Re: [ccp4bb] Methods to improve ligand density of a homodimer?

[Paul Emsley]

On 20/04/2020 20:23, Kyle Gregory wrote:


I am assessing ligand binding and each of the monomers display density at the site but it is not as clear as 
I would like. [] I was wondering if it is feasible, or if there are any 
tools, that can be used to improve density based of the fact there are two molecles present. Is there some 
way to sum (probably the wrong word here) the densities at the binding site?




In Coot:

Calculate -> NCS Maps

The "NCS Average of Chain X Type" is the map you want  (where X is "A" 
typically)

It might be under Extensions in 0.8.9.x

Paul



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

Re: [ccp4bb] Methods to improve ligand density of a homodimer?

[00000c2488af9525-dmarc-request]

I take it that you have 2 molecules in the asymmetric unit. If so, you could try some sort of NCS averaging of the map or just NCS-refinement might help. Not much more here than has been expertly suggested already. Can you give us an idea of the resolution? How many RMS have you contoured the map at?Jon Cooper (pre-polder person)On 20 Apr 2020 20:23, Kyle Gregory <3632e92fcc15-dmarc-requ...@jiscmail.ac.uk> wrote:

Hello all, 





I have a homodimer structure in P1 21 1 spacegroup, the dimer is likely a crystallographic artefact, where it looks like the monomer is rotated by ~ 180 degrees around the Y axis.


I am assessing ligand binding and each of the monomers display density at the site but it is not as clear as I would like. A polder map does help things but I was wondering if it is feasible, or if there are any tools, that can be used to improve density based
 of the fact there are two molecles present. Is there some way to sum (probably the wrong word here) the densities at the binding site?

Kind regards,

Kyle 






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




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

Re: [ccp4bb] Methods to improve ligand density of a homodimer?

[David Schuller]

It sounds like you are looking for MAPROT.

http://www.ccp4.ac.uk/html/maprot.html


On 2020-04-20 15:23, Kyle Gregory wrote:

Hello all,

I have a homodimer structure in P1 21 1 spacegroup, the dimer is 
likely a crystallographic artefact, where it looks like the monomer is 
rotated by ~ 180 degrees around the Y axis.


I am assessing ligand binding and each of the monomers display density 
at the site but it is not as clear as I would like. A polder map does 
help things but I was wondering if it is feasible, or if there are any 
tools, that can be used to improve density based of the fact there are 
two molecles present. Is there some way to sum (probably the wrong 
word here) the densities at the binding site?


Kind regards,

Kyle



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



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




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

Re: [ccp4bb] Methods to improve ligand density of a homodimer?

[Eleanor Dodson]

Hmmm - your space group imposes a too fold screw acid parallel to the Y
axis? If your dimer 2-fold is also parallel to the Y axis you should see  a
strong non crystallographic translation vector? Is that correct? Such
things can complicate density.  Eleanor

On Mon, 20 Apr 2020 at 20:24, Kyle Gregory <
3632e92fcc15-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hello all,
>
> I have a homodimer structure in P1 21 1 spacegroup, the dimer is likely a
> crystallographic artefact, where it looks like the monomer is rotated by ~
> 180 degrees around the Y axis.
>
> I am assessing ligand binding and each of the monomers display density at
> the site but it is not as clear as I would like. A polder map does help
> things but I was wondering if it is feasible, or if there are any tools,
> that can be used to improve density based of the fact there are two
> molecles present. Is there some way to sum (probably the wrong word here)
> the densities at the binding site?
>
> Kind regards,
>
> Kyle
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

[ccp4bb] Methods to improve ligand density of a homodimer?

[Kyle Gregory]

Hello all,

I have a homodimer structure in P1 21 1 spacegroup, the dimer is likely a 
crystallographic artefact, where it looks like the monomer is rotated by ~ 180 
degrees around the Y axis.

I am assessing ligand binding and each of the monomers display density at the 
site but it is not as clear as I would like. A polder map does help things but 
I was wondering if it is feasible, or if there are any tools, that can be used 
to improve density based of the fact there are two molecles present. Is there 
some way to sum (probably the wrong word here) the densities at the binding 
site?

Kind regards,

Kyle



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

Re: [ccp4bb] insect secretion recommendations?

[Olson, Linda]

If your protein is His-tagged: spin down cells @~1500xg, adust pH with tris 
buffer and NaOH to ~pH 7.1 (spin again higher speed if necessary to remove more 
particulates) and pass over chemical resistant Ni-resin.

