[ccp4bb] AW: [ccp4bb] twinning fun

[Herman . Schreuder]

Dear Bert,

The first thing I would do is to calculate the Matthews number: Does at least 
one monomer fit in the P622 asymmetric unit? If not, your crystals are 
definitively twinned.
As mentioned below, I would also check  the /^2 ratio, but I would do 
it with the data processed in P6, since processing true P6 data in P622 will 
produce a twinned ratio even when the P6 data was not twinned. If it turns out, 
that some crystals are twinned and others not, I would  look at the diffraction 
patterns to see if something funny is going on (ice rings, high background, 
strange spot shape etc.). In this case, I would try to solve the structure with 
untwinned crystals. Maybe less fun, but also less hassle, frustration and 
cleaner maps.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Dirk 
Kostrewa
Gesendet: Dienstag, 28. Januar 2014 22:01
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] twinning fun

Dear Bert Van-Den-Berg,

as far as I understand this, if you have true P622, process the data in P6 and 
then test for twinning, both the Britton-test and H-test will indicate perfect 
merohedral twinning.
This is because the Britton-test checks for a sudden increase of negative 
intensities after de-twinning, which happens only at twin fractions close to 
0.5 if the intensities used for de-twinning are the same. But this is true if 
they are related by crystallographic symmetry.
The H-test relates the absolute difference to the sum of the presumably twinned 
intensities, which gives "0" for intensities related by crystallographic 
symmetry, again resulting in twin fractions close to 0.5.
In other words, intensities related by crystallographic symmetry would indicate 
"perfect" twinning in both of these tests.

A better test for perfect merohedral twinning would be the ratio of /^2 
which should be 2 for untwinned and 1.5 for perfectly twinned data, tested in 
the higher space group. These values are reported by data processing programs 
like XDS. Please, be aware that these ratios have rather strange values if you 
have an unusually high background (loop fiber diffraction, ice rings, etc.) or 
extremely weak data.

For a really good discussion of twin tests, see Yeates, Methods. Enzymol. 276, 
344-358, 1997.

Best regards,

Dirk.
Am 28.01.14 18:26, schrieb Bert Van-Den-Berg:
Dear all,

I recently collected several datasets for a protein that needs experimental 
phasing.
The crystals are hexagonal plates, and (automatic) data processing suggests 
with high confidence that the space group is P622. This is where the fun begins.
For some datasets (processed in P622), the intensity distributions are normal, 
and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest that there is 
no twinning (twinning fractions < 0.05). However, for other datasets (same cell 
dimensions), the intensity distributions are not normal (eg Z-scores > 10). 
Given that twinning is not possible in P622, this suggests to me that the real 
space group could be P6 with (near) perfect twinning.

If I now process the "normal L-test P622" datasets in P6, the twin-law based 
tests (britton and H-test in xtriage) give high twin fractions (0.45- 0.5), 
suggesting all my data is twinned.
Does this make sense (ie can one have twinning with "normal" intensity 
distributions)?
If it does, would the "normal L-test" datasets have a higher probability of 
being solvable?

Is there any strategy for experimental phasing of (near) perfect twins? SAD 
would be more suitable than SIR/MIR? (I also have potential heavy atom 
derivatives).

Thanks for any insights!

Bert



--



***

Dirk Kostrewa

Gene Center Munich, A5.07

Department of Biochemistry

Ludwig-Maximilians-Universität München

Feodor-Lynen-Str. 25

D-81377 Munich

Germany

Phone:   +49-89-2180-76845

Fax: +49-89-2180-76999

E-mail:  kostr...@genzentrum.lmu.de

WWW: www.genzentrum.lmu.de

***

Re: [ccp4bb] twinning fun

[Kay Diederichs]

Dear Bert,

as Dirk has pointed out, if P622 is the correct space group, then the twinning 
statistics printed out if you process in P6 are meaningless.

Intensity statistics, like the ratio of /^2 , can be misleading if 
there is (e.g. pseudo-translational) NCS in the crystal; however, the effect of 
NCS on the value of the ratio of /^2 is opposite to that of twinning. 
Thus if a crystal is twinned and has NCS, you might not notice any problem in 
the ratio of /^2 .

The other statistics, like Britton and H-test, present the intensity statistics 
in a different way, but from my understanding do not give substantially 
different information.

The L-test does look at a different kind of information and therefore gives 
additional insight.

If your measurements suffer from high background, diffuse scatter, ice rings, 
smeared reflections, additional crystals in the beam, or any other pathology, 
then all these tests may give distorted answers. In other words, even if 
twinning is not really present, any test designed to convert the deviation of 
data from ideality into an estimate of the twinning fraction will give you an 
alpha > 0. So my experience is: if your data are very good, then the tests give 
good answers; if the data are mediocre or bad, don't necessarily believe the 
numbers. 

Finally, it's not only twinning of P6 that would give you P622, it's also 
twinning of P3x21, P3x12 that gives P6y22.

