Re: [ccp4bb] [off topic] Recovering pET expression plasmid from BL21 strain

[Nicholas Clark]

Hi Javier,

For a few years (during an industry job) we regularly cloned directly into
BL21 (we made our own high competency cells) and confirmed via expression
before plasmid sequencing. Only after sequencing did we transform into a
cloning strain for miniprep and “long term storage”.

We didn’t see significant issues with RecA but more so over time we lost
the DNA to nuclease activity (endA). However, this took months to see any
appreciable loss.

If your goal is only plasmid recovery, as Jon stated, your current plan
should work perfectly. Be sure to use the “optional wash” in your miniprep
kit (e.g., Buffer PB in the Qiagen kit) and transform into Top10 within a
month and you shouldn’t have significant issues.

Best,

Nick Clark

Nicholas D. Clark (He/Him)
PhD Candidate
Malkowski Lab
University at Buffalo
Department of Structural Biology
Jacob's School of Medicine & Biomedical Sciences
955 Main Street, RM 5130
Buffalo, NY 14203

Cell: 716-830-1908


On Sun, Feb 18, 2024 at 7:50 PM Javier Gonzalez  wrote:

> Dear all,
> I'm sure this issue comes up very often, but for the first time in our lab
> we need to recover a pET-type expression plasmid from a BL21-like E. coli
> strain (NEB's T7 Express).
> I know a RecA+ strain is not suitable for plasmid production, but the
> basic plan is to grow and mini-prep the cells to recover the plasmid and
> later transform another E coli strain (Top10) to make frozen stocks and for
> plasmid production.
> Is this a regular practice or is there any known protocol we should follow?
> Any advice will be greatly appreciated.
>
> Best wishes,
> Javier
>
> --
> Dr. Javier M. González
> Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
> Universidad Nacional de Santiago del Estero (UNSE)
> RN9, Km 1125. Villa El Zanjón. (G4206XCP)
> Santiago del Estero. Argentina
> Tel: +54-(0385)-4238352
> Email  Twitter
> 
>
>
> --
>
> 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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Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Flavio Di Pisa]

Dear all,
thank you for your valuable suggestions.

Have a good day,
Flavio.

Il 16/02/24 15:36, Karla J. F. Satchell ha scritto:


Sorry did not mean to go off topic. Original question was requests for 
suggestions of cleavable c-term tag vectors. I only meant to provide 
info on one type recommended by another. Others may have further 
suggestions for Flavio.


*From: *CCP4 bulletin board  on behalf of Karla 
J. F. Satchell 

*Date: *Friday, February 16, 2024 at 7:13 AM
*To: *CCP4BB@JISCMAIL.AC.UK 
*Subject: *Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid 
expression vector for E.Coli with a cleavable his-tag at the c-ter


Hi everyone—

Thought I might chime in.

This technology was developed in my lab based on our studies of toxins 
and we have methods papers and two patent.


The CPD-tag works great for production of protein with minimal 
residual residues on the protein. Our studies indicate that it can 
help solubilize protein during expression. Protein is purified with 
the C-terminal CPD tag, and the entire CPD and 6xHis-tag are released 
by addition of InsP6 (phytic acid) which is very inexpensive.


The only restriction of the recognition sequence is small-Leu-small so 
a small residue plus Leucine does remain on your protein. There does 
also need to be a little flexibility at the end of your protein for 
access to the protease so our vectors add extra Gly-Ala so the 
residual is GAAL. CPD is particularly useful for small proteins if the 
additional residues do not interfere with your assays.


https://pubmed.ncbi.nlm.nih.gov/28056928/ 
<https://urldefense.com/v3/__https:/pubmed.ncbi.nlm.nih.gov/28056928/__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HimFkBThs$>


https://pubmed.ncbi.nlm.nih.gov/31773580/ 
<https://urldefense.com/v3/__https:/pubmed.ncbi.nlm.nih.gov/31773580/__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HijC4KPFp$>


https://patents.google.com/patent/US8257946B2/en 
<https://urldefense.com/v3/__https:/patents.google.com/patent/US8257946B2/en__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HirGMggut$>


https://patents.google.com/patent/US8383400B2/en 
<https://urldefense.com/v3/__https:/patents.google.com/patent/US8383400B2/en__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HiqMuRtc4$>


Here is a link to the original paper on mechanism of action of CPD

https://pubmed.ncbi.nlm.nih.gov/19620709/ 
<https://urldefense.com/v3/__https:/pubmed.ncbi.nlm.nih.gov/19620709/__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HinAiY8dQ$>


These are not commercially available as despite multiple marketing 
attempts, there was little interest mid-2010s from biotech for 
licensing new cloning technologies and we abandoned advanced development


If this technology is interesting to you to add to your commercial 
biotech portfolio, please feel free to reach out to me.


Flavio, we can share our vector, but my institution does require an 
MTA as technology is patented. Most people just use synthetic DNA to 
generate the exact clone they want so we rarely get requests but happy 
to share sequences if you need for your synthetic design.


Karla Satchell

*From: *CCP4 bulletin board  on behalf of 
Srivastava, Dhiraj <bda2be8a675a-dmarc-requ...@jiscmail.ac.uk>

*Date: *Friday, February 16, 2024 at 6:42 AM
*To: *CCP4BB@JISCMAIL.AC.UK 
*Subject: *Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid 
expression vector for E.Coli with a cleavable his-tag at the c-ter


Hi Flavio

      While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease 
cleavage site of your choice. However this strategy will results in 
quite a few extra residues from protease site.


An alternative, which is not commercial but available through addgene, 
is cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in 
solubility and expression.


https://www.addgene.org/38251/ 
<https://urldefense.com/v3/__https:/www.addgene.org/38251/__;!!Dq0X2DkFhyF93HkjWTBQKhk!X4zQW9pYePXhpOWDRmDhx8F9CS1fcLsydhbQbLz2By2Pl6vW6Rnzq4kJp8HcdKp1EvVk5oIaXe__iBvZYFJul3k-1u1NgbhjZdL2xUs9Cg$>


There are other variants of this vector available on addgene that you 
can choose from. Depending on your c terminal sequence, it may leave 
either no extra residues or only one or two residues.


Dhiraj



*From:*CCP4 bulletin board  on behalf of Flavio 
Di Pisa 

*Sent:* Friday, February 16, 2024 3:33 AM
*To:* CCP4BB@JISCMAIL.AC.UK 
*Subject:* [External] [ccp4bb] Off topic : plasmid expression vector 
fo

Re: [ccp4bb] [off topic] Recovering pET expression plasmid from BL21 strain

[Jon Cooper]

This should all go fine. You can maxi- or mini-prep the plasmid DNA from the 
expression strain and transform it back into a cloning strain for sequencing, 
etc.

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

Sent from Proton Mail mobile

 Original Message 
On 19 Feb 2024, 00:49, Javier Gonzalez wrote:

> Dear all,
> I'm sure this issue comes up very often, but for the first time in our lab we 
> need to recover a pET-type expression plasmid from a BL21-like E. coli strain 
> (NEB's T7 Express).
> I know a RecA+ strain is not suitable for plasmid production, but the basic 
> plan is to grow and mini-prep the cells to recover the plasmid and later 
> transform another E coli strain (Top10) to make frozen stocks and for plasmid 
> production.
> Is this a regular practice or is there any known protocol we should follow?
> Any advice will be greatly appreciated.
>
> Best wishes,
> Javier
>
> --
>
> Dr. Javier M. González
> Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
> Universidad Nacional de Santiago del Estero (UNSE)
> RN9, Km 1125. Villa El Zanjón. (G4206XCP)
> Santiago del Estero. Argentina
>
> Tel: +54-(0385)-4238352
> [Email](mailto:bio...@gmail.com) [Twitter](https://twitter.com/_biojmg)
>
> ---
>
> 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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hosted by www.jiscmail.ac.uk, terms & conditions are available at 
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[ccp4bb] [off topic] Recovering pET expression plasmid from BL21 strain

[Javier Gonzalez]

Dear all,
I'm sure this issue comes up very often, but for the first time in our lab
we need to recover a pET-type expression plasmid from a BL21-like E. coli
strain (NEB's T7 Express).
I know a RecA+ strain is not suitable for plasmid production, but the basic
plan is to grow and mini-prep the cells to recover the plasmid and later
transform another E coli strain (Top10) to make frozen stocks and for
plasmid production.
Is this a regular practice or is there any known protocol we should follow?
Any advice will be greatly appreciated.

Best wishes,
Javier

-- 
Dr. Javier M. González
Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET)
Universidad Nacional de Santiago del Estero (UNSE)
RN9, Km 1125. Villa El Zanjón. (G4206XCP)
Santiago del Estero. Argentina
Tel: +54-(0385)-4238352
Email  Twitter 



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=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 
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Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Karla J. F. Satchell]

Sorry did not mean to go off topic. Original question was requests for 
suggestions of cleavable c-term tag vectors. I only meant to provide info on 
one type recommended by another. Others may have further suggestions for Flavio.

From: CCP4 bulletin board  on behalf of Karla J. F. 
Satchell 
Date: Friday, February 16, 2024 at 7:13 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector 
for E.Coli with a cleavable his-tag at the c-ter
Hi everyone—

Thought I might chime in.
This technology was developed in my lab based on our studies of toxins and we 
have methods papers and two patent.

The CPD-tag works great for production of protein with minimal residual 
residues on the protein. Our studies indicate that it can help solubilize 
protein during expression. Protein is purified with the C-terminal CPD tag, and 
the entire CPD and 6xHis-tag are released by addition of InsP6 (phytic acid) 
which is very inexpensive.

The only restriction of the recognition sequence is small-Leu-small so a small 
residue plus Leucine does remain on your protein. There does also need to be a 
little flexibility at the end of your protein for access to the protease so our 
vectors add extra Gly-Ala so the residual is GAAL. CPD is particularly useful 
for small proteins if the additional residues do not interfere with your assays.

https://pubmed.ncbi.nlm.nih.gov/28056928/<https://urldefense.com/v3/__https:/pubmed.ncbi.nlm.nih.gov/28056928/__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HimFkBThs$>
https://pubmed.ncbi.nlm.nih.gov/31773580/<https://urldefense.com/v3/__https:/pubmed.ncbi.nlm.nih.gov/31773580/__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HijC4KPFp$>

https://patents.google.com/patent/US8257946B2/en<https://urldefense.com/v3/__https:/patents.google.com/patent/US8257946B2/en__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HirGMggut$>
https://patents.google.com/patent/US8383400B2/en<https://urldefense.com/v3/__https:/patents.google.com/patent/US8383400B2/en__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HiqMuRtc4$>

Here is a link to the original paper on mechanism of action of CPD
https://pubmed.ncbi.nlm.nih.gov/19620709/<https://urldefense.com/v3/__https:/pubmed.ncbi.nlm.nih.gov/19620709/__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HinAiY8dQ$>

These are not commercially available as despite multiple marketing attempts, 
there was little interest mid-2010s from biotech for licensing new cloning 
technologies and we abandoned advanced development

If this technology is interesting to you to add to your commercial biotech 
portfolio, please feel free to reach out to me.

Flavio, we can share our vector, but my institution does require an MTA as 
technology is patented. Most people just use synthetic DNA to generate the 
exact clone they want so we rarely get requests but happy to share sequences if 
you need for your synthetic design.

Karla Satchell


From: CCP4 bulletin board  on behalf of Srivastava, 
Dhiraj <bda2be8a675a-dmarc-requ...@jiscmail.ac.uk>
Date: Friday, February 16, 2024 at 6:42 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector 
for E.Coli with a cleavable his-tag at the c-ter
Hi Flavio
While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease cleavage 
site of your choice. However this strategy will results in quite a few extra 
residues from protease site.
An alternative, which is not commercial but available through addgene, is 
cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in solubility 
and expression.

https://www.addgene.org/38251/<https://urldefense.com/v3/__https:/www.addgene.org/38251/__;!!Dq0X2DkFhyF93HkjWTBQKhk!X4zQW9pYePXhpOWDRmDhx8F9CS1fcLsydhbQbLz2By2Pl6vW6Rnzq4kJp8HcdKp1EvVk5oIaXe__iBvZYFJul3k-1u1NgbhjZdL2xUs9Cg$>

There are other variants of this vector available on addgene that you can 
choose from. Depending on your c terminal sequence, it may leave either no 
extra residues or only one or two residues.

Dhiraj

From: CCP4 bulletin board  on behalf of Flavio Di Pisa 

Sent: Friday, February 16, 2024 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli 
with a cleavable his-tag at the c-ter

Dear community,

I'm looking for a commercial expression vector for E.Coli with a
cleavable his-tag at the c-ter. Anyone c

Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Karla J. F. Satchell]

Hi everyone—

Thought I might chime in.
This technology was developed in my lab based on our studies of toxins and we 
have methods papers and two patent.

The CPD-tag works great for production of protein with minimal residual 
residues on the protein. Our studies indicate that it can help solubilize 
protein during expression. Protein is purified with the C-terminal CPD tag, and 
the entire CPD and 6xHis-tag are released by addition of InsP6 (phytic acid) 
which is very inexpensive.

The only restriction of the recognition sequence is small-Leu-small so a small 
residue plus Leucine does remain on your protein. There does also need to be a 
little flexibility at the end of your protein for access to the protease so our 
vectors add extra Gly-Ala so the residual is GAAL. CPD is particularly useful 
for small proteins if the additional residues do not interfere with your assays.

https://pubmed.ncbi.nlm.nih.gov/28056928/
https://pubmed.ncbi.nlm.nih.gov/31773580/

https://patents.google.com/patent/US8257946B2/en
https://patents.google.com/patent/US8383400B2/en

Here is a link to the original paper on mechanism of action of CPD
https://pubmed.ncbi.nlm.nih.gov/19620709/

These are not commercially available as despite multiple marketing attempts, 
there was little interest mid-2010s from biotech for licensing new cloning 
technologies and we abandoned advanced development

If this technology is interesting to you to add to your commercial biotech 
portfolio, please feel free to reach out to me.

