Re: [ccp4bb] Crystals Disappearing Overnight

[Dr. Anthony Addlagatta]

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Maria,

>From my experience with co-crystallization experiments, if crystals appear 
>immediately
after setting up the drop in presence of small molecules, they most likely are 
crystals
of small molecules and not of protein. Since you have 8% 8K PEG, initially the 
solution
is heterogeneous with pockets of high viscosity that will precipitate the small
molecules. Over the period of time, solution becomes homogenous that may 
dissolve the
crystals. 

To rule out this, set up two crystallization experiments under similar 
conditions. 

1) only the protein
2) only the adenosine

Regards

Anthony
-
Dr. Anthony Addlagatta
Center for Chemical Biology 
Indian Institute of Chemical Technology [IICT]
Tarnaka, Hyderabad
AP-500 607, INDIA
Tel:91-40-27191812
Web: https://sites.google.com/site/chembioliict/home/dr-anthony-addlagatta-1

-- Original Message ---
From: dusky dew 
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wed, 7 May 2014 00:52:22 -0700
Subject: Re: [ccp4bb] Crystals Disappearing Overnight

> ***
> This message has been scanned by the InterScan for CSC SSM at IICT and found 
> to be
free of known security risks.
> ***
> 
> I tried microbatch and the crystals are not stable. They dissolve
> overnight.  I also have reproducibility issue.  Can this be due to poor
> stability of adenosine?
> 
> Best
> Maria
> 
> On Monday, May 5, 2014, Bob Cudney  wrote:
> > Try the microbatch first to see if the problem is related to ionic
> strength.
> >
> >
> >
> > When possible and practical it is good to change only one variable at a
> time to identify cause and effect.
> >
> >
> >
> > Kind Regards, Bob Cudney
> >
> >
> >
> > Hampton Research
> >
> > 34 Journey
> >
> > Aliso Viejo, CA 92656-3317 USA
> >
> >
> >
> > Telephone 1 949 425 1321 Extension 200
> >
> > Fax 1 949 425 1611
> >
> > E-mail b...@hrmail.com
> >
> > Web www.hamptonresearch.com
> >
> >
> >
> > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> dusky dew
> > Sent: Saturday, May 03, 2014 1:29 AM
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: Re: [ccp4bb] Crystals Disappearing Overnight
> >
> >
> >
> > Thank you all for getting back!
> >
> > I will set up the drops using microbatch method.
> > Regarding the temp, I set them up in lab and put them in incubator. The
> lab temp may be slightly higher. So are they not stable at lower temp? Or
> its the shock?
> > So how can I take care of the temp issue?
> >
> > Thanks again!
> > Maria
> >
> > On Saturday, May 3, 2014, Bob Cudney  wrote:
> >> Hello Maria,
> >>
> >>
> >>
> >> Check to see if there might have been a temperature change between the
> time the crystals were present and when the crystals disappeared.  If your
> sample has temperature dependent solubility, in this relatively low ionic
> strength condition, a temperature change could mean the difference between
> the presence and absence of crystals.  That being said, if the experiment
> is returned to the temperature that produced the crystals, the crystals
> should/might reappear.
> >>
> >>
> >>
> >> If your drop is made by mixing 1 part of protein with 1 part of reagent
> the initial drop concentration would be 10 mM Tris, 150 mM NaCl, 2.5% w/v
> PEG 8,000, 50 mM Sodium cacodylate.  Is this a vapor diffusion experiment?
> If yes, then the reservoir would be 5% w/v PEG 8,000, 100 mM Sodium
> cacodylate.  The ionic strength of your drop would initially be higher than
> the ionic strength in your reservoir.  This means water vapor leaves the
> reservoir and vapor diffuses into the drop, lowering the protein and
> reagent concentration in your drop.  This decrease in relative
> supersaturation could dissolve a crystal.  Your set up would be a reserve
> vapor diffusion.  You say the crystals appeared right after setting the
> experiment so your crystallization is essentially a batch experiment.
> Therefore you might want to change your set up from a vapor diffusion to a
> microbatch experiment under oil.  If you need more information about how to
> perform a microbatch experiment, let me know and I’ll explain.
> >>
> >>
> >>
> >> Hope this helps.
> >>
> >>
> >>
> >> Kind Regards, Bob Cudney
> >>
> >>
> >>
> >> Hampton Research
> >>
> >> 34 Journey
> >>
> >> Aliso Viejo, CA 92656-3317 USA
> >>
> >>
> >>
> >> Telephone 1 949 425 1321 Extension 200
> >>
> >> Fax 1 949 425 1611
> >>
> >> E-mail b...@hrmail.com
> >>
> >> Web www.hamptonresearch.com
> >>
> >>
> >>
> >> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> dusky dew
> >> Sent: Friday, May 02, 2014 4:39 AM
> >> To: CCP4BB@JISCMAIL.AC.UK
> >> Subject: [ccp4bb] Crystals Disappearing Overnight
> >>
> >>
> >>
> >> Dear All,
> >>
> >>
> >>
> >> I am trying to crystallize a protein with Adenosin

[ccp4bb] PDRA opportunity in crystallography software methods development at Diamond Light Source

[Alun Ashton]

We are looking for a high calibre PDRA/software scientist to join our 
scientific software group to work on the development and optimisation of 
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their application on new crystallographic problems, especially small molecule 
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Full details can be found here:
http://www.diamond.ac.uk/Careers/Vacancies/All/DIA0920_CH.html  

Regards,
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___
Alun Ashton, alun.ash...@diamond.ac.uk Tel: +44 1235 778404
Group Leader - Data Analysis Software,www.diamond.ac.uk 
Diamond Light Source, Chilton, Didcot, Oxon, OX11 0DE, U.K.





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[ccp4bb] AW: [ccp4bb] Space group problem

[Herman . Schreuder]

Dear Rain,

Maybe a little late, but here are some more comments:

1) What is the deal? For me, one can only know the space group after the 
structure has been solved. I have seen quite few cases (e.g. twinning, 
non-crystallographic symmetry etc.) that all programs (XDS, pointless) and 
statistics would insist on the wrong space group. Since there is a discussion 
on the space group, the structure has obviously not yet been solved, so why not 
say something like: most probably hexagonal or maybe trigonal? You could even 
give statistics for both possibilities. Based on diffraction data alone, it is 
impossible to determine the space group with certainty (at least for proteins). 

2) Higher than expected Rmeas at high resolution also make me suspicious. In 
such cases I usually compare the statistics of the data processed in P1 vs. the 
data processed in a higher symmetry space group. If there is a big discrepancy 
at high resolution, there may be a mundane explanation like radiation damage or 
anisotropic diffraction, but it might also be that the symmetry which was 
thought to be crystallographic is in fact non-crystallographic. At low 
resolution, this symmetry closely matches crystallographic symmetry, but at 
high resolution, this symmetry deviates more and more resulting in much higher 
Rfactors. So in fact, you may have a lower symmetry space group with almost 
crystallographic non-crystallographic symmetry.

In your case I would tell the referee that since the structure has not yet been 
solved it is impossible to determine the exact space group and leave it at that.

Best regards,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Gesendet: Mittwoch, 7. Mai 2014 21:41
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Space group problem

I thought there was a theoretical Rmeas number for I/sigma=2 that is around 
50%? In my case, the Rmeas is much higher. 
Before, we reported P62(4) and got rejected. 
I'll just change a journal.  Hopefully reviewer will not be too picky about 
that. 
Thanks to everyone!

[ccp4bb] FORTRAN still rules?

[David Schuller]

http://arstechnica.com/science/2014/05/scientific-computings-future-can-any-coding-language-top-a-1950s-behemoth/


 Scientific computing's future: Can any coding language top a 1950s
 behemoth?


   Cutting-edge research still universally involves Fortran; a trio of
   challengers wants in.

---

Includes a JPEG image of a Hollerith card for the younguns  who have 
never seen one.


"Julia may be the first language since Fortran created specifically with 
scientific number crunching in mind."


"Fortran has been consistently regarded as the fastest language 
available for numerical work, and it remains the standard used for 
comparatively benchmarking supercomputers. But what does it mean for a 
language to be fast?"


"Julia's published benchmarks  show it 
performing close to or slightly worse than C, and Fortran, as usual, 
performing better than C for most tasks."


http://julialang.org/benchmarks/

"The epigraph that opens this article notwithstanding, there is a 
reasonable chance that the language of choice for scientific computing 
in another decade will be called "Julia.""


Discuss.

--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu

Re: [ccp4bb] Space group problem

[Kay Diederichs]

Hi,

please post the pointless table  "Analysing rotational symmetry in lattice 
group P 6/m m m". This gives a quite clear information about the 
presence/absence of rotational symmetry axes. 
Look at at the following example

Analysing rotational symmetry in lattice group P 6/m m m
--



Scores for each symmetry element

Nelmt  Lklhd  Z-ccCCN  RmeasSymmetry & operator (in Lattice 
Cell)

  1   0.936   9.71   0.978676  0.077 identity
  2   0.936   9.74   0.97   12213  0.071 *** 2-fold l ( 0 0 1) {-h,-k,l}
  3   0.055   1.58   0.16   12097  0.514 2-fold k ( 0 1 0) {-h,h+k,-l}
  4   0.056   1.74   0.17   12146  0.495 2-fold h ( 1 0 0) {h+k,-k,-l}
  5   0.056   1.73   0.17   12274  0.505 2-fold   ( 1-1 0) {-k,-h,-l}
  6   0.055   1.61   0.16   12238  0.521 2-fold   ( 2-1 0) {h,-h-k,-l}
  7   0.056   1.69   0.17   12148  0.495 2-fold   (-1 2 0) {-h-k,k,-l}
  8   0.056   1.71   0.17   12056  0.511 2-fold   ( 1 1 0) {k,h,-l}
  9   0.248   6.35   0.64   24331  0.326 3-fold l ( 0 0 1) 
{k,-h-k,l}{-h-k,h,l}
 10   0.245   6.32   0.63   24629  0.327 6-fold l ( 0 0 1) 
{h+k,-h,l}{-k,h+k,l}


Here, the 2-fold symmetry is just as good as the identity, and all the other 
symmetries are insignificant, with one exception: there is a pseudo-3fold that 
together with the true 2-fold gives a pseudo-6fold. So the true space group is 
P2 (or P2_1) but it has 3-fold NCS.
I have seen even more interesting cases, where the CC was 0.8 to 0.9 for some 
rotational elements - but even such a seemingly high CC was significantly lower 
than the CC for the identity.
So my advice is: look at this table, and compare the CC values with that of the 
identity operation.