Hope this helps,

Kay




On Tue, 28 Jan 2014 17:26:23 +, Bert Van-Den-Berg 
 wrote:

>Dear all,
>
>I recently collected several datasets for a protein that needs experimental 
>phasing.
>The crystals are hexagonal plates, and (automatic) data processing suggests 
>with high confidence that the space group is P622. This is where the fun 
>begins.
>For some datasets (processed in P622), the intensity distributions are normal, 
>and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest that there is 
>no twinning (twinning fractions < 0.05). However, for other datasets (same 
>cell dimensions), the intensity distributions are not normal (eg Z-scores > 
>10). Given that twinning is not possible in P622, this suggests to me that the 
>real space group could be P6 with (near) perfect twinning.
>
>If I now process the "normal L-test P622" datasets in P6, the twin-law based 
>tests (britton and H-test in xtriage) give high twin fractions (0.45- 0.5), 
>suggesting all my data is twinned.
>Does this make sense (ie can one have twinning with "normal" intensity 
>distributions)?
>If it does, would the "normal L-test" datasets have a higher probability of 
>being solvable?
>
>Is there any strategy for experimental phasing of (near) perfect twins? SAD 
>would be more suitable than SIR/MIR? (I also have potential heavy atom 
>derivatives).
>
>Thanks for any insights!
>
>Bert
>

Re: [ccp4bb] Examples of multiple ASU copies with different conformations

[Kay Diederichs]

Hi Shane,

some crystal forms of trimeric AcrB (a multi-drug resistance secondary 
transporter) have 3 (or 6) monomers in the ASU and these are substantially 
different, which suggests how the protein functions.
One reference is e.g.  Seeger et al. (2006) "Structural Asymmetry of AcrB 
Trimer Suggests a Peristaltic Pump Mechanism " Science 313, 1295-1298 
DOI: 10.1126/science.1131542 (sorry for the self-plug!)

best,

Kay



On Mon, 27 Jan 2014 13:08:33 -0500, Shane Caldwell  
wrote:

>Hi ccp4bb,
>
>I'm putting together a talk for some peers that highlights strengths and
>weaknesses of structural models for the outsider. For one point, I'd like
>to find some examples of proteins that show very different conformations
>between different copies in the ASU. One example I know of is c-Abl (1OPL),
>which crystallizes with both autoinhibited and active forms in the ASU,
>with dramatically different domain organization. I'd like to find some
>additional examples - can anyone suggest some other structures that have
>multiple copies with large structural variations?
>
>Thanks in advance!
>
>Shane Caldwell
>McGill University
>

Re: [ccp4bb] Examples of multiple ASU copies with different conformations

[Tobias Weinert]

On 27 Jan 2014, at 19:08, Shane Caldwell  wrote:

Here is another interesting one:

Perez, C., Koshy, C., Yildiz, Ö., & Ziegler, C. (2012). Alternating-access 
mechanism in conformationally asymmetric trimers of the betaine transporter 
BetP. Nature.

best wishes,

Tobias


> Hi ccp4bb,
> 
> I'm putting together a talk for some peers that highlights strengths and 
> weaknesses of structural models for the outsider. For one point, I'd like to 
> find some examples of proteins that show very different conformations between 
> different copies in the ASU. One example I know of is c-Abl (1OPL), which 
> crystallizes with both autoinhibited and active forms in the ASU, with 
> dramatically different domain organization. I'd like to find some additional 
> examples - can anyone suggest some other structures that have multiple copies 
> with large structural variations?
> 
> Thanks in advance!
> 
> Shane Caldwell
> McGill University
> 
> 

Re: [ccp4bb] refining nucleic acids with Coot

[David Waterman]

For reference, the CCP4 tool is called 'pdb2to3'

-- David


On 14 January 2014 18:32, Almudena Ponce Salvatierra
wrote:

> Thank you all very much for your very helpful advices! I managed to solve
> my problem! :-)
>
> Best,
>
> Almudena.
>
>
> 2014/1/14 Ashley Pike 
>
>>  Hi Almudena - I came across this issue recently as well with an old pdb
>> file. As Paul says you need to convert from v2.3 to v3.2 pdb formats. In
>> addition to the link he gave, you can also accomplish this using the iotbx
>> toolbox that comes with phenix.
>>
>>  iotbx.pdb_remediator file_name=in.pdb output_file=out.pdb
>>
>>
>>
>> will rename * atom names to ' (eg O4* to O4') which can then be
>> regularised in coot.
>>
>>
>>
>> Ashley
>>
>>
>>
>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
>> *Almudena Ponce Salvatierra
>> *Sent:* 14 January 2014 15:52
>> *To:* CCP4BB@JISCMAIL.AC.UK
>> *Subject:* [ccp4bb] refining nucleic acids with Coot
>>
>>
>>
>> Dear all,
>>
>> I am trying to refine an oligonucleotide chain in Coot and I get the
>> following message when I try:
>>
>> "Fail to match (to the dictionary) the following model atom names:
>>
>> G
>>
>> O5* C5* O4* etc
>>
>> That would cause exploding atoms so the refinement did not start"
>>
>> Does anyone know how to solve this?
>>
>> Best wishes,
>>
>> Almudena.
>>
>>
>> --
>>
>> Almudena Ponce-Salvatierra
>>
>> Macromolecular crystallography and Nucleic acid chemistry
>>
>> Max Planck Institute for Biophysical Chemistry
>>
>> Am Fassberg 11 37077 Göttingen
>>
>> Germany
>>
>>
>>
>
>
>
> --
> Almudena Ponce-Salvatierra
> Macromolecular crystallography and Nucleic acid chemistry
> Max Planck Institute for Biophysical Chemistry
>  Am Fassberg 11 37077 Göttingen
> Germany
>
>