Flavio, we can share our vector, but my institution does require an MTA as 
technology is patented. Most people just use synthetic DNA to generate the 
exact clone they want so we rarely get requests but happy to share sequences if 
you need for your synthetic design.

Karla Satchell


From: CCP4 bulletin board  on behalf of Srivastava, 
Dhiraj <bda2be8a675a-dmarc-requ...@jiscmail.ac.uk>
Date: Friday, February 16, 2024 at 6:42 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector 
for E.Coli with a cleavable his-tag at the c-ter
Hi Flavio
While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease cleavage 
site of your choice. However this strategy will results in quite a few extra 
residues from protease site.
An alternative, which is not commercial but available through addgene, is 
cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in solubility 
and expression.

https://www.addgene.org/38251/<https://urldefense.com/v3/__https:/www.addgene.org/38251/__;!!Dq0X2DkFhyF93HkjWTBQKhk!X4zQW9pYePXhpOWDRmDhx8F9CS1fcLsydhbQbLz2By2Pl6vW6Rnzq4kJp8HcdKp1EvVk5oIaXe__iBvZYFJul3k-1u1NgbhjZdL2xUs9Cg$>

There are other variants of this vector available on addgene that you can 
choose from. Depending on your c terminal sequence, it may leave either no 
extra residues or only one or two residues.

Dhiraj

From: CCP4 bulletin board  on behalf of Flavio Di Pisa 

Sent: Friday, February 16, 2024 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli 
with a cleavable his-tag at the c-ter

Dear community,

I'm looking for a commercial expression vector for E.Coli with a
cleavable his-tag at the c-ter. Anyone can help me?

Thank you in advance,

Flavio.



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1<https://urldefense.com/v3/__https:/www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1__;!!Dq0X2DkFhyF93HkjWTBQKhk!X4zQW9pYePXhpOWDRmDhx8F9CS1fcLsydhbQbLz2By2Pl6vW6Rnzq4kJp8HcdKp1EvVk5oIaXe__iBvZYFJul3k-1u1NgbhjZdKw_JbGJA$>

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Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Srivastava, Dhiraj]

Hi Flavio
While i am not aware of any commercial vector with cleavable c 
terminal tag, you can easily make your own by introducing protease cleavage 
site of your choice. However this strategy will results in quite a few extra 
residues from protease site.
An alternative, which is not commercial but available through addgene, is 
cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in solubility 
and expression.

https://www.addgene.org/38251/

There are other variants of this vector available on addgene that you can 
choose from. Depending on your c terminal sequence, it may leave either no 
extra residues or only one or two residues.

Dhiraj

From: CCP4 bulletin board  on behalf of Flavio Di Pisa 

Sent: Friday, February 16, 2024 3:33 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli 
with a cleavable his-tag at the c-ter

Dear community,

I'm looking for a commercial expression vector for E.Coli with a
cleavable his-tag at the c-ter. Anyone can help me?

Thank you in advance,

Flavio.



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] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter

[Flavio Di Pisa]

Dear community,

I'm looking for a commercial expression vector for E.Coli with a 
cleavable his-tag at the c-ter. Anyone can help me?


Thank you in advance,

Flavio.



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Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Irwin Selvam]

Hi,

In my hands, I have found the addition of TCEP (~0.5 mM) helps with TEV 
cleavage efficiency, especially reactions at 4C where TEV is much less 
efficient anyway. The caveat is that my targets may have been "happier" with 
TCEP present, therefore encouraging more efficient cleavage. The variance in 
what you've been able to find online, and reflected in the responses thus far, 
could be down to different labs using different TEV constructs. Older 
constructs seem to be more sensitive to reducing agent presence whereas 
optimised versions of TEV are much more efficient so any drop off is less 
noticeable. It may be worth looking at your cleavage buffer composition too - 
not too much salt, glycerol, detergents etc

As for on-column TEV cleavage, if your TEV is also His-tagged then it will 
likely be less efficient. If you're willing to try a new construct, I have had 
great success with on-column cleavage using target bound to Streptactin XT 
resin. If you want to stick to Ni-based IMAC, Cytiva's Ni Excel, BioRad's 
Profinity and Protein Ark's Fastback Ni Advance all leach less than 
conventional Ni-NTA/IDA under less than ideal buffer conditions.

Good luck!

Irwin

From: CCP4 bulletin board  on behalf of Hughes, Jonathan 

Sent: 02 November 2023 09:17:30
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] AW: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

(this is from a different jon)
if a chelator really is necessary at this stage (post AmS?), in any case use 
IDA instead of EDTA! that's pretty obvious from the affinities of EDTA/NTA/IDA.
cheers
jon

Von: CCP4 bulletin board  Im Auftrag von Jonathan Bailey
Gesendet: Mittwoch, 1. November 2023 19:53
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

I have always performed TEV cleavage after eluting my protein from the Ni-NTA 
column (TEV cleavage performed during overnight dialysis in imidazole free 
buffer as imidazole inhibits TEV activity at high concentrations). I use TCEP 
as reducing agent and have never included EDTA, by this method I've never had a 
problem with membrane or soluble proteins and get almost 100 % cleavage of the 
tag.

Best,

Jon





On Tue, 31 Oct 2023 at 19:21, Rafael Marques 
mailto:rafael_mmsi...@hotmail.com>> wrote:
Hi everyone,

I have been looking on this bb and other websites as well but I could not find 
a veredict. We are suspecting that when I elute my sample from my Ni-NTA 
column, the imidazole concentration (250 mM) is making it to precipitate. Once 
my sample has a cleavable TEV site, I was planning to incubate my loaded resin 
overnight with TEV and get my sample back simply using my lysis buffer. And 
here lies the problem. Most of the TEVs are kept in EDTA and DTT and I wonder 
if they are essential for its protease activity or if I could use another 
reducing agent more compatible with my resin (or maybe do not add both). I saw 
that someone did not have EDTA and used b-mercap. instead of DTT. May I have 
your comments if you guys already faced a similar situation?

Best wishes

__

Rafael Marques da Silva

PhD Student – Structural Biology

University of Leicester

Mestrando em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"




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[ccp4bb] AW: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Hughes, Jonathan]

(this is from a different jon)
if a chelator really is necessary at this stage (post AmS?), in any case use 
IDA instead of EDTA! that's pretty obvious from the affinities of EDTA/NTA/IDA.
cheers
jon

Von: CCP4 bulletin board  Im Auftrag von Jonathan Bailey
Gesendet: Mittwoch, 1. November 2023 19:53
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

I have always performed TEV cleavage after eluting my protein from the Ni-NTA 
column (TEV cleavage performed during overnight dialysis in imidazole free 
buffer as imidazole inhibits TEV activity at high concentrations). I use TCEP 
as reducing agent and have never included EDTA, by this method I've never had a 
problem with membrane or soluble proteins and get almost 100 % cleavage of the 
tag.

Best,

Jon





On Tue, 31 Oct 2023 at 19:21, Rafael Marques 
mailto:rafael_mmsi...@hotmail.com>> wrote:
Hi everyone,

I have been looking on this bb and other websites as well but I could not find 
a veredict. We are suspecting that when I elute my sample from my Ni-NTA 
column, the imidazole concentration (250 mM) is making it to precipitate. Once 
my sample has a cleavable TEV site, I was planning to incubate my loaded resin 
overnight with TEV and get my sample back simply using my lysis buffer. And 
here lies the problem. Most of the TEVs are kept in EDTA and DTT and I wonder 
if they are essential for its protease activity or if I could use another 
reducing agent more compatible with my resin (or maybe do not add both). I saw 
that someone did not have EDTA and used b-mercap. instead of DTT. May I have 
your comments if you guys already faced a similar situation?

Best wishes

__

Rafael Marques da Silva

PhD Student – Structural Biology

University of Leicester

Mestrando em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"




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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Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Jonathan Bailey]

I have always performed TEV cleavage after eluting my protein from the
Ni-NTA column (TEV cleavage performed during overnight dialysis in
imidazole free buffer as imidazole inhibits TEV activity at high
concentrations). I use TCEP as reducing agent and have never included EDTA,
by this method I've never had a problem with membrane or soluble proteins
and get almost 100 % cleavage of the tag.

Best,

Jon





On Tue, 31 Oct 2023 at 19:21, Rafael Marques 
wrote:

> Hi everyone,
>
> I have been looking on this bb and other websites as well but I could not
> find a veredict. We are suspecting that when I elute my sample from my
> Ni-NTA column, the imidazole concentration (250 mM) is making it to
> precipitate. Once my sample has a cleavable TEV site, I was planning to
> incubate my loaded resin overnight with TEV and get my sample back simply
> using my lysis buffer. And here lies the problem. Most of the TEVs are kept
> in EDTA and DTT and I wonder if they are essential for its protease
> activity or if I could use another reducing agent more compatible with my
> resin (or maybe do not add both). I saw that someone did not have EDTA and
> used b-mercap. instead of DTT. May I have your comments if you guys already
> faced a similar situation?
>
> Best wishes
>
> __
>
> Rafael Marques da Silva
>
> PhD Student – Structural Biology
>
> University of Leicester
>
> Mestrando em Física Biomolecular
> Universidade de São Paulo
>
> Bacharel em Ciências Biológicas
> Universidade Federal de São Carlos
>
> phone: +55 16 99766-0021
>
> *   "A sorte acompanha uma mente bem treinada"*
> **
>
> --
>
> 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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Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Prof. Dr. Arne Skerra]

Hi Rafael,

In this case I recommend the use of Zn-charged Chelating Sepharose™ Fast 
Flow (Cytiva), as we have established it in the early days when first 
applying IMAC to the purification of native antibody fragments (see 
PMID: 1367302 and PMID: 8163179).


While the Zn-IDA matrix has slightly lower affinity towards the His6-tag 
than Ni-NTA it provides better selectivity and we still use it today for 
purifying sensitive proteins including the presence of reducing agents.


Importantly, in contrast to Ni(II), Zn(II) is essentially 
redox-inactive, so it does not catalyze the oxidation of thiol groups 
and just forms reversible complexes. Furthermore, Zn(II) is non-toxic 
and does not cause allergic reactions.


Cheers, Arne



Am 31.10.23 um 23:05 schrieb Thomas Edwards:

Hi Rafael, Dom et al,

Indeed nickel eluted with imidazole stays on all your proteins with a 
His tag, and addition of DTT will make almost all proteins go brown 
and crap out at this point. But, as Dom says, addition of EDTA 
*before* the DTT will solve the problem. Most of the time…


If your protein goes brown after a nickel column and you thought it 
was something special to your protein, try again..!


If your protein can’t handle EDTA, try the pH trick suggested in 
another post.


If none of that works, then move to GST or some such…

*/Ed/*
https://www.ularkin.org/team-member/thomas-edwards/
Sent from my iPhone. On the run…


On 31 Oct 2023, at 16:48, Dom Bellini  wrote:

 Hi Rafael,

Once I inherited a protocol with a problem similar to yours and they 
told me that the precipitation was caused by nickel leaking out 
during the elution with 250 mM imidazole. I am not sure whether this 
was true, however, their fix was to place something like 20 ul of 200 
mM EDTA at the bottom of the collection tubes into which the protein 
was eluting. This indeed kept the protein soluble, at least long 
enough to dialyse or gel filtration.


Good luck!

D

On 31 Oct 2023, at 19:21, Rafael Marques 
 wrote:



CAUTION: This email originated from outside of the LMB.
Do not click links or open attachments unless you recognize the 
sender and know the content is safe.

*.-owner-ccp...@jiscmail.ac.uk-.*
Hi everyone,

I have been looking on this bb and other websites as well but I 
could not find a veredict. We are suspecting that when I elute my 
sample from my Ni-NTA column, the imidazole concentration (250 mM) 
is making it to precipitate. Once my sample has a cleavable TEV 
site, I was planning to incubate my loaded resin overnight with TEV 
and get my sample back simply using my lysis buffer. And here lies 
the problem. Most of the TEVs are kept in EDTA and DTT and I wonder 
if they are essential for its protease activity or if I could use 
another reducing agent more compatible with my resin (or maybe do 
not add both). I saw that someone did not have EDTA and used 
b-mercap. instead of DTT. May I have your comments if you guys 
already faced a similar situation?


Best wishes

__

Rafael Marques da Silva

PhD Student – Structural Biology

University of Leicester


Mestrando em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

/           "A sorte acompanha uma mente bem treinada"/
//



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







--
Prof. Dr. Arne Skerra
Lehrstuhl f. Biologische Chemie  |  Technische Universitaet Muenchen
Emil-Erlenmeyer-Forum 5  |  85354 Freising (Weihenstephan)  |  Germany
Phone: +49 8161 71 4351  |  Fax: 4352
eMail: ske...@tum.de  |  http://biologische-chemie.userweb.mwn.de



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Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Artem Evdokimov]

Dear Rafael

In addition to the excellent suggestions already offered by previous
responders, I can attest that Ni-penta resin (sold among others by a
company with an odd name Marvelgent) is resistant to EDTA and DTT, and it
leaks very little Ni (almost none). Plus, it elutes with low inidazole,
owing to the single available chelation point offered by the immobilized Ni
ions.

Depending on the resin and other conditions tou use, there generally is no
reason to worry about the small amount of DTT and EDTA that may be present
in TEV preparations. On the other hand, there is a reason to be slightly
concerned that the leaky Ni ions may inhibit your TEV.

In summary, you have to try it on small scale, the odds are good that
cleavage on resin will work for you. Notably it even works when TEV itself
is his-tagged, presumably owing ro the dynamic nature of His/Ni
interactions (strictly speaking, resin affinity is only about 1 uM, but
avidity and intra resin exchange and recapture keep the bulk of protein
trapped in the resin).