Two caveats are: 
1) a CC may be lower than the identity, due to radiation damage (but that must 
then be quite significant)
2) a CC may be high, but still not mean true crystallographic symmetry, if 
there is twinning

Finally, a different topic: Rmerge and Rmeas refer to precision of unmerged 
data, and their values have no immediate relation to I/sigma, a precision 
indicator for the merged data (in this table). So when somehow trying to 
compare these values, you are comparing apples to oranges - but this is 
meaningless.  

HTH,

Kay

On Wed, 7 May 2014 18:26:24 +0100,   wrote:

>Hi all,
>I have a 360 degree data set collect on home beam.
>I used XDS to integrate the frames in P1. 
>I progressively merge the data from P1 to P2 or P1 to P3 in XDS and attach the 
>log below.
>The cell looks like P3 and pointless suggest P6. But the Rmerge and Rmeas are 
>much higher than normal at I/sigmaI=2. 
>I think P1 might be the true space group. But the Rpim reported by aimless 
>seems high in the high resolution shell. Why is that?
>Thanks!
>
>  LATTICE-  BRAVAIS-   QUALITY  UNIT CELL CONSTANTS (ANGSTROEM & DEGREES)
> REINDEXING TRANSFORMATION
> CHARACTER  LATTICE OF FIT  a  b  c   alpha  beta gamma
>
> *  31aP  0.0  62.5   82.5   82.6  60.1  89.9  90.0   -1  
> 0  0  0  0 -1 -1  0  0 -1  0  0
> *  44aP  0.2  62.5   82.5   82.6 119.9  90.1  90.01  
> 0  0  0  0  1  1  0  0 -1  0  0
> *  41mC  0.6 143.1   82.5   62.5  90.0  90.1  90.00  
> 1 -1  0  0 -1 -1  0 -1  0  0  0
> *  30mC  0.7  82.5  143.1   62.5  89.9  90.0  90.00 
> -1 -1  0  0  1 -1  0  1  0  0  0
> *  35mP  1.0  82.5   62.5   82.6  90.1 119.9  90.00 
> -1 -1  0 -1  0  0  0  0  1  0  0
> *  40oC  1.2  82.5  143.1   62.5  89.9  90.0  90.00 
> -1 -1  0  0 -1  1  0 -1  0  0  0
> *  20mC  2.6 142.9   82.6   62.5  90.0  90.0  90.10 
> -2 -1  0  0  0 -1  0  1  0  0  0
> *  23oC  3.3  82.6  142.9   62.5  90.0  90.0  89.90  
> 0  1  0  0 -2 -1  0  1  0  0  0
> *  25mC  3.3  82.6  142.9   62.5  90.0  90.0  89.90  
> 0  1  0  0 -2 -1  0  1  0  0  0
> *  22hP  3.5  82.5   82.6   62.5  90.1  90.0 119.90  
> 1  1  0  0 -1  0  0  1  0  0  0
>37mC249.8 176.5   62.5   82.5  90.0 117.8  69.31 
> -2  0  0  1  0  0  0  0  1  1  0
>42oI250.0  62.5   82.5  156.1  90.0 113.6  90.0   -1  
> 0  0  0  0 -1 -1  0  1 -1  1  0
>39mC250.6 176.4   62.5   82.6  90.0 117.9  69.21 
> -2 -2  0  1  0  0  0  0  0  1  0
>33mP434.8  62.5   82.5   82.6 119.9  90.1  90.01  
> 0  0  0  0  1  1  0  0 -1  0  0
>34mP435.2  62.5   82.6   82.5 119.9  90.0  90.1   -1  
> 0  0  0  0  1  0  0  0 -1 -1  0
>32oP435.4  62.5   82.5   82.6 119.9  90.1  90.01  
> 0  0  0  0  1  1  0  0 -1  0  0
>21tP437.6  82.5   82.6   62.5  90.1  90.0 119.90  
> 1  1  0  0 -1  0  0  1  0  0  0
>
>
>
>P1:
> SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.

[ccp4bb] AW: [ccp4bb] Space group problem

[Christophe Wirth]

Dear all,

To add another bit to the discussion, I would say that an increase of Rmerge 
and Rmeas is just expected in such a case, isn't it?

According to your tables, in P1, the multiplicity is about 4. In P2, it's about 
7. In P3, it's 10. And in P6, it's approaching 20. I would say that this leads 
us to the now "classical" problem those statistics have with high multiplicity 
datasets. 

Best,

Christophe




--
Christophe Wirth, PhD
Centre for Biological Signalling Studies (bioss) 
Institute for Biochemistry and Molecular Biology
University of Freiburg
Stefan-Meier-Str. 17
D-79104 Freiburg, Germany
Tel: +49 (0) 761 203 52 77
--





 








-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Gesendet: Mittwoch, 7. Mai 2014 19:26
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] Space group problem

Hi all,
I have a 360 degree data set collect on home beam.
I used XDS to integrate the frames in P1. 
I progressively merge the data from P1 to P2 or P1 to P3 in XDS and attach the 
log below.
The cell looks like P3 and pointless suggest P6. But the Rmerge and Rmeas are 
much higher than normal at I/sigmaI=2. 
I think P1 might be the true space group. But the Rpim reported by aimless 
seems high in the high resolution shell. Why is that?
Thanks!

  LATTICE-  BRAVAIS-   QUALITY  UNIT CELL CONSTANTS (ANGSTROEM & DEGREES)
REINDEXING TRANSFORMATION
 CHARACTER  LATTICE OF FIT  a  b  c   alpha  beta gamma

 *  31aP  0.0  62.5   82.5   82.6  60.1  89.9  90.0   -1  0 
 0  0  0 -1 -1  0  0 -1  0  0
 *  44aP  0.2  62.5   82.5   82.6 119.9  90.1  90.01  0 
 0  0  0  1  1  0  0 -1  0  0
 *  41mC  0.6 143.1   82.5   62.5  90.0  90.1  90.00  1 
-1  0  0 -1 -1  0 -1  0  0  0
 *  30mC  0.7  82.5  143.1   62.5  89.9  90.0  90.00 -1 
-1  0  0  1 -1  0  1  0  0  0
 *  35mP  1.0  82.5   62.5   82.6  90.1 119.9  90.00 -1 
-1  0 -1  0  0  0  0  1  0  0
 *  40oC  1.2  82.5  143.1   62.5  89.9  90.0  90.00 -1 
-1  0  0 -1  1  0 -1  0  0  0
 *  20mC  2.6 142.9   82.6   62.5  90.0  90.0  90.10 -2 
-1  0  0  0 -1  0  1  0  0  0
 *  23oC  3.3  82.6  142.9   62.5  90.0  90.0  89.90  0 
 1  0  0 -2 -1  0  1  0  0  0
 *  25mC  3.3  82.6  142.9   62.5  90.0  90.0  89.90  0 
 1  0  0 -2 -1  0  1  0  0  0
 *  22hP  3.5  82.5   82.6   62.5  90.1  90.0 119.90  1 
 1  0  0 -1  0  0  1  0  0  0
37mC249.8 176.5   62.5   82.5  90.0 117.8  69.31 -2 
 0  0  1  0  0  0  0  1  1  0
42oI250.0  62.5   82.5  156.1  90.0 113.6  90.0   -1  0 
 0  0  0 -1 -1  0  1 -1  1  0
39mC250.6 176.4   62.5   82.6  90.0 117.9  69.21 -2 
-2  0  1  0  0  0  0  0  1  0
33mP434.8  62.5   82.5   82.6 119.9  90.1  90.01  0 
 0  0  0  1  1  0  0 -1  0  0
34mP435.2  62.5   82.6   82.5 119.9  90.0  90.1   -1  0 
 0  0  0  1  0  0  0 -1 -1  0
32oP435.4  62.5   82.5   82.6 119.9  90.1  90.01  0 
 0  0  0  1  1  0  0 -1  0  0
21tP437.6  82.5   82.6   62.5  90.1  90.0 119.90  1 
 1  0  0 -1  0  0  1  0  0  0



P1:
 SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
 RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR  R-FACTOR 
COMPARED I/SIGMA   R-meas  CC(1/2)  Anomal  SigAno   Nano
   LIMIT OBSERVED  UNIQUE  POSSIBLE OF DATA   observed  expected
  Corr

 8.4545001165  1288   90.5%   2.2%  2.4% 
4498   51.19  2.6%99.9*100.8381030
 6.2771741844  1872   98.5%   3.1%  3.0% 
7173   35.92  3.6%99.9*11*   0.9061671
 5.2090092288  2324   98.5%   4.5%  4.3% 
9008   26.88  5.2%99.8* 40.8352168
 4.55   105972686  2744   97.9%   5.2%  5.2%
10595   22.65  6.0%99.7*-20.7762557
 4.09   120513051  3147   96.9%   7.5%  7.5%
12048   16.93  8.7%99.4* 20.7792915
 3.75   127403226  3342   96.5%  14.8% 14.5%
127389.37 17.1%98.2* 10.7603078
 3.48   143443631  3738   97.1%  22.2% 22.2%
143426.36 25.7%95.8* 00.7853471
 3.26   150793813  3948   96.6%  47.3% 48.5%
150762.99 54.8%83.7*-10.7143655
 3.08   140883797  4242   89.5%  99.7%105.6%
139451.30116.0%58.6

Re: [ccp4bb] AW: [ccp4bb] Space group problem

[Kay Diederichs]

On Thu, 8 May 2014 16:56:01 +0200, Christophe Wirth 
 wrote:

>Dear all,
>
>To add another bit to the discussion, I would say that an increase of Rmerge 
>and Rmeas is just expected in such a case, isn't it?
>
>According to your tables, in P1, the multiplicity is about 4. In P2, it's 
>about 7. In P3, it's 10. And in P6, it's approaching 20. I would say that this 
>leads us to the now "classical" problem those statistics have with high 
>multiplicity datasets. 

Rmeas does not have this problem, only Rmerge has it!

But there are other factors which produce higher (and more realistic!) Rmeas in 
the higher-symmetry spacegroup, like radiation damage and absorption or other 
systematic differences that can not be removed by scaling.