Re: [ccp4bb] twinning fun

[Keller, Jacob]

Try looking into tetartohedral twinning as well--I think I may have such a 
crystal, and it's tough going. And as Kay pointed out, try the various P3's. 
Since I have not yet been successful in figuring my similar case out, what do 
people on the list recommend as an approach to figuring this out--just trying 
every possible space group with various parameters? I would think there should 
be some actual advantages at the phasing step to having twins, such as a sort 
of "NCS" of intensities rather than amplitudes, weighted by twin fraction, but 
it doesn't seem that any software uses this. Perhaps there is a reason for that?

A paper on tetartohedral twinning I saw:

Acta Crystallogr D Biol Crystallogr. 2012 Apr;68(Pt 4):418-24. doi: 
10.1107/S0907444912006737. Epub 2012 Mar 16.
Tetartohedral twinning could happen to you too.
Roversi P, Blanc E, Johnson S, Lea SM.
Author information
Abstract
Tetartohedral crystal twinning is discussed as a particular case of 
(pseudo)merohedral twinning when the number of twinned domains is four. 
Tetartohedrally twinned crystals often possess pseudosymmetry, with the 
rotational part of the pseudosymmetry operators coinciding with the twinning 
operators. Tetartohedrally twinned structures from the literature are reviewed 
and the recent structure determination of tetartohedrally twinned triclinic 
crystals of human complement factor I is discussed.
PMID: 22505261 [PubMed - indexed for MEDLINE] PMCID: PMC3322600 Free PMC Article

JPK



-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kay 
Diederichs
Sent: Wednesday, January 29, 2014 4:17 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] twinning fun

Dear Bert,

as Dirk has pointed out, if P622 is the correct space group, then the twinning 
statistics printed out if you process in P6 are meaningless.

Intensity statistics, like the ratio of /^2 , can be misleading if 
there is (e.g. pseudo-translational) NCS in the crystal; however, the effect of 
NCS on the value of the ratio of /^2 is opposite to that of twinning. 
Thus if a crystal is twinned and has NCS, you might not notice any problem in 
the ratio of /^2 .

The other statistics, like Britton and H-test, present the intensity statistics 
in a different way, but from my understanding do not give substantially 
different information.

The L-test does look at a different kind of information and therefore gives 
additional insight.

If your measurements suffer from high background, diffuse scatter, ice rings, 
smeared reflections, additional crystals in the beam, or any other pathology, 
then all these tests may give distorted answers. In other words, even if 
twinning is not really present, any test designed to convert the deviation of 
data from ideality into an estimate of the twinning fraction will give you an 
alpha > 0. So my experience is: if your data are very good, then the tests give 
good answers; if the data are mediocre or bad, don't necessarily believe the 
numbers. 

Finally, it's not only twinning of P6 that would give you P622, it's also 
twinning of P3x21, P3x12 that gives P6y22.

Hope this helps,

Kay




On Tue, 28 Jan 2014 17:26:23 +, Bert Van-Den-Berg 
 wrote:

>Dear all,
>
>I recently collected several datasets for a protein that needs experimental 
>phasing.
>The crystals are hexagonal plates, and (automatic) data processing suggests 
>with high confidence that the space group is P622. This is where the fun 
>begins.
>For some datasets (processed in P622), the intensity distributions are normal, 
>and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest that there is 
>no twinning (twinning fractions < 0.05). However, for other datasets (same 
>cell dimensions), the intensity distributions are not normal (eg Z-scores > 
>10). Given that twinning is not possible in P622, this suggests to me that the 
>real space group could be P6 with (near) perfect twinning.
>
>If I now process the "normal L-test P622" datasets in P6, the twin-law based 
>tests (britton and H-test in xtriage) give high twin fractions (0.45- 0.5), 
>suggesting all my data is twinned.
>Does this make sense (ie can one have twinning with "normal" intensity 
>distributions)?
>If it does, would the "normal L-test" datasets have a higher probability of 
>being solvable?
>
>Is there any strategy for experimental phasing of (near) perfect twins? SAD 
>would be more suitable than SIR/MIR? (I also have potential heavy atom 
>derivatives).
>
>Thanks for any insights!
>
>Bert
>

Re: [ccp4bb] twinning fun

[Eleanor Dodson]

Dont forget that with twinning in apparent point group PG6/mmm the
true SG may be P6i or P3i21
See the twinning notes: http://www.ccp4.ac.uk/dist/html/twinning.html


Detecting twinning can be problematic -

My rule of thumb, following the procedure od ctruncate::

0) Check the matthews coefficient for likely number of molecules.
Half a molecule must mean you are assigning too high a symmetry count.
Lots of molecules means you need to check for non-crystallographic
translation etc.