Good luck!

Artem

On Tue, Oct 31, 2023, 3:21 PM Rafael Marques 
wrote:

> Hi everyone,
>
> I have been looking on this bb and other websites as well but I could not
> find a veredict. We are suspecting that when I elute my sample from my
> Ni-NTA column, the imidazole concentration (250 mM) is making it to
> precipitate. Once my sample has a cleavable TEV site, I was planning to
> incubate my loaded resin overnight with TEV and get my sample back simply
> using my lysis buffer. And here lies the problem. Most of the TEVs are kept
> in EDTA and DTT and I wonder if they are essential for its protease
> activity or if I could use another reducing agent more compatible with my
> resin (or maybe do not add both). I saw that someone did not have EDTA and
> used b-mercap. instead of DTT. May I have your comments if you guys already
> faced a similar situation?
>
> Best wishes
>
> __
>
> Rafael Marques da Silva
>
> PhD Student – Structural Biology
>
> University of Leicester
>
> Mestrando em Física Biomolecular
> Universidade de São Paulo
>
> Bacharel em Ciências Biológicas
> Universidade Federal de São Carlos
>
> phone: +55 16 99766-0021
>
> *   "A sorte acompanha uma mente bem treinada"*
> **
>
> --
>
> 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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Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Thomas Edwards]

Hi Rafael, Dom et al,

Indeed nickel eluted with imidazole stays on all your proteins with a His tag, 
and addition of DTT will make almost all proteins go brown and crap out at this 
point. But, as Dom says, addition of EDTA before the DTT will solve the 
problem. Most of the time…

If your protein goes brown after a nickel column and you thought it was 
something special to your protein, try again..!

If your protein can’t handle EDTA, try the pH trick suggested in another post.

If none of that works, then move to GST or some such…

Ed
https://www.ularkin.org/team-member/thomas-edwards/
Sent from my iPhone. On the run…

On 31 Oct 2023, at 16:48, Dom Bellini  wrote:

 Hi Rafael,

Once I inherited a protocol with a problem similar to yours and they told me 
that the precipitation was caused by nickel leaking out during the elution with 
250 mM imidazole. I am not sure whether this was true, however, their fix was 
to place something like 20 ul of 200 mM EDTA at the bottom of the collection 
tubes into which the protein was eluting. This indeed kept the protein soluble, 
at least long enough to dialyse or gel filtration.

Good luck!

D

On 31 Oct 2023, at 19:21, Rafael Marques  wrote:


CAUTION: This email originated from outside of the LMB.
Do not click links or open attachments unless you recognize the sender and know 
the content is safe.
.-owner-ccp...@jiscmail.ac.uk-.

Hi everyone,

I have been looking on this bb and other websites as well but I could not find 
a veredict. We are suspecting that when I elute my sample from my Ni-NTA 
column, the imidazole concentration (250 mM) is making it to precipitate. Once 
my sample has a cleavable TEV site, I was planning to incubate my loaded resin 
overnight with TEV and get my sample back simply using my lysis buffer. And 
here lies the problem. Most of the TEVs are kept in EDTA and DTT and I wonder 
if they are essential for its protease activity or if I could use another 
reducing agent more compatible with my resin (or maybe do not add both). I saw 
that someone did not have EDTA and used b-mercap. instead of DTT. May I have 
your comments if you guys already faced a similar situation?

Best wishes

__

Rafael Marques da Silva

PhD Student – Structural Biology

University of Leicester

Mestrando em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"




To unsubscribe from the CCP4BB list, click the following link:
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Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[David Briggs]

Hi Rafael,

For completeness -  there are alternatives to imidazole for eluting proteins 
from Ni-NTA resins:

EDTA - (strips Ni2+, and therefore everything bound to the Ni2+) - 50mM should 
be sufficient, in your favourite buffer/salt system. Obviously not appropriate 
for metalloproteins. You'll need to recharge the column/resin afterwards.

Low pH - Citrate or Acetate buffer with a pH lower than 5.5 (lower still for 
multimers) with an appropriate salt concentration. Obviously test this on a 
small scale first to see if your protein tolerates the drop in pH. You can 
reduce the time of exposure to low pH by eluting the protein straight into some 
1M Tris or HEPES to bring the pH back up to something more neutral.

Hth,

Dave


Dr David C. Briggs CSci MRSB

Principal Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

about.me/david_briggs<http://about.me/david_briggs>


From: CCP4 bulletin board  on behalf of Rafael Marques 

Sent: Tuesday, October 31, 2023 7:21:21 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage


External Sender: Use caution.

Hi everyone,

I have been looking on this bb and other websites as well but I could not find 
a veredict. We are suspecting that when I elute my sample from my Ni-NTA 
column, the imidazole concentration (250 mM) is making it to precipitate. Once 
my sample has a cleavable TEV site, I was planning to incubate my loaded resin 
overnight with TEV and get my sample back simply using my lysis buffer. And 
here lies the problem. Most of the TEVs are kept in EDTA and DTT and I wonder 
if they are essential for its protease activity or if I could use another 
reducing agent more compatible with my resin (or maybe do not add both). I saw 
that someone did not have EDTA and used b-mercap. instead of DTT. May I have 
your comments if you guys already faced a similar situation?

Best wishes

__

Rafael Marques da Silva

PhD Student – Structural Biology

University of Leicester

Mestrando em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"




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

The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 Midland Road London NW1 1AT



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Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Dom Bellini]

Hi Rafael,

Once I inherited a protocol with a problem similar to yours and they told me 
that the precipitation was caused by nickel leaking out during the elution with 
250 mM imidazole. I am not sure whether this was true, however, their fix was 
to place something like 20 ul of 200 mM EDTA at the bottom of the collection 
tubes into which the protein was eluting. This indeed kept the protein soluble, 
at least long enough to dialyse or gel filtration. 

Good luck!

D

> On 31 Oct 2023, at 19:21, Rafael Marques  wrote:
> 
> 
> CAUTION: This email originated from outside of the LMB.
> Do not click links or open attachments unless you recognize the sender and 
> know the content is safe.
> .-owner-ccp...@jiscmail.ac.uk-.
> Hi everyone, 
> 
> I have been looking on this bb and other websites as well but I could not 
> find a veredict. We are suspecting that when I elute my sample from my Ni-NTA 
> column, the imidazole concentration (250 mM) is making it to precipitate. 
> Once my sample has a cleavable TEV site, I was planning to incubate my loaded 
> resin overnight with TEV and get my sample back simply using my lysis buffer. 
> And here lies the problem. Most of the TEVs are kept in EDTA and DTT and I 
> wonder if they are essential for its protease activity or if I could use 
> another reducing agent more compatible with my resin (or maybe do not add 
> both). I saw that someone did not have EDTA and used b-mercap. instead of 
> DTT. May I have your comments if you guys already faced a similar situation? 
> 
> Best wishes
> 
> __
> 
> Rafael Marques da Silva
> PhD Student – Structural Biology
> University of Leicester
> 
> Mestrando em Física Biomolecular
> Universidade de São Paulo
> 
> Bacharel em Ciências Biológicas
> Universidade Federal de São Carlos
> 
> phone: +55 16 99766-0021
> 
>"A sorte acompanha uma mente bem treinada"
> 
> 
> 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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Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Nikolay Dobrev]

Hi Rafael,
the simple answer is that the EDTA and DTT( or any other reducing
agent) is not required for the TEV activity.
You can have your TEV protease in 20 mM Tris pH 7.5 (RT) and 150 mM
NaCl, plus either 10% Glycerol or 50% glycerol depending on storage
conditions preference - 80 or -20C respectively.

Normally you need to take care that you are dilating the TEV several
fold (at least 5) in order to drop the Glycerol concentration below
10% not to have an inhibitory effect on the activity.
Also your on-column cleavage buffer should not contain higher than 300
mM salt, otherwise the TEV activity will be decreased as well.

Go ahead and give it a try.

Good luck!
Kind regards,
Nikolay

On Tue, Oct 31, 2023 at 3:21 PM Rafael Marques
 wrote:
>
> Hi everyone,
>
> I have been looking on this bb and other websites as well but I could not 
> find a veredict. We are suspecting that when I elute my sample from my Ni-NTA 
> column, the imidazole concentration (250 mM) is making it to precipitate. 
> Once my sample has a cleavable TEV site, I was planning to incubate my loaded 
> resin overnight with TEV and get my sample back simply using my lysis buffer. 
> And here lies the problem. Most of the TEVs are kept in EDTA and DTT and I 
> wonder if they are essential for its protease activity or if I could use 
> another reducing agent more compatible with my resin (or maybe do not add 
> both). I saw that someone did not have EDTA and used b-mercap. instead of 
> DTT. May I have your comments if you guys already faced a similar situation?
>
> Best wishes
>
> __
>
> Rafael Marques da Silva
>
> PhD Student – Structural Biology
>
> University of Leicester
>
>
> Mestrando em Física Biomolecular
> Universidade de São Paulo
>
> Bacharel em Ciências Biológicas
> Universidade Federal de São Carlos
>
> phone: +55 16 99766-0021
>
>"A sorte acompanha uma mente bem treinada"
> 
>
> 
>
> 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] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Rafael Marques]

Hi everyone,

I have been looking on this bb and other websites as well but I could not find 
a veredict. We are suspecting that when I elute my sample from my Ni-NTA 
column, the imidazole concentration (250 mM) is making it to precipitate. Once 
my sample has a cleavable TEV site, I was planning to incubate my loaded resin 
overnight with TEV and get my sample back simply using my lysis buffer. And 
here lies the problem. Most of the TEVs are kept in EDTA and DTT and I wonder 
if they are essential for its protease activity or if I could use another 
reducing agent more compatible with my resin (or maybe do not add both). I saw 
that someone did not have EDTA and used b-mercap. instead of DTT. May I have 
your comments if you guys already faced a similar situation?

Best wishes

__

Rafael Marques da Silva

PhD Student – Structural Biology

University of Leicester

Mestrando em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"




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[ccp4bb] [Off-topic] Research Coordinator position available immediately

[PULSARSTRIAN]

There is a Research coordinator position available immediately at
University of Oklahoma.



*Research Coordinator* *-* *Job Number:*  *230192*

*Organization:* Chemistry/Biochemistry

*Job Location:* Oklahoma-Norman-Norman Campus

*Schedule:* Full-time

*Work Schedule:* 8 AM - 5 PM Monday - Friday

*Salary Range:* Targeted salary $45,000 based on experience

*Benefits Provided:* Yes

*Required Attachments:* Resume, Cover Letter

*Job Description*

---

The Chemistry department is looking for a Research Coordinator who will
provide assistance to research staff for Dr. Peters by conducting simple
research experiments, collecting data and samples, keeping records,
conducting literature searches, analyzing data, and preparing reports.

*Job Requirements*

*Required Education:*  Bachelors degree, *AND: *

   - 24 months research or laboratory experience.

*Equivalency Substitution: *Will accept 48 months experience in lieu of the
bachelors degree for a total of 72 months of experience

*Skills*:

   - Proficient navigating and maintaining databases.
   - Ability to speak, read and write clear, concise English.
   - Strong oral and written communication skills.
   - Proficient in Microsoft Office.
   - Highly organized and able to handle multiple projects and deadlines.
   - Able to produce reports and complete work within deadlines.
   - Must be able to read and interpret policy as well as State and Federal
   regulations

*Certifications*: None

*Advertised Physical Requirements:*

   - Ability to engage in repetitive motions. Must be able to crouch,
   crawl, and reach.
   - Research or laboratory environment or in the field.

*Departmental Preferences:* None

*Supervision:* None

The link to apply to this job is below:

https://ou.taleo.net/careersection/2/jobdetail.ftl?job=230192=GMT-05%3A00=America%2FChicago



Kindly reach out to me for any informal queries: bhanu.jagili...@ou.edu



Regards,

Bhanu



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[ccp4bb] [Off-topic] NMR manager position available

[PULSARSTRIAN]

There is a NMR manager position available at University of Oklahoma.

*NMR Manager position at University of Oklahoma  *

*Organization:* Chemistry/Biochemistry

*Job Location:* Oklahoma-Norman-Norman Campus

*Schedule:* Full-time

*Work Schedule:* Monday - Friday 8-5

*Salary Range:* Target Salary $85,000 based on experience

*Benefits Provided:* Yes

*Required Attachments:* Resume, Cover Letter



Job can be applied via following link:

https://ou.taleo.net/careersection/2/jobdetail.ftl?job=222915=GMT-08%3A00=PST8PDT

Regards,

Bhanu



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Re: [ccp4bb] Off topic - 3D models demonstrating drug binding to students

[Bryan Lepore]

These magnetically-linking atomically accurate single-atom models look interesting - but I understand they might be too detailed or might shift around inadvertently :Snatoms Online Store - Magnetic Molecular Models for Educationsnatoms.comI also cannot say I used them (but I am always looking for an excuse to get some!).-Bryan W. Lepore

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Re: [ccp4bb] Off topic - 3D models demonstrating drug binding to students

[Dias, Joao M.]

Hi Ed,
The future is already here... and virtual reality is your friend.
This is a field that will likely be revolutionized in the near future, and your 
students would get an advantage by being exposed to these new technologies.
You could check the following:
https://www.labcompare.com/10-Featured-Articles/577506-VR-for-Science-Drug-Discovery-and-More-in-the-Virtual-World/

https://nanome.ai/
I wonder how they will respond to an academic collaboration, but you might be 
very surprised.
Let me know if you need more info or if you want me to make the introduction.

