[ccp4bb] Permanent position on the cryo-electron microscopy facility at Université Paris-Saclay

[THOMPSON Andrew]

Dear all 
I would like to draw your attention to the following opportunity. 
Regards 
Andy 




A permanent position is open to join the cryo-electron microscopy facility at 
Université Paris-Saclay (cryoEM@UPSaclay) in the south of Paris, France. 

cryoEM@UPSaclay is a joint facility between the French synchrotron source 
SOLEIL and the Institute for Integrative Biology of the Cell (I2BC) from CEA 
Joliot Saclay. 

The facility provides state-of-the-art resources in biological cryoEM to the 
Paris-Saclay community, and beyond to academic and private users. We recently 
purchased two microscopes: a Titan Krios G4 and a Glacios2, to be installed at 
SOLEIL and I2BC, respectively. The open position is for a research engineer 
employed by CEA, the French Alternative Energies and Atomic Energy Commission. 
CEA is a key player in fundamental research, development and innovation in 
physics and life sciences, including research in low-carbon energies. The 
successful applicant will operate the Titan Krios at SOLEIL for single particle 
analysis and electron tomography (cryoET). 

He/She will plan and execute experiments for in-house and external users, 
manage and analyse the data, and assist users in performing these steps as 
required. He/She will also take part in the development of new methods in these 
rapidly-expanding fields, and interact with the engineering facilities at 
SOLEIL and at I2BC. Finally, the applicant will be an active contributor to 
develop correlative microscopy, including light microscopy and X-ray imaging 
such as soft X-ray tomography, taking advantage of the opportunities afforded 
by the synchrotron environment. The applicant should have a PhD in physical or 
biological sciences and experiences in cryoEM, with a demonstrated proficiency 
in the use of electron microscopes. 

Experience at a cryoEM facility and in interfacing with users is an important 
plus; expertise in cryo-ET and/or cryo-CLEM is highly desirable. 

Contacts: [ mailto:stephane.bressane...@i2bc.paris-saclay.fr | 
stephane.bressane...@i2bc.paris-saclay.fr ] and [ 
mailto:andrew.thomp...@synchrotron-soleil.fr | 
andrew.thomp...@synchrotron-soleil.fr ] 



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Re: [ccp4bb] Regarding the correct space group identification

[THOMPSON Andrew]

Dear Sayan 
So much has been said about this already. What struck me, in looking at the 
second of your images, is that there is almost certain to be spot overlap (to 
the left / centre of the image) and a chance that the correct unit cell may be 
larger than the indexing suggests. The lower beam divergence at a synchrotron, 
combined with fine sliced images, may tell a different story about the 
indexing. 
Regards 
Andy 


De: "Sayan Saha"  
À: CCP4BB@JISCMAIL.AC.UK 
Envoyé: Jeudi 28 Juillet 2022 18:11:54 
Objet: Re: [ccp4bb] Regarding the correct space group identification 

Dear Sir, 

The detector-to-crystal distance was 190 mm. The Oscillation range was 1.0 
degree. Please find attached two diffraction images. 
With best regards, 
Sayan Saha. 


On Thu, Jul 28, 2022 at 7:31 PM Kay Diederichs < [ 
mailto:kay.diederi...@uni-konstanz.de | kay.diederi...@uni-konstanz.de ] > 
wrote: 


Dear Sayan, 

On Thu, 28 Jul 2022 15:12:30 +0530, Sayan Saha < [ mailto:ssaha43...@gmail.com 
| ssaha43...@gmail.com ] > wrote: 

>Dear Sir, 
> 
>1. There are no ice-rings. However, diffraction spots seem to be 
>overlapping. This can be seen during the data processing, as the space 
>group (C2 or P222) varies even in the consecutive frames. 

spot overlap results in inaccurate intensity values. Inaccurate intensities 
result in high Rwork/Rfree. 

Why do the spots overlap? High mosaicity? Detector distance too small? 
Oscillation range too high (0.1° is typically adequate)? 