In other words: in an ideal experiment Rmeas should be the same for the correct 
space group and its sub-groups. In a real experiment, however, Rmeas in a 
high-symmetry space group "sees" the differences resulting from systematic 
errors, whereas Rmeas in a low-symmetry space group often does not "see" it - 
simply because it does not compare those reflections which suffer from the 
error.

best,

Kay 

>
>Best,
>
>Christophe
>
>
>
>
>--
>Christophe Wirth, PhD
>Centre for Biological Signalling Studies (bioss) 
>Institute for Biochemistry and Molecular Biology
>University of Freiburg
>Stefan-Meier-Str. 17
>D-79104 Freiburg, Germany
>Tel: +49 (0) 761 203 52 77
>--
>
>
>
>
>
> 
>
>
>
>
>
>
>
>
>-Ursprüngliche Nachricht-
>Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Field>
>Gesendet: Mittwoch, 7. Mai 2014 19:26
>An: CCP4BB@JISCMAIL.AC.UK
>Betreff: [ccp4bb] Space group problem
>
>Hi all,
>I have a 360 degree data set collect on home beam.
>I used XDS to integrate the frames in P1. 
>I progressively merge the data from P1 to P2 or P1 to P3 in XDS and attach the 
>log below.
>The cell looks like P3 and pointless suggest P6. But the Rmerge and Rmeas are 
>much higher than normal at I/sigmaI=2. 
>I think P1 might be the true space group. But the Rpim reported by aimless 
>seems high in the high resolution shell. Why is that?
>Thanks!
>
>  LATTICE-  BRAVAIS-   QUALITY  UNIT CELL CONSTANTS (ANGSTROEM & DEGREES)
> REINDEXING TRANSFORMATION
> CHARACTER  LATTICE OF FIT  a  b  c   alpha  beta gamma
>
> *  31aP  0.0  62.5   82.5   82.6  60.1  89.9  90.0   -1  
> 0  0  0  0 -1 -1  0  0 -1  0  0
> *  44aP  0.2  62.5   82.5   82.6 119.9  90.1  90.01  
> 0  0  0  0  1  1  0  0 -1  0  0
> *  41mC  0.6 143.1   82.5   62.5  90.0  90.1  90.00  
> 1 -1  0  0 -1 -1  0 -1  0  0  0
> *  30mC  0.7  82.5  143.1   62.5  89.9  90.0  90.00 
> -1 -1  0  0  1 -1  0  1  0  0  0
> *  35mP  1.0  82.5   62.5   82.6  90.1 119.9  90.00 
> -1 -1  0 -1  0  0  0  0  1  0  0
> *  40oC  1.2  82.5  143.1   62.5  89.9  90.0  90.00 
> -1 -1  0  0 -1  1  0 -1  0  0  0
> *  20mC  2.6 142.9   82.6   62.5  90.0  90.0  90.10 
> -2 -1  0  0  0 -1  0  1  0  0  0
> *  23oC  3.3  82.6  142.9   62.5  90.0  90.0  89.90  
> 0  1  0  0 -2 -1  0  1  0  0  0
> *  25mC  3.3  82.6  142.9   62.5  90.0  90.0  89.90  
> 0  1  0  0 -2 -1  0  1  0  0  0
> *  22hP  3.5  82.5   82.6   62.5  90.1  90.0 119.90  
> 1  1  0  0 -1  0  0  1  0  0  0
>37mC249.8 176.5   62.5   82.5  90.0 117.8  69.31 
> -2  0  0  1  0  0  0  0  1  1  0
>42oI250.0  62.5   82.5  156.1  90.0 113.6  90.0   -1  
> 0  0  0  0 -1 -1  0  1 -1  1  0
>39mC250.6 176.4   62.5   82.6  90.0 117.9  69.21 
> -2 -2  0  1  0  0  0  0  0  1  0
>33mP434.8  62.5   82.5   82.6 119.9  90.1  90.01  
> 0  0  0  0  1  1  0  0 -1  0  0
>34mP435.2  62.5   82.6   82.5 119.9  90.0  90.1   -1  
> 0  0  0  0  1  0  0  0 -1 -1  0
>32oP435.4  62.5   82.5   82.6 119.9  90.1  90.01  
> 0  0  0  0  1  1  0  0 -1  0  0
>21tP437.6  82.5   82.6   62.5  90.1  90.0 119.90  
> 1  1  0  0 -1  0  0  1  0  0  0
>
>
>
>P1:
> SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
> RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR  R-FACTOR 
> COMPARED I/SIGMA   R-meas  CC(1/2)  Anomal  SigAno   Nano
>   LIMIT OBSERVED  UNIQUE  POSSIBLE OF DATA   observed  expected   
>Corr
>
> 8.4545001165  1288   90.5%   2.2%  2.4% 
> 4498   51.19  2.6%99.9*100.8381030
> 6.2771741844  1872   98.5%   3.1%  3.0% 
> 7173   35.92  3.6%99.9*11*   0.9061671
> 5.209009 

[ccp4bb] postdoctoral position at UW-Madison

[Yongna Xing]

 
Dear all,
 
 
 
You are invited to apply for a postdoctoral position in the structural biology 
lab of McArdle Cancer Institute of University of Wisconsin at Madison. We are 
interested in understanding the structural basis and mechanisms of 
cancer-related cell signaling proteins and protein complexes. One important 
focus of the study is to elucidate mechanisms of protein chaperones in 
modulating the function of signaling proteins with important relevance to 
cancer, such as protein phosphatase 2A (PP2A) complexes, viral kinases, nuclear 
receptors, and RNA interaction proteins, using X-ray crystallography, NMR, FRET 
imaging and diverse biochemical, biophysical, and cell biology approaches. Some 
results related to this aspect can be found in our recent publications 
(Molecular Cell 41, 1–12, 2011; Nat. Commun., 4:1699, 2013; Cell Research, 
24(2):190-203, 2014).
 
 
 
The lab is equipped with a state-of-the-art setup for x-ray crystallographic 
studies, including crystallization robot, and advanced imaging system for FRET 
study in mammalian cells. The researcher will work in a multidisciplinary 
setup, including cell biology, biophysics, proteomics, and protein 
biochemistry, in addition to protein crystallography. 
 
Candidates should hold a PhD in protein biochemistry and crystallography and 
have a good working knowledge of molecular biology techniques. Experience in 
NMR spectrometry is highly appealing. The candidates should be highly 
motivated, goal-driven, and dedicated individuals who can think critically and 
work both independently and collaboratively with good verbal and written 
communication skills. It is also important that the candidates are creative in 
problem solving and have a strong desire to seek answers to questions related 
to the molecular basis of cellular processes. Experience in cell culture 
techniques and writing scientific research papers would be advantageous. 
Postdoctoral training grant is available for eligible candidates. Eligibility 
to training grant is appealing, but not essential.
 
 
 
This is a full-time position with a starting salary of $41,000 – 45,000 per 
year depending on skills and experience.
 
 
 
Review of applications will begin immediately and continue until the positions 
are filled.
 
 
 
To apply: Please send a cover letter, CV and the names and contact information 
for three references to Dr. Yongna Xing, 1400 University Avenue, McArdle 
Laboratory for Cancer Research, University of Wisconsin-Madison-Madison, WI 
53706. Email: yx...@oncology.wisc.edu.
 
 
 
Yongna Xing, Ph.D.

[ccp4bb] Post-doc fellowship in Sweden

[Erik Johansson]

Post-doctoral fellowship (two years) on Structural Studies of DNA Polymerase 
Epsilon

We are looking for a highly motivated postdoctoral research fellow to join our 
laboratory at Umeå University, Sweden. We are interested in structure-function 
studies of yeast DNA polymerase epsilon (Pol ε) using a combination of x-ray 
crystallography, biochemistry and genetics. The candidate will participate in a 
project with the aim to mechanistically understand how Pol ε synthesizes DNA 
with high fidelity and high processivity. These studies expands to experiments 
addressing how Pol ε is directed to the leading strand in yeast. We recently 
published a high-resolution structure of the catalytic core of Pol ε 
(www.nature.com/nsmb/journal/v21/n1/full/nsmb.2712.html
 ). Our present aim is to obtain new high-resolution structures of Pol ε to 
learn more about the details in how Pol ε function. The work will include 
plasmid construction, over-expression and purification of proteins that will be 
studied. The structural work will be complemented with biochemical studies of 
Pol ε and other proteins which participate at the eukaryotic replication fork 
or in DNA repair.

Qualifications:
Successful candidates should have received a Ph.D. degree, preferably a 
background in structural biology. Good written and oral communication skills 
are essential.

Preferred start is at the earliest possible date.

Applicants should submit
•   a Curriculum Vitae
•   a statement of previous research achievements (max 1-2 pages)
•   a publication list
•   the names and contact information for three references

Documents sent electronically should be in MS Word or PDF format.

We are looking forward to receiving your application, preferably before May 
29th.

For more information, contact Erik Johansson at 
erik.johans...@medchem.umu.se.
Dept of Medical Biochemistry and Biophysics
Web-page:  
www.medchem.umu.se/english/research/principal-investigators/johansson/

[ccp4bb] Aimless and multiple crystals

[Katherine Sippel]

Hi all,

I have two nice, but a hair under-complete data sets that I am trying to
merge together. If I run aimless on them separately everything looks
beautiful. When I merge them together the CC1/2 is still good but all the
various and sundry Rs sky rocket. When I go to the log file and look at the
Rs in respect to batch there is a jump between the first and second data
set, so clearly they are not scaling well in respect to one another. I
suspect that there is some obvious scripting command that I am missing, but
I couldn't find it in the Aimless documentation or the bb archives. Any
help would be appreciated.

Cheers,
Katherine

-- 
"Nil illegitimo carborundum"* - *Didactylos

Re: [ccp4bb] Aimless and multiple crystals

[Katherine Sippel]

I forgot to mention the space group is P1. Sorry about that.

Thanks,
Katherine


On Thu, May 8, 2014 at 12:02 PM, Katherine Sippel <
katherine.sip...@gmail.com> wrote:

> Hi all,
>
> I have two nice, but a hair under-complete data sets that I am trying to
> merge together. If I run aimless on them separately everything looks
> beautiful. When I merge them together the CC1/2 is still good but all the
> various and sundry Rs sky rocket. When I go to the log file and look at the
> Rs in respect to batch there is a jump between the first and second data
> set, so clearly they are not scaling well in respect to one another. I
> suspect that there is some obvious scripting command that I am missing, but
> I couldn't find it in the Aimless documentation or the bb archives. Any
> help would be appreciated.
>
> Cheers,
> Katherine
>
> --
> "Nil illegitimo carborundum"* - *Didactylos
>



-- 
"Nil illegitimo carborundum"* - *Didactylos

Re: [ccp4bb] Aimless and multiple crystals

[Craig Bingman]

How close are the cell constants?