Good luck,
Joao

Joao M. Dias, Ph.D.
Principal Scientist
Pfizer
Structural and Molecular Sciences
Building 220/ room 3263, MS-8220-3224
445 Eastern Point Rd.
Groton, CT 06340



From: CCP4 bulletin board  On Behalf Of Edward Snell
Sent: Wednesday, February 15, 2023 3:13 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [EXTERNAL] [ccp4bb] Off topic - 3D models demonstrating drug binding 
to students

Dear CCP4,

I apologize if this is off topic but I thought this may be a good community to 
ask. Before we re-invent the wheel this end, we are looking for any commercial 
source of three-dimensional macromolecular models that can be used to teach how 
drugs are designed to fit to protein targets. Our audience is mid to advanced 
high-school students and we are looking for materials that can withstand being 
passed around the students and can teach basic concepts of how structure can 
inform ligand binding.

There are commercial sources of these models for macromolecules that we are 
aware of, but few if any that allow us to manipulate a drug and show how it 
fits to the model. We would love to find ones where a student may have name 
recognition of the drug concerned and we can show how it would fit to the 
target of that drug.

Any experience in this area and a source of such models would be greatly 
appreciated. We enjoy our interactions with these students and have the 
advantage of a professional high-school science teacher/science curriculum 
developer on staff with Nicole Terranova (copied on the email). Our program 
related to this is being developed and if anyone has any experiences with this 
nature of teaching, any offline feedback would be appreciated.

Thank you,

Eddie

Edward Snell Ph.D.
President and CEO | Hauptman-Woodward Medical Research Institute
Director | NSF BioXFEL Science and Technology Center
Professor, Materials Design and Innovation | University at Buffalo, SUNY
Fellow of the American Crystallographic Association - The Structural Science 
Society
p: +1 716 898 8631 | f: +1 716 898 8660
e: esn...@hwi.buffalo.edu<mailto:esn...@hwi.buffalo.edu>
skype: eddie.snell
Hauptman-Woodward Medical Research Institute
700 Ellicott Street | Buffalo, NY 14203-1102
hwi.buffalo.edu<https://urldefense.com/v3/__https:/hwi.buffalo.edu/__;!!H9nueQsQ!9I3n8FaljxVxUlya3-fNm9prByPB0RNfVyoMOonG6xlREiNN0LgYqtvzrdCbgMScGtWFMkx8uIVATPGcZswt$>

[hwi-logo-primary-horizontal]





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[ccp4bb] Off topic - 3D models demonstrating drug binding to students

[Edward Snell]

Dear CCP4,

I apologize if this is off topic but I thought this may be a good community to 
ask. Before we re-invent the wheel this end, we are looking for any commercial 
source of three-dimensional macromolecular models that can be used to teach how 
drugs are designed to fit to protein targets. Our audience is mid to advanced 
high-school students and we are looking for materials that can withstand being 
passed around the students and can teach basic concepts of how structure can 
inform ligand binding.

There are commercial sources of these models for macromolecules that we are 
aware of, but few if any that allow us to manipulate a drug and show how it 
fits to the model. We would love to find ones where a student may have name 
recognition of the drug concerned and we can show how it would fit to the 
target of that drug.

Any experience in this area and a source of such models would be greatly 
appreciated. We enjoy our interactions with these students and have the 
advantage of a professional high-school science teacher/science curriculum 
developer on staff with Nicole Terranova (copied on the email). Our program 
related to this is being developed and if anyone has any experiences with this 
nature of teaching, any offline feedback would be appreciated.

Thank you,

Eddie

Edward Snell Ph.D.

President and CEO | Hauptman-Woodward Medical Research Institute
Director | NSF BioXFEL Science and Technology Center
Professor, Materials Design and Innovation | University at Buffalo, SUNY
Fellow of the American Crystallographic Association - The Structural Science 
Society

p: +1 716 898 8631 | f: +1 716 898 8660
e: esn...@hwi.buffalo.edu
skype: eddie.snell

Hauptman-Woodward Medical Research Institute
700 Ellicott Street | Buffalo, NY 14203-1102
hwi.buffalo.edu


[hwi-logo-primary-horizontal]





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Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

[Dom Bellini - MRC LMB]

Hi Oleksiy,

If the Akta Start is just for loading affinity columns (without air 
sensor or software), why not to get a Cyvita peristaltic pump 
 
for £3k rather than £9k?


They are really good work horses in my experience; they are also 
overpriced but still much cheaper than the rest?


BW,

D



On 16/09/2022 15:48, Kovtun, Oleksiy wrote:

Hi Eike,

Instead of AKTA Pure I purchased a combo of AKTA Go (€24k with air 
sensor) and AKta start (€9k with frac collector). It provides two 
parallel workstations with AKTA start handling the dirty job of 
loading lysates onto affinity cartridges and the GO doing clean SEC 
stages. For multiple proteins isolation, this combo beats AKTA Pure 
flat out for half price. Note that AKTA Start can’t be equipped with 
an air sensor, and sample loading need to be done by time control. 
However, we didn’t find it to be an issue.
Also, AKTA Start needs a separate version of Uncocrn suite for an 
extra €1k. So we skipped that and control the system via the built-in 
touchscreen.


As for the longevity, we need to see. Six months into active operation 
went smooth—the only malfunction was the Windows update breaking 
network card drivers.


Best,

Oleksiy


Dr Oleksiy Kovtun
Max-Planck Research Group Leader
Molecular Mechanisms of Membrane Trafficking
Max-Planck-Institute for Multidisciplinary Sciences
City-Campus, Hermann-Rein-Str. 3, 37075 Göttingen
Germany
Email: oleksiy.kov...@mpinat.mpg.de 
https://www.mpinat.mpg.de/kovtun
Phone:+49-551-3899-410


On 16. Sep 2022, at 09:39, Schulz, Eike-Christian 
 wrote:


Dear all,
I am considering to purchase a chromatography system for routine 
protein purification. The device is supposed to be used in a 
multi-user environment, hence ease of use, ease of training and ease 
of maintenance is important. I am rather looking for a robust system 
people like to use than a system that comes with many bells and 
whistles that no one dares to touch.


  * Is there any particularly _/bad/_ experience with either system?

  * Is there any specific advantage of one system over the other?

  * Does anyone have experience about (long-term) stability /
performance of the systems?

  * Do you think the higher price-point of the Aekta systems is
justified?

With best regards,
Eike



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--
Dom Bellini, Xray Crystallography Facility (1S205)
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH
Phone 01223 267839



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Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

[Xiao, Chuan]

We have a AKTA start and a AKTA Pure. Don’t like the AKTA start at all. The 
software is buggy (command not performed correctly as it should do) but the 
touch screen is OK. By now I have changed two failed/leaking valves and the UV 
detector for the AKTA start with very light usage. AKTA start uses a 
peristaltic pump that cannot reach high pressure. The gradient is controlled by 
timing between two valves since both buffers are sucked instead of pumped out 
of A and B before mixer. I will not suggest anyone to buy AKTA start. Not a 
very good system. The company will not provide any service for you. You need to 
do all the fixing yourself. If you take out the tubing (as suggested by the 
manual) for the peristaltic pump when not using, the solution will be siphoned 
out to waste line. Poorly designed. Watch those solenoid vales, they are leaky. 
AKTA Pure is a good system but expensive.

Agree with many of you. After changing to Cytiva, the customer service is 
horrible and the whole company become a money sucker. Once I was asking some 
suggestion about configuration changed for AKTA Pure. Two months passed without 
response then asking me how satisfied I was for the service. I gave zero. Then 
they contacted me and give me $3000 quote for remote consulting. I ended up 
just reimaging back the hard drive to recover the setting.

I am still maintaining an very old AKTA FPLC (dark grey machine) using parts 
from eBay. It is still a very good system.

Best,

[cid:image001.jpg@01D73AFE.96DB0800]

From: CCP4 bulletin board  On Behalf Of Kovtun, Oleksiy
Sent: Friday, September 16, 2022 8:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Hi Eike,

Instead of AKTA Pure I purchased a combo of AKTA Go (€24k with air sensor) and 
AKta start (€9k with frac collector). It provides two parallel workstations 
with AKTA start handling the dirty job of loading lysates onto affinity 
cartridges and the GO doing clean SEC stages. For multiple proteins isolation, 
this combo beats AKTA Pure flat out for half price. Note that AKTA Start can’t 
be equipped with an air sensor, and sample loading need to be done by time 
control. However, we didn’t find it to be an issue.
Also, AKTA Start needs a separate version of Uncocrn suite for an extra €1k. So 
we skipped that and control the system via the built-in touchscreen.

As for the longevity, we need to see. Six months into active operation went 
smooth—the only malfunction was the Windows update breaking network card 
drivers.

Best,

Oleksiy


Dr Oleksiy Kovtun
Max-Planck Research Group Leader
Molecular Mechanisms of Membrane Trafficking
Max-Planck-Institute for Multidisciplinary Sciences
City-Campus, Hermann-Rein-Str. 3, 37075 Göttingen
Germany
Email: oleksiy.kov...@mpinat.mpg.de<mailto:oleksiy.kov...@mpinat.mpg.de>
https://www.mpinat.mpg.de/kovtun
Phone:+49-551-3899-410


On 16. Sep 2022, at 09:39, Schulz, Eike-Christian 
mailto:eike.sch...@mpsd.mpg.de>> wrote:

Dear all,

I am considering to purchase a chromatography system for routine protein 
purification. The device is supposed to be used in a multi-user environment, 
hence ease of use, ease of training and ease of maintenance is important. I am 
rather looking for a robust system people like to use than a system that comes 
with many bells and whistles that no one dares to touch.


  *   Is there any particularly _bad_ experience with either system?


  *   Is there any specific advantage of one system over the other?


  *   Does anyone have experience about (long-term) stability / performance of 
the systems?


  *   Do you think the higher price-point of the Aekta systems is justified?


With best regards,

Eike





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Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

[Kovtun, Oleksiy]

Hi Eike,

Instead of AKTA Pure I purchased a combo of AKTA Go (€24k with air sensor) and 
AKta start (€9k with frac collector). It provides two parallel workstations 
with AKTA start handling the dirty job of loading lysates onto affinity 
cartridges and the GO doing clean SEC stages. For multiple proteins isolation, 
this combo beats AKTA Pure flat out for half price. Note that AKTA Start can’t 
be equipped with an air sensor, and sample loading need to be done by time 
control. However, we didn’t find it to be an issue.
Also, AKTA Start needs a separate version of Uncocrn suite for an extra €1k. So 
we skipped that and control the system via the built-in touchscreen.

As for the longevity, we need to see. Six months into active operation went 
smooth—the only malfunction was the Windows update breaking network card 
drivers.

Best,

Oleksiy


Dr Oleksiy Kovtun
Max-Planck Research Group Leader
Molecular Mechanisms of Membrane Trafficking
Max-Planck-Institute for Multidisciplinary Sciences
City-Campus, Hermann-Rein-Str. 3, 37075 Göttingen
Germany
Email: oleksiy.kov...@mpinat.mpg.de
https://www.mpinat.mpg.de/kovtun
Phone:+49-551-3899-410


On 16. Sep 2022, at 09:39, Schulz, Eike-Christian 
mailto:eike.sch...@mpsd.mpg.de>> wrote:

Dear all,

I am considering to purchase a chromatography system for routine protein 
purification. The device is supposed to be used in a multi-user environment, 
hence ease of use, ease of training and ease of maintenance is important. I am 
rather looking for a robust system people like to use than a system that comes 
with many bells and whistles that no one dares to touch.


  *   Is there any particularly _bad_ experience with either system?



  *   Is there any specific advantage of one system over the other?



  *   Does anyone have experience about (long-term) stability / performance of 
the systems?



  *   Do you think the higher price-point of the Aekta systems is justified?



With best regards,

Eike






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Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

[Oganesyan, Vaheh]

Hi Stephan,

You’re mostly correct, however, gradients made on instrument with one pump are 
less reliable.

Thank you.

Vaheh

From: CCP4 bulletin board  On Behalf Of Stephan Rempel
Sent: Friday, September 16, 2022 7:38 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Hi,

I had some experience for about 2-3 years with the BioRad systems before I 
moved on and they worked just fine like the AKTAs. At the time, the price point 
was a huge plus over the AKTA Pure and in my opinion for protein purification 
there was no reason to fork out the money for an AKTA Pure because you would 
never use the two pumps anyway. We had the NGC quest with multi wavelength 
detection for protein labeling, which worked fine. I liked the NGCs a lot, they 
were reliable, and I would be happy to get one any time.

In the meantime, Cytiva has released the AKTA Go, which is a scaled down 
version of the Pure with only one pump. And I would give this one a thought if 
you only want to purify proteins.

It is a great (!) little system for protein purification and the price is very 
reasonable in my opinion. Our version with the additional inlet sample valve 
(allows for scouting over night), air detector (very convenient when loading 
large lysate volumes on IMAC columns), and column valve was in the region of 
20-25k. You also have the choice between a simpler fraction collector and the 
one from the AKTA Pure. The only caveat is that they only offer a fixed 
wavelength detector. After two years, we had not a single fault (they have been 
on the market for 2.5-3 years). I very much like the Unicorn software.

I hope this helps,
Stephan.





Stephan Rempel, PhD
Senior Scientist, FoRx Therapeutics AG

Lichtstrasse 35, WSJ-350.3.10
4056 Basel, Switzerland
stephan.rem...@forxtherapeutics.com<mailto:stephan.rem...@forxtherapeutics.com>
www.forxtherapeutics.com<http://www.forxtherapeutics.com/>

  [cid:image001.png@01D8C9B6.8D851690]

From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Marko Hyvonen
Sent: Friday, 16 September 2022 13:17
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Hello Eike,

A couple of comments on the AKTA Pures (while I have not experience with the 
BioRad systems).

