concerning the comparison of different  structures.  
Suppose you have a crystal structure containing a few domains.  You also have 
another structure of the same, but in a different condition (with a bound 
ligand, a mutation, or simply a different crystallization condition,...).  
After careful superpositions, you notice that one of the domains has shifted 
over a particular distance compared to the other domains, say  1-1.5 Angstrom.  
 This is a shift of the entire domain.  Now how can you know that this is a 
'significant' change?  Say the overall resolution of the structures is lower 
than the observed distance (2.5A for example).

Now saying that a 1.5 Angstrom movement of an entire domain is not relevant at 
this resolution would seem wrong: we're not talking about some electron density 
protruding a bit more in one structure versus another, but all of the density 
has moved in a concerted fashion.  So this would seem 'real', and not due to 
noise.   I'm not talking about the fact that this movement was artificially 
caused by crystal packing or something similar. Just for whatever the reason 
(whether packing, pH, ligand binding, ...), you simply observe the movement.

So the question is: how you can state that a particular movement was 
'significantly large' compared to the resolution limit?  In particular, what is 
the theoretical framework that allows you to state that some movement is 
signifcant? This type of question of course also applies to other methods such 
as cryo-EM.  Is a 7A movement of an entire domain 'significant' in a 10A map? 
If it is, how do we quantify the significance?

If anybody has a great reference or just an individual opinion, I'd like to 
hear about it.

Regards,

Filip Van Petegem

--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email:  
filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/

Re: [ccp4bb] Movements of domains

[Jacob Keller]

Just to clarify: I think the question is about the mathematical sense
of "significance," and not the functional or physiological
significance, right? If I understand the question correctly, wouldn't
the reasoning be that admittedly each atom in the model has a certain
positional error, but all together, it would be very unlikely for all
atoms to be skewed in the same direction?

Jacob



On Mon, Nov 21, 2011 at 4:04 PM, Filip Van Petegem
 wrote:
> Dear crystallographers,
> I have a general question concerning the comparison of different
>  structures.  Suppose you have a crystal structure containing a few domains.
>  You also have another structure of the same, but in a different condition
> (with a bound ligand, a mutation, or simply a different crystallization
> condition,...).  After careful superpositions, you notice that one of the
> domains has shifted over a particular distance compared to the other
> domains, say  1-1.5 Angstrom.   This is a shift of the entire domain.  Now
> how can you know that this is a 'significant' change?  Say the overall
> resolution of the structures is lower than the observed distance (2.5A for
> example).
> Now saying that a 1.5 Angstrom movement of an entire domain is not relevant
> at this resolution would seem wrong: we're not talking about some electron
> density protruding a bit more in one structure versus another, but all of
> the density has moved in a concerted fashion.  So this would seem 'real',
> and not due to noise.   I'm not talking about the fact that this movement
> was artificially caused by crystal packing or something similar. Just for
> whatever the reason (whether packing, pH, ligand binding, ...), you simply
> observe the movement.
> So the question is: how you can state that a particular movement was
> 'significantly large' compared to the resolution limit?  In particular, what
> is the theoretical framework that allows you to state that some movement is
> signifcant? This type of question of course also applies to other methods
> such as cryo-EM.  Is a 7A movement of an entire domain 'significant' in a
> 10A map? If it is, how do we quantify the significance?
> If anybody has a great reference or just an individual opinion, I'd like to
> hear about it.
> Regards,
> Filip Van Petegem
>
> --
> Filip Van Petegem, PhD
> Assistant Professor
> The University of British Columbia
> Dept. of Biochemistry and Molecular Biology
> 2350 Health Sciences Mall - Rm 2.356
> Vancouver, V6T 1Z3
>
> phone: +1 604 827 4267
> email: filip.vanpete...@gmail.com
> http://crg.ubc.ca/VanPetegem/
>



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

Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

[Michael Thompson]

- Forwarded Message -
From: "Michael Thompson" 
To: "e dodson" 
Sent: Monday, November 21, 2011 11:30:17 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

A question regarding the plea for less MR (which I support):

There have been several recent instances in which I have used the solution of 
an isomorphous structure to do rigid body refinement for a new crystal (as 
described by Eleanor). It has always produced good results. My question is 
about how to best handle the free set of reflections when doing this? I have 
heard a number of differing opinions about whether or not it is important to 
carry the freeR flags from the original structure over to the new data set. I 
have heard equally convincing arguments from both sides, so my young and 
impressionable mind does not know who to believe. I was hoping I could get an 
opinion from the "advocates for less MR."

Sorry for hijacking this thread, but hopefully it will provide some insight 
that is relevant to the original post.

Thanks!

Mike




- Original Message -
From: "Eleanor Dodson" 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Monday, November 21, 2011 2:23:21 AM GMT -08:00 US/Canada Pacific
Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

Just a plea for less molecular replacement.

If you get a new crystal of a known protein with the  same cell 
dimension as youur old crystal, the most likely scenario is that it has 
the same group, and you really should not try MR - use the previous 
solution as input to do rigid body refinement, and then
  a) the R factor will tell you if this is a reasonable hypothesis (it 
usually is..) and
b) you dont have this awful problem of not being able to compare the 
solutions..