On May 8, 2014, at 12:17 PM, Katherine Sippel  
wrote:

> I forgot to mention the space group is P1. Sorry about that.
> 
> Thanks,
> Katherine
> 
> 
> On Thu, May 8, 2014 at 12:02 PM, Katherine Sippel 
>  wrote:
> Hi all,
> 
> I have two nice, but a hair under-complete data sets that I am trying to 
> merge together. If I run aimless on them separately everything looks 
> beautiful. When I merge them together the CC1/2 is still good but all the 
> various and sundry Rs sky rocket. When I go to the log file and look at the 
> Rs in respect to batch there is a jump between the first and second data set, 
> so clearly they are not scaling well in respect to one another. I suspect 
> that there is some obvious scripting command that I am missing, but I 
> couldn't find it in the Aimless documentation or the bb archives. Any help 
> would be appreciated. 
> 
> Cheers,
> Katherine 
> 
> -- 
> "Nil illegitimo carborundum" - Didactylos
> 
> 
> 
> -- 
> "Nil illegitimo carborundum" - Didactylos

[ccp4bb] stalled refinement after MR solution

[Yarrow Madrona]

Hello CCP4 community,

I am stumped and would love some help. I have a molecular replacement
solution that has Rfree stuck around 40% while Rwork is aorund 30%. The
model is actually the same enzyme with a similar inhibitor bound. Relevant
information is below.

-Yarrow

I have solved a structure in a P21 spacegroup:

51.53 88.91 89.65, beta = 97.1.

Processing stats (XDS) are very good with low Rmerge (~5% overall) and good
completeness.

I don't think twinning is an option with these unit cell dimensions. My
data was highly aniosotropic. I ran the data through the UCLA anisotropic
server to scale in the B- direction (
http://services.mbi.ucla.edu/anisoscale/)

I get a small (a little over 5) patterson peak suggesting there is not much
t-NCS to worry about. However, the output structure does have 2 fold
symmetry (see below) and as Dale Tronrud pointed out, there is always tNCS
in a P21 space group with two monomers related by a 2-fold axis.
I calculated the translation to be unit cell fractions of 0.36 0.35, 0.32.

rota_matrix   -0.9860   -0.1636   -0.0309
rota_matrix   -0.16590.95110.2605
rota_matrix   -0.01320.2620   -0.9650
tran_orth  34.3310  -24.0033  107.0457

center_orth   15.76077.2426   77.7512

*Phaser stats:*

*  SOLU SET  RFZ=20.3 TFZ=19.5 PAK=0 LLG=1314 RF++ TFZ=63.8 PAK=0 LLG=4745
LLG=4947*

Re: [ccp4bb] Refining Metal Ion Occupancy

[Chris Fage]

Hi Everyone,

Thank you for the advice, especially Pavel's. My issue has been
resolved. I lowered the occupancy and B-factors of the metal ions in
the .pdb and ran phenix.refine for 10 cycles. This removed most of the
negative Fo-Fc density. Anisotropic refinement of the metal ion
B-factors was also effective, dropping Rwork and Rfree by ~1% each. To
do this, I edited the "Individual B-factor refinement" values as
follows.
Isotropic atoms: not (element Zn)
Anisotropic atoms: element Zn

Best,
Chris


On 5/6/14, Chris Fage  wrote:
> I have used CNS before, but not for this sort of refinement. I see in
> the bindividual.inp file that I can "select atoms to be included"--it
> is defaulted at "known and not hydrogen". Do you know the proper
> nomenclature for selecting a Zn ion in chains A and B?
>
> Thanks,
> Chris
>
> On 5/6/14, Steven Herron  wrote:
>>
>> Refining the occupancy will help your R-factor and flatten your density,
>> but you need to be careful to also refine the B-factor of the metal
>> ion.  Don't refine both the occupancy and the B-factor during the same
>> run (the two are correlated at this resolution), refine the occupancy of
>> just the metal ion and then refine the B-factor of just the metal ion
>> (repeat as needed).  I used X-plor/CNS to do my refinements, so it was
>> easy to refine the occupancy (or B-factor) of just the metal ion.  After
>> a few rounds of refinement both parameters will stop changing and you
>> will have your answer.  The final B-factor of the metal ion should be
>> similar to the amino acid residues that are coordinated to it.
>>
>> Soaking in several different ion concentrations and collecting
>> additional datasets is also a good idea (if you have the time).  I did
>> this type of experiment once before  (see: JBC 278(14):12271-7. [ Apr 4,
>> 2003]) (or: http://www.ncbi.nlm.nih.gov/pubmed/12540845).  I soaked in
>> several different Ca2+ ion concentrations and was able to determine the
>> binding affinity for that calcium ion using crystallography.
>>
>> To make sure I was not stuck in a local minima, I would modify either
>> the occupancy of the B-factor of the metal while keeping the other fixed
>> and do a refinement.  I even tried both large and small changes (both
>> increases and decreases in value).  It always came back to the earlier
>> answer.
>>
>> Different Ca2+ ion concentrations can give some additional insight into
>> the metal binding site.  Between the no-Ca2+ structure and the high-Ca2+
>> structure there was a conserved Asp-residue that changed conformation.
>> So, I soaked in the appropriate amount of Ca2+ to see the residue in
>> both positions.  There was a high correlation between the asp residue
>> orientation and the Ca2+ ion occupancy.
>>
>> Steven Herron
>> sherron_...@yahoo.com
>>
>>
>>
>>
>> On 5/6/2014 11:02 AM, Chris Fage wrote:
>>> Hi Everyone,
>>>
>>> In my 2.5-angstrom structure, there is negative Fo-Fc density
>>> surrounding a metal ion after refining in Phenix. From anomalous
>>> diffraction I am certain of the metal's identity and position in each
>>> monomer. Also, the ion is appropriately coordinated by nearby side
>>> chains. Should I be refining the occupancy of the ion in attempt to
>>> "flatten" the negative density? I am considering soaking the metal ion
>>> into crystals or cocrystallizing and collecting additional datasets.
>>>
>>> Thanks for your help!
>>>
>>> Regards,
>>> Chris
>>
>>
>

Re: [ccp4bb] Aimless and multiple crystals

[Katherine Sippel]

That is probably the culprit. The two unit cell dimensions are...

dataset #1: 68.8295   75.9597   83.3998   65.4000   88.8500   64.9000
dataset #2: 68.3005   75.7100   83.1299   65.3601   88.8900   65.2600

I can't believe I didn't look at that. I know better. Thanks for all of
your quick responses.

Katherine



On Thu, May 8, 2014 at 12:18 PM, Craig Bingman wrote:

> How close are the cell constants?
>
> On May 8, 2014, at 12:17 PM, Katherine Sippel 
> wrote:
>
> I forgot to mention the space group is P1. Sorry about that.
>
> Thanks,
> Katherine
>
>
> On Thu, May 8, 2014 at 12:02 PM, Katherine Sippel <
> katherine.sip...@gmail.com> wrote:
>
>> Hi all,
>>
>> I have two nice, but a hair under-complete data sets that I am trying to
>> merge together. If I run aimless on them separately everything looks
>> beautiful. When I merge them together the CC1/2 is still good but all the
>> various and sundry Rs sky rocket. When I go to the log file and look at the
>> Rs in respect to batch there is a jump between the first and second data
>> set, so clearly they are not scaling well in respect to one another. I
>> suspect that there is some obvious scripting command that I am missing, but
>> I couldn't find it in the Aimless documentation or the bb archives. Any
>> help would be appreciated.
>>
>> Cheers,
>> Katherine
>>
>> --
>> "Nil illegitimo carborundum"* - *Didactylos
>>
>
>
>
> --
> "Nil illegitimo carborundum"* - *Didactylos
>
>
>


-- 
"Nil illegitimo carborundum"* - *Didactylos

Re: [ccp4bb] Refining Metal Ion Occupancy

[Tim Gruene]

Hi Chris,

this certainly improved your model with respect to the data including
all their errors. How do you know this did not make your model worse
with respect to chemistry, respectively to what is inside your crystal?

Best,
Tim

On 05/08/2014 07:26 PM, Chris Fage wrote:
> Hi Everyone,
> 
> Thank you for the advice, especially Pavel's. My issue has been
> resolved. I lowered the occupancy and B-factors of the metal ions in
> the .pdb and ran phenix.refine for 10 cycles. This removed most of the
> negative Fo-Fc density. Anisotropic refinement of the metal ion
> B-factors was also effective, dropping Rwork and Rfree by ~1% each. To
> do this, I edited the "Individual B-factor refinement" values as
> follows.
> Isotropic atoms: not (element Zn)
> Anisotropic atoms: element Zn
> 
> Best,
> Chris
> 
> 
> On 5/6/14, Chris Fage  wrote:
>> I have used CNS before, but not for this sort of refinement. I see in
>> the bindividual.inp file that I can "select atoms to be included"--it
>> is defaulted at "known and not hydrogen". Do you know the proper
>> nomenclature for selecting a Zn ion in chains A and B?
>>
>> Thanks,
>> Chris
>>
>> On 5/6/14, Steven Herron  wrote:
>>>
>>> Refining the occupancy will help your R-factor and flatten your density,
>>> but you need to be careful to also refine the B-factor of the metal
>>> ion.  Don't refine both the occupancy and the B-factor during the same
>>> run (the two are correlated at this resolution), refine the occupancy of
>>> just the metal ion and then refine the B-factor of just the metal ion
>>> (repeat as needed).  I used X-plor/CNS to do my refinements, so it was
>>> easy to refine the occupancy (or B-factor) of just the metal ion.  After
>>> a few rounds of refinement both parameters will stop changing and you
>>> will have your answer.  The final B-factor of the metal ion should be
>>> similar to the amino acid residues that are coordinated to it.
>>>
>>> Soaking in several different ion concentrations and collecting
>>> additional datasets is also a good idea (if you have the time).  I did
>>> this type of experiment once before  (see: JBC 278(14):12271-7. [ Apr 4,
>>> 2003]) (or: http://www.ncbi.nlm.nih.gov/pubmed/12540845).  I soaked in
>>> several different Ca2+ ion concentrations and was able to determine the
>>> binding affinity for that calcium ion using crystallography.
>>>
>>> To make sure I was not stuck in a local minima, I would modify either
>>> the occupancy of the B-factor of the metal while keeping the other fixed
>>> and do a refinement.  I even tried both large and small changes (both
>>> increases and decreases in value).  It always came back to the earlier
>>> answer.
>>>
>>> Different Ca2+ ion concentrations can give some additional insight into
>>> the metal binding site.  Between the no-Ca2+ structure and the high-Ca2+
>>> structure there was a conserved Asp-residue that changed conformation.
>>> So, I soaked in the appropriate amount of Ca2+ to see the residue in
>>> both positions.  There was a high correlation between the asp residue
>>> orientation and the Ca2+ ion occupancy.
>>>
>>> Steven Herron
>>> sherron_...@yahoo.com
>>>
>>>
>>>
>>>
>>> On 5/6/2014 11:02 AM, Chris Fage wrote:
 Hi Everyone,

 In my 2.5-angstrom structure, there is negative Fo-Fc density
 surrounding a metal ion after refining in Phenix. From anomalous
 diffraction I am certain of the metal's identity and position in each
 monomer. Also, the ion is appropriately coordinated by nearby side
 chains. Should I be refining the occupancy of the ion in attempt to
 "flatten" the negative density? I am considering soaking the metal ion
 into crystals or cocrystallizing and collecting additional datasets.