It would be good to see the data, otherwise we can only speculate. 

Space group does not change from one frame to the next. If you use XDS, a good 
guide to decide between higher and lower-symmetry space groups is to compare 
their ISa values. 

best, 
Kay 

> 
>2. Crystal packing of C2 and P22121 seem to be similar (please see the 
>attached images). 
> 
>3. Forgot to mention in my previous email that we have already processed 
>the data in P1 and MR solution could be found only in P1 (Phaser was used 
>with an option in all possible space groups of that point group). 
> 
>Please let me know if any other information is required. 
> 
>With best regards, 
>Sayan Saha. 
> 
> 
>On Thu, Jul 28, 2022 at 1:26 PM Schreuder, Herman /DE < 
> [ mailto:herman.schreu...@sanofi.com | herman.schreu...@sanofi.com ] > wrote: 
> 
>> Dear Sayan, 
>> 
>> 
>> 
>> If a subunit is correctly oriented, but the translation is incorrect, 
>> density for a ligand may still show up in the binding site of the protein. 
>> It might be that one of the 2-fold axes, you think is crystallographic, is 
>> in fact non crystallographic and a few Angstroms away from the 
>> crystallographic position. 
>> 
>> 
>> 
>> What I would do: 
>> 
>> 1. Check the images: are there ice-rings or other artifacts that could 
>> cause scaling problems that would lead to high Rw/Rf values? In that case, 
>> there is not much you can do. 
>> 2. Compare the C2 and P22121 solutions: do they have the same overall 
>> crystal packing (CS+NCS), or are they different? Do they have the same 
>> Rw/Rf values? Can we learn anything from the differences in overall crystal 
>> packing? 
>> 3. Process, run MR and refine in P1. Do you get lower R-factors? If 
>> so, then run Zanuda to find out the real space group. 
>> 
>> 
>> 
>> Best, 
>> 
>> Herman 
>> 
>> 
>> 
>> *Von:* CCP4 bulletin board < [ mailto:CCP4BB@JISCMAIL.AC.UK | 
>> CCP4BB@JISCMAIL.AC.UK ] > *Im Auftrag von *Sayan 
>> Saha 
>> *Gesendet:* Donnerstag, 28. Juli 2022 08:15 
>> *An:* [ mailto:CCP4BB@JISCMAIL.AC.UK | CCP4BB@JISCMAIL.AC.UK ] 
>> *Betreff:* [ccp4bb] Regarding the correct space group identification 
>> 
>> 
>> 
>> Dear All, 
>> 
>> 
>> 
>> We have collected home-source X-ray intensity data for a protein at 2.6 
>> Angstrom. The data can be processed in either C2 (a=120, b=80, c=85 and 
>> beta=115) or P222 (P22121, a=80, b=85, c=110). MR solution can be obtained 
>> in both the space groups. However, the solution can be refined with an 
>> Rw/Rf of 29/32% only. The protein is bound to a ligand (co-crystallization) 
>> for which a clear density can be observed. 
>> 
>> 
>> 
>> Any help and suggestion in this regard would be very helpful. 
>> 
>> 
>> 
>> With best regards, 
>> 
>> Sayan Saha. 
>> 
>> 
>> 
>> 
>> -- 
>> 
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> 
> 
> 
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Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[THOMPSON Andrew]

Just thinking out loud and following up Tom's post  - Could prediction be a 
guide to sample preparation for detailed binding studies?
Andy

De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Luca Pellegrini 
[lp...@cam.ac.uk]
Envoyé : vendredi 4 décembre 2020 10:15
À : CCP4BB@JISCMAIL.AC.UK
Objet : Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

Exciting times, indeed. I haven’t looked through the results myself, but it 
does look like an extraordinary advance. I wonder though how this advance 
correlates with ‘understanding’ how proteins folds. Can these outstanding 
results be distilled in a set of improved principles for how proteins fold? Ot 
put it another way, should we invite the AlphaFold programmers to deliver the 
conclusive lecture on the theory of protein folding? Or maybe we should invite 
the algorithm to present its results…