  Eleanor

On 11/20/2011 03:57 PM, Napoleão Valadares wrote:
> Thank you all for the replies. Felix Frolow, Dan Leahy, Hans
> Brandstetter, Boaz Shaanan and Tim Gruene you really helped a lot.
>
> I think I understand it now, I always thought the "one ring to rule them
> all" translated in the crystallography realms to "one origin to rule
> them all". That probably means I have a long road in front of me.
>
> I'm still half confused, I definitely need to read more, as much as I
> read about symmetry and space groups I never seem to improve or get a
> better understanding, but I'll keep trying.
>
> About the same origin:
> The pdbs of both Solution-1 and Solution-2 present the same space group
> and cell, as observed opening the pdbs as text files or in pymol. When I
> open both maps on coot they are not superposed but present the same cell
> and origin.
>
> If I open both solutions on pymol they clash. If I generate the symmetry
> mates of both solutions none of them are superposed, instead they clash.
> But I think they are related as you all pointed, I'll check it out.
>
> Thank you all for your kind answers and your patience with a beginner.
> Regards from a sunny Brazil,
> Napo
>
>
> On 11/20/2011 2:58 AM, Felix Frolow wrote:
>> Napoleao,
>> It is so called alternative origins play a game with you. You do not
>> change your structure by shifting 1/2 translation (or even combination
>> of these translations)
>> into directions of the main axes of your unit cell. Structure factors
>> after this operation stay the same, however phases change
>> systematically, producing however the same
>> map features.
>> Would I be a begin crystallographer now, I would read a bit more old
>> fashioned books
>> on crystallography such as probably Jensen and Stout…
>> FF
>> Dr Felix Frolow
>> Professor of Structural Biology and Biotechnology
>> Department of Molecular Microbiology
>> and Biotechnology
>> Tel Aviv University 69978, Israel
>>
>> Acta Crystallographica F, co-editor
>>
>> e-mail: mbfro...@post.tau.ac.il 
>> Tel: ++972-3640-8723
>> Fax: ++972-3640-9407
>> Cellular: 0547 459 608
>>
>> On Nov 20, 2011, at 07:42 , Napoleão Valadares wrote:
>>
>>> Hello,
>>> I'm observing a very strange phenomena (at least to me, I'm a
>>> beginner). It is related to symmetry (I think).
>>>
>>> I got a data set at 1.95A (I/Sigma 3.5, R-Factor and R-meas < 35% in
>>> the last shell) and a partially refined solution with R/Rfree 22/24,
>>> 166 aminoacids observed and around 30 solvent molecules. I'll call
>>> this Solution-1. The refinement was smooth, the densities were very
>>> clearly "asking" for the correct missing side chains and the map
>>> looks good.
>>>
>>> The space group I'm using is P212121, pointless and XDS agree with
>>> that (but me and pointless both have a long history of being wrong
>>> about space groups). Phenix.xtriage says there's no twinning.
>>>
>>> I took Solution-1 and used it as a template in a molecular
>>> replacement in the same space group (P212121) using the same mtz as
>>> the one used to refine the template. I got a different (not
>>> superposed in space) solution (called Solution-2, scores by Phaser
>>> RFZ=24.2 TFZ=33.0 PAK=0 LLG=1413 LLG

Re: [ccp4bb] Movements of domains

[Filip Van Petegem]

Hello Jacob,

that's correct, I'm only looking at the mathematical significance, not the
biological one.  I follow the same reasoning - it is highly improbably for
all atoms to be skewed in the same direction.

In a case I'm currently looking at, I'm particularly dealing with cryo-EM
data, not X-ray structures, but with the same underlying principles: what
are the odds that all pixels of the map move together in the same
direction?

As mentioned for X-ray structures, a Luzzati analysis may give information
about the positional errors, but there should be an increased resolution
when comparing domain movements, because it's unlikely for all atoms to
have an error in the same direction.

Filip

On Mon, Nov 21, 2011 at 2:16 PM, Jacob Keller <
j-kell...@fsm.northwestern.edu> wrote:

> Just to clarify: I think the question is about the mathematical sense
> of "significance," and not the functional or physiological
> significance, right? If I understand the question correctly, wouldn't
> the reasoning be that admittedly each atom in the model has a certain
> positional error, but all together, it would be very unlikely for all
> atoms to be skewed in the same direction?
>
> Jacob
>
>
>
> On Mon, Nov 21, 2011 at 4:04 PM, Filip Van Petegem
>  wrote:
> > Dear crystallographers,
> > I have a general question concerning the comparison of different
> >  structures.  Suppose you have a crystal structure containing a few
> domains.
> >  You also have another structure of the same, but in a different
> condition
> > (with a bound ligand, a mutation, or simply a different crystallization
> > condition,...).  After careful superpositions, you notice that one of the
> > domains has shifted over a particular distance compared to the other
> > domains, say  1-1.5 Angstrom.   This is a shift of the entire domain.
>  Now
> > how can you know that this is a 'significant' change?  Say the overall
> > resolution of the structures is lower than the observed distance (2.5A
> for
> > example).
> > Now saying that a 1.5 Angstrom movement of an entire domain is not
> relevant
> > at this resolution would seem wrong: we're not talking about some
> electron
> > density protruding a bit more in one structure versus another, but all of
> > the density has moved in a concerted fashion.  So this would seem 'real',
> > and not due to noise.   I'm not talking about the fact that this movement
> > was artificially caused by crystal packing or something similar. Just for
> > whatever the reason (whether packing, pH, ligand binding, ...), you
> simply
> > observe the movement.
> > So the question is: how you can state that a particular movement was
> > 'significantly large' compared to the resolution limit?  In particular,
> what
> > is the theoretical framework that allows you to state that some movement
> is
> > signifcant? This type of question of course also applies to other methods
> > such as cryo-EM.  Is a 7A movement of an entire domain 'significant' in a
> > 10A map? If it is, how do we quantify the significance?
> > If anybody has a great reference or just an individual opinion, I'd like
> to
> > hear about it.
> > Regards,
> > Filip Van Petegem
> >
> > --
> > Filip Van Petegem, PhD
> > Assistant Professor
> > The University of British Columbia
> > Dept. of Biochemistry and Molecular Biology
> > 2350 Health Sciences Mall - Rm 2.356
> > Vancouver, V6T 1Z3
> >
> > phone: +1 604 827 4267
> > email: filip.vanpete...@gmail.com
> > http://crg.ubc.ca/VanPetegem/
> >
>
>
>
> --
> ***
> Jacob Pearson Keller
> Northwestern University
> Medical Scientist Training Program
> email: j-kell...@northwestern.edu
> ***
>



-- 
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/

Re: [ccp4bb] Movements of domains

[James Stroud]

On Nov 21, 2011, at 3:04 PM, Filip Van Petegem wrote:

> So the question is: how you can state that a particular movement was 
> 'significantly large' compared to the resolution limit?

I can think of a different but related question. How significant is a 
particular movement compared to a measured coordinate error? One way to measure 
the coordinate error in this example is to least-squares superpose the two 
instances of the domain in question and calculate the rmsd.

This makes the calculation of significance independent of the resolution of the 
data set.

James

Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

[Parthasarathy Sampathkumar]

Hi Mike,

Often, I generate independent freeR set (especially in cases where "soak"
dataset is of different resolution (usually worse) compared to the native
dataset and do following two things to get rid-off the bias: 1. add a noise
to the coordinates (this can be done using PDBSET). 2. set the Bvalues to
the wilson B of the "soak" dataset (within Refmac5 before rigidbody
refinement).

HTH,
Partha


On Mon, Nov 21, 2011 at 5:47 PM, Michael Thompson wrote:

> - Forwarded Message -
> From: "Michael Thompson" 
> To: "e dodson" 
> Sent: Monday, November 21, 2011 11:30:17 AM GMT -08:00 US/Canada Pacific
> Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase
>
> A question regarding the plea for less MR (which I support):
>
> There have been several recent instances in which I have used the solution
> of an isomorphous structure to do rigid body refinement for a new crystal
> (as described by Eleanor). It has always produced good results. My question
> is about how to best handle the free set of reflections when doing this? I
> have heard a number of differing opinions about whether or not it is
> important to carry the freeR flags from the original structure over to the
> new data set. I have heard equally convincing arguments from both sides, so
> my young and impressionable mind does not know who to believe. I was hoping
> I could get an opinion from the "advocates for less MR."
>
> Sorry for hijacking this thread, but hopefully it will provide some
> insight that is relevant to the original post.
>
> Thanks!
>
> Mike
>
>
>
>
> - Original Message -
> From: "Eleanor Dodson" 
> To: CCP4BB@JISCMAIL.AC.UK
> Sent: Monday, November 21, 2011 2:23:21 AM GMT -08:00 US/Canada Pacific
> Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase
>
> Just a plea for less molecular replacement.
>
> If you get a new crystal of a known protein with the  same cell
> dimension as youur old crystal, the most likely scenario is that it has
> the same group, and you really should not try MR - use the previous
> solution as input to do rigid body refinement, and then
>  a) the R factor will tell you if this is a reasonable hypothesis (it
> usually is..) and
> b) you dont have this awful problem of not being able to compare the
> solutions..
>
>  Eleanor
>
> On 11/20/2011 03:57 PM, Napoleão Valadares wrote:
> > Thank you all for the replies. Felix Frolow, Dan Leahy, Hans
> > Brandstetter, Boaz Shaanan and Tim Gruene you really helped a lot.
> >
> > I think I understand it now, I always thought the "one ring to rule them
> > all" translated in the crystallography realms to "one origin to rule
> > them all". That probably means I have a long road in front of me.
> >
> > I'm still half confused, I definitely need to read more, as much as I
> > read about symmetry and space groups I never seem to improve or get a
> > better understanding, but I'll keep trying.
> >
> > About the same origin:
> > The pdbs of both Solution-1 and Solution-2 present the same space group
> > and cell, as observed opening the pdbs as text files or in pymol. When I
> > open both maps on coot they are not superposed but present the same cell
> > and origin.
> >
> > If I open both solutions on pymol they clash. If I generate the symmetry
> > mates of both solutions none of them are superposed, instead they clash.
> > But I think they are related as you all pointed, I'll check it out.
> >
> > Thank you all for your kind answers and your patience with a beginner.
> > Regards from a sunny Brazil,
> > Napo
> >
> >
> > On 11/20/2011 2:58 AM, Felix Frolow wrote:
> >> Napoleao,
> >> It is so called alternative origins play a game with you. You do not
> >> change your structure by shifting 1/2 translation (or even combination
> >> of these translations)
> >> into directions of the main axes of your unit cell. Structure factors
> >> after this operation stay the same, however phases change
> >> systematically, producing however the same
> >> map features.
> >> Would I be a begin crystallographer now, I would read a bit more old
> >> fashioned books
> >> on crystallography such as probably Jensen and Stout…
> >> FF
> >> Dr Felix Frolow
> >> Professor of Structural Biology and Biotechnology
> >> Department of Molecular Microbiology
> >> and Biotechnology
> >> Tel Aviv University 69978, Israel
> >>
> >> Acta Crystallographica F, co-editor
> >>
> >> e-mail: mbfro...@post.tau.ac.il 
> >> Tel: ++972-3640-8723
> >> Fax: ++972-3640-9407
> >> Cellular: 0547 459 608
> >>
> >> On Nov 20, 2011, at 07:42 , Napoleão Valadares wrote:
> >>
> >>> Hello,
> >>> I'm observing a very strange phenomena (at least to me, I'm a
> >>> beginner). It is related to symmetry (I think).
> >>>
> >>> I got a data set at 1.95A (I/Sigma 3.5, R-Factor and R-meas < 35% in
> >>> the last shell) and a partially refined solution with R/Rfree 22/24,
> >>> 166 aminoacids observed and around 30 solvent molecules. I'll call
> >>> this Solution-1. The refin

Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase

[Dale Tronrud]

   I'll jump in here, and avoid the question entirely.

   Since running MR on an isomorphous crystal gives the same answer as just
stuffing in the model and running some rigid body refinement, however you decide
to handle your R free flags, you should do the same thing in both cases.  The
model is the same.

Dale Tronrud

On 11/21/11 14:47, Michael Thompson wrote:
> - Forwarded Message -
> From: "Michael Thompson" 
> To: "e dodson" 
> Sent: Monday, November 21, 2011 11:30:17 AM GMT -08:00 US/Canada Pacific
> Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase
> 
> A question regarding the plea for less MR (which I support):
> 
> There have been several recent instances in which I have used the solution of 
> an isomorphous structure to do rigid body refinement for a new crystal (as 
> described by Eleanor). It has always produced good results. My question is 
> about how to best handle the free set of reflections when doing this? I have 
> heard a number of differing opinions about whether or not it is important to 
> carry the freeR flags from the original structure over to the new data set. I 
> have heard equally convincing arguments from both sides, so my young and 
> impressionable mind does not know who to believe. I was hoping I could get an 
> opinion from the "advocates for less MR."
> 
> Sorry for hijacking this thread, but hopefully it will provide some insight 
> that is relevant to the original post.
> 
> Thanks!
> 
> Mike
> 
> 
> 
> 
> - Original Message -
> From: "Eleanor Dodson" 
> To: CCP4BB@JISCMAIL.AC.UK
> Sent: Monday, November 21, 2011 2:23:21 AM GMT -08:00 US/Canada Pacific
> Subject: Re: [ccp4bb] LESS MR pleae.. 1.95A, different phase
> 
> Just a plea for less molecular replacement.
> 
> If you get a new crystal of a known protein with the  same cell 
> dimension as youur old crystal, the most likely scenario is that it has 
> the same group, and you really should not try MR - use the previous 
> solution as input to do rigid body refinement, and then
>   a) the R factor will tell you if this is a reasonable hypothesis (it 
> usually is..) and
> b) you dont have this awful problem of not being able to compare the 
> solutions..
> 
>   Eleanor
> 
> On 11/20/2011 03:57 PM, Napoleão Valadares wrote:
>> Thank you all for the replies. Felix Frolow, Dan Leahy, Hans
>> Brandstetter, Boaz Shaanan and Tim Gruene you really helped a lot.
>>
>> I think I understand it now, I always thought the "one ring to rule them
>> all" translated in the crystallography realms to "one origin to rule
>> them all". That probably means I have a long road in front of me.
>>
>> I'm still half confused, I definitely need to read more, as much as I
>> read about symmetry and space groups I never seem to improve or get a
>> better understanding, but I'll keep trying.
>>
>> About the same origin:
>> The pdbs of both Solution-1 and Solution-2 present the same space group
>> and cell, as observed opening the pdbs as text files or in pymol. When I
>> open both maps on coot they are not superposed but present the same cell
>> and origin.
>>
>> If I open both solutions on pymol they clash. If I generate the symmetry
>> mates of both solutions none of them are superposed, instead they clash.
>> But I think they are related as you all pointed, I'll check it out.
>>
>> Thank you all for your kind answers and your patience with a beginner.
>> Regards from a sunny Brazil,
>> Napo
>>
>>
>> On 11/20/2011 2:58 AM, Felix Frolow wrote:
>>> Napoleao,
>>> It is so called alternative origins play a game with you. You do not
>>> change your structure by shifting 1/2 translation (or even combination
>>> of these translations)
>>> into directions of the main axes of your unit cell. Structure factors
>>> after this operation stay the same, however phases change
>>> systematically, producing however the same
>>> map features.
>>> Would I be a begin crystallographer now, I would read a bit more old
>>> fashioned books
>>> on crystallography such as probably Jensen and Stout…
>>> FF
>>> Dr Felix Frolow
>>> Professor of Structural Biology and Biotechnology
>>> Department of Molecular Microbiology
>>> and Biotechnology
>>> Tel Aviv University 69978, Israel
>>>
>>> Acta Crystallographica F, co-editor
>>>
>>> e-mail: mbfro...@post.tau.ac.il 
>>> Tel: ++972-3640-8723
>>> Fax: ++972-3640-9407
>>> Cellular: 0547 459 608
>>>
>>> On Nov 20, 2011, at 07:42 , Napoleão Valadares wrote:
>>>
 Hello,
 I'm observing a very strange phenomena (at least to me, I'm a
 beginner). It is related to symmetry (I think).

