Re: [ccp4bb] [External] Re: [ccp4bb] protease inhibitor and 3c protease

[Nikolay Dobrev]

Dear Dhiraj,
yes, on-column cleavage works as well quite efficiently with 3C protease.
I am performing it in a cold room (8-10C) for 1h normally reaches similar 
efficiency.
Depending on your tags (if the 3C has a His-tag) you can add even more protease 
1:50 then for sure 1h is more than enough.
The only thing you need to ensure is that upon cleavage your protein does not 
bind to the IMAC resin due to internal Histidines. If that is the case you can, 
just collect the flow-through and pass it over the beads couple of more times 
to ensure efficient binding of the His-3C protease then you should have a 
highly pure target.
Please note, I am referring to Ni-NTA Agarose beads, which you use in bach mode.
The efficiency will be lower most like if you are using pre-packed columns.

I hope that is helpfull.
Best, Nikolay


> On 02/03/2022 6:56 PM Srivastava, Dhiraj  
> wrote:
> 
> 
> Thanks Nikolay
>  I am doing on column cleavage, so I am 
> incubating it overnight at 4-degree C. Do you think even on column digestion 
> is also going to be done in one hour.
> 
> Thank you
> Dhiraj
> 
> 
> -
> From: CCP4 bulletin board  on behalf of Nikolay 
> Dobrev 
> Sent: Thursday, February 3, 2022 11:53 AM
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: [External] Re: [ccp4bb] protease inhibitor and 3c protease
> 
> Dear Dhiraj,
> the 3C protease is from the Cysteine protease family.
> Therefore for sure, you can add EDTA (if it is not indented for follow up 
> IMAC purification) and that will inhibit most of the metalloprotease. 
> Furthermore, you can resort to Serine type protease inhibitors like PMSF or 
> Aprotinin.
> 
> One side not, the 3C cleavage does not need to be long, as it is an 
> extremely potent protease. If the cleavage sequence is well accessible in 
> less than 1 hour at 4*C you can obtain >95% cleavage when you use 1:100 ratio 
> protease to substrate. Increasing the temperature speeds up the cleavage 
> dramatically and at room temperature, you can obtain the same efficiency for 
> 10 mins approx.
> I am assuming you are interested in using the 3C protease to cleave a tag 
> from recombinantly expressed protein.
> 
> 
> Kind regards,
> Nikolay
> 
> 
> > > On 02/03/2022 6:25 PM Srivastava, Dhiraj 
>  wrote:
> > 
> > 
> > Hi 
> > sorry for off topic question. does anyone know which protease 
> > inhibitors we can include safely while cleaving with 3C protease? 
> > 
> > Thank you
> > Dhiraj
> > 
> > 
> > -
> > 
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> > 
> > > 
> Nikolay Dobrev 
> Postdoctoral Fellow @ Wilmanns group
> EMBL Hamburg, c/o DESY, Building 25A,
> Notkestraße 85, 22607 Hamburg, Germany
> T +49 40 89902 165 | M +49 173 684 0532
> twitter.com/emblevents https://twitter.com/emblevents 
> |http://facebook.com/embl.org  | http://youtube.com/user/emblmedia
> Visit http://www.embl.org/events  for a complete list of all EMBL events.
> 
> 
> 
> -
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> 
> 
> 
> -
> 
> To unsubscribe from the CCP4BB list, click the following link:
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> 

Nikolay Dobrev 
Postdoctoral Fellow @ Wilmanns group
EMBL Hamburg, c/o DESY, Building 25A,
Notkestraße 85, 22607 Hamburg, Germany
T +49 40 89902 165 | M +49 173 684 0532
twitter.com/emblevents https://twitter.com/emblevents 
|http://facebook.com/embl.org  | http://youtube.com/user/emblmedia
Visit http://www.embl.org/events  for a complete list of all EMBL events.



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Re: [ccp4bb] [External] Re: [ccp4bb] protease inhibitor and 3c protease

[Srivastava, Dhiraj]

Thanks Nikolay
 I am doing on column cleavage, so I am incubating it 
overnight at 4-degree C. Do you think even on column digestion is also going to 
be done in one hour.

Thank you
Dhiraj

From: CCP4 bulletin board  on behalf of Nikolay Dobrev 

Sent: Thursday, February 3, 2022 11:53 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] Re: [ccp4bb] protease inhibitor and 3c protease

Dear Dhiraj,
the 3C protease is from the Cysteine protease family.
Therefore for sure, you can add EDTA (if it is not indented for follow up IMAC 
purification) and that will inhibit most of the metalloprotease. Furthermore, 
you can resort to Serine type protease inhibitors like PMSF or Aprotinin.