1) Look at the /^2 plot after correction for anisotropy
If it isnt reasonably  straight with resolution you probably have some
data problems, and these can make all the tests pretty useless.

2) Is there a NC translation - truncate tells you that.
If not, and the data is OK,  you are unlikely to have twinning if
/^2 for acentrics is ~ 2, and  the L test looks OK.
H test and Britten tests a bit more influenced by other NC symmetry
considerations

3) If there IS NC translation /^2 for acentrics will probably
be > 2  but the L test is still pretty reliable.

Good luck Eleanor

experimental phasing is tricky with perfect twinning but it has been
done. Sorry I have forgotten reference though..
Eleanor



On 29 January 2014 09:17, Kay Diederichs  wrote:
> Dear Bert,
>
> as Dirk has pointed out, if P622 is the correct space group, then the 
> twinning statistics printed out if you process in P6 are meaningless.
>
> Intensity statistics, like the ratio of /^2 , can be misleading if 
> there is (e.g. pseudo-translational) NCS in the crystal; however, the effect 
> of NCS on the value of the ratio of /^2 is opposite to that of 
> twinning. Thus if a crystal is twinned and has NCS, you might not notice any 
> problem in the ratio of /^2 .
>
> The other statistics, like Britton and H-test, present the intensity 
> statistics in a different way, but from my understanding do not give 
> substantially different information.
>
> The L-test does look at a different kind of information and therefore gives 
> additional insight.
>
> If your measurements suffer from high background, diffuse scatter, ice rings, 
> smeared reflections, additional crystals in the beam, or any other pathology, 
> then all these tests may give distorted answers. In other words, even if 
> twinning is not really present, any test designed to convert the deviation of 
> data from ideality into an estimate of the twinning fraction will give you an 
> alpha > 0. So my experience is: if your data are very good, then the tests 
> give good answers; if the data are mediocre or bad, don't necessarily believe 
> the numbers.
>
> Finally, it's not only twinning of P6 that would give you P622, it's also 
> twinning of P3x21, P3x12 that gives P6y22.
>
> Hope this helps,
>
> Kay
>
>
>
>
> On Tue, 28 Jan 2014 17:26:23 +, Bert Van-Den-Berg 
>  wrote:
>
>>Dear all,
>>
>>I recently collected several datasets for a protein that needs experimental 
>>phasing.
>>The crystals are hexagonal plates, and (automatic) data processing suggests 
>>with high confidence that the space group is P622. This is where the fun 
>>begins.
>>For some datasets (processed in P622), the intensity distributions are 
>>normal, and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest that 
>>there is no twinning (twinning fractions < 0.05). However, for other datasets 
>>(same cell dimensions), the intensity distributions are not normal (eg 
>>Z-scores > 10). Given that twinning is not possible in P622, this suggests to 
>>me that the real space group could be P6 with (near) perfect twinning.
>>
>>If I now process the "normal L-test P622" datasets in P6, the twin-law based 
>>tests (britton and H-test in xtriage) give high twin fractions (0.45- 0.5), 
>>suggesting all my data is twinned.
>>Does this make sense (ie can one have twinning with "normal" intensity 
>>distributions)?
>>If it does, would the "normal L-test" datasets have a higher probability of 
>>being solvable?
>>
>>Is there any strategy for experimental phasing of (near) perfect twins? SAD 
>>would be more suitable than SIR/MIR? (I also have potential heavy atom 
>>derivatives).
>>
>>Thanks for any insights!
>>
>>Bert
>>

[ccp4bb] Post-doctoral positions in Structural Microbiology in Stockholm, Sweden

[Adnane Achour]

Post-doctoral positions in Structural Microbiology with a focus on 
Streptococcus pneumonia-associated virulence factors at the Science for Life 
Laboratory in Stockholm, Sweden.

We are currently seeking for two highly motivated and multi-talented 
post-doctoral fellows to work on a collaborative project in the Achour 
laboratory within SciLifeLab (SciLifeLab.se). This is an 
ideal position for a post-doc who is looking to bolster experience and/or 
publication record for several years before moving to a faculty position.
The selected post-doctoral fellow will focus on determining the 
three-dimensional structures of Streptococcus pneumonia-associated virulence 
factors alone and/or in complex with ligands/inhibitors (Schulte et al, Open 
Biology 2014; Mellroth et al, mBio 2014). A successful PhD in a relevant 
scientific discipline and proficiency in structural and molecular biology are 
required. The post-doc fellows will have a lot of autonomy, but are also 
expected to provide theoretical and technical assistance with overall 
laboratory computational and crystallographic projects. The candidates should 
have a strong will to work on challenging and significant protein complexes in 
a multidisciplinary setting. Main skill sets required are facility in cloning 
and protein production, practical aspects of protein crystallization, structure 
determination and computation. Familiarity with protein expression systems, 
cell culture, various biochemical and biophysical techniques to study 
protein-protein interactions are also highly desirable. Besides determining the 
three-dimensional structures of protein complexes, the candidates will also be 
provided with the possibility to assess biophysically their interactions using 
e.g. Surface Plasmon Resonance (SPR), isothermal titration calorimetry (ITC) or 
microscale thermophoresis. A large panel of other biophysical technologies is 
also available within SciLifeLab.
The positions are initially offered for a period of two years (1+ 1year), but 
if successful, the post-doctoral fellows will also be offered a portfolio of 
their own projects in the areas of structure and/or protein engineering, as 
well as possibility to establish an own research group. The applicants should 
send a CV, with a complete list of publications (only published manuscripts) as 
well as a letter in which they describe the basis of their interest in the 
proposed project. Three professional references should also be sent in which 
the scientific and social qualities of the candidate are described. All 
required information should be sent by e-mail to 
adnane.ach...@ki.se.
More information about the Achour group can be found in 
scilifelab.se. The positions are available from the 
beginning of April 2014, and will remain open until filled. The initial 
appointment is for 1 year. Consideration of applications starts when this 
advertisement appears. This call closes on February 26th, 2014.
The newly created SciLifeLab, which is a joint effort between four Swedish 
universities, including the Royal Institute of Technology (KTH), the Karolinska 
Institute (KI), Stockholm University (SU) and Uppsala University (UU), serves 
as a Swedish national infrastructure to support infrastructure and technically 
advanced research in the life science area. The infrastructure at SciLifeLab 
includes state-of-the-art technologies for high-throughput molecular 
biosciences.
Adnane Achour