 I got a data set at 1.95A (I/Sigma 3.5, R-Factor and R-meas < 35% in
 the last shell) and a partially refined solution with R/Rfree 22/24,
 166 aminoacids observed and around 30 solvent molecules. I'll call
 this Solution-1. The refinement was smooth, the densities were very
 clearly "asking" for the correct missing side chains and the map
 looks good

Re: [ccp4bb] Movements of domains

[Dale Tronrud]

   This is a subtle problem and performing an analysis of this type
of error is confusing.  Most of the tools we use to analyze errors
begin with the assumption that the "errors" are random and uncorrelated.
These include Luzzati and Fo-Fc maps.

   My solution is to perform a null hypothesis test.  If you run
two refinements starting from the same model, in one allowing the
RB shift and in the other forbidding it, which fits the data better?
If the difference in likelihood is quite small then you cannot
distinguish between a RB shifted model and one w/o the shift and
that shift must be insignificant (in a statistical sense.)  If the
likelihood is better when the shift is allowed then the shift is
significant.

   In my experience RB shifts of a couple tens of an Angstrom
are very significant even with 4 A resolution data.  X-ray diffraction
is exquisitely sensitive to this sort of motion.

Dale Tronrud

On 11/21/11 14:52, Filip Van Petegem wrote:
> Hello Jacob,
> 
> that's correct, I'm only looking at the mathematical significance, not
> the biological one.  I follow the same reasoning - it is highly
> improbably for all atoms to be skewed in the same direction.
> 
> In a case I'm currently looking at, I'm particularly dealing with
> cryo-EM data, not X-ray structures, but with the same underlying
> principles: what are the odds that all pixels of the map move together
> in the same direction?  
> 
> As mentioned for X-ray structures, a Luzzati analysis may give
> information about the positional errors, but there should be an
> increased resolution when comparing domain movements, because it's
> unlikely for all atoms to have an error in the same direction.
> 
> Filip
> 
> On Mon, Nov 21, 2011 at 2:16 PM, Jacob Keller
> mailto:j-kell...@fsm.northwestern.edu>>
> wrote:
> 
> Just to clarify: I think the question is about the mathematical sense
> of "significance," and not the functional or physiological
> significance, right? If I understand the question correctly, wouldn't
> the reasoning be that admittedly each atom in the model has a certain
> positional error, but all together, it would be very unlikely for all
> atoms to be skewed in the same direction?
> 
> Jacob
> 
> 
> 
> On Mon, Nov 21, 2011 at 4:04 PM, Filip Van Petegem
> mailto:filip.vanpete...@gmail.com>> wrote:
> > Dear crystallographers,
> > I have a general question concerning the comparison of different
> >  structures.  Suppose you have a crystal structure containing a
> few domains.
> >  You also have another structure of the same, but in a different
> condition
> > (with a bound ligand, a mutation, or simply a different
> crystallization
> > condition,...).  After careful superpositions, you notice that one
> of the
> > domains has shifted over a particular distance compared to the other
> > domains, say  1-1.5 Angstrom.   This is a shift of the entire
> domain.  Now
> > how can you know that this is a 'significant' change?  Say the overall
> > resolution of the structures is lower than the observed distance
> (2.5A for
> > example).
> > Now saying that a 1.5 Angstrom movement of an entire domain is not
> relevant
> > at this resolution would seem wrong: we're not talking about some
> electron
> > density protruding a bit more in one structure versus another, but
> all of
> > the density has moved in a concerted fashion.  So this would seem
> 'real',
> > and not due to noise.   I'm not talking about the fact that this
> movement
> > was artificially caused by crystal packing or something similar.
> Just for
> > whatever the reason (whether packing, pH, ligand binding, ...),
> you simply
> > observe the movement.
> > So the question is: how you can state that a particular movement was
> > 'significantly large' compared to the resolution limit?  In
> particular, what
> > is the theoretical framework that allows you to state that some
> movement is
> > signifcant? This type of question of course also applies to other
> methods
> > such as cryo-EM.  Is a 7A movement of an entire domain
> 'significant' in a
> > 10A map? If it is, how do we quantify the significance?
> > If anybody has a great reference or just an individual opinion,
> I'd like to
> > hear about it.
> > Regards,
> > Filip Van Petegem
> >
> > --
> > Filip Van Petegem, PhD
> > Assistant Professor
> > The University of British Columbia
> > Dept. of Biochemistry and Molecular Biology
> > 2350 Health Sciences Mall - Rm 2.356
> > Vancouver, V6T 1Z3
> >
> > phone: +1 604 827 4267 
> > email: filip.vanpete...@gmail.com 
> > http://crg.ubc.ca/VanPetegem/
> >
> 
> 
> 
> --
> ***
> Jacob Pearson Keller
> Northwestern Univer