One side not, the 3C cleavage does not need to be long, as it is an extremely 
potent protease. If the cleavage sequence is well accessible in less than 1 
hour at 4*C you can obtain >95% cleavage when you use 1:100 ratio protease to 
substrate. Increasing the temperature speeds up the cleavage dramatically and 
at room temperature, you can obtain the same efficiency for 10 mins approx.
I am assuming you are interested in using the 3C protease to cleave a tag from 
recombinantly expressed protein.


Kind regards,
Nikolay

On 02/03/2022 6:25 PM Srivastava, Dhiraj  wrote:


Hi
sorry for off topic question. does anyone know which protease inhibitors we 
can include safely while cleaving with 3C protease?

Thank you
Dhiraj



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Nikolay Dobrev
Postdoctoral Fellow @ Wilmanns group
EMBL Hamburg, c/o DESY, Building 25A,
Notkestraße 85, 22607 Hamburg, Germany
T +49 40 89902 165 | M +49 173 684 0532
twitter.com/emblevents | 
facebook.com/embl.org | 
youtube.com/user/emblmedia
Visit www.embl.org/events for a complete list of 
all EMBL events.




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https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



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Re: [ccp4bb] protease inhibitor and 3c protease

[Nikolay Dobrev]

Dear Dhiraj,
the 3C protease is from the Cysteine protease family.
Therefore for sure, you can add EDTA (if it is not indented for follow up IMAC 
purification) and that will inhibit most of the metalloprotease. Furthermore, 
you can resort to Serine type protease inhibitors like PMSF or Aprotinin.

One side not, the 3C cleavage does not need to be long, as it is an extremely 
potent protease. If the cleavage sequence is well accessible in less than 1 
hour at 4*C you can obtain >95% cleavage when you use 1:100 ratio protease to 
substrate. Increasing the temperature speeds up the cleavage dramatically and 
at room temperature, you can obtain the same efficiency for 10 mins approx.
I am assuming you are interested in using the 3C protease to cleave a tag from 
recombinantly expressed protein.


Kind regards,
Nikolay


> On 02/03/2022 6:25 PM Srivastava, Dhiraj  
> wrote:
> 
> 
> Hi 
> sorry for off topic question. does anyone know which protease 
> inhibitors we can include safely while cleaving with 3C protease? 
> 
> Thank you
> Dhiraj
> 
> 
> -
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
> 

Nikolay Dobrev 
Postdoctoral Fellow @ Wilmanns group
EMBL Hamburg, c/o DESY, Building 25A,
Notkestraße 85, 22607 Hamburg, Germany
T +49 40 89902 165 | M +49 173 684 0532
twitter.com/emblevents https://twitter.com/emblevents 
|http://facebook.com/embl.org  | http://youtube.com/user/emblmedia
Visit http://www.embl.org/events  for a complete list of all EMBL events.



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[ccp4bb] protease inhibitor and 3c protease

[Srivastava, Dhiraj]

Hi
sorry for off topic question. does anyone know which protease inhibitors we 
can include safely while cleaving with 3C protease?

Thank you
Dhiraj



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Re: [ccp4bb] Updating Ubuntu 18 LTS to 20?

[Domen Zafred]

Dear Boaz
I'm replying to the bulletin, for others to also benefit from the
conversation.
I was hoping for more (positive) replies and decided I will not upgrade it
for now. I will wait for the 22 or 24 LTS release and then very likely buy
a new SSD for a clean installation, keeping the current set-up intact on
the old drive.
Hope that helps.
BW
Domen

On Sun, Jan 30, 2022 at 4:17 PM Boaz Shaanan  wrote:

> Hi Domen,
>
> Since I am also currently using 18.04, I would be most interested to hear
> about your experience on the upgrade to 20.04, if and when you do this, and
> of course which way you decided to go with the upgrade (I saw the replies
> you received regarding the upgrade modes).
> I would be most grateful.
>
> All the best,
>
>  Boaz
>
>
>
>
>
>
>
>
>
>
>
> *Boaz Shaanan, Ph.D. Dept. of Life
> Sciences  Ben-Gurion University of the
> Negev  Beer-Sheva
> 84105
> Israel
> E-mail:
> bshaa...@bgu.ac.il  Phone: 972-8-647-2220
>Fax:   972-8-647-2992 or 972-8-646-1710*
>
>
>
>
>
> --
> *From:* CCP4 bulletin board  on behalf of Domen
> Zafred 
> *Sent:* Friday, January 14, 2022 10:59 PM
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* [ccp4bb] Updating Ubuntu 18 LTS to 20?
>
> Hi all,
>
> has anyone updated the Ubuntu 18.04 LTS Bionic Beaver to the never 20.04?
> That means with programs we usually have installed (java, phython, xds,
> ccp4 & coot, phenix, R, data warrior, pymol, nomacine, etc)? Any troubles,
> recommendations?
> 22 LTS is coming out this year, I'm not sure if jumping from 18 to 22
> would be a good idea, but 18 is getting old.
>
> Thanks for any hints
>
> Domen
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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[ccp4bb] 2022 3DEM GRC/GRS announcement

[Yanhe Zhao]

Dear CCP4bb community,



The 2022 GRC and GRS for 3DEM will be held at the Rey Don Jaime Grand Hotel in 
Castelldefels, Spain, on June 18-19 (GRS) and June 19-24, 2022 (GRC). These 
meetings will highlight the latest technical and scientific developments in our 
field.