 Thanks for your help!

 Regards,
 Chris
>>>
>>>
>>
> 

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

GPG Key ID = A46BEE1A



signature.asc
Description: OpenPGP digital signature

Re: [ccp4bb] Refining Metal Ion Occupancy

[Chris Fage]

Hi Tim,

That's a good question. The negative density vanished and the model
itself has not changed much. I still plan to set up
soaking/cocrystallization experiments with the metal ion. I am hoping
this will increase occupancy. That is the only solution that comes to
mind.

Regards,
Chris

On 5/8/14, Tim Gruene  wrote:
> Hi Chris,
>
> this certainly improved your model with respect to the data including
> all their errors. How do you know this did not make your model worse
> with respect to chemistry, respectively to what is inside your crystal?
>
> Best,
> Tim
>
> On 05/08/2014 07:26 PM, Chris Fage wrote:
>> Hi Everyone,
>>
>> Thank you for the advice, especially Pavel's. My issue has been
>> resolved. I lowered the occupancy and B-factors of the metal ions in
>> the .pdb and ran phenix.refine for 10 cycles. This removed most of the
>> negative Fo-Fc density. Anisotropic refinement of the metal ion
>> B-factors was also effective, dropping Rwork and Rfree by ~1% each. To
>> do this, I edited the "Individual B-factor refinement" values as
>> follows.
>> Isotropic atoms: not (element Zn)
>> Anisotropic atoms: element Zn
>>
>> Best,
>> Chris
>>
>>
>> On 5/6/14, Chris Fage  wrote:
>>> I have used CNS before, but not for this sort of refinement. I see in
>>> the bindividual.inp file that I can "select atoms to be included"--it
>>> is defaulted at "known and not hydrogen". Do you know the proper
>>> nomenclature for selecting a Zn ion in chains A and B?
>>>
>>> Thanks,
>>> Chris
>>>
>>> On 5/6/14, Steven Herron  wrote:

 Refining the occupancy will help your R-factor and flatten your
 density,
 but you need to be careful to also refine the B-factor of the metal
 ion.  Don't refine both the occupancy and the B-factor during the same
 run (the two are correlated at this resolution), refine the occupancy
 of
 just the metal ion and then refine the B-factor of just the metal ion
 (repeat as needed).  I used X-plor/CNS to do my refinements, so it was
 easy to refine the occupancy (or B-factor) of just the metal ion.
 After
 a few rounds of refinement both parameters will stop changing and you
 will have your answer.  The final B-factor of the metal ion should be
 similar to the amino acid residues that are coordinated to it.

 Soaking in several different ion concentrations and collecting
 additional datasets is also a good idea (if you have the time).  I did
 this type of experiment once before  (see: JBC 278(14):12271-7. [ Apr
 4,
 2003]) (or: http://www.ncbi.nlm.nih.gov/pubmed/12540845).  I soaked in
 several different Ca2+ ion concentrations and was able to determine the
 binding affinity for that calcium ion using crystallography.

 To make sure I was not stuck in a local minima, I would modify either
 the occupancy of the B-factor of the metal while keeping the other
 fixed
 and do a refinement.  I even tried both large and small changes (both
 increases and decreases in value).  It always came back to the earlier
 answer.

 Different Ca2+ ion concentrations can give some additional insight into
 the metal binding site.  Between the no-Ca2+ structure and the
 high-Ca2+
 structure there was a conserved Asp-residue that changed conformation.
 So, I soaked in the appropriate amount of Ca2+ to see the residue in
 both positions.  There was a high correlation between the asp residue
 orientation and the Ca2+ ion occupancy.

 Steven Herron
 sherron_...@yahoo.com




 On 5/6/2014 11:02 AM, Chris Fage wrote:
> Hi Everyone,
>
> In my 2.5-angstrom structure, there is negative Fo-Fc density
> surrounding a metal ion after refining in Phenix. From anomalous
> diffraction I am certain of the metal's identity and position in each
> monomer. Also, the ion is appropriately coordinated by nearby side
> chains. Should I be refining the occupancy of the ion in attempt to
> "flatten" the negative density? I am considering soaking the metal ion
> into crystals or cocrystallizing and collecting additional datasets.
>
> Thanks for your help!
>
> Regards,
> Chris


>>>
>>
>
> --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
>
> GPG Key ID = A46BEE1A
>
>

[ccp4bb] Comments needed CBF X-axis definition ambiguity

[Herbert J. Bernstein]

Dear Colleagues,

  Please pardon the shotgun distribution of this query.  It may only
directly concern a few beam-line scientists and software developers,
but comments from all interested parties are welcome.

  Many people have happily used the IUCr imgCIF dictionary definitions
in data collection and processing software for many years.  Just today,
however, we discovered that there is an ambiguity in the interpretation
of the CBF laboratory standard coordinate frame definition that comes
from two alternate readings of the definition of the X-axis.  Before
we put clarifying wording in the dictionary and resolve the ambiguity,
we would appreciate knowing which of the two interpretations is currently
in major use so that the resolution will be as non-disruptive as possible.

  The imgCIF dictionary says:

Axis 1 (*X*): The*X*-axis is aligned to the mechanical axis pointing from
 the sample or specimen along the  principal axis of the goniometer or
 sample positioning system if the sample positioning system has an axis
 that intersects the origin and which form an angle of more than 22.5
 degrees with the beam axis.

Without any intention of saying which of the following intepretations
is the original intention of this definition by the ordering, here is
what people have gotten from this:

Interpretation 1:  If you treat the sample as the origin, the +X axis runs
from the sample along the pin _into_ the sample holder; or

Interpretation 2:  If you treat the sample as the origin, the -X axis runs
from the sample along the pin _into_ the sample holder;

There are important implications for processing software on the handedness
of the resulting scan rotations, so we would appreciate whatever guidance
any of you can provide as to how you have been reading this spec.

Please send your comments to this list, or, if you prefer, to me personally
at yaya...@gmail.com

My apologies to the community for not having resolved this sooner, but
we only became aware today that some people had been reading the spec one way,
and others the other way.

With deepest apologies,
  Herbert J. Bernstein

--

Herbert J. Bernstein
Professor of Mathematics and Computer Science
Dowling College, Brookhaven Campus, A210/B205
1300 William Floyd Parkway, Shirley, NY, 11967

+1-631-244-1328
Lab: +1-631-244-1935
Cell: +1-631-428-1397
y...@dowling.edu


/Users/yaya/Documents/Eudora Folder/Signature Folder/Alternate

Re: [ccp4bb] stalled refinement after MR solution

[Randy Read]

Hi Yarrow,

If Dale said that, he probably wasn’t saying what he meant clearly enough!  The 
NCS 2-fold axis has to be parallel to the crystallographic 2-fold (screw) axis 
to generate tNCS.  In your case, the NCS is a 2-fold approximately parallel to 
the y-axis, but it’s nearly 9 degrees away from being parallel to y.  That 
explains why the Patterson peak is so small, and there will be very little 
disruption from the statistical effects of tNCS.

The anisotropy could be an issue.  It might be interesting to look at the 
R-factors for the stronger subset of the data.  It can make sense to apply an 
elliptical cutoff of the data using the anisotropy server (though Garib says 
that having systematically incomplete data can create problems for Refmac), but 
I hope you’re not using the anisotropically scaled data for refinement.  The 
determination of the anisotropic B-factors by Phaser without a model 
(underlying the anisotropy server) will not be as accurate as what Refmac or 
phenix.refine can do with a model.

Finally, as Phil Evans always says, the space group is just a hypothesis, so 
you should always be willing to go back and look at the evidence for the space 
group if something doesn’t work as expected.

Best wishes,

Randy Read

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

On 8 May 2014, at 18:11, Yarrow Madrona  wrote:

> Hello CCP4 community,
> 
> I am stumped and would love some help. I have a molecular replacement 
> solution that has Rfree stuck around 40% while Rwork is aorund 30%. The model 
> is actually the same enzyme with a similar inhibitor bound. Relevant 
> information is below.
> 
> -Yarrow
> 
> I have solved a structure in a P21 spacegroup:
> 
> 51.53 88.91 89.65, beta = 97.1.
> 
> Processing stats (XDS) are very good with low Rmerge (~5% overall) and good 
> completeness.
> 
> I don't think twinning is an option with these unit cell dimensions. My data 
> was highly aniosotropic. I ran the data through the UCLA anisotropic server 
> to scale in the B- direction (http://services.mbi.ucla.edu/anisoscale/)
> 
> I get a small (a little over 5) patterson peak suggesting there is not much 
> t-NCS to worry about. However, the output structure does have 2 fold symmetry 
> (see below) and as Dale Tronrud pointed out, there is always tNCS in a P21 
> space group with two monomers related by a 2-fold axis.
> I calculated the translation to be unit cell fractions of 0.36 0.35, 0.32.
> 
> rota_matrix   -0.9860   -0.1636   -0.0309
> rota_matrix   -0.16590.95110.2605
> rota_matrix   -0.01320.2620   -0.9650
> tran_orth  34.3310  -24.0033  107.0457
> 
> center_orth   15.76077.2426   77.7512
> 
> Phaser stats:
>   SOLU SET  RFZ=20.3 TFZ=19.5 PAK=0 LLG=1314 RF++ TFZ=63.8 PAK=0 LLG=4745 
> LLG=4947
> 
> 
> 
>   
> 

Re: [ccp4bb] stalled refinement after MR solution

[Keller, Jacob]

The b and c cell constants look remarkably similar

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Randy Read
Sent: Thursday, May 08, 2014 3:41 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] stalled refinement after MR solution

Hi Yarrow,

If Dale said that, he probably wasn't saying what he meant clearly enough!  The 
NCS 2-fold axis has to be parallel to the crystallographic 2-fold (screw) axis 
to generate tNCS.  In your case, the NCS is a 2-fold approximately parallel to 
the y-axis, but it's nearly 9 degrees away from being parallel to y.  That 
explains why the Patterson peak is so small, and there will be very little 
disruption from the statistical effects of tNCS.