[ccp4bb] Open Post-doc positions at the Science for Life Laboratory (scilifelab.se) in Stockholm, Sweden.

[Adnane Achour]

Open Post-doc positions in Structural Immunology with a focus on enhanced TCR 
recognition of MHC molecules at the Science for Life Laboratory 
(scilifelab.se) in Stockholm, Sweden.

We are currently seeking for two highly motivated and multi-talented 
post-doctoral fellows to work on a collaborative project in the Achour 
laboratory within SciLifeLab (SciLifeLab.se). This is an 
ideal position for a post-doc who is looking to bolster experience and/or 
publication record for several years before moving to a faculty position.

The selected post-doctoral fellow(s) will focus on determining the 
three-dimensional structures of T cell receptors in complex with MHC class I or 
class II, presenting wild-type and altered versions of epitopes related to 
autoimmune and/or cancer diseases. The overall aim of the project is to 
establish and understand the mechanisms underlying enhanced TCR recognition of 
infected and/or cancer cells, as well as to provide a structural basis for the 
induction of autoimmune responses.

A successful PhD in a relevant scientific discipline and proficiency in 
structural and molecular biology are required. The post-doc fellows will have a 
lot of autonomy, but are also expected to provide theoretical and technical 
assistance with overall laboratory computational and crystallographic projects. 
The candidates should have a strong will to work on challenging and significant 
protein complexes in a multidisciplinary setting. Main skill sets required are 
facility in cloning and protein production, practical aspects of protein 
crystallization, structure determination and computation. Familiarity with 
protein expression systems, cell culture, various biochemical and biophysical 
techniques to study protein-protein interactions are also highly desirable. 
Besides determining the three-dimensional structures of protein complexes, the 
candidates will also be provided with the possibility to assess biophysically 
their interactions using e.g. Surface Plasmon Resonance (SPR), isothermal 
titration calorimetry (ITC) or microscale thermophoresis. A large panel of 
other biophysical technologies is also available within SciLifeLab.

The positions are initially offered for a period of two years (1+ 1year), but 
if successful, the post-doctoral fellows will also be offered a portfolio of 
their own projects in the areas of structure and/or protein engineering, as 
well as possibility to establish an own research group. The applicants should 
send a CV, with a complete list of publications (only published manuscripts) as 
well as a letter in which they describe the basis of their interest in the 
proposed project. Three professional references should also be sent in which 
the scientific and social qualities of the candidate are described. All 
required information should be sent by e-mail to 
adnane.ach...@ki.se.

The newly created SciLifeLab, which is a joint effort between four Swedish 
universities, including the Royal Institute of Technology (KTH), the Karolinska 
Institute (KI), Stockholm University (SU) and Uppsala University (UU), serves 
as a Swedish national infrastructure to support infrastructure and technically 
advanced research in the life science area. The infrastructure at SciLifeLab 
includes state-of-the-art technologies for high-throughput molecular 
biosciences.

More information about the Achour group can be found in 
scilifelab.se. The positions are available from the 
beginning of April 2014, and will remain open until filled. The initial 
appointment is for 1 year. Consideration of applications starts when this 
advertisement appears. This call closes on February 26th, 2014.

Adnane Achour

Re: [ccp4bb] twinning fun

[Randy Read]

Also, if you have translational NCS then recent versions of Phaser can correct 
for the statistical effects and give you /^2 moment tests that are 
diagnostic of twinning.  This works pretty well for 2-fold tNCS (i.e. one major 
Patterson peak corresponding to one or more pairs of molecules separated by the 
same translation).  If there's higher order tNCS, then this works less well in 
the current version.  We give some examples in the paper describing the 
algorithm: http://journals.iucr.org/d/issues/2013/02/00/dz5268/dz5268.pdf.