[ccp4bb] sugar and coot

[Jan van Agthoven]

Hi everyone!
Does anyone know if there is a way of auto-refining a sugar in Coot?
Jan

Re: [ccp4bb] Movements of domains

[James Stroud]

On Nov 21, 2011, at 3:52 PM, Filip Van Petegem wrote:

> As mentioned for X-ray structures, a Luzzati analysis may give information 
> about the positional errors, but there should be an increased resolution when 
> comparing domain movements, because it's unlikely for all atoms to have an 
> error in the same direction.


Here's how I think about it:

If you use the empirical coordinate error that I described previously, you can 
use simple statistics to calculate how likely you are to get a coordinated 
movement (relative to a fixed landmark).

I can use a 1-d case as an example. In this 1-d case, let's pretend that we 
have a domain of N=25 atoms where atom 2 is about 1 away from atom 1 and atom 3 
is 2 away from atom 1 and one away from atom 2, etc, with a standard deviation 
of 1 for the position of the atoms. If atom 1 for domain A is at 1, this is just

A_j = j

Then you can have domain B that has moved +1 compared to domain A:

B_j = j+1

Since we have an alignment (B_j -> A_j), then we can calculate the movement, X:

X = mean(B) - mean(A)

We can also calculate the error of the ensemble (aka the error of the mean):

sigmaE = std( (B - mean(B)) - (A - mean(B)) ) / sqrt(25)

Then, we can calculate how likely it is we observe the movement X by tail 
integration of the cumulative normal distribution. We will justify this for the 
3-d case because the least squares superposition (from which we estimate the 
coordinate error) assumes normality.

Here is a simulation of this scenario in python:

py> import numpy
py> from scipy.special import ndtr
py> a = numpy.array([numpy.random.normal(j) for j in xrange(25)])
py> b = numpy.array([numpy.random.normal(j+1) for j in xrange(25)])
py> a
array([  1.38125295,  -0.27126096,   1.7597104 ,   1.36242299,
 3.88327659,   4.33063307,   5.00544708,   7.0258,
 7.83945228,   9.72101719,  10.36231633,  10.29176378,
11.78497375,  12.16082056,  14.31057296,  13.25941344,
17.93779336,  18.05626047,  18.62148347,  20.52756478,
19.73362283,  21.83953268,  22.28038617,  23.24545481,  22.96192518])
py> b
array([  3.32750181,   2.42664791,   3.23309368,   4.32882699,
 6.59985764,   6.49597664,   5.27921723,   7.8573831 ,
 9.98722475,  10.65225383,  11.69970159,  11.67435798,
12.16191254,  13.69297801,  14.21845382,  17.21423427,
16.89347161,  17.68778305,  17.89371115,  18.7679351 ,
20.84842496,  20.69249899,  23.97436807,  23.54011453,  26.84986504])
py> X = b.mean() - a.mean()
py> sigma_ensemble = ((b - b.mean()) - (a - a.mean())).std() / math.sqrt(25)
py> X_standardized = (X - 0) / sigma_ensemble
py> 2 * ndtr(-abs(X_standardized))
0.00011596192653578624

This means, for the 1-d scenario I describe, (using the random arrays generated 
above), the movement is expected about once for every 10,000 "experiments", 
providing a p-value, or estimate of significance. Note that the 2 comes from 
the fact that the cumulative distribution has 2 tails.

A 3-D calculation using the rmsd as the coordinate error would be similar 
except that you use Euclid's formula to calculate the distances in higher 
dimensions (instead of the absolute value of a simple subtraction as in 1-d).

James

[ccp4bb] Coot, RH6.1 x86 linux

[Santarsiero, Bernard D.]

I just installed CCP4-6.2.0 and COOT on a Red Hat v6.1, x86, linux
workstation. Some of the menus, along the right edge, are gray buttons on
gray background. Where is the preferences file to change the colors of the
menu buttons? The menus across the top of the window are fine.

Also, idiffdisp is unable to view a file. It loads the idiffdisp window,
but gives error messages. Is there a log file that I can scan? I think I'm
missing some library files for x86.