We have a couple of AKTA Pures and we (and another lab close to us) had a 
catastrophic failure of the 3-wavelength detector “block”. Price tag for the 
replacement part (apparently a black box  with nothing accessible for repair – 
happy to hear if this was misinformation!) was over £12,000. Someone commented 
that they had even more eye-watering price tag for an upgrade to triple 
wavelength. We switched to a single wavelength detector at £5k and I will get 
these from now on only.

I really dislike the database system used in new Unicorn for storing data etc: 
another black box. I much prefer the way older Unicorns worked, but maybe that 
is just me being old and grumpy and stuck in my ways. That reminds also that we 
tend to source computers separately. Much better systems available for less 
money and with MUCH smaller footprint.

The price tags on Pures (like almost everything else with Cytiva) are in my 
opinion a reflection of the market dominance. I wish there was more competition 
(bring back Biocad!). Service contracts with Cytiva are also ridiculously 
expensive – we have, luckily,  a very knowledgeable company here that does all 
the servicing and maintenance, at less than half the price.

Having just complained, I do like AKTA Pures and there is much to like in them. 
Time will tell if these will be as robust as the original “grey” AKTA 
purifiers. Several of ours Purifiers still running perfectly after over 15 
years. As long as the detector works and flow is accurate, the proteins elute 
just the same from the columns. However, once you have even one of the shinier 
systems around, those old ones start collecting dust very soon.

Hth, Marko


Marko Hyvonen
Department of Biochemistry
University of Cambridge
https://hyvonen.bioc.cam.ac.uk<https://hyvonen.bioc.cam.ac.uk>
@HyvonenGroup
mh...@cam.ac.uk<mailto:mh...@cam.ac.uk>




From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Schulz, Eike-Christian
Sent: 16 September 2022 08:40
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Dear all,

I am considering to purchase a chromatography system for routine protein 
purification. The device is supposed to be used in a multi-user environment, 
hence ease of use, ease of training and ease of maintenance is important. I am 
rather looking for a robust system people like to use than a system that comes 
with many bells and whistles that no one dares to touch.


·   Is there any particularly _bad_ experience wit

Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

[Mark J. van Raaij]

Hi Eike,
we bought a Biorad back in 2005 or so, an Akta Purifier in 2011 and recently an 
Akta Go (2020). All work(ed) well, the latter two are in use. Don't know about 
the Biorad, it's in Santiago de Compostela. Both the Biorad and Purifier have 
been used a lot, the Akta Go not that much so far. Just because the programs 
are set up for the Purifier I think, not because people like it more per se.
For "normal" protein purifications all are fine. We've had breakdowns in both 
the Biorad and Purifier, which could be fixed with not too much spending.
I'm not sure about this kind of equipment in a multi-user environment, best to 
keep it in one group I think, otherwise it's difficult people feel responsible 
enough to use them properly. This is even more important for columns, they are 
easily ruined if just once an incorrectly prepared sample is run through.
Price: we got very good deals on the two Akta systems we bought, not more 
expensive than a comparable Biorad. But Cytiva service/repair is very expensive.
Good luck,
Mark

Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. +34 91 585 4616 (internal 432092)
Section Editor Acta Crystallographica F
https://journals.iucr.org/f/
https://namedrop.io/markvanraaij

> On 16 Sep 2022, at 09:39, Schulz, Eike-Christian  
> wrote:
> 
> Dear all, 
>  
> I am considering to purchase a chromatography system for routine protein 
> purification. The device is supposed to be used in a multi-user environment, 
> hence ease of use, ease of training and ease of maintenance is important. I 
> am rather looking for a robust system people like to use than a system that 
> comes with many bells and whistles that no one dares to touch.
>  
> Is there any particularly _bad_ experience with either system?
>  
> Is there any specific advantage of one system over the other?
>  
> Does anyone have experience about (long-term) stability / performance of the 
> systems?
>  
> Do you think the higher price-point of the Aekta systems is justified?
>  
>  
> With best regards, 
>  
> Eike
>  
>  
>  
> 
> 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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Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

[Stephan Rempel]

Hi,

I had some experience for about 2-3 years with the BioRad systems before I 
moved on and they worked just fine like the AKTAs. At the time, the price point 
was a huge plus over the AKTA Pure and in my opinion for protein purification 
there was no reason to fork out the money for an AKTA Pure because you would 
never use the two pumps anyway. We had the NGC quest with multi wavelength 
detection for protein labeling, which worked fine. I liked the NGCs a lot, they 
were reliable, and I would be happy to get one any time.

In the meantime, Cytiva has released the AKTA Go, which is a scaled down 
version of the Pure with only one pump. And I would give this one a thought if 
you only want to purify proteins.

It is a great (!) little system for protein purification and the price is very 
reasonable in my opinion. Our version with the additional inlet sample valve 
(allows for scouting over night), air detector (very convenient when loading 
large lysate volumes on IMAC columns), and column valve was in the region of 
20-25k. You also have the choice between a simpler fraction collector and the 
one from the AKTA Pure. The only caveat is that they only offer a fixed 
wavelength detector. After two years, we had not a single fault (they have been 
on the market for 2.5-3 years). I very much like the Unicorn software.

I hope this helps,
Stephan.





Stephan Rempel, PhD
Senior Scientist, FoRx Therapeutics AG

Lichtstrasse 35, WSJ-350.3.10
4056 Basel, Switzerland
stephan.rem...@forxtherapeutics.com<mailto:stephan.rem...@forxtherapeutics.com>
www.forxtherapeutics.com<http://www.forxtherapeutics.com/>

  [cid:image001.png@01D8C9D1.8E327950]

From: CCP4 bulletin board  On Behalf Of Marko Hyvonen
Sent: Friday, 16 September 2022 13:17
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Hello Eike,

A couple of comments on the AKTA Pures (while I have not experience with the 
BioRad systems).

We have a couple of AKTA Pures and we (and another lab close to us) had a 
catastrophic failure of the 3-wavelength detector “block”. Price tag for the 
replacement part (apparently a black box  with nothing accessible for repair – 
happy to hear if this was misinformation!) was over £12,000. Someone commented 
that they had even more eye-watering price tag for an upgrade to triple 
wavelength. We switched to a single wavelength detector at £5k and I will get 
these from now on only.

I really dislike the database system used in new Unicorn for storing data etc: 
another black box. I much prefer the way older Unicorns worked, but maybe that 
is just me being old and grumpy and stuck in my ways. That reminds also that we 
tend to source computers separately. Much better systems available for less 
money and with MUCH smaller footprint.

The price tags on Pures (like almost everything else with Cytiva) are in my 
opinion a reflection of the market dominance. I wish there was more competition 
(bring back Biocad!). Service contracts with Cytiva are also ridiculously 
expensive – we have, luckily,  a very knowledgeable company here that does all 
the servicing and maintenance, at less than half the price.

Having just complained, I do like AKTA Pures and there is much to like in them. 
Time will tell if these will be as robust as the original “grey” AKTA 
purifiers. Several of ours Purifiers still running perfectly after over 15 
years. As long as the detector works and flow is accurate, the proteins elute 
just the same from the columns. However, once you have even one of the shinier 
systems around, those old ones start collecting dust very soon.

Hth, Marko


Marko Hyvonen
Department of Biochemistry
University of Cambridge
https://hyvonen.bioc.cam.ac.uk
@HyvonenGroup
mh...@cam.ac.uk<mailto:mh...@cam.ac.uk>




From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Schulz, Eike-Christian
Sent: 16 September 2022 08:40
To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Dear all,

I am considering to purchase a chromatography system for routine protein 
purification. The device is supposed to be used in a multi-user environment, 
hence ease of use, ease of training and ease of maintenance is important. I am 
rather looking for a robust system people like to use than a system that comes 
with many bells and whistles that no one dares to touch.


· Is there any particularly _bad_ experience with either system?


· Is there any specific advantage of one system over the other?


· Does anyone have experience about (long-term) stability / performance 
of the systems?


· Do you think the higher price-point of the Aekta systems is justified?


With best regards,

Eike






To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-b

Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

[Marko Hyvonen]

Hello Eike,

A couple of comments on the AKTA Pures (while I have not experience with the 
BioRad systems).

We have a couple of AKTA Pures and we (and another lab close to us) had a 
catastrophic failure of the 3-wavelength detector "block". Price tag for the 
replacement part (apparently a black box  with nothing accessible for repair - 
happy to hear if this was misinformation!) was over £12,000. Someone commented 
that they had even more eye-watering price tag for an upgrade to triple 
wavelength. We switched to a single wavelength detector at £5k and I will get 
these from now on only.

I really dislike the database system used in new Unicorn for storing data etc: 
another black box. I much prefer the way older Unicorns worked, but maybe that 
is just me being old and grumpy and stuck in my ways. That reminds also that we 
tend to source computers separately. Much better systems available for less 
money and with MUCH smaller footprint.

The price tags on Pures (like almost everything else with Cytiva) are in my 
opinion a reflection of the market dominance. I wish there was more competition 
(bring back Biocad!). Service contracts with Cytiva are also ridiculously 
expensive - we have, luckily,  a very knowledgeable company here that does all 
the servicing and maintenance, at less than half the price.

Having just complained, I do like AKTA Pures and there is much to like in them. 
Time will tell if these will be as robust as the original "grey" AKTA 
purifiers. Several of ours Purifiers still running perfectly after over 15 
years. As long as the detector works and flow is accurate, the proteins elute 
just the same from the columns. However, once you have even one of the shinier 
systems around, those old ones start collecting dust very soon.

Hth, Marko


Marko Hyvonen
Department of Biochemistry
University of Cambridge
https://hyvonen.bioc.cam.ac.uk
@HyvonenGroup
mh...@cam.ac.uk<mailto:mh...@cam.ac.uk>




From: CCP4 bulletin board  On Behalf Of Schulz, 
Eike-Christian
Sent: 16 September 2022 08:40
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Dear all,

I am considering to purchase a chromatography system for routine protein 
purification. The device is supposed to be used in a multi-user environment, 
hence ease of use, ease of training and ease of maintenance is important. I am 
rather looking for a robust system people like to use than a system that comes 
with many bells and whistles that no one dares to touch.


·   Is there any particularly _bad_ experience with either system?


·   Is there any specific advantage of one system over the other?


·   Does anyone have experience about (long-term) stability / performance 
of the systems?


·   Do you think the higher price-point of the Aekta systems is justified?


With best regards,

Eike






To unsubscribe from the CCP4BB list, click the following link:
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[ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

[Schulz, Eike-Christian]

Dear all,

I am considering to purchase a chromatography system for routine protein 
purification. The device is supposed to be used in a multi-user environment, 
hence ease of use, ease of training and ease of maintenance is important. I am 
rather looking for a robust system people like to use than a system that comes 
with many bells and whistles that no one dares to touch.


  *   Is there any particularly _bad_ experience with either system?


  *   Is there any specific advantage of one system over the other?


  *   Does anyone have experience about (long-term) stability / performance of 
the systems?


  *   Do you think the higher price-point of the Aekta systems is justified?


With best regards,

Eike






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

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Re: [ccp4bb] Off-topic question related to ITC binding studies

[ABHISHEK SUMAN]

Hi Gergő,

Thank you for discussing it in detail. It is a great help. Based on the
curve fit, we also believe that the two proteins bind independently to the
DNA duplex with the same affinity. The DNA indeed contains 2 sites for
protein binding. As far as the dimerization of protein is concerned, we
have already confirmed, through different approaches, that the protein
doesn't behave as a dimer in the solution.

Regards
Abhishek


*Dr. Abhishek Suman*

*Ph.D (Structural Biology)*

Indian Institute of Technology Hyderabad

Kandi 502 284 Sangareddy

Telangana INDIA

Contact: +91 91002 74548, +91 80843 11898

Email: abhisheks.i...@gmail.com



P* Please don't print this e-mail or attachment unless necessary. Preserve
trees on the planet.*


On Wed, Aug 17, 2022 at 12:24 AM Gergő Gógl  wrote:

> Dear Abhishek,
>
> When you use a function with "one set of sites" and an “independent”
> model, you most likely used some sort of quadratic binding formula. In case
> the true binding equation is like you wrote [2A + B <-> (A2)B], then it
> is not expected to look like a quadratic, but rather like a cubic formula.
>
> {Just solve this: Kd=( ( Atot - 0.5*[(A2)B] )^2 * (Btot - [(A2)B]) ) / ( [
> (A2)B] ) and you will see that the resulting function is cubic for the
> concentration of the complex ([(A2)B]).}
>
> If you indeed had a perfect sigmoidal observation with no systematic
> divergence from the fitted quadratic curve, I would not find any strong
> indication that the binding function should be considered as cubic.
> Instead, I would assume that:
>
> -the protein binding to the DNA follows a simple bimolecular binding model
> and the DNA just happens to be able to bind two molecules of proteins
> independently with the same affinity (within the detection thresholds of
> the assay). [A + B_site1 <-> AB_site1 and A + B_site2 <-> AB_site2]
> -or the protein binds as a stable dimer to the DNA molecule and therefore
> the binding can be considered as pseudo bimolecular. (DNA+dimer <->complex)
> In this case, you should change the protein concentration to "protein dimer
> concentration" during fitting. [(A2) + B <-> (A2)B]
>
> In either case, your dissociation constant has a dimension of M and not
> M^2.
>
> If your binding model really follows the reaction you proposed and you do
> not like the first option where the affinities of the sites are identical,
> you should use a "two independent set of sites" model. In this case, you
> will end up with 2 independent affinities that are not identical and their
> dimension will be in M. (If they are close, you are better if you use a one
> site and assume that the sites are not distinguishable.)
>
> If your sites are not independent and you have strong cooperativity, you
> will need to use more complex biochemistry (such as sequential binding) and
> your Kd dimension may be different, but this is most likely not the case
> because you had a decent fit with a simple.
>
> I hope I could help and did not miss something important. Please let me
> know if others propose something different!
>
> Best,
> Gergo
>
> ABHISHEK SUMAN <2fccd9428006-dmarc-requ...@jiscmail.ac.uk> ezt írta
> (időpont: 2022. aug. 16., K, 19:40):
>
>> Hello everyone!
>>
>>
>>
>> Hope this email finds you well. I have an off-topic question regarding
>> ITC binding studies, which was asked by a reviewer.
>>
>>
>>
>> We performed an ITC binding study (using Affinity ITC, TA Instruments) to
>> evaluate protein-DNA interaction which resulted in a perfect sigmoidal
>> curve. We used the ‘one set of sites’ binding algorithm (“independent”
>> model) for curve fitting and to calculate binding and thermodynamic
>> parameters. The study suggested two copies of the protein binding to a
>> single duplex DNA, i.e., the stoichiometry of protein:DNA is 2:1 (N=2). The
>> ITC calculated the KD (equilibrium molar dissociation constant) in μM
>> (micromolar). But the reviewer is asking to report the KD in
>> (micromolar)^2 instead of micromolar mentioning that the binding reaction
>> is 2A + B <-> (A2)B and the complex is (A2)B and not AB. Though we're
>> trying to explain to the reviewer that we couldn't find any software that
>> can compute the KD in (micromolar)^2 for the stoichiometry of 2 but he
>> is not agreeing to it. We have used the NanoAnalyze software from the TA
>> instrument. This software does not have a model to measure the KD in
>> (micromolar)^2.
>>
>>
>> I would be grateful if you could help me to resolve this problem or at
>> least let me know what explanation might be appropriate to answer the
>> reviewer’s concern that it’s a general practice to report the KD in the
>> Molar irrespective of stoichiometry.
>>
>>
>>
>> Thanks in advance.
>>
>>
>>
>> Regards
>>
>> Abhishek
>>
>>
>> *Dr. Abhishek Suman*
>>
>> *Ph.D (Structural Biology)*
>>
>> Indian Institute of Technology Hyderabad
>>
>> Kandi 502 284 Sangareddy
>>
>> Telangana INDIA
>>
>> Contact: +91 