Linda Olson, PhD
Assistant Professor/
x-Ray Facility Manager
Dept. Biochemistry
Medical College of Wisconsin
8701 Watertown Plank Rd
Milwaukee, WI 53226

phone 414-955-8545
fax  414-456-6510

From: CCP4 bulletin board  on behalf of Gloria Borgstahl 

Sent: Monday, April 20, 2020 10:56 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] insect secretion recommendations?

ATTENTION: This email originated from a sender outside of MCW. Use caution when 
clicking on links or opening attachments.

Hi Friends,  We are secreting Spike Ecto domain into the media from insect 
cells for purification.  As we scale up I am wondering what is recommended for 
collecting the media from large volumes of culture.  Centrifugation?  
Filtration of some kind?  I imagine we need to be gentle to not lyse the insect 
cells.  Thank you, Gloria



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



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

Re: [ccp4bb] insect secretion recommendations?

[Artem Evdokimov]

Gentle spin followed by TFF - a cheap peristaltic pump plus $200 filter
cartridge will do the trick.

Artem

On Mon, Apr 20, 2020, 11:56 AM Gloria Borgstahl 
wrote:

> Hi Friends,  We are secreting Spike Ecto domain into the media from insect
> cells for purification.  As we scale up I am wondering what is recommended
> for collecting the media from large volumes of culture.  Centrifugation?
> Filtration of some kind?  I imagine we need to be gentle to not lyse the
> insect cells.  Thank you, Gloria
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

Re: [ccp4bb] insect secretion recommendations?

[Xavier Brazzolotto]

I used to do a low g centrifugation (100g for about 10min), collect the 
supernatant and filter it onto Polycap units (Whatman) 0,8µm/0,2µm (the size 
will depend on your volume) using a large peristaltic pump (Masterflex).

> Le 20 avr. 2020 à 17:56, Gloria Borgstahl  a écrit :
> 
> Hi Friends,  We are secreting Spike Ecto domain into the media from insect 
> cells for purification.  As we scale up I am wondering what is recommended 
> for collecting the media from large volumes of culture.  Centrifugation?  
> Filtration of some kind?  I imagine we need to be gentle to not lyse the 
> insect cells.  Thank you, Gloria
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 
Xavier Brazzolotto, PhD
xavier.brazzolo...@chemdef.fr

Département de Toxicologie et Risques Chimiques
Unité Neurotoxiques
Institut de Recherche Biomédicale des Armées
1 Place du Général Valérie André, BP 73
91223 Brétigny sur Orge
France

Phone   +33 (0) 1 78 65 14 00
Alt Phone   +33 (0) 4 57 42 87 19
Cell+33 (0) 6 58 36 39 09

The information in this e-mail may be privileged and confidential, intended 
only for the use of the addressee(s) above. Any unauthorized use or disclosure 
of this information is prohibited. If you have received this e-mail by mistake, 
please delete it and immediately contact the sender.

This is not an official email address of the French Ministry of the Armed 
Forces. It does not allow diffusion of protected informations and does not 
necessarily complies to the ministry cyber security policy.




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

[ccp4bb] insect secretion recommendations?

[Gloria Borgstahl]

Hi Friends,  We are secreting Spike Ecto domain into the media from insect
cells for purification.  As we scale up I am wondering what is recommended
for collecting the media from large volumes of culture.  Centrifugation?
Filtration of some kind?  I imagine we need to be gentle to not lyse the
insect cells.  Thank you, Gloria



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

[ccp4bb] Membrane protein crystallographer position at Evotec

[Stephanie Duclos]

Dear CCP4 community,


Evotec (UK) Ltd is currently seeking to add to its Structural Biology 
Department. The group works closely with our Discovery Chemistry Department and 
with clients to develop novel small molecule drugs. It is at the forefront of 
new science and technology, and is seeking to expand as business needs grow.
If you are interested, please apply using this link:
https://evotecgroup.wd3.myworkdayjobs.com/Evotec_Career_Site/job/Abingdon-Evotec/Senior-Scientist---Membrane-Protein-Crystallography_REQ-01707

Best wishes,

Stephanie

STATEMENT OF CONFIDENTIALITY.

This email and any attachments may contain confidential, proprietary, 
privileged and/or private information.  
If received in error, please notify us immediately by reply email and then 
delete this email and any attachments from your system. Thank you!


https://www.evotec.com/en/about/site-information/data-protection-uk

Evotec (UK) Ltd is a limited company registered in England and Wales. 
Registration number:2674265. Registered office: 114 Innovation Drive, Milton 
Park, Abingdon, Oxfordshire, OX14 4RZ, United Kingdom.



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