Re: [ccp4bb] cell discrepancies and stuck refinement using different XDS-versions

[Keller, Jacob]

>>But there is no rule without exception,

Well, occasionally there is.

JPK




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Re: [ccp4bb] cell discrepancies and stuck refinement using different XDS-versions

[Kay Diederichs]

Dear all,
I post this again to CCP4BB because some people informed me that they 
received an empty email from me. This time I use my email client; 
previously I used the Jiscmail web interface - does anybody know what 
went wrong? Hopefully it works this time. Sorry if you get this twice.

Kay

Dear Sabine,

indeed, the differences between the last XDS versions are in the area of 
indexing, with  BUILT=20180409 being the most robust (and recommended) 
one. This gives the desired results in all my tests, and GlobalPhasing 
has successfully tested it with ~60 difficult cases. But there is no 
rule without exception, which means that it could nevertheless give the 
wrong answer in your case.


According to CCP4 othercell, the two cells that you cite are related by 
the [h-2l,-h,k] reindexing operator. However the cell volumes differ by 
slightly more than a factor of 2 , namely 232274 / 110672 = 2.1 which I 
find astonishing because typically such different indexing results 
result in a pseudo-centering situation, where the indexing results 
differ in that one tries to explain the weak and strong reflections, 
whereas the other only explains the strong reflections. What exactly 
happens in your case I don't know; I'd have to see the data and the 
detailed output of XDS. It could also be a case of two lattices (from 
two crystals) superimposed.


You can probably get the small cell with  BUILT=20180409 if you either
a) specify it in XDS.INP (together with SPACE_GROUP_NUMBER=1)
b) or remove the weak reflections from SPOT.XDS
c) or by adjusting some parameters in XDS.INP

To really understand what is going on in your case, you could
a) run the checkcentering program (from 
ftp://turn5.biologie.uni-konstanz.de/pub/linux_bin ) which will analyze 
your data w.r.t. pseudo-centering (similar to what pointless does)
b) look, in a representation of reciprocal space, at the spots that are 
used for indexing and that are indexed, or not. For this, you need the 
spot2pdb program (same download directory). Run it
grep -s allow-duplicate-sequence-numbers ~/.coot || echo 
"(allow-duplicate-sequence-numbers)" >>~/.coot

spot2pdb
coot SPOT-*.pdb
as suggested in 
https://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/XDSGUI#tools


BTW it is normal that R factors with tNCS are higher ("stuck") than 
without; this is due to the wider distribution of reflection intensities 
in the case of tNCS (many weak ones, and many strong ones; less with 
intermediate intensity). Most people would say that the space group and 
cell where the refinement proceeds best must be the correct one; however 
if that does not explain all the reflections of the lattice then this is 
not quite satisfactory.


best wishes,

Kay


On Mon, 6 Aug 2018 16:50:56 +0200, Sabine Schneider 
 wrote:


>Dear all,
>
>We are encountering a differences in indexing using different XDS
>versions, which results in either 2 or 4 mols/asu (SG P1; structure
>determination via MR (30% seq ID model)) and either successful
>refinement or stuck R/Rfree
>
>- the data were collected at ESRF ID30A3 (Aiger detector) and extend to
>about 2.3A resolution (CC1/2 ~50%)
>
>The XDS version build 20180126 (and or autoprocessing at the ESRF via
>autoproc, dials, xdsapp or grenade -> here also xds version from
>20180126 used) gives us:
>
>P1  38.6550.7661.11 110.057  99.945  90.197
>XDS complains and I need to use "DEFPIX INTEGRATE CORRECT"
>-> 2mols/asu, structure refines to R/Rfree of 22/25
>
>In contrast the actual XDS-Version BUILT=20180409 results in:
>P1  39.2   51.3  116.8  86.3  82.3  89.8
>but XDS runs smoothly.
>-> 4mols/asu, tNCS, R/Rfree stuck at 28/32
>If I feed XDS with the smaller cell above, it fails.
>
>(The smaller cell is also found by Xia2/dials via the CCP4i2 interface.)
>
>Thus I am wondering, what are the differences between the two
>XDS-versions? (I remember vaguely that there was a tread about different
>XDS-versions, but couldn't find it..)
>
>Cheers Sabine
>
>
>--
>--
>Dr. Sabine Schneider
>Research Group Leader
>Technical University of Munich
>Department of Chemistry
>Chair of Biochemistry
>Lichtenbergstr. 4
>85748 Garching
>Germany
>Tel.: +49 (0) 89 289 13759
>Fax: +49 (0) 89 289 13363
>http://www.biochemie.ch.tum.de/index.php?id=919
>
>--
>--
>Dr. Sabine Schneider
>Research Group Leader
>Technical University of Munich
>Department of Chemistry
>Chair of Biochemistry
>Lichtenbergstr. 4
>85748 Garching
>Germany
>Tel.: +49 (0) 89 289 13759
>Fax: +49 (0) 89 289 13363
>http://www.biochemie.ch.tum.de/index.php?id=919
>
>
>
>To unsubscribe from the CCP4BB list, click the following link:
>https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
--
Kay Diederichshttp://strucbio.biologie.uni-konstanz.de
email: kay.diederi...@uni-konstanz.deTel +49 7531 88 4049 Fax 3183
Fachbereich 