[ccp4bb] RES: [ccp4bb] Off-topic question related to ITC binding studies

[Rafael Marques]

I am not a proper chemist, but I remember that during my college classes my 
professor emphasized that ^x is a measured value and not really related to 
stoichiometry. If someone has something to add…

Best

Rafael Marques da Silva
Mestre em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"


De: ABHISHEK SUMAN<mailto:2fccd9428006-dmarc-requ...@jiscmail.ac.uk>
Enviado:terça-feira, 16 de agosto de 2022 14:41
Para: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>
Assunto: [ccp4bb] Off-topic question related to ITC binding studies

Hello everyone!

Hope this email finds you well. I have an off-topic question regarding ITC 
binding studies, which was asked by a reviewer.

We performed an ITC binding study (using Affinity ITC, TA Instruments) to 
evaluate protein-DNA interaction which resulted in a perfect sigmoidal curve. 
We used the ‘one set of sites’ binding algorithm (“independent” model) for 
curve fitting and to calculate binding and thermodynamic parameters. The study 
suggested two copies of the protein binding to a single duplex DNA, i.e., the 
stoichiometry of protein:DNA is 2:1 (N=2). The ITC calculated the KD 
(equilibrium molar dissociation constant) in μM (micromolar). But the reviewer 
is asking to report the KD in (micromolar)^2 instead of micromolar mentioning 
that the binding reaction is 2A + B <-> (A2)B and the complex is (A2)B and not 
AB. Though we're trying to explain to the reviewer that we couldn't find any 
software that can compute the KD in (micromolar)^2 for the stoichiometry of 2 
but he is not agreeing to it. We have used the NanoAnalyze software from the TA 
instrument. This software does not have a model to measure the KD in 
(micromolar)^2.

I would be grateful if you could help me to resolve this problem or at least 
let me know what explanation might be appropriate to answer the reviewer’s 
concern that it’s a general practice to report the KD in the Molar irrespective 
of stoichiometry.

Thanks in advance.

Regards
Abhishek

Dr. Abhishek Suman
Ph.D (Structural Biology)
Indian Institute of Technology Hyderabad
Kandi 502 284 Sangareddy
Telangana INDIA
Contact: +91 91002 74548, +91 80843 11898
Email: abhisheks.i...@gmail.com<mailto:abhisheks.i...@gmail.com>

P Please don't print this e-mail or attachment unless necessary. Preserve trees 
on the planet.


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[ccp4bb] Off-topic question related to ITC binding studies

[ABHISHEK SUMAN]

Hello everyone!



Hope this email finds you well. I have an off-topic question regarding ITC
binding studies, which was asked by a reviewer.



We performed an ITC binding study (using Affinity ITC, TA Instruments) to
evaluate protein-DNA interaction which resulted in a perfect sigmoidal
curve. We used the ‘one set of sites’ binding algorithm (“independent”
model) for curve fitting and to calculate binding and thermodynamic
parameters. The study suggested two copies of the protein binding to a
single duplex DNA, i.e., the stoichiometry of protein:DNA is 2:1 (N=2). The
ITC calculated the KD (equilibrium molar dissociation constant) in μM
(micromolar). But the reviewer is asking to report the KD in (micromolar)^2
instead of micromolar mentioning that the binding reaction is 2A + B <->
(A2)B and the complex is (A2)B and not AB. Though we're trying to explain
to the reviewer that we couldn't find any software that can compute the KD
in (micromolar)^2 for the stoichiometry of 2 but he is not agreeing to it.
We have used the NanoAnalyze software from the TA instrument. This software
does not have a model to measure the KD in (micromolar)^2.


I would be grateful if you could help me to resolve this problem or at
least let me know what explanation might be appropriate to answer the
reviewer’s concern that it’s a general practice to report the KD in the
Molar irrespective of stoichiometry.



Thanks in advance.



Regards

Abhishek


*Dr. Abhishek Suman*

*Ph.D (Structural Biology)*

Indian Institute of Technology Hyderabad

Kandi 502 284 Sangareddy

Telangana INDIA

Contact: +91 91002 74548, +91 80843 11898

Email: abhisheks.i...@gmail.com



P* Please don't print this e-mail or attachment unless necessary. Preserve
trees on the planet.*

-- 


Disclaimer:- This footer text is to convey that this email is sent by one 
of the users of IITH. So, do not mark it as SPAM.



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Re: [ccp4bb] Off topic: Are all storage dewars equal?

[Nukri Sanishvili]

Hi Carmien,
It appears to me that the dewars in your first link are those made by
Taylor Wharton. You can do a search with that name and hopefully find less
expensive options. We've used them for many years and they are good.
Another one, you could try, is VME https://mvebio.com/aluminum-dewars/
We've used them as well and they are good too.

Just make sure to order a dewar with a wide neck/wide mouth so that your
puck holder fits in.
Also, for holding just 7 pucks, you could get away with smaller and less
expensive dewar. However, smaller ones will need adding of LNG more
frequently, so you will have to pick your poison.
Good luck!
Nukri

On Tue, Jul 5, 2022 at 4:13 AM Carmien Tolmie 
wrote:

> Hello everyone,
>
> We want to buy a storage dewar that can house seven unipucks. I have seen
> dewars on the website of the crystallography suppliers (such as the HC35
> https://www.moleculardimensions.com/products/high-capacity-cryogenic-refrigerators),
> which is significantly more expensive than the run-off-the-mill cryogenic
> storage dewars (such as the Biocane73
> https://www.thermofisher.com/order/catalog/product/CK509X5).
>
> Is there something that makes the dewars from the crystallography
> suppliers special? Or can you use any type of cryogenic storage dewar to
> store the pucs?
>
> Thank you very much for your input.
>
> Best wishes,
>
> Carmien
>
>
>
> --
>
> 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] Off topic: Are all storage dewars equal?

[Carmien Tolmie]

Hello everyone,

We want to buy a storage dewar that can house seven unipucks. I have seen
dewars on the website of the crystallography suppliers (such as the HC35
https://www.moleculardimensions.com/products/high-capacity-cryogenic-refrigerators),
which is significantly more expensive than the run-off-the-mill cryogenic
storage dewars (such as the Biocane73
https://www.thermofisher.com/order/catalog/product/CK509X5).

Is there something that makes the dewars from the crystallography suppliers
special? Or can you use any type of cryogenic storage dewar to store the
pucs?

Thank you very much for your input.

Best wishes,

Carmien



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Re: [ccp4bb] Off-topic: Mol* Viewer re-centring hack

[David Armstrong]

Hi Jon,

Glad to see you are making use of the Mol* viewer. I am also replying 
on-list as others will hopefully find my response useful.


For your specific case, I would recommend using the 'selection mode' - 
activated by clicking the cursor icon at the top right of the viewport. 
This mode changes the left-click action to a 'selection' of regions of 
the 3D structure, depending on the picking level defined in the 
selection toolbar (e.g. residue, atom, chain etc.). Once this mode is 
activated, clicking away from the structure will no longer reset the 
view - it will also enable you to create bespoke selections for e.g. 
visualisations or measurements.


There is more information on selections in Mol* and much more in the 
Mol* Viewer documentation at https://molstar.org/viewer-docs


There is also a recent webinar by one of my PDBe colleagues James 
Tolchard, which covers use of the PDBe implementation of the Mol* 
viewer. This may also give a good background for those new to using 
Mol*. You can access the webinar at https://youtu.be/13qSb1_3VWE


Please feel free to contact me off-list if anyone is interest in 
learning more about the Mol* implementation at PDBe and queries about 
the use of the viewer.


Kind Regards,
David Armstrong

On 28/06/2022 10:36, Read, Jon wrote:


I have been using Mol* recently which looks like a really nice tool 
for basic model viewing.


When you click off an atom (which is the normal action for clearing a 
selection in model viewers), it changes the zoom, orientation and 
re-slabs it.


Has anyone found a hack to stop it re-centring/zooming/slabbing when 
you click off an atom.


*Jon Read*



AstraZeneca UK Limited is a company incorporated in England and Wales 
with registered number:03674842 and its registered office at 1 Francis 
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affiliates may process information, personal data and monitor 
communications, please see our privacy notice at www.astrazeneca.com 






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--
David Armstrong
Outreach and Training Lead
PDBe
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European Molecular Biology Laboratory
Wellcome Trust Genome Campus
Hinxton
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[ccp4bb] Off-topic: Mol* Viewer re-centring hack

[Read, Jon]

I have been using Mol* recently which looks like a really nice tool for basic 
model viewing.

When you click off an atom (which is the normal action for clearing a selection 
in model viewers), it changes the zoom, orientation and re-slabs it.

Has anyone found a hack to stop it re-centring/zooming/slabbing when you click 
off an atom.

Jon Read


AstraZeneca UK Limited is a company incorporated in England and Wales with 
registered number:03674842 and its registered office at 1 Francis Crick Avenue, 
Cambridge Biomedical Campus, Cambridge, CB2 0AA.

This e-mail and its attachments are intended for the above named recipient only 
and may contain confidential and privileged information. If they have come to 
you in error, you must not copy or show them to anyone; instead, please reply 
to this e-mail, highlighting the error to the sender and then immediately 
delete the message. For information about how AstraZeneca UK Limited and its 
affiliates may process information, personal data and monitor communications, 
please see our privacy notice at 
www.astrazeneca.com



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[ccp4bb] Off-topic - Cryojet5 - boiloff heater spare

[Kristof Van Hecke]

Dear, 

My apologies for the off-topic post.

Our (11-years old) Oxford Instruments Cryojet5 LN2 cryocooling has broken down 
and to repair this, we are looking to purchase a simple ‘boiloff heater’ 
element for the shield flow dewar leg. 
Oxford Instruments informed us that this product was discontinued in 2015 and 
they are no longer able to supply spares for it. 


Therefore, if anyone has spare parts for a Cryojet5, please get back to me? 


Thank you very much. 

Kristof Van Hecke
Ghent University, Belgium



  





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[ccp4bb] Off topic: docking for biological insight

[Andrew Lovering]

Dear ccp4 community,
When I see the (improving) accuracy of docking in generating drug like leads, I 
see predominantly synthetic compounds used.

It occurred to me that it might be informative to use docking of naturally 
occurring compounds (amino acids, sugars, lipids, factors etc) to find pockets 
of interest in structures of cryptic function. So I have two questions:
1) which software does this best, or perhaps makes this easiest?
2) are they good validated examples of this in the literature that you know of?

Many thanks in advance for your opinions and insights
Andy



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Re: [ccp4bb] off topic -- pymol error message

[Robbie Joosten]

This means that you have an O-umlaut in your PDB file. That should never 
happen! PDB files should only have basic ASCII characters, not UTF-8. 

Cheers,
Robbie

> -Original Message-
> From: CCP4 bulletin board  On Behalf Of 陈成
> Sent: Tuesday, April 5, 2022 13:12
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] off topic -- pymol error message
> 
> Hi all,
> 
> What does this message means when loading a pdb file in pymol:
> 
> 'utf-8' codec can't decode byte 0xd6 in position 37: invalid continuation byte
> 
> Thank you!
> 
> Chen
> 
> 
> 
> 
> 
> --
> Cheng Chen, Associate Professor
> 
> School of Life Sciences, Building 15, Tianjin University
> 
> No.92 Weijin Road, Nankai District, Tianjin 300072, China
> 
> 天津市南开区卫津路92号,天津大学生命科学学院15教学楼, 邮编:300072
> 
> 
> 
> 
> 
> 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] off topic -- pymol error message

[陈成]

Hi all,


What does this message means when loading a pdb file in pymol:


'utf-8' codec can't decode byte 0xd6 in position 37: invalid continuation byte


Thank you!