Best wishes,

Randy Read

On 29 Jan 2014, at 13:30, Eleanor Dodson  wrote:

> Dont forget that with twinning in apparent point group PG6/mmm the
> true SG may be P6i or P3i21
> See the twinning notes: http://www.ccp4.ac.uk/dist/html/twinning.html
> 
> 
> Detecting twinning can be problematic -
> 
> My rule of thumb, following the procedure od ctruncate::
> 
> 0) Check the matthews coefficient for likely number of molecules.
> Half a molecule must mean you are assigning too high a symmetry count.
> Lots of molecules means you need to check for non-crystallographic
> translation etc.
> 
> 
> 1) Look at the /^2 plot after correction for anisotropy
> If it isnt reasonably  straight with resolution you probably have some
> data problems, and these can make all the tests pretty useless.
> 
> 2) Is there a NC translation - truncate tells you that.
> If not, and the data is OK,  you are unlikely to have twinning if
> /^2 for acentrics is ~ 2, and  the L test looks OK.
> H test and Britten tests a bit more influenced by other NC symmetry
> considerations
> 
> 3) If there IS NC translation /^2 for acentrics will probably
> be > 2  but the L test is still pretty reliable.
> 
> Good luck Eleanor
> 
> experimental phasing is tricky with perfect twinning but it has been
> done. Sorry I have forgotten reference though..
> Eleanor
> 
> 
> 
> On 29 January 2014 09:17, Kay Diederichs  
> wrote:
>> Dear Bert,
>> 
>> as Dirk has pointed out, if P622 is the correct space group, then the 
>> twinning statistics printed out if you process in P6 are meaningless.
>> 
>> Intensity statistics, like the ratio of /^2 , can be misleading if 
>> there is (e.g. pseudo-translational) NCS in the crystal; however, the effect 
>> of NCS on the value of the ratio of /^2 is opposite to that of 
>> twinning. Thus if a crystal is twinned and has NCS, you might not notice any 
>> problem in the ratio of /^2 .
>> 
>> The other statistics, like Britton and H-test, present the intensity 
>> statistics in a different way, but from my understanding do not give 
>> substantially different information.
>> 
>> The L-test does look at a different kind of information and therefore gives 
>> additional insight.
>> 
>> If your measurements suffer from high background, diffuse scatter, ice 
>> rings, smeared reflections, additional crystals in the beam, or any other 
>> pathology, then all these tests may give distorted answers. In other words, 
>> even if twinning is not really present, any test designed to convert the 
>> deviation of data from ideality into an estimate of the twinning fraction 
>> will give you an alpha > 0. So my experience is: if your data are very good, 
>> then the tests give good answers; if the data are mediocre or bad, don't 
>> necessarily believe the numbers.
>> 
>> Finally, it's not only twinning of P6 that would give you P622, it's also 
>> twinning of P3x21, P3x12 that gives P6y22.
>> 
>> Hope this helps,
>> 
>> Kay
>> 
>> 
>> 
>> 
>> On Tue, 28 Jan 2014 17:26:23 +, Bert Van-Den-Berg 
>>  wrote:
>> 
>>> Dear all,
>>> 
>>> I recently collected several datasets for a protein that needs experimental 
>>> phasing.
>>> The crystals are hexagonal plates, and (automatic) data processing suggests 
>>> with high confidence that the space group is P622. This is where the fun 
>>> begins.
>>> For some datasets (processed in P622), the intensity distributions are 
>>> normal, and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest 
>>> that there is no twinning (twinning fractions < 0.05). However, for other 
>>> datasets (same cell dimensions), the intensity distributions are not normal 
>>> (eg Z-scores > 10). Given that twinning is not possible in P622, this 
>>> suggests to me that the real space group could be P6 with (near) perfect 
>>> twinning.
>>> 
>>> If I now process the "normal L-test P622" datasets in P6, the twin-law 
>>> based tests (britton and H-test in xtriage) give high twin fractions (0.45- 
>>> 0.5), suggesting all my data is twinned.
>>> Does this make sense (ie can one have twinning with "normal" intensity 
>>> distributions)?
>>> If it does, would the "normal L-test" datasets have a higher probability of 
>>> being solvable?
>>> 
>>> Is there any strategy for experimental phasing of (near) perfect twins? SAD 
>>> would be more suitable than SIR/MIR? (I also have potential heavy atom 
>>> derivatives).
>>> 
>>> Thanks for any insights!
>>> 
>>> Bert
>>> 

--
Randy J. Read

Re: [ccp4bb] making high res image in pymol

[A K]

Thank you all for the suggestions. I lowered the hash-max and could get ray
tracing to complete for line or even ribbon modes (to be honest line mode
worked even without lowering the hash-max). But as Matthew pointed out, I
could not get it to work in the cartoon mode. I think I am restricted here
with my hardware limitations...
Alex


On Tue, Jan 28, 2014 at 2:20 PM, Yong Wang  wrote:

>  Hi Alex,
>
>
>
> If you don't mind forgoing the ray tracing, you may try the draw command
> to specifically set the resolution (and antialiasing) to your needs, and
> then save the image.
>
>
>
> Yong
>
>
>
>
> --
>
> *Yong Wang, Ph.D. Research
> Advisor, Discovery Chemistry Research*
>
> Eli Lilly & Company Phone:
> 317-655-9145
>
> Lilly Corporate Center  DC 0403  Fax:  317-651-6333
>
> Indianapolis, IN  46285
> wang_y...@lilly.com
>
>
>
> CONFIDENTIALITY NOTICE:  This e-mail message from Eli Lilly and Company
> (including all attachments) is for the sole use of the intended
> recipient(s) and may contain confidential and privileged information.  Any
> unauthorized review, use, disclosure, copying or distribution is strictly
> prohibited.  If you are not the intended recipient, please contact the
> sender by reply e-mail and destroy all copies of the original message.
>
>
>
>
>
>
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *A
> K
>
> *Sent:* Tuesday, January 28, 2014 10:27 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] making high res image in pymol
>
>
>
> Hi all,
>
> I am trying to generate a high resolution figure of a molecule together
> with its symmetry mates (250 A readius) for a poster. If I try to ray it,
> the pymol session crashes (perhaps too many molecules are open). Using png
> xxx.png, dpi=300 or dpi=600 command doesn't make any difference; the image
> is still kind of low resolution for A0 or A1. Any idea how I can generate
> this in pymol? (I am using the free version of pymol)
>
> Thank you in advance,
>
> Alex
>

Re: [ccp4bb] twinning fun

[Keller, Jacob]

Two more papers on twinning I found informative:

===

Acta Cryst. (2003). D59, 2004-2016[ doi:10.1107/S0907444903021085 ]

Twinned crystals and anomalous phasing
Z. Dauter
Abstract: Merohedral or pseudomerohedral twinning of crystals cannot be 
identified from inspection of the diffraction patterns. Several methods for the 
identification of twinning and the estimation of the twin fraction are suitable 
for macromolecular crystals and all are based on the statistical properties of 
the measured diffraction intensities. If the crystal twin fraction is estimated 
and is not too close to 0.5, the diffraction data can be detwinned; that is, 
related to the individual crystal specimen. However, the detwinning procedure 
invariably introduces additional inaccuracies to the estimated intensities, 
which substantially increase when the twin fraction approaches 0.5. In some 
cases, a crystal structure can be solved with the original twinned data by 
standard techniques such as molecular replacement, multiple isomorphous 
replacement or multiwavelength anomalous diffraction. Test calculations on data 
collected from a twinned crystal of gpD, the bacteriophage [lambda] capsid 
protein, show that the single-wavelength anomalous diffraction (SAD) method can 
be used to solve its structure even if the data set corresponds to a perfectly 
twinned crystal with a twin fraction of 0.5.

Keywords: twinning; merohedral; pseudomerohedral; anomalous scattering; SAD.

===

Acta Crystallogr D Biol Crystallogr. 2008 Jan;64(Pt 1):99-107. Epub 2007 Dec 5.
Surprises and pitfalls arising from (pseudo)symmetry.
Zwart PH, Grosse-Kunstleve RW, Lebedev AA, Murshudov GN, Adams PD.
Author information
Abstract
It is not uncommon for protein crystals to crystallize with more than a single 
molecule per asymmetric unit. When more than a single molecule is present in 
the asymmetric unit, various pathological situations such as twinning, 
modulated crystals and pseudo translational or rotational symmetry can arise. 
The presence of pseudosymmetry can lead to uncertainties about the correct 
space group, especially in the presence of twinning. The background to certain 
common pathologies is presented and a new notation for space groups in unusual 
settings is introduced. The main concepts are illustrated with several examples 
from the literature and the Protein Data Bank.
PMID: 18094473 [PubMed - indexed for MEDLINE] PMCID: PMC2394827 Free PMC Article

[ccp4bb] Scripting error running Phaser_EP/HYSS?

[Roger Rowlett]

Is there a scripting error in 
ccp4-6.4.0/share/ccp4i/scripts/phaser_EP.script? CCP4i jobs fail with an 
argument format error, e.g.,


Error interpreting command line argument as a parameter definition: 
"data-label=F_whatever(+)"

Improper definition name "data-label"

I think phenix requires underscores, not hyphens, e.g. "data_label", 
"pdb_only" etc. Even the phenix documentation page is conflicted, 
showing both "data-label" and "data_label". Maybe is isn't supposed to 
matter, but in Phenix 1.8.4, it apparently does matter.


Is this something that could be fixed in the next CCP4 update? Or should 
I direct this to the phenix developers?


Cheers,

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

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

[ccp4bb] coloring by RMSD

[Colussi, Timothy]

I was wondering if anybody knows of a program or script that you can run that 
will calculate local RMSDs between 2 superposed pdbs and color based on those 
local RMSDs or allow for the ability to color based on RMSD? I have a molecule 
that is similar in some aspects to another molecule but also very different in 
others and thought this would be a good way to show this graphically.