[ccp4bb] Postdoc position in structural biology

[Davis, Jamaine]

Postdoctoral position investigating the impact of genetic variations on 
three-dimensional structure/function of proteins available in the laboratory of 
Jamaine Davis at Meharry Medical College in Nashville, TN



There is an immediate availability in the laboratory of Jamaine Davis for an 
enthusiastic postdoc who is interested in the molecular recognition of protein 
complexes essential for genome maintenance. The successful candidate will 
investigate DNA repair protein structure and complex assembly mechanisms using 
X-ray crystallography and/or single-particle cryo-EM, in addition to performing 
functional assays. The work in the lab is generally geared towards developing a 
mechanistic understanding of the DNA damage response using functional and 
structural techniques. The ideal candidate for this position is a protein 
biochemist who has experience in X-ray crystallography and wants to gain 
experience in cryo-EM.



We are located on the campus of Meharry Medical College within vibrant 
Nashville, aka Music City. Nashville has been rated #12 on Forbes list of best 
places for businesses and careers. Our lab is also a member of the Center for 
Structural Biology at Vanderbilt University 
(http://structbio.vanderbilt.edu/faculty/davis.php),where we have access to the 
Vanderbilt Cryo Electron Microscopy Facility (V-CEM). The V-CEM facility houses 
high-resolution transmission electron microscopes (TEMs) and cameras needed to 
collect data for single particle analysis and tomography.



Qualifications and experience: Candidates should hold a Ph.D. in a relevant 
field and have a solid background in protein biochemistry. Candidates with 
prior experience in protein expression/purification, mutagenesis, structure 
determination (e.g., X-ray, NMR) and protein-nucleic acid interaction are 
preferred.  Experience with cryo-EM is advantageous. Excellent verbal and 
written English communication skills, and ability to work in close 
collaboration with other researchers are required.



Qualified applicants should send a cover letter, CV, and the names of three 
references by email to Jamaine Davis at jda...@mmc.edu.



Jamaine S. Davis, Ph.D. | Assistant Professor
Department of Biochemistry & Cancer Biology | School of Medicine
Meharry Medical College | 1023 21st Avenue N | 2007 WBS Bldg | Nashville, TN 
37208
Of: 615-963-2847 | Lab: 615-327-6383
jda...@mmc.edu



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Re: [ccp4bb] tCNS and space group determination

[Randy Read]

Dear Marcelo,

First I would look at the data to see if you have ice rings, because the peak 
in mean intensity and second moment of the intensity at about 2.25A resolution 
suggests an ice ring problem.  If so, you should make sure you don't 
contaminate the data with spurious large intensities.

Second, the statistics (e.g. the second moments plot after tNCS correction in 
Phaser) would be consistent with a scenario in which you have pseudosymmetry 
along with a twin operator that parallels the pseudosymmetry.  If that's true, 
it's hard to be sure of the symmetry.  For instance, if the structure really is 
monoclinic, can you be sure you chose the correct axis to be the 2-fold?

Since you have a good model that gives clear MR solutions even in P21, you can 
probably process the data in P1 and solve it with 8 copies in the unit cell.  
Then you can look at the symmetry of the MR solution (e.g. in Zanuda) and see 
whether it obeys any higher symmetry than P1.

Good luck!