Chen





--
Cheng Chen, Associate ProfessorSchool of Life Sciences, Building 15, Tianjin 
University
No.92 Weijin Road, Nankai District, Tianjin 300072, China
天津市南开区卫津路92号,天津大学生命科学学院15教学楼, 邮编:300072









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Re: [ccp4bb] Off topic - gene toxic for expression strains

[Artem Evdokimov]

Hi there,

Somehow I've missed the original email :) Sorry!

There are options for expressing really toxic genes, some of which have
already been mentioned and others perhaps not:

1. tight regulation of expression (promoter, repressor, other regulatory
elements, or a combination thereof). Beyond using araBAD, xylose promoter,
rhamnose promoter, etc. there is one more thing that can be done, which is
quite old school: phage induction. Yes, it means what it says - you add
actual phage to the otherwise normal E.coli and the phage brings
polymerase. This way there is no tangible expression except leakage which
for T7 is close to non-existent.

2. change expression to a different organism: Bacillus, Pseudomonas or
insect/mammalian cells

3. counter-expression of antisense RNA from a weaker promoter: this method
is not exactly easy, but it does work -- you need a weak promoter (e.g.
unmodified lac or trp) that would drive expression of antisense RNA.
Easiest way to go about this is to put the antisense promoter on the 3' end
of the gene, facing 'backwards'. Then, during induction, the sense-strand
promoter is much more active and the sense RNA will win over the antisense.

Good luck!

Artem

P.S. if you use 2% glucose in the media be prepared to fight acidification
and the accumulation of Acetate in the spent medium, both of which can be a
problem - sometimes severe - for 'weaker' E. coli strains. Strongly
buffered medium is a must.
- Cosmic Cats approve of this message


On Mon, Apr 4, 2022 at 9:16 AM Nikolay Dobrev <
nikolay.dob...@embl-hamburg.de> wrote:

> Hi Andy,
> just to follow up on Christian suggestion, which is exactly the way to go.
>
> In case you are using an already pET based vector, simply try BL21-AI (
> https://www.thermofisher.com/order/catalog/product/C607003), which has
> the T7 RNA polymerase under arabinose promoter should do the trick.
> Also, 2% Glucose is a must in this kind of situation.
> I have been dealing with several toxic proteins (from the family of
> restriction enzymes :) ) and BL21-AI was a way to go.
> Please also have a look if your pET backbone has the extra copy of the
> lacI, which makes a difference in leakage expression.
>
> As a sum up:
> 1) try BL21-AI (for the induction of the target protein you will need both
> Arabinose (T7 RNA pol induction) and IPTG for the T7 promoter)
> 2) or BL21 with pLysS or pLysE
> both simple keep 2 % Glucose and also directly from trafo goto liquid
> culture in parallel of the plating approach.
>
> Let me know if you need any further tips.
>
>
> *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.
>
>
> On 04/04/2022 10:32 AM Christian Roth  wrote:
>
>
> Hi Andy,
> have you tried another promotor? Arabinose is much tighter, just to be
> sure that it is really not leaking.
>
> Cheers
> Christian
>
>
> On Mon, Apr 4, 2022 at 10:20 AM Andrew Lovering 
> wrote:
>
> Dear Board,
>
>
>
> Perhaps off-topic, but in the wider scope it’s relevant to many on here.
>
>
>
> We have a gene that we are able to clone, and propagate in DH5a etc
> non-expression cells (hence nucleotide sequence is non-toxic)
>
>
>
> But, when we attempt to transfer to an expression strain we get no
> colonies
>
>
>
> We have tried pLemo, Glucose addition, 30 degrees, C41/C43 and still no joy
>
>
>
> We’d welcome any suggestions here – it’s a fun protein
>
>
>
> Thanks
>
> Andy
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
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>
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Re: [ccp4bb] Off topic - gene toxic for expression strains

[Nikolay Dobrev]

Hi Andy,
just to follow up on Christian suggestion, which is exactly the way to go.

In case you are using an already pET based vector, simply try BL21-AI 
(https://www.thermofisher.com/order/catalog/product/C607003), which has the T7 
RNA polymerase under arabinose promoter should do the trick.
Also, 2% Glucose is a must in this kind of situation.
I have been dealing with several toxic proteins (from the family of restriction 
enzymes :) ) and BL21-AI was a way to go.
Please also have a look if your pET backbone has the extra copy of the lacI, 
which makes a difference in leakage expression.

As a sum up:
1) try BL21-AI (for the induction of the target protein you will need both 
Arabinose (T7 RNA pol induction) and IPTG for the T7 promoter)
2) or BL21 with pLysS or pLysE 
both simple keep 2 % Glucose and also directly from trafo goto liquid culture 
in parallel of the plating approach.

Let me know if you need any further tips.


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.



> On 04/04/2022 10:32 AM Christian Roth  wrote:
> 
> 
> Hi Andy,
> have you tried another promotor? Arabinose is much tighter, just to be 
> sure that it is really not leaking.
> 
> Cheers
> Christian
> 
> 
> On Mon, Apr 4, 2022 at 10:20 AM Andrew Lovering  mailto:a.lover...@bham.ac.uk > wrote:
> 
> > > 
> > Dear Board,
> > 
> >  
> > 
> > Perhaps off-topic, but in the wider scope it’s relevant to many on 
> > here.
> > 
> >  
> > 
> > We have a gene that we are able to clone, and propagate in DH5a etc 
> > non-expression cells (hence nucleotide sequence is non-toxic)
> > 
> >  
> > 
> > But, when we attempt to transfer to an expression strain we get no 
> > colonies
> > 
> >  
> > 
> > We have tried pLemo, Glucose addition, 30 degrees, C41/C43 and 
> > still no joy
> > 
> >  
> > 
> > We’d welcome any suggestions here – it’s a fun protein
> > 
> >  
> > 
> > Thanks
> > 
> > Andy
> > 
> > 
> > 
> > -
> > 
> > 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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> 



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Re: [ccp4bb] Off topic - gene toxic for expression strains

[Christian Roth]

Hi Andy,
have you tried another promotor? Arabinose is much tighter, just to be sure
that it is really not leaking.

Cheers
Christian


On Mon, Apr 4, 2022 at 10:20 AM Andrew Lovering 
wrote:

> Dear Board,
>
>
>
> Perhaps off-topic, but in the wider scope it’s relevant to many on here.
>
>
>
> We have a gene that we are able to clone, and propagate in DH5a etc
> non-expression cells (hence nucleotide sequence is non-toxic)
>
>
>
> But, when we attempt to transfer to an expression strain we get no
> colonies
>
>
>
> We have tried pLemo, Glucose addition, 30 degrees, C41/C43 and still no joy
>
>
>
> We’d welcome any suggestions here – it’s a fun protein
>
>
>
> Thanks
>
> Andy
>
> --
>
> 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] Off topic - gene toxic for expression strains

[Andrew Lovering]

Dear Board,

Perhaps off-topic, but in the wider scope it's relevant to many on here.

We have a gene that we are able to clone, and propagate in DH5a etc 
non-expression cells (hence nucleotide sequence is non-toxic)

But, when we attempt to transfer to an expression strain we get no colonies

We have tried pLemo, Glucose addition, 30 degrees, C41/C43 and still no joy

We'd welcome any suggestions here - it's a fun protein

Thanks
Andy



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[ccp4bb] Off-topic: Replacement CCD camera for Rigaku Dekstop Minstrel UV imager

[Christoph Parthier]

Hi,

I know the chance is low, but in a desperate try to fix our long-serving Rigaku 
crystal imager I'm wondering whether anyone is aware of any decommissioned 
(Desktop) Minstrel systems around which could provide spare sparts. 
Unfortunately Rigaku crystal automation systems are no longer supported . 

In particular, we are in need of the built-in replacement CCD camera (a 
QImaging Retiga 4000R, 
https://www.photometrics.com/wp-content/uploads/2020/03/Retiga-4000R-Datasheet.pdf),
 which is (not surprisingly) also not available any longer. 

As the Rigaku Minstrel system is rather closed (and the software no longer 
developed) I'm not sure if other cameras would work, too (it's connected via 
Firewire and has a standard C-mount, 2048x2048 pixels). Does anyone have 
experience or heard of other cameras working in a Minstrel system?

Any help would be greatly appreciated (please contact off the list)!

Thanks
Christoph

-- 
- - - - - - - - - - - - - - - - - - - - - - - - - -
Dr. Christoph Parthier
Arbeitsgruppe Röntgenkristallografie

Institut für Biochemie/Biotechnologie
Martin-Luther-Universität Halle-Wittenberg
Kurt-Mothes-Str. 3a, Raum 3.47
06120 Halle (Saale), Germany
Tel: ++49-345-5524898 Fax: ++49-345-5527014
E-Mail: christoph.parth...@biochemtech.uni-halle.de
- - - - - - - - - - - - - - - - - - - - - - - - - -



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Re: [ccp4bb] Off-topic: happy 2022

[Bernhard Rupp]

For those who want to riddle it out (and as structural biologists hopefully

don’t come across mirror and glide planes) a ppt where you can

take the International Tables and superimpose the tetragonal PG diagrams on

the Kaleidoscope. Find the unit cell first, and then ferret out the rest.

 

https://www.dropbox.com/s/kpss03zueie2sbk/p4_plane_groups.pptx?dl=0

 

Cheers, BR




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Re: [ccp4bb] Off-topic: happy 2022

[Ian Tickle]

Looks like no. 12 p4g to me:
http://www2.clarku.edu/faculty/djoyce/wallpaper/wall12.html

Happy New Year.

On Sat, 1 Jan 2022 at 20:52, Bernhard Rupp  wrote:

> ..and which one of the 17 plane groups do we see here?
>
> Cherrs br
>
> On Sat, Jan 1, 2022, 12:47 Phoebe A. Rice  wrote:
>
>> Apologies for the attachment, but this seems to have become an annual
>> tradition.
>>
>> Best wishes and thanks to this great international community of
>> structural biologists!
>>
>>   (and thanks to the KaleidoPaint app)
>>
>>
>>
>> [image: Background pattern Description automatically generated]
>>
>> --
>>
>> 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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Re: [ccp4bb] Off Topic: CCP4MG Help

[CCP4BB]

Hi

Or make your first object residues 1-50 & 60-100 and the second object 50-60 
(i.e. don't leave a gap between them). ISTR there's a way to join two atoms, 
but I can't remember off-hand.

Harry
--
Dr Harry Powell

> On 5 Nov 2021, at 19:10, Denis Rousseau  
> wrote:
> 
> Hi Matthew
> 
> Try making one set from 1-100 and a separate set from 50-60.  Then change the 
> color of the 50-60. It worked on my computer.
> 
> Best
> 
> Denis
> 
> From: CCP4 bulletin board  on behalf of Whitley, 
> Matthew J 
> Sent: Friday, November 5, 2021 2:21 PM
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: [ccp4bb] Off Topic: CCP4MG Help
>  
> CAUTION: This email comes from an external source; the attachments and/or 
> links may compromise our secure environment. Do not open or click on 
> suspicious emails. Please click on the “Phish Alert” button on the top right 
> of the Outlook dashboard to report any suspicious emails.
> Hello all,
> 
> Looking for advice from any CCP4MG users.
> 
> I am making some structural figures and have run into a problem.  I posted a 
> question to the CCP4MG-specific email list quite a while ago but received no 
> responses, so I don't know if that list is still active.
> 
> Essentially, I want to change the color of a small stretch of residues within 
> the larger protein chain.  For example, I want to display a protein running 
> from residues 1-100 in gray using the ribbons representation, and I want to 
> change only residues 50-60 to red, also in the ribbons representation.
> 
> If I try to accomplish this in CCP4MG by creating two different display 
> objects, {1-49, 61-100} and {50-60}, I can change the colors as desired, but 
> gaps appear at the interfaces between the segments of the ribbons 
> representation, i.e. between residues 49 and 50, and then between residues 60 
> and 61.  Is there a way to plug those gaps such that the structure is 
> smoothly connected from 1-100 in the ribbons representation?  When I display 
> the structure using the cylinders representation, everything is smoothly 
> connected with the correct colors, but when I change to ribbons 
> representation, the gaps appear.  Any ideas?
> 
> Alternatively, there's got to be a way simply to change the color of a subset 
> of residues within a chain without having to create an individual display 
> object for each segment, but if it exists it has escaped me.
> 
> Thanks for any advice you can provide.
> 
> Matthew
> 
> ---
> Matthew J. Whitley, Ph.D.
> Research Instructor
> Department of Pharmacology & Chemical Biology
> University of Pittsburgh School of Medicine
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
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> 
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Re: [ccp4bb] Off Topic: CCP4MG Help

[Denis Rousseau]

Hi Matthew

Try making one set from 1-100 and a separate set from 50-60.  Then change the 
color of the 50-60. It worked on my computer.

Best

Denis


From: CCP4 bulletin board  on behalf of Whitley, Matthew 
J 
Sent: Friday, November 5, 2021 2:21 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Off Topic: CCP4MG Help

CAUTION: This email comes from an external source; the attachments and/or links 
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Hello all,

Looking for advice from any CCP4MG users.

I am making some structural figures and have run into a problem.  I posted a 
question to the CCP4MG-specific email list quite a while ago but received no 
responses, so I don't know if that list is still active.

Essentially, I want to change the color of a small stretch of residues within 
the larger protein chain.  For example, I want to display a protein running 
from residues 1-100 in gray using the ribbons representation, and I want to 
change only residues 50-60 to red, also in the ribbons representation.

If I try to accomplish this in CCP4MG by creating two different display 
objects, {1-49, 61-100} and {50-60}, I can change the colors as desired, but 
gaps appear at the interfaces between the segments of the ribbons 
representation, i.e. between residues 49 and 50, and then between residues 60 
and 61.  Is there a way to plug those gaps such that the structure is smoothly 
connected from 1-100 in the ribbons representation?  When I display the 
structure using the cylinders representation, everything is smoothly connected 
with the correct colors, but when I change to ribbons representation, the gaps 
appear.  Any ideas?

