[ccp4bb] Six year Post-doc at the MX beamline (XALOC) at the ALBA synchrotron, deadline 15/11

[Roeland Boer]

Dear community,

We have an opening for a post-doc at the MX beamline XALOC for a total 
duration of up to six years. We are looking for a highly motivated 
person interested in understanding biology on a structural level and 
that would like to contribute to the field through lab work, methods 
development in crystallography or instrumental improvements on the 
beamline hardware.


ALBA synchrotron has a strong structural biology group, incorporating a 
well-equped biolab (including an AKTA, mosquito crystallization robot, 
crystal farm etc) two MX beamlines,  a Glacios TEM, an X-ray imaging 
beamline and the ability to perform SAXS experiments.


Feel free to drop me a line if you are interested, the full job 
advertisement can be found here: 
https://public.cells.es/jobs/#!/jobs/postdoctoral-researcher-position-at-the-xaloc-beamline


Best regards,

Roeland.



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[ccp4bb] CryoeM RA post Newcastle

[Arnaud Basle]

Dear All,

I am looking for enthusiastic specialists in CryoEM (experience from grid 
preparation to model building) to study molecular mechanisms of transcription – 
various complexes of RNA polymerase with nucleic acids, different factors and 
fellow cellular machineries.

There is a number of projects at various stages of progress, and some projects 
that are yet to start. The positions are available from 01/01/2024. As 
advertised, they are currently quite short (until 31/08/2025) but are 
potentially extendable. The positions will be based in the world-renowned 
Centre for Bacterial Cell Biology of Newcastle University. Some more details 
can be found via these links:

https://jobs.ncl.ac.uk/job/Newcastle-Research-AssistantAssociate/1004031501/
https://www.jobs.ac.uk/job/DDZ793/research-assistant-associate

or by contacting me, Nikolay Zenkin, directly (my email is also in the links).
If you know a good candidate who can start soon, please direct them to me. I 
would also be grateful if you could communicate this advert to the relevant 
people in your structural biology departments.

Prof. Nikolay Zenkin

Cheers,
Arnaud Basle on behalf of Prof. Nikolay Zenkin




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[ccp4bb] problem in sugar refinement with CCPEM

[Gianluca Cioci]

Dear All,

I am trying to refine a cryoEM structure containing a simple glucose 
disaccharide using CCPEM + CCP4-8.0


but I get the following error from Refmac Servalcat :

ERROR : atom :O1   BGC 1  Z4_  is absent in the library
ATTENTION: atom:O1   BGC 2  Z5_  is missing in the structure

and interestingly, the Coot version coming with CCPEM (0.9.8.5) is also 
not able to refine my sugar...


However, If I use CCPEM with the older CCP4-7.1, the refinement works fine.

Any idea ?

With many thanks,

Gia



--
Dr. Gianluca CIOCI
Toulouse Biotechnology Institute (TBI)
https://www.toulouse-biotechnology-institute.fr/en/poles/equipe-cimes/
PICT - Plateforme Intégrée de Criblage de Toulouse
http://www.pict.ipbs.fr/

Tel: +33 (0)5 61 55 97 68
E-mail: ci...@insa-toulouse.fr

TBI - INSA Toulouse
135 avenue de Rangueil
31077 Toulouse CEDEX 04
http://www.toulouse-biotechnology-institute.fr



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Re: [ccp4bb] What could these crystals be?

[Dr. Kevin M Jude]

It’s easy to do on a 0.2 µm spin filter, just pipet the crystals onto the 
filter with excess mother liquor, wash with several spins of mother-liquor, and 
elute with SDS loading buffer. Run all the washes and elution on a gel.

--
Kevin M. Jude, PhD
Structural Biology Research Specialist, Garcia Lab
Howard Hughes Medical Institute
Stanford University School of Medicine
Beckman B177, 279 Campus Drive, Stanford CA 94305


From: CCP4 bulletin board  on behalf of MARCHOT Pascale 

Date: Wednesday, November 8, 2023 at 9:28 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] What could these crystals be?

... and reveal by WB if the protein is tagged.



The trickiest part of all this will be to rinse the "xtal(s)" as extensively as 
possible in a protein&DNA-free solution before dissolving it for SDS-PAGE/WB or 
MS or whichever method you select, in order to avoid contamination by the 
protein-DNA complex present in the drop solution. Paradoxically, the good news 
is that if the xtal collapses/melts easily during the rinsing steps, it will 
mean that it is protein.



Best wishes - Pascale




De : CCP4 bulletin board  de la part de Jurgen Bosch 

Envoyé : mercredi 8 novembre 2023 18:08
À : CCP4BB@JISCMAIL.AC.UK
Objet : Re: [ccp4bb] What could these crystals be?

You could also run them on a gel if you don’t want to shot with canons on small 
birds.