The anisotropy could be an issue.  It might be interesting to look at the 
R-factors for the stronger subset of the data.  It can make sense to apply an 
elliptical cutoff of the data using the anisotropy server (though Garib says 
that having systematically incomplete data can create problems for Refmac), but 
I hope you're not using the anisotropically scaled data for refinement.  The 
determination of the anisotropic B-factors by Phaser without a model 
(underlying the anisotropy server) will not be as accurate as what Refmac or 
phenix.refine can do with a model.

Finally, as Phil Evans always says, the space group is just a hypothesis, so 
you should always be willing to go back and look at the evidence for the space 
group if something doesn't work as expected.

Best wishes,

Randy Read

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

On 8 May 2014, at 18:11, Yarrow Madrona  wrote:

> Hello CCP4 community,
> 
> I am stumped and would love some help. I have a molecular replacement 
> solution that has Rfree stuck around 40% while Rwork is aorund 30%. The model 
> is actually the same enzyme with a similar inhibitor bound. Relevant 
> information is below.
> 
> -Yarrow
> 
> I have solved a structure in a P21 spacegroup:
> 
> 51.53 88.91 89.65, beta = 97.1.
> 
> Processing stats (XDS) are very good with low Rmerge (~5% overall) and good 
> completeness.
> 
> I don't think twinning is an option with these unit cell dimensions. My data 
> was highly aniosotropic. I ran the data through the UCLA anisotropic server 
> to scale in the B- direction (http://services.mbi.ucla.edu/anisoscale/)
> 
> I get a small (a little over 5) patterson peak suggesting there is not much 
> t-NCS to worry about. However, the output structure does have 2 fold symmetry 
> (see below) and as Dale Tronrud pointed out, there is always tNCS in a P21 
> space group with two monomers related by a 2-fold axis.
> I calculated the translation to be unit cell fractions of 0.36 0.35, 0.32.
> 
> rota_matrix   -0.9860   -0.1636   -0.0309
> rota_matrix   -0.16590.95110.2605
> rota_matrix   -0.01320.2620   -0.9650
> tran_orth  34.3310  -24.0033  107.0457
> 
> center_orth   15.76077.2426   77.7512
> 
> Phaser stats:
>   SOLU SET  RFZ=20.3 TFZ=19.5 PAK=0 LLG=1314 RF++ TFZ=63.8 PAK=0 LLG=4745 
> LLG=4947
> 
> 
> 
>   
> 

[ccp4bb] 2014 Machines on Genes Conference

[Eichman, Brandt F]

FASEB Summer Research Conference
Machines on Genes: Nucleic Acids Enzymes
June 22-27, 2014
Base Village Conference Center
Snowmass Village, Colorado

Abstract deadline: May 22, 2014

Co-Organizers
Thomas Carell   Ludwig-Maximilians University
Brandt Eichman   Vanderbilt University
Jeffrey Kieft   University of Colorado

Session topics
Translation and the Ribosome
DNA Damage and Repair
Transcription and RNA Modification
DNA Replication
Catalytic Nucleic Acids
Translocation and Remodeling
Chromatin and Epigenetics
Multifunctional Machines

Program: https://secure.faseb.org/FASEB/meetings/summrconf/Programs/11574.pdf
Registration: 
https://secure.faseb.org/FASEB/meetings/summrconf/selectTopic.aspx?Sortfrom=2013
**Travel Grants (13 available): 
http://www.faseb.org/Science-Research-Conferences/Awards-and-Travel-Grants.aspx

This meeting will focus on the architectures and mechanics of proteins and 
protein-nucleic acid complexes that maintain and decode the information stored 
within DNA and RNA. The 2014 meeting marks a consolidation of the long-running 
FASEB Nucleic Acids Enzymes Conference with the Biochemical Society Machines on 
Genes Conference, which has taken place in the UK twice since 2010. Sessions 
will cover the mechanisms of important nucleic acid transactions and will 
highlight the use of emerging biophysical methods.

In addition to an exciting list of invited speakers, a number of oral 
presentations will be selected from the abstracts. Funds have been secured to 
provide 13 travel scholarships for students and postdocs. The meeting will also 
feature several organized panel discussions to help junior colleagues navigate 
various topics related to careers in science. Ample time has been designated 
for informal discussion over poster presentations and recreational activities 
in order to provide participants opportunities to exchange ideas and formulate 
new collaborations.

We hope to see you in Snowmass this summer ~

Brandt, Jeff, and Thomas



Brandt Eichman
Associate Professor of Biological Sciences and Biochemistry
Vanderbilt University
Nashville, TN 37232
615.936.5233
http://structbio.vanderbilt.edu/eichman

Re: [ccp4bb] stalled refinement after MR solution

[Yarrow Madrona]

Sorry Dale. I left that out. I thought that it was almost parallel or very
close. I guess that is not enough. This is after all an exact science. My
fault.


On Thu, May 8, 2014 at 10:54 AM, Dale Tronrud  wrote:

> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
>
>
>   By the way...  I did not say what you claim I said.  I said there
> is always translational ncs in P21 when there is a ncs 2-fold PARALLEL
> to the crystallographic screw.  Your 2-fold appears to be off-set
> from the screw axis enough that Phaser is not detecting tNCS, which
> means it is not strong enough for it to be a problem for Phaser.
> Whatever your problem is, it is not translational NCS.
>
>Is there anyone in your area that could help you?  In situations
> like this you need to go back to square one and reevaluate every
> decision you made starting from the images.  This is nearly impossible
> to do remotely.  We do not know your protein.  We do not know how
> your crystal relates to the crystal which you are using as your MR
> probe.  All of the thousand things that can go wrong are unknown to
> us.  Unless you are willing to dump your entire project onto the
> BB there is little we can do for you.
>
> Dale
>
> On 05/08/2014 10:11 AM, Yarrow Madrona wrote:
> > Hello CCP4 community,
> >
> > I am stumped and would love some help. I have a molecular
> > replacement solution that has Rfree stuck around 40% while Rwork is
> > aorund 30%. The model is actually the same enzyme with a similar
> > inhibitor bound. Relevant information is below.
> >
> > -Yarrow
> >
> > I have solved a structure in a P21 spacegroup:
> >
> > 51.53 88.91 89.65, beta = 97.1.
> >
> > Processing stats (XDS) are very good with low Rmerge (~5% overall)
> > and good completeness.
> >
> > I don't think twinning is an option with these unit cell
> > dimensions. My data was highly aniosotropic. I ran the data through
> > the UCLA anisotropic server to scale in the B- direction
> > (http://services.mbi.ucla.edu/anisoscale/)
> >
> > I get a small (a little over 5) patterson peak suggesting there is
> > not much t-NCS to worry about. However, the output structure does
> > have 2 fold symmetry (see below) and as Dale Tronrud pointed out,
> > there is always tNCS in a P21 space group with two monomers related
> > by a 2-fold axis. I calculated the translation to be unit cell
> > fractions of 0.36 0.35, 0.32.
> >
> > rota_matrix   -0.9860   -0.1636   -0.0309 rota_matrix   -0.1659
> > 0.95110.2605 rota_matrix   -0.01320.2620   -0.9650
> > tran_orth  34.3310  -24.0033  107.0457
> >
> > center_orth   15.76077.2426   77.7512 * * *Phaser stats:*
> >
> > *  SOLU SET  RFZ=20.3 TFZ=19.5 PAK=0 LLG=1314 RF++ TFZ=63.8 PAK=0
> > LLG=4745 LLG=4947*
> >
> >
> > *  *
> >
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v2.0.14 (GNU/Linux)
>
> iEYEARECAAYFAlNrxPAACgkQU5C0gGfAG11ivgCglp//CaOwgKyP3j73GRwH0tX8
> n28AnijNVEjm/wXz19Hn8EEdyatTpedw
> =aOOF
> -END PGP SIGNATURE-
>

Re: [ccp4bb] stalled refinement after MR solution

[Yarrow Madrona]

Hi Randy,

Again, sorry about the mis-quote. From a quick look I thought the NCS
2-fold was parallel or close enough. I didn't know that 10 degrees would
change the patterson that much. I have tried refinement with both sets of
data and get the same results. I will investigate the space group
assignment again.


On Thu, May 8, 2014 at 12:40 PM, Randy Read  wrote:

> Hi Yarrow,
>
> If Dale said that, he probably wasn’t saying what he meant clearly enough!
>  The NCS 2-fold axis has to be parallel to the crystallographic 2-fold
> (screw) axis to generate tNCS.  In your case, the NCS is a 2-fold
> approximately parallel to the y-axis, but it’s nearly 9 degrees away from
> being parallel to y.  That explains why the Patterson peak is so small, and
> there will be very little disruption from the statistical effects of tNCS.
>
> The anisotropy could be an issue.  It might be interesting to look at the
> R-factors for the stronger subset of the data.  It can make sense to apply
> an elliptical cutoff of the data using the anisotropy server (though Garib
> says that having systematically incomplete data can create problems for
> Refmac), but I hope you’re not using the anisotropically scaled data for
> refinement.  The determination of the anisotropic B-factors by Phaser
> without a model (underlying the anisotropy server) will not be as accurate
> as what Refmac or phenix.refine can do with a model.
>
> Finally, as Phil Evans always says, the space group is just a hypothesis,
> so you should always be willing to go back and look at the evidence for the
> space group if something doesn’t work as expected.
>
> Best wishes,
>
> Randy Read
>
> -
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical ResearchTel: +44 1223 336500
> Wellcome Trust/MRC Building Fax: +44 1223 336827
> Hills Road
>  E-mail: rj...@cam.ac.uk
> Cambridge CB2 0XY, U.K.
> www-structmed.cimr.cam.ac.uk
>
> On 8 May 2014, at 18:11, Yarrow Madrona  wrote:
>
> > Hello CCP4 community,
> >
> > I am stumped and would love some help. I have a molecular replacement
> solution that has Rfree stuck around 40% while Rwork is aorund 30%. The
> model is actually the same enzyme with a similar inhibitor bound. Relevant
> information is below.
> >
> > -Yarrow
> >
> > I have solved a structure in a P21 spacegroup:
> >
> > 51.53 88.91 89.65, beta = 97.1.
> >
> > Processing stats (XDS) are very good with low Rmerge (~5% overall) and
> good completeness.
> >
> > I don't think twinning is an option with these unit cell dimensions. My
> data was highly aniosotropic. I ran the data through the UCLA anisotropic
> server to scale in the B- direction (
> http://services.mbi.ucla.edu/anisoscale/)
> >
> > I get a small (a little over 5) patterson peak suggesting there is not
> much t-NCS to worry about. However, the output structure does have 2 fold
> symmetry (see below) and as Dale Tronrud pointed out, there is always tNCS
> in a P21 space group with two monomers related by a 2-fold axis.
> > I calculated the translation to be unit cell fractions of 0.36 0.35,
> 0.32.
> >
> > rota_matrix   -0.9860   -0.1636   -0.0309
> > rota_matrix   -0.16590.95110.2605
> > rota_matrix   -0.01320.2620   -0.9650
> > tran_orth  34.3310  -24.0033  107.0457
> >
> > center_orth   15.76077.2426   77.7512
> >
> > Phaser stats:
> >   SOLU SET  RFZ=20.3 TFZ=19.5 PAK=0 LLG=1314 RF++ TFZ=63.8 PAK=0
> LLG=4745 LLG=4947
> >
> >
> >
> >
> >
>

Re: [ccp4bb] stalled refinement after MR solution

[Yarrow Madrona]

Hi Jacob. I am worried that I would dramatically suffer in data
completeness. I am not sure how reliable the data is when you are have 50%
completeness. These crystals are also pretty much impossible to reproduce
at the moment.