Tim Colussi

Re: [ccp4bb] twinning fun

[Jrh]

Dear Bert,
In my own review:-
http://www.tandfonline.com/doi/abs/10.1080/08893110802360925?journalCode=gcry20#.UulGyGtYCSM
molecular replacement emerged in my mind as the most robust option for 
structure determination in such a case, apart from finding an untwinned crystal 
form of course.
Best wishes,
John

Prof John R Helliwell DSc FInstP CPhys FRSC CChem F Soc Biol.
Chair School of Chemistry, University of Manchester, Athena Swan Team.
http://www.chemistry.manchester.ac.uk/aboutus/athena/index.html
 
 

On 28 Jan 2014, at 17:26, Bert Van-Den-Berg  
wrote:

> Dear all,
> 
> I recently collected several datasets for a protein that needs experimental 
> phasing.
> The crystals are hexagonal plates, and (automatic) data processing suggests 
> with high confidence that the space group is P622. This is where the fun 
> begins.
> For some datasets (processed in P622), the intensity distributions are 
> normal, and the L-test (aimless, xtriage) and Z-scores (xtriage) suggest that 
> there is no twinning (twinning fractions < 0.05). However, for other datasets 
> (same cell dimensions), the intensity distributions are not normal (eg 
> Z-scores > 10). Given that twinning is not possible in P622, this suggests to 
> me that the real space group could be P6 with (near) perfect twinning.
> 
> If I now process the "normal L-test P622" datasets in P6, the twin-law based 
> tests (britton and H-test in xtriage) give high twin fractions (0.45- 0.5), 
> suggesting all my data is twinned.
> Does this make sense (ie can one have twinning with "normal" intensity 
> distributions)? 
> If it does, would the "normal L-test" datasets have a higher probability of 
> being solvable?
> 
> Is there any strategy for experimental phasing of (near) perfect twins? SAD 
> would be more suitable than SIR/MIR? (I also have potential heavy atom 
> derivatives).
> 
> Thanks for any insights!
> 
> Bert

Re: [ccp4bb] coloring by RMSD

[Sampson, Jared]

Hi Tim -

There is a script on the PyMOL Wiki that does just this:  
http://pymolwiki.org/index.php/ColorByRMSD.

Cheers,
Jared

--
Jared Sampson
Xiangpeng Kong Lab
NYU Langone Medical Center
550 First Avenue
New York, NY 10016
212-263-7898
http://kong.med.nyu.edu/






On Jan 29, 2014, at 12:43 PM, "Colussi, Timothy" 
mailto:timothy.colu...@ucdenver.edu>>
 wrote:

I was wondering if anybody knows of a program or script that you can run that 
will calculate local RMSDs between 2 superposed pdbs and color based on those 
local RMSDs or allow for the ability to color based on RMSD? I have a molecule 
that is similar in some aspects to another molecule but also very different in 
others and thought this would be a good way to show this graphically.

Tim Colussi


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[ccp4bb] Post-doc opening at MacCHESS

[Marian Szebenyi]

Postdoctoral Associate: "Applications of Pressure to Structural Biology"

The Macromolecular Diffraction Facility of the Cornell High-Energy Synchrotron 
Source (MacCHESS) has an opening for a post-doctoral associate to continue 
development of the pressure cryocooling method (Kim et al. Acta Cryst. D61, 
881-890 (2005), Kim et al. J. Appl. Crystallog. 46, 234-241 (2013)) and to apply 
pressure cryocooling to areas such as trapping of intermediates in biochemical 
reactions, preparation of samples for diffraction and imaging experiments, and 
elucidation of the effects of pressure on macromolecular structure. Applicants 
should have a Ph.D. degree in structural biology, biophysics, or a related 
field. Experience in hands-on development of sample-handling methods is 
desirable, and experience working at a synchrotron source is a plus. In addition 
to pursuing his/her own study of pressure effects, the successful candidate will 
be expected to collaborate with research groups wishing to apply pressure 
cryocooling to their samples.


The Cornell High-Energy Synchrotron Source (CHESS) serves a world-wide user base 
of structural biologists, chemists, physicists, and engineers. MacCHESS is an 
NIH-supported National Resource providing support for structural biology at 
CHESS. MacCHESS is a heavily team-oriented environment. Good clear communication 
skills are a must, including fluency in the English language. This position is 
renewable for up to 3 years total, contingent upon availability of funds and 
employee performance.


Direct inquiries and applications (include cover letter, CV including 
publications, and detailed summary of research experience and interests, and 
arrange to have at least three letters of reference sent) to:


Dr. Marian Szebenyi
CHESS, 200L Wilson Lab
Cornell University
Ithaca, NY 14853
E-mail: dm...@cornell.edu

Applications must be received by March 31, 2014. Starting date is negotiable.
Cornell is an equal opportunity employer.

[ccp4bb] preparation of citrate buffer pH3-6.5

[sreetama das]

Dear All,

           We have obtained many tiny protein crystals in a condition 
containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are too 
small for mounting in loops.

           We intend to vary the salt concentration & pH to obtain larger 
crystals.

           Could anyone direct us to some links, or provide us with a method 
(with calculations) to calculate the amounts of citric acid & trisodium citrate 
required to obtain buffers in a range of pH 3 - 6.5?
           I have come across online buffer calculators and links where the 
amounts of the components required are mentioned in grams, but none explaining 
how those values were arrived at.

Thanks & regards,
sreetama