Jürgen



On Nov 8, 2023, at 11:33 AM, M T  wrote:

Dear Careina,

If you have easy access to mass spectrometry, you can try to fish/rince your « 
pumpkin seeds » and send them to mass to try to identify what is inside to see 
if it needs optimization or not.

Best.


Le 8 nov. 2023 à 16:03, 02531c126adf-dmarc-requ...@jiscmail.ac.uk a écrit :

Hi all
We have been trying with no success to crystalize a protein. Recently we got 
these strange shape "crystals". They are hard and flat but they do not diffract 
at all. Any ideas as to what could cause this?
Careina



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[ccp4bb] Making thioester bond in Chimera/ChimeraX

[Firdous Tarique]

Hi

Does anybody know how to make thioester bonds in Chimera or ChimeraX ?

I am unable to make a bond between Cys321 in mol A with Gly1 in mol B
(S-glycyl-L-cysteine) using Chimera or ChimeraX.

I tried Isolde in ChimeraX and Build Structure in Chimera to make a bond
between selected atoms but so far have been unsuccessful. I don't know if I
am missing something.

Any help would be appreciated.

Thanks

Firdous



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Re: [ccp4bb] What could these crystals be?

[Kevin Jin]

Dear Careina,

I usually see such kind of "Quasi-crystal" when the folding force was not
properly adjusted. Of course, you need to verify they are protein ppt
before any further modifications.

For these melon-seed-like crystals,  the sharp-end indicated that some
crystallization occurred. However, the condition needs to be further
modified.

1. For buffer, you need to try different buffers within the similar pH
range 7.0 ~ 7.8. For Tris, the temperature is important.  Different buffer
salts could give you crystals with different morphologies. In general,
sulphate salt is harder. Sodium salt or other cations from buffer salts
should also be counted for the total Sodium/cation concentration. PEG
usually could bring down the pH value.

2. For NaCl, you may try  NH4 salts, Li Salts, Na salts, K salts with
different anion combinations, including Cl, NO3, SO4.

3. For PEG3350, you may try PEG with different molecular weights, 1K -6K.
In your case, I will expect better crystallization with lower MW PEG, since
higher MW may introduce more hydrophobic factors resulting in oil-like
effects. Anyway, you need to try both for comparison.

Different polymers have different pH values, like MME 1.5K, PVP 1.5K, PAS
5.1K. For PAS 5.1K, the pH value is 7.0, and I obtained nice crystals of my
target which I could only get quasi-xtals under PEG 3k.

4. Try 5mM  ZnCl2, which may work too.

Well, there must be a lot of modification strategies ahead. All of my
suggestions here are to fine tune the ionic strength in the conditions
since you are very close to get xals.

Good luck !

Kevin.

PS. You may try Coca or Pepsi + PEG 3K too. I got a hit for DEAD-Box
complex before.






On Wed, Nov 8, 2023 at 7:18 AM stephen.c...@rc-harwell.ac.uk <
8f3604def7f0-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hi Careina,
>
> Without knowing what's in your protein buffer or crystallisation condition
> it's hard to comment.
>
> Best wishes,
> Steve
>
> Dr Stephen Carr
> Research Complex at Harwell (RCaH)
> Rutherford Appleton Laboratory
> Harwell Oxford
> Didcot
> Oxon OX11 0FA
> United Kingdom
> Email stephen.c...@rc-harwell.ac.uk
> tel 01235 567717
> --
> *From:* CCP4 bulletin board  on behalf of
> careinaedgo...@yahoo.com <02531c126adf-dmarc-requ...@jiscmail.ac.uk>
> *Sent:* 08 November 2023 15:00
> *To:* ccp4bb 
> *Subject:* [ccp4bb] What could these crystals be?
>
> Hi all
> We have been trying with no success to crystalize a protein. Recently we
> got these strange shape "crystals". They are hard and flat but they do not
> diffract at all. Any ideas as to what could cause this?
> Careina
>
> --
>
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[ccp4bb] Video link for RSC meeting on "British X-ray Crystallographers."

[Jon Cooper]

Message from the conference organiser:

Dear all,

I am very glad to be able to tell you that all the talks given on 18th October 
have been put up on the RSC Historical Group YouTube playlist:

[https://www.youtube.com/playlist?list=PLLnAFJxOjzZu7N0f5-nVtHcLNxU2tKmpC](https://outlook.office.com/owa/redir.aspx?REF=KhF6iKBxs0JkDI3WrY44a3dAn3iCufRejcJaQY1nDL7uQ25cduHbCAFodHRwczovL3d3dy55b3V0dWJlLmNvbS9wbGF5bGlzdD9saXN0PVBMTG5BRkp4T2p6WnU3TjBmNS1uVnRIY0xOeFUydEttcEM.)

My thanks for Professor Stephen Neidle for recording his talk without an 
audience in the following week and toKathryn Espino of RSC Networks for 
uploading them to YouTube.