On Thu, May 8, 2014 at 1:30 PM, Keller, Jacob wrote:

>  Since your search model is so good, why not go down to p1 to see what’s
> going on, then re-merge if necessary?
>
>
>
> JPK
>
>
>
> *From:* yarrowmadr...@gmail.com [mailto:yarrowmadr...@gmail.com] *On
> Behalf Of *Yarrow Madrona
> *Sent:* Thursday, May 08, 2014 4:29 PM
> *To:* Keller, Jacob
>
> *Subject:* Re: [ccp4bb] stalled refinement after MR solution
>
>
>
> I have had problems in the past with a and c cell being equal and having
> pseudo-merhohedral twining where the space group looked like C2221 but the
> true space group was P21 (near perfect 2fold NCS). But I didn't think
> twining was possible in this case.
>
>
>
> On Thu, May 8, 2014 at 12:43 PM, Keller, Jacob 
> wrote:
>
> The b and c cell constants look remarkably similar
>
> JPK
>
>
> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> Randy Read
> Sent: Thursday, May 08, 2014 3:41 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] stalled refinement after MR solution
>
> Hi Yarrow,
>
> If Dale said that, he probably wasn't saying what he meant clearly enough!
>  The NCS 2-fold axis has to be parallel to the crystallographic 2-fold
> (screw) axis to generate tNCS.  In your case, the NCS is a 2-fold
> approximately parallel to the y-axis, but it's nearly 9 degrees away from
> being parallel to y.  That explains why the Patterson peak is so small, and
> there will be very little disruption from the statistical effects of tNCS.
>
> The anisotropy could be an issue.  It might be interesting to look at the
> R-factors for the stronger subset of the data.  It can make sense to apply
> an elliptical cutoff of the data using the anisotropy server (though Garib
> says that having systematically incomplete data can create problems for
> Refmac), but I hope you're not using the anisotropically scaled data for
> refinement.  The determination of the anisotropic B-factors by Phaser
> without a model (underlying the anisotropy server) will not be as accurate
> as what Refmac or phenix.refine can do with a model.
>
> Finally, as Phil Evans always says, the space group is just a hypothesis,
> so you should always be willing to go back and look at the evidence for the
> space group if something doesn't work as expected.
>
> Best wishes,
>
> Randy Read
>
> -
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical ResearchTel: +44 1223 336500
> Wellcome Trust/MRC Building Fax: +44 1223 336827
> Hills Road
>  E-mail: rj...@cam.ac.uk
> Cambridge CB2 0XY, U.K.
> www-structmed.cimr.cam.ac.uk
>
> On 8 May 2014, at 18:11, Yarrow Madrona  wrote:
>
> > Hello CCP4 community,
> >
> > I am stumped and would love some help. I have a molecular replacement
> solution that has Rfree stuck around 40% while Rwork is aorund 30%. The
> model is actually the same enzyme with a similar inhibitor bound. Relevant
> information is below.
> >
> > -Yarrow
> >
> > I have solved a structure in a P21 spacegroup:
> >
> > 51.53 88.91 89.65, beta = 97.1.
> >
> > Processing stats (XDS) are very good with low Rmerge (~5% overall) and
> good completeness.
> >
> > I don't think twinning is an option with these unit cell dimensions. My
> data was highly aniosotropic. I ran the data through the UCLA anisotropic
> server to scale in the B- direction (
> http://services.mbi.ucla.edu/anisoscale/)
> >
> > I get a small (a little over 5) patterson peak suggesting there is not
> much t-NCS to worry about. However, the output structure does have 2 fold
> symmetry (see below) and as Dale Tronrud pointed out, there is always tNCS
> in a P21 space group with two monomers related by a 2-fold axis.
> > I calculated the translation to be unit cell fractions of 0.36 0.35,
> 0.32.
> >
> > rota_matrix   -0.9860   -0.1636   -0.0309
> > rota_matrix   -0.16590.95110.2605
> > rota_matrix   -0.01320.2620   -0.9650
> > tran_orth  34.3310  -24.0033  107.0457
> >
> > center_orth   15.76077.2426   77.7512
> >
> > Phaser stats:
> >   SOLU SET  RFZ=20.3 TFZ=19.5 PAK=0 LLG=1314 RF++ TFZ=63.8 PAK=0
> LLG=4745 LLG=4947
> >
> >
> >
> >
> >
>
>
>

Re: [ccp4bb] stalled refinement after MR solution

[Yarrow Madrona]

Hi Brent,

I forgot to mention the resolution and other statistics. Here they are (XDS
-unmerged data P2 below). Overall completeness is 93.4(85.2)% for 2.2A.
I do have some anisotropy in the b-direction. I have tried running phaser
with data scaled by the UCLA anisotropy server and obtained the same
results.

-Yarrow

SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF RESOLUTION
 RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR  R-FACTOR
COMPARED I/SIGMA   R-meas  CC(1/2)  Anomal  SigAno   Nano LIMIT
OBSERVED  UNIQUE  POSSIBLE OF DATA   observed  expected Corr

 6.4047881487  1718   86.6%   2.7%  2.9%
  4675   33.78  3.2%99.8*20*   0.895 873
 4.6086442667  2852   93.5%   3.0%  3.1%
  8511   30.77  3.6%99.8* 90.8561461
 3.78   106593301  3598   91.7%   3.0%  3.1%
 10559   31.05  3.6%99.8* 00.7891747
 3.28   123983874  4261   90.9%   3.7%  3.7%
 12319   25.46  4.4%99.7*-40.7791981
 2.94   149704587  4766   96.2%   4.8%  4.9%
 14886   20.15  5.8%99.7*-20.7792476
 2.69   167185129  5292   96.9%   6.7%  6.9%
 16638   15.44  8.1%99.4*-20.7672681
 2.49   178425536  5722   96.7%   9.3%  9.4%
 17734   11.89 11.1%98.9*-20.7552779
 2.33   197436017  6133   98.1%  12.0% 12.6%
 196239.27 14.3%98.4*-40.7373132
 2.20   167775566  6533   85.2%  17.4% 16.9%
 164206.77 21.2%96.8*-80.7492334
total  122539   38164 40875   93.4%   5.3%  5.4%
121365   17.41  6.4%99.7*-20.776   19464


 NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES  134095
 NUMBER OF REJECTED MISFITS   11536
 NUMBER OF SYSTEMATIC ABSENT REFLECTIONS  0
 NUMBER OF ACCEPTED OBSERVATIONS 122559
 NUMBER OF UNIQUE ACCEPTED REFLECTIONS38170



On Thu, May 8, 2014 at 10:48 AM, Segelke, Brent W. wrote:

>  You didn’t report your resolution and completeness. Also, is there
> anisotropy in your data?
>
>
>
> Brent
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Yarrow
> Madrona
> *Sent:* Thursday, May 08, 2014 10:12 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] stalled refinement after MR solution
>
>
>
> Hello CCP4 community,
>
>
>
> I am stumped and would love some help. I have a molecular replacement
> solution that has Rfree stuck around 40% while Rwork is aorund 30%. The
> model is actually the same enzyme with a similar inhibitor bound. Relevant
> information is below.
>
>
>
> -Yarrow
>
>
>
> I have solved a structure in a P21 spacegroup:
>
>
>
> 51.53 88.91 89.65, beta = 97.1.
>
>
>
> Processing stats (XDS) are very good with low Rmerge (~5% overall) and
> good completeness.
>
>
>
> I don't think twinning is an option with these unit cell dimensions. My
> data was highly aniosotropic. I ran the data through the UCLA anisotropic
> server to scale in the B- direction (
> http://services.mbi.ucla.edu/anisoscale/)
>
>
>
> I get a small (a little over 5) patterson peak suggesting there is not
> much t-NCS to worry about. However, the output structure does have 2 fold
> symmetry (see below) and as Dale Tronrud pointed out, there is always tNCS
> in a P21 space group with two monomers related by a 2-fold axis.
>
> I calculated the translation to be unit cell fractions of 0.36 0.35, 0.32.
>
>
>
> rota_matrix   -0.9860   -0.1636   -0.0309
>
> rota_matrix   -0.16590.95110.2605
>
> rota_matrix   -0.01320.2620   -0.9650
>
> tran_orth  34.3310  -24.0033  107.0457
>
>
>
> center_orth   15.76077.2426   77.7512
>
>
>
> *Phaser stats:*
>
> *  SOLU SET  RFZ=20.3 TFZ=19.5 PAK=0 LLG=1314 RF++ TFZ=63.8 PAK=0 LLG=4745
> LLG=4947*
>
>
>
>
>

Re: [ccp4bb] stalled refinement after MR solution

[Dale Tronrud]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1


   Refinement of a model with only 50% completeness is problematic, but
you have four copies of a molecule (in P1) so your molecular
replacement is only looking for 24 parameters.  You should be able to
get a solution with 50% completeness.