Bernie

-- 
Bernard D. Santarsiero
Research Professor
Center for Pharmaceutical Biotechnology and the
 Department of Medicinal Chemistry and Pharmacognosy
Center for Structural Biology
Center for Clinical and Translational Science
University of Illinois at Chicago
MC870  3070MBRB  900 South Ashland Avenue
Chicago, IL 60607-7173  USA
(312) 413-0339 (office)
(312) 413-9303 (FAX)
http://www.uic.edu/labs/bds

Re: [ccp4bb] Movements of domains

[James Stroud]

On Nov 21, 2011, at 5:23 PM, James Stroud wrote:
> except that you use Euclid's formula to calculate the distances in higher 
> dimensions

I meant to say "Euclidian distance". "Euclid's formula" has a specific meaning 
that is different.

Re: [ccp4bb] sugar and coot

[Dirk]

Hi,

I'm not sure if that is what you are looking for, but if you want to use 
the "real space refine zone" function in coot with sugars that is possible.
For a monosaccharide you will need a cif dictionary with the restraint 
definitions - in some cases present in the refmac monomer database, but 
be careful not all cif files there are correct. If none is present or if 
your monomer is disrupted during refinement you can look at the small 
molecule databases (Hic-Up etc) or calculate one with PRODRG  
(http://davapc1.bioch.dundee.ac.uk/prodrg/). Load it with "File" "Import 
CIF dictionary and real space refinement should work.
If you try to refine a polysaccharide it's a bit more difficult since 
coot will ignore any glycosidic bond restraints - if two sugars are 
close by and one has well defined density and the other one not it might 
even place them on top of each other.

I like to use the anchor tool on the bonding atoms in those cases.
The other option for a polysaccharide is to use PRODRG to generate a 
single "monomer" out of your polysaccharide.


In any case at the end you have to inspect the geometry manually since 
the restraint definitions are often incomplete.


Greetings

Dirk


Am 22.11.11 01:16, schrieb Jan van Agthoven:

Hi everyone!
Does anyone know if there is a way of auto-refining a sugar in Coot?
Jan

Re: [ccp4bb] Movements of domains

[Jacob Keller]

I am curious how all of this can be more than splitting hairs, i.e.,
under what conditions can this 1Ang domain motion mean something
biologically significant? Proteins are pretty flexible, after all,
especially between domains.

JPK

On Mon, Nov 21, 2011 at 6:41 PM, James Stroud  wrote:
> On Nov 21, 2011, at 5:23 PM, James Stroud wrote:
>> except that you use Euclid's formula to calculate the distances in higher 
>> dimensions
>
> I meant to say "Euclidian distance". "Euclid's formula" has a specific 
> meaning that is different.
>



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

Re: [ccp4bb] Movements of domains

[James Stroud]

On Nov 21, 2011, at 6:34 PM, Jacob Keller wrote:
> I am curious how all of this can be more than splitting hairs, i.e.,
> under what conditions can this 1Ang domain motion mean something
> biologically significant?

To engage in the discussion, I think we had to accept this:

On Nov 21, 2011, at 3:04 PM, Filip Van Petegem wrote:
> I'm not talking about the fact that this movement was artificially caused by 
> crystal packing or something similar.
> Just for whatever the reason (whether packing, pH, ligand binding, ...), you 
> simply observe the movement.   

So the point of the discussion, as I understand it, is to figure out whether 
the movement warrants further consideration in the first place, i.e. whether it 
is significant with respect to the error of the models.

I think it doesn't take too much energy to discount the attempt to quantify the 
statistical significance by claiming that one can't imagine how such a change 
might be biologically significant. I'm really not privy to the structures in 
question, so I am in no position to make this judgement.

James

Re: [ccp4bb] Movements of domains

[Bernhard Rupp (Hofkristallrat a.D.)]

> If the difference in likelihood is quite small then you cannot distinguish
between a RB shifted model and one w/o the shift and that shift must be
insignificant (in a statistical sense.)  If the likelihood is better when
the shift is allowed then the shift is significant.

That of course is correct and leads to the interesting (and in part
previously discussed) question how to quantify (log) likelihood ratios in
terms of significance. I am not sure that this is trivial. More likely
better, very likely better, kinda really likely better? Having said that I
also want to caution the normal distribution & statistical test fans, who
think that a p value or similar has any more meaning. Whether p=0.05 means
something (other than a statistical metric) is equally fuzzy; it just
conveys a false sense of precision and erudition. 

May I quote:

"The scientist must be the judge of his own hypotheses, not the
statistician."
 
A.F.W. Edwards (1992) in Likelihood - An account of the statistical concept
of likelihood and its application to scientific inference , p. 34.