Best wishes,
Luca

Luca Pellegrini, PhD
Department of Biochemistry
University of Cambridge
Cambridge CB2 1GA
UK



On 3 Dec 2020, at 11:17, Isabel Garcia-Saez 
mailto:isabel.gar...@ibs.fr>> wrote:

Dear all,

Just commenting that after the stunning performance of AlphaFold that uses AI 
from Google maybe some of us we could dedicate ourselves to the noble art of 
gardening, baking, doing Chinese Calligraphy, enjoying the clouds pass or 
everything together (just in case I have already prepared my subscription to 
Netflix).

https://www.nature.com/articles/d41586-020-03348-4

Well, I suppose that we still have the structures of complexes (at the moment). 
I am wondering how the labs will have access to this technology in the future 
(would it be for free coming from the company DeepMind - Google?). It seems 
that they have already published some code. Well, exciting times.

Cheers,

Isabel


Isabel Garcia-Saez PhD
Institut de Biologie Structurale
Viral Infection and Cancer Group (VIC)-Cell Division Team
71, Avenue des Martyrs
CS 10090
38044 Grenoble Cedex 9
France
Tel.: 00 33 (0) 457 42 86 15
e-mail: isabel.gar...@ibs.fr
FAX: 00 33 (0) 476 50 18 90
http://www.ibs.fr/




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Re: [ccp4bb] 8/ 2263 spots indexed XDS

[THOMPSON Andrew]

Hi Almudena
I wouldn't claim to be a great expert on XDS, but, based on the experience of 
analyzing a load of data on the beamline,  there are many things you can try -
The most important ones are to use lots (all?) your images for the spot search 
(SPOT_RANGE) , and not to hesitate cutting the resolution limit for the search 
to something very low where the spots are not streaky (3, 4, 5 Angstrom?). 
There may be "good regions" and "bad regions", so you can get the integration 
"going" from a good region. Once things are indexed and analyse OK you can add 
the other data back.
Other interesting keywords will be
STRONG_PIXEL (might need to be bigger or smaller!), 
MAXIMUM_NUMBER_OF_PIXELS_IN_A_SPOT (same) and DELPHI (once you have indexed)
Since you have processed one data set, you can give XDS the correct unit cell, 
that always helps.
Once you have got the integration going, *save* the processing directory as, 
with terrible looking spots, you can get "driven away" from the right indexing 
by the post refinement.
I always add data in "block by block".
I have successfully processed some pretty awful looking data with XDS, but it 
needs a bit of determination and quite a lot of time (hours, not minutes), so 
only invest if the data set is just unique
Good luck
Andy


De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Almudena Ponce 
Salvatierra [maps.fa...@gmail.com]
Envoyé : vendredi 29 novembre 2019 12:48
À : CCP4BB@JISCMAIL.AC.UK
Objet : [ccp4bb] 8/ 2263 spots indexed XDS

Dear all,

I have some data sets that don't want to be processed :p

In one of them, when I look at IDXREF.LP I see virtually none of the found 
spots were indexed and the reason is that they are "too far from the expected 
position". The spots are smeary and elongated, so not the prettiest.

I have managed to process so far only one data set with decent statistics from 
another crystal harvested from the same drop, where the diffraction spots look 
better.

I am trying to find in the xds wiki the keyword I should fine tune in order to 
make those spots indexable.

Could you help me please?

Thank you very much in advance.