Dale Tronrud

On 5/8/2014 1:43 PM, Yarrow Madrona wrote:
> Hi Jacob. I am worried that I would dramatically suffer in data 
> completeness. I am not sure how reliable the data is when you are
> have 50% completeness. These crystals are also pretty much
> impossible to reproduce at the moment.
> 
> 
> On Thu, May 8, 2014 at 1:30 PM, Keller, Jacob
> mailto:kell...@janelia.hhmi.org>>
> wrote:
> 
> Since your search model is so good, why not go down to p1 to see 
> what’s going on, then re-merge if necessary?
> 
> __ __
> 
> JPK
> 
> __ __
> 
> *From:*yarrowmadr...@gmail.com  
> [mailto:yarrowmadr...@gmail.com ] 
> *On Behalf Of *Yarrow Madrona *Sent:* Thursday, May 08, 2014 4:29
> PM *To:* Keller, Jacob
> 
> 
> *Subject:* Re: [ccp4bb] stalled refinement after MR solution
> 
> __ __
> 
> I have had problems in the past with a and c cell being equal and 
> having pseudo-merhohedral twining where the space group looked
> like C2221 but the true space group was P21 (near perfect 2fold
> NCS). But I didn't think twining was possible in this case.
> 
> __ __
> 
> On Thu, May 8, 2014 at 12:43 PM, Keller, Jacob 
> mailto:kell...@janelia.hhmi.org>>
> wrote:
> 
> The b and c cell constants look remarkably similar
> 
> JPK
> 
> 
> -Original Message- From: CCP4 bulletin board
> [mailto:CCP4BB@JISCMAIL.AC.UK ] On
> Behalf Of Randy Read Sent: Thursday, May 08, 2014 3:41 PM To:
> CCP4BB@JISCMAIL.AC.UK  Subject: Re:
> [ccp4bb] stalled refinement after MR solution
> 
> Hi Yarrow,
> 
> If Dale said that, he probably wasn't saying what he meant clearly 
> enough!  The NCS 2-fold axis has to be parallel to the 
> crystallographic 2-fold (screw) axis to generate tNCS.  In your 
> case, the NCS is a 2-fold approximately parallel to the y-axis,
> but it's nearly 9 degrees away from being parallel to y.  That
> explains why the Patterson peak is so small, and there will be very
> little disruption from the statistical effects of tNCS.
> 
> The anisotropy could be an issue.  It might be interesting to look 
> at the R-factors for the stronger subset of the data.  It can make 
> sense to apply an elliptical cutoff of the data using the
> anisotropy server (though Garib says that having systematically
> incomplete data can create problems for Refmac), but I hope you're
> not using the anisotropically scaled data for refinement.  The
> determination of the anisotropic B-factors by Phaser without a
> model (underlying the anisotropy server) will not be as accurate as
> what Refmac or phenix.refine can do with a model.
> 
> Finally, as Phil Evans always says, the space group is just a 
> hypothesis, so you should always be willing to go back and look at 
> the evidence for the space group if something doesn't work as
> expected.
> 
> Best wishes,
> 
> Randy Read
> 
> - Randy J. Read Department of Haematology, University of
> Cambridge Cambridge Institute for Medical ResearchTel: +44 1223
> 336500  Wellcome Trust/MRC Building
> Fax: +44 1223 336827  Hills Road
>  E-mail: rj...@cam.ac.uk  Cambridge CB2
> 0XY, U.K. www-structmed.cimr.cam.ac.uk
> 
> 
> On 8 May 2014, at 18:11, Yarrow Madrona  > wrote:
> 
>> Hello CCP4 community,
>> 
>> I am stumped and would love some help. I have a molecular
> replacement solution that has Rfree stuck around 40% while Rwork
> is aorund 30%. The model is actually the same enzyme with a
> similar inhibitor bound. Relevant information is below.
>> 
>> -Yarrow
>> 
>> I have solved a structure in a P21 spacegroup:
>> 
>> 51.53 88.91 89.65, beta = 97.1.
>> 
>> Processing stats (XDS) are very good with low Rmerge (~5%
>> overall)
> and good completeness.
>> 
>> I don't think twinning is an option with these unit cell
> dimensions. My data was highly aniosotropic. I ran the data
> through the UCLA anisotropic server to scale in the B- direction 
> (http://services.mbi.ucla.edu/anisoscale/)
>> 
>> I get a small (a little over 5) patterson peak suggesting there
>> is
> not much t-NCS to worry about. However, the output structure does 
> have 2 fold symmetry (see below) and as Dale Tronrud pointed out, 
> there is always tNCS in a P21 space group with two monomers
> related by a 2-fold axis.
>> I calculated the translation to be unit cell fractions of 0.36
> 0.35, 0.32.
>> 
>> rota_matrix   -0.9860   -0.1636   -0.0309 rota_matrix   -0.1659
>> 0.95110.2605 rota_matrix   -0.01320.2620   -0.9650 
>> tran_orth  34.3310  -24.0033  107.0457
>> 
>> center_orth   15.76077.2426 

[ccp4bb] Postdoctoral Position available at the University of Georgia

[Scott Pegan]

Seeking Post-Doctoral candidates for a position in my laboratory located in
the University of Georgia's Department of Pharmaceutical & Biomedical
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http://pbs.rx.uga.edu/index.php/people/faculty/scott_pegan/

Recent graduates encouraged. X-ray, or NMR, experience a plus. Ideal start
date would be July 1, 2014. Interested parties please send a single PDF
file with your CV, with publication list included, and a minimum of
information for two references.  Please submit to spe...@uga.edu.

Scott

Re: [ccp4bb] stalled refinement after MR solution

[Yarrow Madrona]

Thank you for the tips Brent.

I will try what you suggest. There are actually multiple lattices. And I
actually only processed the dominant one in XDS.
On May 8, 2014 2:26 PM, "Segelke, Brent W."  wrote:

>  Completeness looks pretty good and you have a good enough resolution
> that you should be able to get this eventually.
>
>
>
> A couple of things to look at to start. First, suspect your data
> processing. As others have already said, double check your space group. You
> could even do an initial sanity check by expanding your MR solution into P1
> and refining with strong NCS in P1 reduced data. You will automatically get
> a lower R because you are introducing more parameters, but if you use
> strong NCS and both R and Rfree are better behaved, then something is going
> on with the merging (e.g., wrong space group).
>
>
>
> Look in your log and see if there are an unusual number of reflection or
> frames thrown out, it should be only a few percentage. If you have more
> than 10-15% rejected, something probably went awry. Tweak your error model
> and/or summing box, etc.
>
>
>
> You could also try refining to a less ambitious resolution to start,
> assuming anisotropy will affect the higher resolution to a greater degree.
>
>
>
> Hope this helps.
>
>
>
> Brent
>
>
>
> *From:* yarrowmadr...@gmail.com [mailto:yarrowmadr...@gmail.com] *On
> Behalf Of *Yarrow Madrona
> *Sent:* Thursday, May 08, 2014 1:50 PM
> *To:* Segelke, Brent W.; CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] stalled refinement after MR solution
>
>
>
> Hi Brent,
>
>
>
> I forgot to mention the resolution and other statistics. Here they are
> (XDS -unmerged data P2 below). Overall completeness is 93.4(85.2)% for 2.2A.
>
> I do have some anisotropy in the b-direction. I have tried running phaser
> with data scaled by the UCLA anisotropy server and obtained the same
> results.
>
>
>
> -Yarrow
>
>
>
> SUBSET OF INTENSITY DATA WITH SIGNAL/NOISE >= -3.0 AS FUNCTION OF
> RESOLUTION
>
>  RESOLUTION NUMBER OF REFLECTIONSCOMPLETENESS R-FACTOR  R-FACTOR
> COMPARED I/SIGMA   R-meas  CC(1/2)  Anomal  SigAno   Nano LIMIT
> OBSERVED  UNIQUE  POSSIBLE OF DATA   observed  expected Corr
>
>
>
>  6.4047881487  1718   86.6%   2.7%  2.9%
>   4675   33.78  3.2%99.8*20*   0.895 873
>
>  4.6086442667  2852   93.5%   3.0%  3.1%
>   8511   30.77  3.6%99.8* 90.8561461
>
>  3.78   106593301  3598   91.7%   3.0%  3.1%
>  10559   31.05  3.6%99.8* 00.7891747
>
>  3.28   123983874  4261   90.9%   3.7%  3.7%
>  12319   25.46  4.4%99.7*-40.7791981
>
>  2.94   149704587  4766   96.2%   4.8%  4.9%
>  14886   20.15  5.8%99.7*-20.7792476
>
>  2.69   167185129  5292   96.9%   6.7%  6.9%
>  16638   15.44  8.1%99.4*-20.7672681
>
>  2.49   178425536  5722   96.7%   9.3%  9.4%
>  17734   11.89 11.1%98.9*-20.7552779
>
>  2.33   197436017  6133   98.1%  12.0% 12.6%
>  196239.27 14.3%98.4*-40.7373132
>
>  2.20   167775566  6533   85.2%  17.4% 16.9%
>  164206.77 21.2%96.8*-80.7492334
>
> total  122539   38164 40875   93.4%   5.3%  5.4%
>   121365   17.41  6.4%99.7*-20.776   19464
>
>
>
>
>
>  NUMBER OF REFLECTIONS IN SELECTED SUBSET OF IMAGES  134095
>
>  NUMBER OF REJECTED MISFITS   11536
>
>  NUMBER OF SYSTEMATIC ABSENT REFLECTIONS  0
>
>  NUMBER OF ACCEPTED OBSERVATIONS 122559
>
>  NUMBER OF UNIQUE ACCEPTED REFLECTIONS38170
>
>
>
>
>
> On Thu, May 8, 2014 at 10:48 AM, Segelke, Brent W. 
> wrote:
>
> You didn’t report your resolution and completeness. Also, is there
> anisotropy in your data?
>
>
>
> Brent
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Yarrow
> Madrona
> *Sent:* Thursday, May 08, 2014 10:12 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] stalled refinement after MR solution
>
>
>
> Hello CCP4 community,
>
>
>
> I am stumped and would love some help. I have a molecular replacement
> solution that has Rfree stuck around 40% while Rwork is aorund 30%. The
> model is actually the same enzyme with a similar inhibitor bound. Relevant
> information is below.
>
>
>
> -Yarrow
>
>
>
> I have solved a structure in a P21 spacegroup:
>
>
>
> 51.53 88.91 89.65, beta = 97.1.
>
>
>
> Processing stats (XDS) are very good with low Rmerge (~5% overall) and
> good completeness.
>
>
>
> I don't think twinning is an option with these unit cell dimensions. My
> data was highly aniosotropic. I ran the data through the UCLA anisotropic
> server to scale in the B- direction (
> ht