Re: [ccp4bb] microfluidizer

2025-03-25 Thread M T 
Dear Cyprian,

I recommend to users to use a Potter-Elvehjem homogenizer before to use the 
LM20, or even (in case of non fragile protein) to do some cycle of sonicator.

I advice also to pay attention to the anti-drop ring of the used bottle. 
Because in case of old one, you may have some plastic particles which falls in 
the sample and clog the LM20 cell. Of course, dust is not welcome neither…

To unclog the cell there is no other choice than to invert it. In case of 
severe clogging, the water-bath sonicator can help (gentle heating and 
sonication), before to retry to unclog the cell mounted in a reverse way.

Best.

Michel.

> Le 25 mars 2025 à 07:09, Cyprian Cukier  a écrit :
> 
> 
> Dear Community,
>  
> First of all, apologies for the slightly off-topic question, which is related 
> to protein purification rather than crystallography.
>  
> I am seeking your advice on preparing the cell pellets for the lysis process. 
> We have an LM20 microfluidizer and we experience quite frequent clogging of 
> the system that is tedious to remove. This happens particularly with the E. 
> coli pellets, but from time to time the eukaryotic cells are also 
> problematic. Does anyone have well-established protocols on how to handle the 
> cell pellets before applying them to the lysis process in the microfluidizer?
>  
> I should add that of course we follow the manufacturer's recommendations to 
> have a homogenous suspension, which is not too dense.
>  
> I would also appreciate any advice on how you clean your systems (regularly 
> or in case of clogging).
>  
> Best regards
> Cyprian
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Unexplained electron density

2025-03-25 Thread David J. Schuller 
"The protein was neither crystallized nor purified with any substrate"


On returning to an old project, I discovered that a site believed to be empty 
was actually at around 25% occupancy in the crystal structure, with the native 
ligand. Since the ligand was not added to the crystallization mix, that means 
it was pulled through the purification procedure from the expression system.

Modern purification procedures are much shorter and quicker than in the old 
days. Ammonium sulfate cuts, multiple ion exchange columns and extensive 
dialysis have been replaced by affinity columns. And if you are replacing 
extensive dialysis with centrifugal filters, you shouldn't be surprised if it 
isn't as effective.

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


From: CCP4 bulletin board  on behalf of Manjula Ramu 

Sent: Tuesday, March 25, 2025 16:07
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Unexplained electron density

Dear All,


I have a trimeric protein structure with unexplained electron density in the 
catalytic pocket. The protein was neither crystallized nor purified with any 
substrate, and the buffer and crystallization conditions contained only Tris, 
NaCl, ammonium sulfate, and 3% 2-propanol.

I have attached the images of the electron density and would appreciate any 
insights on identifying this unknown feature.



Thanks and Regards,

Manjula





To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] Unexplained electron density

2025-03-25 Thread Parthasarathy Sampathkumar 
Hi Manjula,
Since this is an enzyme, if you are able to share its expected
functionality then this community might be able to help better.

Best wishes,
Partha

On Tue, Mar 25, 2025 at 1:08 PM Manjula Ramu  wrote:

> Dear All,
>
> I have a trimeric protein structure with unexplained electron density in
> the catalytic pocket. The protein was neither crystallized nor purified
> with any substrate, and the buffer and crystallization conditions contained
> only Tris, NaCl, ammonium sulfate, and 3% 2-propanol.
>
> I have attached the images of the electron density and would appreciate
> any insights on identifying this unknown feature.
>
> 
>
> Thanks and Regards,
>
> *Manjula*
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

[ccp4bb] Open positions in the Structural Biology and Biophysics Unit at Fondazione Ri.MED

2025-03-25 Thread caterina alfano 
Dear all

I would like to bring to your attention two open positions in the
Structural Biology and Biophysics Unit at Fondazione Ri.MED
(*https://www.fondazionerimed.eu/?lang=en
*):

1. Postdoctoral Research Fellowship in Structural Biology applied to Drug
Discovery (*https://www.fondazionerimed.eu/open-position/?lang=en
*, Rif. BIST-NAB/25).
*DEADLINE 06/04/2025*

2. Permanent Position as Senior NMR Specialist
(*https://www.fondazionerimed.eu/open-position/?lang=en
*, Rif. SPIN/25).
*DEADLINE 30/04/2025*

I would appreciate it if you could share this announcement with potential
candidates.

Best regards,

Caterina

**



*Caterina Alfano, PhD*

*Group Leader Structural Biology and Biophysics*

*Fondazione Ri.MED*

c/o ATeN Center – University of Palermo

Viale delle Scienze, Ed. 18/A

90128, Palermo, Italy

Office   +39 091 23893179

calf...@fondazionerimed.com







To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/

Re: [ccp4bb] microfluidizer

2025-03-25 Thread Kovtun, Oleksiy 
Dear all,

A note on high pressure homogeniser designs.

Clogging is the main problem with all fixed geometry disruption channel 
instruments. When selecting a cell disruptor for my laboratory I came across 
the Stansted high pressure variable geometry chamber homogeniser. The 
"disruption" gap is formed between the conical surface of the chamber and a 
needle on an "air suspension". This solution allows particles to push the 
needle off and temporarily widen the chamber gap, preventing blockages.

We have not had a single clogging incident with their SPCH-10 homogeniser after 
3 years of use.

https://www.homogenisingsystems.com/laboratory_high_pressure_homogenizers.html





Oleksiy
sent from mobile

On 25 Mar 2025 8:44 am, M T  wrote:
Dear Cyprian,

I recommend to users to use a Potter-Elvehjem homogenizer before to use the 
LM20, or even (in case of non fragile protein) to do some cycle of sonicator.

I advice also to pay attention to the anti-drop ring of the used bottle. 
Because in case of old one, you may have some plastic particles which falls in 
the sample and clog the LM20 cell. Of course, dust is not welcome neither…

To unclog the cell there is no other choice than to invert it. In case of 
severe clogging, the water-bath sonicator can help (gentle heating and 
sonication), before to retry to unclog the cell mounted in a reverse way.

Best.

Michel.

Le 25 mars 2025 à 07:09, Cyprian Cukier  a écrit :



Dear Community,



First of all, apologies for the slightly off-topic question, which is related 
to protein purification rather than crystallography.



I am seeking your advice on preparing the cell pellets for the lysis process. 
We have an LM20 microfluidizer and we experience quite frequent clogging of the 
system that is tedious to remove. This happens particularly with the E. coli 
pellets, but from time to time the eukaryotic cells are also problematic. Does 
anyone have well-established protocols on how to handle the cell pellets before 
applying them to the lysis process in the microfluidizer?



I should add that of course we follow the manufacturer's recommendations to 
have a homogenous suspension, which is not too dense.



I would also appreciate any advice on how you clean your systems (regularly or 
in case of clogging).



Best regards

Cyprian



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1

This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list 
hosted by www.jiscmail.ac.uk, terms & conditions are available at 
https://www.jiscmail.ac.uk/policyandsecurity/