Best wishes,

Almu



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Re: [ccp4bb] Se-SAD phasing

[THOMPSON Andrew]

Dear Mario
The data seem very good but you are looking for an awful lot of sites. Keep 
trying with SHELX, but there are some keywords that you might use which may 
help (see extract from SHELX doc). This worked for me for a couple of 
structures with > 100 sites. You should also try with and without Patterson 
seeding.  You may need many thousand trials I would also start with the 
data cutoff somewhere between 3.5 - 4 A (to get rid of anisotropy) . You could 
also try manually modifying the Emin value to use just your strongest signal. 
Don't expect the solution to come out easily - you may have to try different 
shelx runs for days before getting a single solution.
At the same time, you really do need to be absolutely certain of the space 
group ..any tests you can do
Good luck
Andy


For large selenomethionine substructures (which behave more like equal atom ab 
initiostructure solution of small molecules) it may be worth increasing the 
number of Pattersonpeaks used for the Patterson seeding (e.g. PATT 200; the 
default is 100) and adding theinstructions WEED 0.3 (random omit maps) and SKIP 
0.5 (uranium atom removal). Thelatter two are the defaults when PLOP is present 
but are switched off by default if PLOP isabsent. When PATS is used, WEED 
produces a much smaller additional improvement in thehit ratio than when PATS 
is absent. For small substructures (<10 sites), WEED and SKIP cando more harm 
than good by eliminating too many correct sites at once.

De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] De la part de Mario 
Tyago Murakami
Envoyé : jeudi 18 juillet 2019 16:19
À : CCP4BB@JISCMAIL.AC.UK
Objet : [ccp4bb] Se-SAD phasing

Dear all,

I am trying to solve the phases of the following SeMet data, but so far 
unsuccessfully. Suggestions are very welcome. Please see below some details 
about the case.

The statistics below is from a merged data from  different kappas of the same 
crystal to increase redundancy. We used the fixed energy 12675 eV since the 
fluorescence detector was not working at the used beamline to get best energy 
values for this crystal.
Xtriage did not indicate any crystallographic pathology, except moderate 
anisotropy.
The unit cells parameters are 118.72   151.82   167.05  90.000  90.000  90.000 
(P212121) containing from 8 to 12 molecules in the asymmetric unit. The protein 
has ~28.5 kDa and 10 Met residues, excluding those from the N- and C-termini, 
probably with low occupancy. Thus, something 80 to 120 scatterers are expected.
Phenix_anomalous_signal indicates a probability of 99% to solve it and the 
anomalous signal is theoretically in a very good range.
I have tried SHELXD with different resolutions and number of sites. I have also 
used Hyss. But all attempts failed.

Thanks in advance
Mario

[cid:image001.png@01D53D5A.1C2140F0]


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[ccp4bb] Beamline manager post available, SOLEIL PROXIMA 1.

[THOMPSON Andrew]

Hello everyone,
Just to draw your attention to this opening for a beamline manager on the 
PROXIMA 1 beamline at the SOLEIL synchrotron. Gif sur Yvette, France.
Anyone interested should look at the job description on the SOLEIL web pages 
(http://www.synchrotron-soleil.fr/portal/page/portal/Soleil/OffresEmplois/Beamline-Manager-PXI-permanent-position)
Kind regards
Andy Thompson

Re: [ccp4bb] tricky mr problem

[THOMPSON Andrew]

We often use a S-SAD type data collection (there would be no need for 
enormous redundancy) which, if your model is correct, should show up the S 
positions in the same way as a Se-met would show up the Se positions. Good way 
of validating your model and a help to tracing.
Cheers
Andy

De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Ethan A Merritt 
[merr...@u.washington.edu]
Envoyé : lundi 23 septembre 2013 23:25
À : CCP4BB@JISCMAIL.AC.UK
Objet : Re: [ccp4bb] tricky mr problem

On Monday, 23 September, 2013 22:01:32 RHYS GRINTER wrote:
 Hi all,

 I have been attempting to find a MR solution for a low resolution data set 
 (3.9A), with pretty poor merging stats of a 22 strand membrane beta barrel 
 I'm working on.

 I've created a trimmed poly-alanine from a structure of 17% identity, that 
 gives a solution with a Tfz of 14.3 with two molecules per asu (llg is around 
 900). I'm guessing this in a genuine solution, but the map is too poor to 
 build into.

 Does anyone have any advice as to proceed from here? It may be just a case of 
 needing better resolution data to work with, but would this indicate that 
 Selenomet derivative crystals won't be needed for this structure?