Randy Read

> On 7 Aug 2018, at 10:35, Marcelo Liberato  wrote:
> 
> Dear CCP4 members, 
> 
> I need your help to figure out what is going on with my data. 
> I've integrated my data set in space groups P1, P2 and P222. All with cell 
> parameters: 45 89 149 90 90 90.
> 
> Aimless always indicates P212121 as the correct space group and the 
> resolution is about 2.0 A.
> 
> I have tried MR (Phaser) with all these space groups (and the alternative 
> ones) using a trimmed model (I've cut some loops) with 37 % identity (besides 
> the low identity, this enzyme belongs to a protein family with high 
> structural similarity). In most cases MR resulted in TFZ between 12-19 and 
> LLG 200-400. 
> However, refinement with refmac (with and without twin) generally results in 
> Rwork/Rfree ~ 0.4400/0.4800. 
> The best scenario was obtained when data was integrated in P21. MR resulted 
> in TFZ=19 and LLG=450, and refmac (with twin) resulted in Rwork/Rfree = 
> 0.4129/0.4517. This data was used in Autobuild (Phenix), twin law h,-k,-l,  
> resulting in Rwork/Rfree = 0.3525/0.4040. The final model seems to fit the 
> electron density, but the map is not very good and there is a lot of bias. 
> 
> All data generated by aimless and MR in the different space groups were 
> analyzed by Xtriage. In all cases, "Translational pseudo-symmetry is very 
> likely present in these data" with an off-origin peak (38 % of origin peak) 
> at fractional coordinates (0.0, 0.5, 0.13). 
> 
> Now, I am stuck and I have no idea how to solve this problem. Could you 
> please help me with this?
> 
> The log files of aimless, MR, refmac and Xtriage of the "best scenario" are 
> attached. 
> 
> Many thanks
> 
> Marcelo Liberato
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> <49_refmac5.log><46_phaser_MR.log><45_aimless.log>

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 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




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[ccp4bb] Help PDBe to enrich structure data with functional annotations

[Mihaly Varadi]

Dear All,

We would be grateful if you could spend a moment of your time filling 
out this short survey on PDBe-KB 
[https://goo.gl/forms/6jcmDZNXIQm6IZFj2], to help us understand what 
types of additional structure annotations would be useful to you.


PDBe-KB (Protein Data Bank in Europe - Knowledge Base) 
[https://pdbe-kb.org] is a community-driven resource managed by the PDBe 
team, collating functional annotations and predictions for structure 
data in the PDB archive. PDBe-KB is a collaborative effort between PDBe 
and a diverse group of bioinformatics resources and research teams.


Thank you for you time,
Mihaly Varadi

--
Mihaly Varadi, PhD
Scientific Programmer

European Bioinformatics Institute (EMBL-EBI)
Main Building, A2-32, Wellcome Trust Genome Campus, Hinxton, Cambridge, 
UK

Office: +44 (0)1223 494 278
E-mail: mvar...@ebi.ac.uk
Website: www.ebi.ac.uk/about/people/mihaly-varadi



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[ccp4bb] PhD position in structural biology available at University of Essex, UK

[Prischi, Filippo]

Ph. D. Student Position
“Flipping the switch; AtS6K2 signalling during stress”

A 3-year Leverhulme Trust funded PhD studentship is available, starting from 
February 2019, to work in the laboratory of Dr Filippo Prischi (University of 
Essex, UK) and Dr Marta Carroni (SciLife laboratories, Sweden). The successful 
applicant will be joining an internationally-renowned Research Centre

Scientific background
All living organisms have adopted ways to maintain internal equilibrium and 
respond to stress factors. Consequently, they have evolved tightly regulated 
signalling pathways, which can sense changes in the environment and elicit a 
response. In plants, like in most eukaryotes, the p70 ribosomal S6 kinases 
(S6Ks) pathway coordinates cell growth, cell proliferation, and stress 
response. Studies in Arabidopsis thaliana have shown that, similarly to in 
humans, the S6K family is composed of two members, called AtS6K1 and AtS6K2, 
which function differently. Little is known about AtS6K2 specific roles, 
despite initial evidences suggesting that it regulates responses to 
environmental stresses and developmental cues. The aim of this project is to 
unravel how AtS6K2 enables the plant to adapt to changes in the environment.

Research methodology
The successful candidate will carry out a biophysical, biochemical and 
structural characterisation of AtS6K2. Protein complexes structures will be 
solved using X-Ray crystallography and single particle cryo-electron microscopy 
(cryo-EM). The atomic details obtained from the 3D structures will provide 
unique insights into how AtS6K2 is regulated, and will contextualise and 
rationalise in vivo and biophysical data, thus providing structure-function 
relationship. This project will set the base for future studies on the human 
kinase and on plant productivity.