GRC:   
https://www.grc.org/three-dimensional-electron-microscopy-conference/2022/
GRS:   
https://www.grc.org/three-dimensional-electron-microscopy-grs-conference/2022/



Last year's GRC for 3DEM, was one of only a few GRC meetings selected to take 
place - a testament to the dedication and strength of our community. I am 
hopeful that the 2022 3DEM GRC and GRS will also take place as planned.



If you would like to attend, the process is two-step: You need to apply to 
attend. I will start accepting applicants mid-February. If you are accepted, 
then you are sent information to register. If you are not accepted, you will be 
put on a waiting list. The GRC and GRS for 3DEM are traditionally highly 
over-subscribed (at least by a factor of 2x, this year maybe even higher). Many 
highly qualified people will have to be put on the waiting list. However, the 
pandemic and travel restrictions may cause cancellations - like last year - 
thus, I expect that acceptances from the waiting list will be made until close 
to the meeting (please be patient).

IMPORTANT, please provide the following information in your application:

  *   Poster title and abstract (preference will be given to people presenting 
posters [if a meaningful poster title is provided] over those not presenting 
posters).
  *   If you are a student, postdoc or staff member of a research group, PLEASE 
provide the name of the PI of your group (failure to do so will delay review of 
our application).
  *   Please provide information about your research activities, and other 
factors that could be considered during the decision-making process (e.g., 
urgency because you need to apply for a VISA to get to Spain, if you were 
accepted for the 2021 meeting but had to cancel, and so on).

(* if you have already applied, but did not provide this information, please go 
into your application at https://www.grc.org/my-grc/login/ and add it; you 
should be able to edit your poster title and abstract, i.e. you can add 
information like the name of your PI to your poster abstract (which will only 
be visible to the conference chairs).

We aim at a balanced and diverse representation of our community with regard to 
geography, gender, career state, types of research, etc., as well as getting 
wide representation from many different research groups. Please understand that 
making decisions about acceptance for this highly over-subscribed meeting is a 
very difficult task and I apologize sincerely to those who we will not be able 
to accept, because the number of attendees is strictly limited by the GRC and 
venue.

The GRS (for early-stage researchers) has a separate application process. 
Please go to that site to apply there. Acceptance to the GRS does not guarantee 
acceptance to the GRC, or vice-versa. They are separate processes.

You can review special Covid-related information for the 
GRC
 and the 
Venue
 following these links; the information will continue to be updated.

With best regards,
Daniela
(2022 3DEM GRC Chair)

---

Daniela Nicastro, PhD

Professor

UT Southwestern Medical Center

Department of Cell Biology
NL Building (North Campus), Room NL5-108B

6000 Harry Hines Blvd.

Dallas, TX 75390-9039



phone: 001-214-648-3925

email: 
daniela.nicas...@utsouthwestern.edu

https://www.utsouthwestern.edu/labs/nicastro/


__

UT Southwestern

Medical Center

The future of medicine, today.




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[ccp4bb] Senior Scientist position in Structural Biology - Bicycle Therapeutics

[Gustavo Arruda Bezerra]

Dear all,

Bicycle Therapeutics is recruiting a Senior Scientist to join the newly formed 
Structural Biology team. This is a great opportunity to help drive early-stage 
drug discovery programs employing revolutionary technologies to treat a range 
of therapeutic areas.


As one of the founding members, the successful candidate will have the 
opportunity to help shape the future development of the team in addition to 
positively influencing a wide range of drug discovery programs, both internal 
and partnered. You will employ different structural methods to furnish insights 
into target biology and map protein-ligand interactions to power 
structure-guided drug design of our unique Bicycle® peptides. You will work 
closely with cross-functional project teams and make use of CROs and academic 
partners to generate impactful structural data that drives project success and 
scientific publications.

Applications via the link below:
https://jobs.smartrecruiters.com/BicycleTherapeutics/743999801449111-senior-scientist-structural-biology

Best regards,
Gustavo

Team Leader for Protein Crystallography
Centre for Medicines Discovery (CMD)
University of Oxford
Department of Biochemistry
South Parks Road
Oxford OX1 3QU
United Kingdom




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