Apart from whether you need SeMet for phasing, when you are having trouble 
fitting into
poor maps it can help a lot to have the location of methionines pinned down by 
peaks
in an anomalous difference Fourier map.

Ethan

[ccp4bb] Postdoctoral Positions for collaborative DIAMOND Light Source / Synchrotron - SOLEIL Initiative

[THOMPSON Andrew]

Dear All,

We’d like to draw your attention to three PDRA posts (two at Diamond Light 
Source and one at Soleil) forming part of a collaborative initiative between 
Diamond Light Source in Didcot, UK and the Soleil synchrotron in Gif sur 
Yvette, France to develop methodology and automated analysis pipelines for 
synchrotron macromolecular crystallography:

They are

Diamond Light Source
•   Postdoctoral Researcher in Experimental Phasing Analysis for 
macromolecular crystallography 
(http://diamond.ac.uk/Home/Jobs/Current/DIA0786_CB.html)
•   Postdoctoral Researcher in Multicrystal X-ray Data Analysis for 
macromolecular crystallography 
(http://diamond.ac.uk/Home/Jobs/Current/DIA0785_CB.html)

Soleil
•   Postdoctoral Researcher in Scaling methods and analysis for 
macromolecular crystallography 
(http://www.synchrotron-soleil.fr/images/File/soleil/DivisionAdministration/Personnel/2012/SOLEIL_postDoc_PROXIMA%201_2013.pdf
 )

All three researchers will work closely with beamline scientists and existing 
software developers at Diamond and Soleil to deliver integrated, expert system 
pipelines to assist beamline users in the measurement and analysis of 
diffraction data with a view to providing interpreted electron density at the 
beamline in minutes.


Gwyndaf Evans and Andrew Thompson

[ccp4bb] Reminder : Workshop on Advanced Data Collection with Multi-axis Goniometer and Single-photon Counting Detector

[THOMPSON Andrew]

Just a reminder that the closing date (30th September) for registering for this 
BioSTRUCT-X funded meeting is fast approaching. The meeting is aimed at 
synchrotron users wishing to get the most out of multi-circle goniometers / 
pixel array detectors.




Workshop on Advanced Data Collection with Multi-axis Goniometer and 
Single-photon Counting Detector
http://indico.psi.ch/multiaxisgonio2012
Swiss Light Source, Paul Scherrer Institut, Switzerland
Nov. 6th - 8th, 2012

This workshop will address aspects including application of multi-axis 
goniometer for difficult phasing experiments, data collection optimization for 
single-photon counting detectors, as well as data processing optimization to 
take full advantage of crystal geometry and detector properties. Hardware and 
software developments, as well as user applications will be covered.

The workshop contains both lectures and practicals. Data collection practicals 
will be performed at beamline X06DA at the Swiss Light Source on a multi-axis 
goniometer PRIGo and a PILATUS 2M detector. Data processing tutorials will be 
on the following programs: AutoPROC (XDS/SCALA), PROTEUM, and CrysAlisPro.

There will be no registration fee and all local expenses will be covered, but 
participants are requested to take care of their travel arrangements.

The workshop is limited to 20 participants.

http://indico.psi.ch/multiaxisgonio2012
This workshop is funded by BioStruct-X (WP10.7).

Speakers
Gerard Bricogne, Global Phasing Ltd. 
Sandor Brockhauser, ESRF
Kay Diederichs, Uni. Konstanz
Gwyndaf Evans, Diamond Light Source
Pierre Legrand, SOLEIL
Andrew McCarthy, ESRF
Marcus Muller, Dectris, Ltd.
Vincent Olieric, SLS
Michael Ruf, Bruker AXS
Thomas Schneider, PETRAIII
Clemens Schulze-Briese, Dectris, Ltd.
Tadeusz Skarzynski, Agilent Technologies
Andrew Thompson, SOLEIL
Clemens Vonrhein, Global Phasing Ltd.
Sandro Waltersperger, SLS
Uwe Mueller, BESSY