Training
This project is highly interdisciplinary and the successful candidate will 
develop skills in recombinant protein expression, protein purification, protein 
extraction from leaves, structural biology (X-RAY, SAXS and cryo-EM), and 
biochemical characterisation (SEC-MALS, fluorescence spectroscopy and SPR). As 
part of the scholarship the student will spend up to six months at the cryo-EM 
facility of SciLife in Stockholm to carry out data collection and analysis. In 
addition to hands-on practical research skills, generic professional skills 
development will be supported internally via Proficio, the innovative 
professional development scheme available at University of Essex, or externally 
via Diamond Light Source, CCP4 and BAC training courses.

Person specification
Candidates should have a background in biochemistry and an interest in 
structural biology. Candidates must have, by December 2018, a degree in 
biological sciences, biochemistry, or a related area (a first or upper second 
class honours degree is desirable). A further qualification such as M.Sc. or 
M.Res. is advantageous

Starting date: 1st of January 2019 or later.

Funding
The studentship covers tuition fees only for UK/EU nationals and provides a 
tax-free stipend of £14,777 per year.

To apply, please send a copy of your CV, and covering letter describing why you 
are suitable for this PhD studentship directly to Dr Filippo Prischi 
(fpris...@essex.ac.uk) by 1st October 2018.


~~
Dr Filippo Prischi, PhD FHEA
Lecturer in Biochemistry
School of Biological Sciences
University of Essex, Wivenhoe Park
Colchester, CO4 3SQ, UK

T  +44 (0)1206 873370
E  fpris...@essex.ac.uk
► http://www.essex.ac.uk/bs/staff/profile.aspx?ID=4512
► https://scholar.google.co.uk/citations?hl=en=j8hfZGIJ
► http://filippoprischilab.org/
► http://www.aisuk.org



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[ccp4bb] Postdoc position for Long-wavelength MX at Diamond Light Source

[armin.wag...@diamond.ac.uk]

Are you interested in probing and pushing the boundaries of long-wavelength 
macromolecular crystallography? Then read on…

We are seeking a highly motivated PDRA to join the I23 beamline team at Diamond 
Light Source (http://www.diamond.ac.uk/Instruments/Mx/I23.html) and carry out 
innovative research using long-wavelength macromolecular crystallography. As 
the beamline is tunable over a large wavelength range not accessible at any 
other MX beamline (down to λ = 5.9 Å), this position allows setting up novel 
experiments to push the boundaries of MX at synchrotrons. Potential projects 
are the investigation of the optimal wavelength for native phasing as function 
of crystal size, testing and improving of analytical absorption correction 
software based on X-ray tomography, evaluation of different data collection and 
processing / scaling protocols or establishing MAD experiments using the strong 
anomalous signal from the uranium M(V)-edge. Candidates are encouraged to 
indicate their personal research interest exploiting the unique capabilities of 
the instrument in the application.

More details can be found here:
https://vacancies.diamond.ac.uk/vacancy/post-doctoral-research-associate-355285.html

Please don’t hesitate to get into touch for further information.

Best regards,

 Armin




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Re: [ccp4bb] X-server failed after installing coot

[Paul Emsley]

On 07/08/2018 08:29, syed ibrahim wrote:


I am running Linux CentOS in my system. Recently I installed latest version of 
coot on my system. After successful installation, I restarted the system. But 
failed to recover Xserver screen. I was trying to restart Xserver from command 
mode but failed with following error message.

"You have a driver of ABI version of 23.0. that is not supported by this NVIDIA 
driver. Please check http://www.nvidia.com/ for driver updates or downgrade to an X 
server with a supported driver ABI.

NVidia: Use the -ignoreABI option to override this check. Fatal server error.


Any suggestions please


NVidia give you a suggestion, don't they?

FYI, I doubt that this server failure has anything to do with Coot.

Paul.



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[ccp4bb] X-server failed after installing coot

[syed ibrahim]

Dear All

I am running Linux CentOS in my system. Recently I installed latest version of 
coot on my system. After successful installation, I restarted the system. But 
failed to recover Xserver screen. I was trying to restart Xserver from command 
mode but failed with following error message.

"You have a driver of ABI version of 23.0. that is not supported by this NVIDIA 
driver. Please check http://www.nvidia.com/ for driver updates or downgrade to 
an X server with a supported driver ABI.

NVidia: Use the -ignoreABI option to override this check. Fatal server error.

No screen found.
Unable to connect to X server. Connection refused server error."