Brrr

On 11/21/11 14:52, Filip Van Petegem wrote:
> Hello Jacob,
> 
> that's correct, I'm only looking at the mathematical significance, not 
> the biological one.  I follow the same reasoning - it is highly
> improbably for all atoms to be skewed in the same direction.
> 
> In a case I'm currently looking at, I'm particularly dealing with 
> cryo-EM data, not X-ray structures, but with the same underlying
> principles: what are the odds that all pixels of the map move together 
> in the same direction?
> 
> As mentioned for X-ray structures, a Luzzati analysis may give 
> information about the positional errors, but there should be an 
> increased resolution when comparing domain movements, because it's 
> unlikely for all atoms to have an error in the same direction.
> 
> Filip
> 
> On Mon, Nov 21, 2011 at 2:16 PM, Jacob Keller 
>  >
> wrote:
> 
> Just to clarify: I think the question is about the mathematical sense
> of "significance," and not the functional or physiological
> significance, right? If I understand the question correctly, wouldn't
> the reasoning be that admittedly each atom in the model has a certain
> positional error, but all together, it would be very unlikely for all
> atoms to be skewed in the same direction?
> 
> Jacob
> 
> 
> 
> On Mon, Nov 21, 2011 at 4:04 PM, Filip Van Petegem
> mailto:filip.vanpete...@gmail.com>>
wrote:
> > Dear crystallographers,
> > I have a general question concerning the comparison of different
> >  structures.  Suppose you have a crystal structure containing a
> few domains.
> >  You also have another structure of the same, but in a different
> condition
> > (with a bound ligand, a mutation, or simply a different
> crystallization
> > condition,...).  After careful superpositions, you notice that one
> of the
> > domains has shifted over a particular distance compared to the other
> > domains, say  1-1.5 Angstrom.   This is a shift of the entire
> domain.  Now
> > how can you know that this is a 'significant' change?  Say the
overall
> > resolution of the structures is lower than the observed distance
> (2.5A for
> > example).
> > Now saying that a 1.5 Angstrom movement of an entire domain is not
> relevant
> > at this resolution would seem wrong: we're not talking about some
> electron
> > density protruding a bit more in one structure versus another, but
> all of
> > the density has moved in a concerted fashion.  So this would seem
> 'real',
> > and not due to noise.   I'm not talking about the fact that this
> movement
> > was artificially caused by crystal packing or something similar.
> Just for
> > whatever the reason (whether packing, pH, ligand binding, ...),
> you simply
> > observe the movement.
> > So the question is: how you can state that a particular movement was
> > 'significantly large' compared to the resolution limit?  In
> particular, what
> > is the theoretical framework that allows you to state that some
> movement is
> > signifcant? This type of question of course also applies to other
> methods
> > such as cryo-EM.  Is a 7A movement of an entire domain
> 'significant' in a
> > 10A map? If it is, how do we quantify the significance?
> > If anybody has a great reference or just an individual opinion,
> I'd like to
> > hear about it.
> > Regards,
> > Filip Van Petegem
> >
> > --
> > Filip Van Petegem, PhD
> > Assistant Professor
> > The University of British Columbia
> > Dept. of Biochemistry and Molecular Biology
> > 2350 Health Sciences Mall - Rm 2.356
> > Vancouver, V6T 1Z3
> >
> > phone: +1 604 827 4267 
> > email: filip.vanpete...@gmail.com

Re: [ccp4bb] Movements of domains

[Vellieux Frederic]

A mixture between mathematical significance and biological significance 
as a part of the reply:


you should also take into account the thermal vibrations of the atoms 
present in that domain, i.e. the "thermal ellipsoids" when you have one 
of the representations of anisotropic temperature factors (when these 
can be obtained, high enough resolution), together with the associated 
density smearing. Especially if you observe correlated thermal 
ellipsoids. If you have a small "motion" but that this motion can be (at 
least in good part) "explained" by the inherent thermal "flexibility" of 
all atoms in that domain then perhaps you can question the significance 
of this domain motion (at least in the publication).


Fred.

Filip Van Petegem wrote:

Dear crystallographers,

I have a general question concerning the comparison of different 
 structures.  Suppose you have a crystal structure containing a few 
domains.  You also have another structure of the same, but in a 
different condition (with a bound ligand, a mutation, or simply a 
different crystallization condition,...).  After careful 
superpositions, you notice that one of the domains has shifted over a 
particular distance compared to the other domains, say  1-1.5 
Angstrom.   This is a shift of the entire domain.  Now how can you 
know that this is a 'significant' change?  Say the overall resolution 
of the structures is lower than the observed distance (2.5A for example).


Now saying that a 1.5 Angstrom movement of an entire domain is not 
relevant at this resolution would seem wrong: we're not talking about 
some electron density protruding a bit more in one structure versus 
another, but all of the density has moved in a concerted fashion.  So 
this would seem 'real', and not due to noise.   I'm not talking about 
the fact that this movement was artificially caused by crystal packing 
or something similar. Just for whatever the reason (whether packing, 
pH, ligand binding, ...), you simply observe the movement.   

So the question is: how you can state that a particular movement was 
'significantly large' compared to the resolution limit?  In 
particular, what is the theoretical framework that allows you to state 
that some movement is signifcant? This type of question of course also 
applies to other methods such as cryo-EM.  Is a 7A movement of an 
entire domain 'significant' in a 10A map? If it is, how do we quantify 
the significance?


If anybody has a great reference or just an individual opinion, I'd 
like to hear about it.


Regards,

Filip Van Petegem

--
Filip Van Petegem, PhD
Assistant Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.com 
http://crg.ubc.ca/VanPetegem/