Best wishes,

Peter Morris

Best wishes, Jon Cooper.
jon.b.coo...@protonmail.com

Sent with [Proton Mail](https://proton.me/) secure email.



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Re: [ccp4bb] [ccpem] Making thioester bond in Chimera/ChimeraX

[Eric Pettersen]

Hello Firdous,
Probably the problem is that mol A and mol B are two different models.  You 
can't add a bond in Chimera or ChimeraX until after combining them into one 
model first.

ChimeraX combine command:


ChimeraX bond command or Build Structure tool, Adjust Bonds section:



For the best chance of getting an answer to a question about ChimeraX, please 
use the chimerax-us...@cgl.ucsf.edu address:


I hope this helps,

--Eric

Eric Pettersen
UCSF Computer Graphics Lab

> From: Collaborative Computational Project in Electron cryo-Microscopy 
> mailto:cc...@jiscmail.ac.uk>> on behalf of Firdous 
> Tarique mailto:kahkashantari...@gmail.com>>
> Date: Thursday, November 9, 2023 at 12:27 PM
> To: cc...@jiscmail.ac.uk   >
> Subject: [ccpem] Making thioester bond in Chimera/ChimeraX
> 
> Hi
>  
> Does anybody know how to make thioester bonds in Chimera or ChimeraX ?
>  
> I am unable to make a bond between Cys321 in mol A with Gly1 in mol B 
> (S-glycyl-L-cysteine) using Chimera or ChimeraX.
>  
> I tried Isolde in ChimeraX and Build Structure in Chimera to make a bond 
> between selected atoms but so far have been unsuccessful. I don't know if I 
> am missing something.
>  
> Any help would be appreciated.
>  
> Thanks
>  
> Firdous
>  



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Re: [ccp4bb] What could these crystals be?

[sadik sattar]

Hi Careina,

I hope this email finds you well. I wanted to share my experience with a 
similar issue I faced in the past concerning one of my proteins, and I've 
attached a picture of my round crystals.

Despite numerous attempts with various screens and additives, I consistently 
encountered these round crystals. Interestingly, PEG3350 as the precipitant 
seemed to be a common factor in most of these instances. The crystals would 
diffract to approximately 6 or 7A in a home source.

To have a deeper understanding of the situation, I  extensively washed the 
crystals with the mother liquid and then ran them on the gel. The results 
revealed two distinct bands: one corresponding to the expected size of my 
protein and another smaller band. Subsequent Mass-spec confirmed that the 
smaller band represented a truncated version of my protein. It seemed that the 
protein was being cleaved during the course of crystallization and the cleaved 
protein is forming crystals with the intact protein. 

Unfortunately, despite my efforts, I couldn't prevent the unintended cleavage 
of the protein at that time, leading me to abandon the project. I wish I had a 
more definitive solution for you.

Feel free to reach out if you have any questions or if there's anything else I 
can assist you with.

Best regards,
Sadik. 



> On Nov 8, 2023, at 10:00 AM, careinaedgo...@yahoo.com 
> <02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Hi all
> We have been trying with no success to crystalize a protein. Recently we got 
> these strange shape "crystals". They are hard and flat but they do not 
> diffract at all. Any ideas as to what could cause this?
> Careina
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 
> 




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Re: [ccp4bb] What could these crystals be?

[Kevin Jin]

I had the same issue before. PAS should be a decent polymer for
modification.

On Thu, Nov 9, 2023 at 7:04 PM sadik sattar  wrote:

> Hi Careina,
>
> I hope this email finds you well. I wanted to share my experience with a
> similar issue I faced in the past concerning one of my proteins, and I've
> attached a picture of my round crystals.
>
> Despite numerous attempts with various screens and additives, I
> consistently encountered these round crystals. Interestingly, PEG3350 as
> the precipitant seemed to be a common factor in most of these instances.
> The crystals would diffract to approximately 6 or 7A in a home source.
>
> To have a deeper understanding of the situation, I  extensively washed the
> crystals with the mother liquid and then ran them on the gel. The results
> revealed two distinct bands: one corresponding to the expected size of my
> protein and another smaller band. Subsequent Mass-spec confirmed that the
> smaller band represented a truncated version of my protein. It seemed that
> the protein was being cleaved during the course of crystallization and the
> cleaved protein is forming crystals with the intact protein.
>
> Unfortunately, despite my efforts, I couldn't prevent the unintended
> cleavage of the protein at that time, leading me to abandon the project. I
> wish I had a more definitive solution for you.
>
> Feel free to reach out if you have any questions or if there's anything
> else I can assist you with.
>
> Best regards,
> Sadik.
>
>
> On Nov 8, 2023, at 10:00 AM, careinaedgo...@yahoo.com <
> 02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:
>
> Hi all
> We have been trying with no success to crystalize a protein. Recently we
> got these strange shape "crystals". They are hard and flat but they do not
> diffract at all. Any ideas as to what could cause this?
> Careina
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
> 
>
>
>
> --
>
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