Any suggestions please

Thank you

Syed



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Re: [ccp4bb] cell discrepancies and stuck refinement using different XDS-versions

[Kay Diederichs]

Dear Sabine,

indeed, the differences between the last XDS versions are in the area of 
indexing, with  BUILT=20180409 being the most robust (and recommended) one. 
This gives the desired results in all my tests, and GlobalPhasing has 
successfully tested it with ~60 difficult cases. But there is no rule without 
exception, which means that it could nevertheless give the wrong answer in your 
case.

According to CCP4 othercell, the two cells that you cite are related by the 
[h-2l,-h,k] reindexing operator. However the cell volumes differ by slightly 
more than a factor of 2 , namely 232274 / 110672 = 2.1 which I find astonishing 
because typically such different indexing results result in a pseudo-centering 
situation, where the indexing results differ in that one tries to explain the 
weak and strong reflections, whereas the other only explains the strong 
reflections. What exactly happens in your case I don't know; I'd have to see 
the data and the detailed output of XDS. It could also be a case of two 
lattices (from two crystals) superimposed.

You can probably get the small cell with  BUILT=20180409 if you either
a) specify it in XDS.INP (together with SPACE_GROUP_NUMBER=1)
b) or remove the weak reflections from SPOT.XDS
c) or by adjusting some parameters in XDS.INP

To really understand what is going on in your case, you could
a) run the checkcentering program (from 
ftp://turn5.biologie.uni-konstanz.de/pub/linux_bin ) which will analyze your 
data w.r.t. pseudo-centering (similar to what pointless does)
b) look, in a representation of reciprocal space, at the spots that are used 
for indexing and that are indexed, or not. For this, you need the spot2pdb 
program (same download directory). Run it 
grep -s allow-duplicate-sequence-numbers ~/.coot || echo 
"(allow-duplicate-sequence-numbers)" >>~/.coot 
spot2pdb
coot SPOT-*.pdb
as suggested in 
https://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/XDSGUI#tools

BTW it is normal that R factors with tNCS are higher ("stuck") than without; 
this is due to the wider distribution of reflection intensities in the case of 
tNCS (many weak ones, and many strong ones; less with intermediate intensity). 
Most people would say that the space group and cell where the refinement 
proceeds best must be the correct one; however if that does not explain all the 
reflections of the lattice then this is not quite satisfactory.

best wishes,

Kay


On Mon, 6 Aug 2018 16:50:56 +0200, Sabine Schneider 
 wrote:

>Dear all,
>
>We are encountering a differences in indexing using different XDS 
>versions, which results in either 2 or 4 mols/asu (SG P1; structure 
>determination via MR (30% seq ID model)) and either successful 
>refinement or stuck R/Rfree
>
>- the data were collected at ESRF ID30A3 (Aiger detector) and extend to 
>about 2.3A resolution (CC1/2 ~50%)
>
>The XDS version build 20180126 (and or autoprocessing at the ESRF via 
>autoproc, dials, xdsapp or grenade -> here also xds version from 
>20180126 used) gives us:
>
>P1  38.65    50.76    61.11 110.057  99.945  90.197
>XDS complains and I need to use "DEFPIX INTEGRATE CORRECT"
>-> 2mols/asu, structure refines to R/Rfree of 22/25
>
>In contrast the actual XDS-Version BUILT=20180409 results in:
>P1  39.2   51.3  116.8  86.3  82.3  89.8
>but XDS runs smoothly.
>-> 4mols/asu, tNCS, R/Rfree stuck at 28/32
>If I feed XDS with the smaller cell above, it fails.
>
>(The smaller cell is also found by Xia2/dials via the CCP4i2 interface.)
>
>Thus I am wondering, what are the differences between the two 
>XDS-versions? (I remember vaguely that there was a tread about different 
>XDS-versions, but couldn't find it..)
>
>Cheers Sabine
>
>
>-- 
>--
>Dr. Sabine Schneider
>Research Group Leader
>Technical University of Munich
>Department of Chemistry
>Chair of Biochemistry
>Lichtenbergstr. 4
>85748 Garching
>Germany
>Tel.: +49 (0) 89 289 13759
>Fax: +49 (0) 89 289 13363
>http://www.biochemie.ch.tum.de/index.php?id=919
>
>-- 
>--
>Dr. Sabine Schneider
>Research Group Leader
>Technical University of Munich
>Department of Chemistry
>Chair of Biochemistry
>Lichtenbergstr. 4
>85748 Garching
>Germany
>Tel.: +49 (0) 89 289 13759
>Fax: +49 (0) 89 289 13363
>http://www.biochemie.ch.tum.de/index.php?id=919
>
>
>
>To unsubscribe from the CCP4BB list, click the following link:
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