Alternatively, there's got to be a way simply to change the color of a subset 
of residues within a chain without having to create an individual display 
object for each segment, but if it exists it has escaped me.

Thanks for any advice you can provide.

Matthew

---
Matthew J. Whitley, Ph.D.
Research Instructor
Department of Pharmacology & Chemical Biology
University of Pittsburgh School of Medicine




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[ccp4bb] Off Topic: CCP4MG Help

[Whitley, Matthew J]

Hello all,

Looking for advice from any CCP4MG users.

I am making some structural figures and have run into a problem.  I posted a 
question to the CCP4MG-specific email list quite a while ago but received no 
responses, so I don't know if that list is still active.

Essentially, I want to change the color of a small stretch of residues within 
the larger protein chain.  For example, I want to display a protein running 
from residues 1-100 in gray using the ribbons representation, and I want to 
change only residues 50-60 to red, also in the ribbons representation.

If I try to accomplish this in CCP4MG by creating two different display 
objects, {1-49, 61-100} and {50-60}, I can change the colors as desired, but 
gaps appear at the interfaces between the segments of the ribbons 
representation, i.e. between residues 49 and 50, and then between residues 60 
and 61.  Is there a way to plug those gaps such that the structure is smoothly 
connected from 1-100 in the ribbons representation?  When I display the 
structure using the cylinders representation, everything is smoothly connected 
with the correct colors, but when I change to ribbons representation, the gaps 
appear.  Any ideas?

Alternatively, there's got to be a way simply to change the color of a subset 
of residues within a chain without having to create an individual display 
object for each segment, but if it exists it has escaped me.

Thanks for any advice you can provide.

Matthew

---
Matthew J. Whitley, Ph.D.
Research Instructor
Department of Pharmacology & Chemical Biology
University of Pittsburgh School of Medicine




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[ccp4bb] (off topic) Job posting - Associate Director, Protein Science

[Artem Evdokimov]

https://boards.greenhouse.io/roivantsciences/jobs/3549863

Finally posted! Thank you for looking.

Artem

- Cosmic Cats approve of this message



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Re: [ccp4bb] off-topic: pH meters

[Roger Rowlett]

The meter doesn't matter so much as the electrode. For the meter, anything
with at least 3 pH multipoint calibration is sufficient for most purposes.
I usually bought something from our preferred university vendor a with very
large LCD displays for my aging eyes.

For protein work, and especially if using anything with Tris buffers, a
refillable, double junction Ag/AgCl reference combination electrode is a
must. Making stock 1M Tris buffer concentrate will wack out single junction
combination electrodes, sometimes permanently. I liked the Fisher
Accutuph/Accuphast electrodes because they were much more difficult for my
students to break, and they last for years. (My students never succeeded in
breaking one.) Gel-filled electrodes are garbage. Refillable double
junction electrodes are the way to go for stable readings, especially in
Tris, and minimizing heavy metal contamination.

Roger Rowlett
Gordon & Dorothy Kline Professoe, Emeritus
Department of Chemistry
Colgate University

On Mon, Sep 20, 2021, 4:57 PM Patrick Loll  wrote:

> Fellow protein biochemists:
>
> My ~30 year-old Beckman pH meter is finally showing its age, and I’m
> looking for a high-quality replacement that doesn’t cost insane amounts of
> money. My initial thoughts gravitate toward Mettler (it’s a name I trust,
> and some of their low-end instruments aren’t absurdly expensive); would
> anybody care to recommend other options?
>
> Much obliged for any suggestions (the more obsessive, the better).
>
> Cheers,
>
> Pat
>
>
>
> ---
> Patrick J. Loll, Ph. D.  (he, him, his)
> Professor of Biochemistry & Molecular Biology
> Drexel University College of Medicine
> Room 10-102 New College Building
> 245 N. 15th St., Mailstop 497
> Philadelphia, PA  19102  USA
>
> (215) 762-7706
> pjl...@gmail.com
> pj...@drexel.edu
>
> 
>
> 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] off-topic: pH meters

[Patrick Loll]

Fellow protein biochemists:

My ~30 year-old Beckman pH meter is finally showing its age, and I’m looking 
for a high-quality replacement that doesn’t cost insane amounts of money. My 
initial thoughts gravitate toward Mettler (it’s a name I trust, and some of 
their low-end instruments aren’t absurdly expensive); would anybody care to 
recommend other options?

Much obliged for any suggestions (the more obsessive, the better).

Cheers,

Pat


---
Patrick J. Loll, Ph. D.  (he, him, his)
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu



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Re: [ccp4bb] Off topic, protein characterization and binding job opportunity in Boston area

[Yanfeng Zhou]

Here is the right link -

https://www.linkedin.com/jobs/view/2662640332/?refId=cCDvahCrRPGWslMQWMgEOQ%3D%3D

Thank you everyone for pointing this out.



On Mon, Aug 23, 2021 at 4:18 PM Yanfeng Zhou 
wrote:

> Hi,
>
> Sorry for the off topic post. If anyone is passionate about working in an
> exciting biotech powered by machine learning, structural biology, and
> protein engineering, around Boston in the US, please check the link below.
>
>
> https://www.linkedin.com/jobs/view/2662640332/efId=cCDvahCrRPGWslMQWMgEOQ%3D%3D
>
> *Who we are*
>
> Seismic Therapeutic is a VC-backed biotechnology startup focused on the
> intersection of structure, protein engineering, machine learning and
> immunology founded by Tim Springer, Jeff Ravetch, Debora Marks and Alan
> Crane.
>
> *What we offer you*
>
> As a member of our new and quickly growing company, you’ll help us shape
> Seismic from the very beginning into a startup that not only takes its
> scientific mission seriously but also provides a positive and supportive
> workplace environment and culture. Seismic will have the opportunity to
> benefit from the insight, diversity, and talent that you’ll bring.
> Together, we’ll help bring therapeutics to the patients who need them most.
>
> *Our mission*
>
> Seismic is determined to help patients in need, starting with novel
> approaches to protein engineering, machine learning and immunology, and
> ending with clinic-ready drugs. At our core, our focus is on phenomenal
> science, meritocracy, a superstar team, supportive culture, and
> patient-centric goals. Seismic has the tools, skills, ideas and people to
> make this a reality – all we need is you! Success will enable treatment for
> millions of patients with currently incurable, often disabling and deadly
> diseases, and we invite you to become a part of that.
>
> *Where?*
>
> Seismic is located in the dynamic biotech community in Watertown,
> alongside other new companies that are turning ideas into reality and
> changing the biotech and medical scenes for the better. We are thrilled to
> expand our Team with hardworking, highly qualified, highly motivated
> individuals to join us in our lab space.
>
> *Available position*
>
> Scientist/Senior Scientist, Biochemistry and protein interactions
>
> General role: In coordination with scientific leadership, you would play a
> central role in designing and implementing Seismic’s pipeline, with
> particular focus on processes occurring during biotherapeutic candidate
> generation, screening and analysis. This role will involve exploring
> customized solutions in the field of protein-protein interactions, followed
> by detailed qualitative and quantitative sample analysis, leading into
> high-throughput screening and characterization techniques. Successfully
> meeting these challenges will require not only a broad range of protein
> biochemistry skills, independent thinking, and an aptitude for
> troubleshooting, but also a strong, creative drive to develop new assays as
> the need arises.
>
> Your work in this role would be in collaboration with scientists and
> computational experts throughout Seismic, learning and contributing to many
> aspects of our pipeline, including the iterative design and production of
> biotherapeutics. You would be responsible for understanding project goals
> and for creatively optimizing solutions to ensure their success. Over time
> you would take on additional responsibilities and contribute to new
> projects.
>
> *Responsibilities*:
>
> ● Design and implement protocols for characterizing protein-protein
> interactions.
> ● Design and optimize qualitative and quantitative biophysical and
> biochemical studies of biotherapeutic molecules, including assay
> development and protein QC.
> ● Share recommendations and insight for pipeline improvement toward
> higher-throughput and efficiency
> ● Collaborate with a diverse team of wet-lab scientists, protein
> engineers, and computational scientists
> ● Maintain an in depth understanding of the biochemistry, molecular
> structure and wider role of each candidate through critical analysis of the
> literature.
>
> *Basic qualifications:*
>
> ● PhD in chemistry, biology, biochemistry, biomedical engineering, or
> related field
> ● 5+ years hands-on wet lab experience, including at least two of the
> following:
> o Strong expertise in characterizing protein-protein interaction using
> label free technologies such as interferometry (Octet) or surface plasmon
> resonance (Biacore, Carterra)
> o Direct experience with independent acquisition of kinetics and affinity
> characterization datasets.
> o Detailed experience in data analysis and reporting in kinetics and
> affinity measurements.
> o Capable at optimizing high-throughput protocols.
> o Solid experience with protein biochemical analysis, including assay
> development (eg. ELISA and ECLIA, etc) and protein quality control
> characterizations.
> ● Strong collaborative, organizational, and 

[ccp4bb] Off topic, protein characterization and binding job opportunity in Boston area

[Yanfeng Zhou]

Hi,

Sorry for the off topic post. If anyone is passionate about working in an
exciting biotech powered by machine learning, structural biology, and
protein engineering, around Boston in the US, please check the link below.

https://www.linkedin.com/jobs/view/2662640332/efId=cCDvahCrRPGWslMQWMgEOQ%3D%3D

*Who we are*

Seismic Therapeutic is a VC-backed biotechnology startup focused on the
intersection of structure, protein engineering, machine learning and
immunology founded by Tim Springer, Jeff Ravetch, Debora Marks and Alan
Crane.

*What we offer you*

As a member of our new and quickly growing company, you’ll help us shape
Seismic from the very beginning into a startup that not only takes its
scientific mission seriously but also provides a positive and supportive
workplace environment and culture. Seismic will have the opportunity to
benefit from the insight, diversity, and talent that you’ll bring.
Together, we’ll help bring therapeutics to the patients who need them most.

*Our mission*

Seismic is determined to help patients in need, starting with novel
approaches to protein engineering, machine learning and immunology, and
ending with clinic-ready drugs. At our core, our focus is on phenomenal
science, meritocracy, a superstar team, supportive culture, and
patient-centric goals. Seismic has the tools, skills, ideas and people to
make this a reality – all we need is you! Success will enable treatment for
millions of patients with currently incurable, often disabling and deadly
diseases, and we invite you to become a part of that.

*Where?*

Seismic is located in the dynamic biotech community in Watertown, alongside
other new companies that are turning ideas into reality and changing the
biotech and medical scenes for the better. We are thrilled to expand our
Team with hardworking, highly qualified, highly motivated individuals to
join us in our lab space.

*Available position*

Scientist/Senior Scientist, Biochemistry and protein interactions

General role: In coordination with scientific leadership, you would play a
central role in designing and implementing Seismic’s pipeline, with
particular focus on processes occurring during biotherapeutic candidate
generation, screening and analysis. This role will involve exploring
customized solutions in the field of protein-protein interactions, followed
by detailed qualitative and quantitative sample analysis, leading into
high-throughput screening and characterization techniques. Successfully
meeting these challenges will require not only a broad range of protein
biochemistry skills, independent thinking, and an aptitude for
troubleshooting, but also a strong, creative drive to develop new assays as
the need arises.

Your work in this role would be in collaboration with scientists and
computational experts throughout Seismic, learning and contributing to many
aspects of our pipeline, including the iterative design and production of
biotherapeutics. You would be responsible for understanding project goals
and for creatively optimizing solutions to ensure their success. Over time
you would take on additional responsibilities and contribute to new
projects.

*Responsibilities*:

● Design and implement protocols for characterizing protein-protein
interactions.
● Design and optimize qualitative and quantitative biophysical and
biochemical studies of biotherapeutic molecules, including assay
development and protein QC.
● Share recommendations and insight for pipeline improvement toward
higher-throughput and efficiency
● Collaborate with a diverse team of wet-lab scientists, protein engineers,
and computational scientists
● Maintain an in depth understanding of the biochemistry, molecular
structure and wider role of each candidate through critical analysis of the
literature.

*Basic qualifications:*

● PhD in chemistry, biology, biochemistry, biomedical engineering, or
related field
● 5+ years hands-on wet lab experience, including at least two of the
following:
o Strong expertise in characterizing protein-protein interaction using
label free technologies such as interferometry (Octet) or surface plasmon
resonance (Biacore, Carterra)
o Direct experience with independent acquisition of kinetics and affinity
characterization datasets.
o Detailed experience in data analysis and reporting in kinetics and
affinity measurements.
o Capable at optimizing high-throughput protocols.
o Solid experience with protein biochemical analysis, including assay
development (eg. ELISA and ECLIA, etc) and protein quality control
characterizations.
● Strong collaborative, organizational, and presentation skills
● Ability to design and improve upon advanced protocols and procedures
● Ability to direct Research Associates, Technicians and CRO’s
● Ability to document, maintain, and share an organized account of lab work
and results



*Preferred qualifications:*
● 2+ years post PhD experience
● Understanding in immunology research and industrial biologics R
● Mentoring and managing 

Re: [ccp4bb] off-topic: structural motif / domain comparison

[Bernhard Rupp]

A lesser known service with very powerful search across domains and chains is 

TopSearch by Manfred Sippl & Cie.:

https://topsearch.services.came.sbg.ac.at/

 

Its training set includes PDB entries up to 2018.

 

Best, BR

 

From: CCP4 bulletin board  On Behalf Of Sam Tang
Sent: Thursday, August 5, 2021 23:43
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off-topic: structural motif / domain comparison

 

Dear all

 

Sorry for an off-topic question here. I wonder if anyone may be aware of any 
search program which allows one to 'blast' a protein domain just like we 
'blast' a protein sequence? For example I have an epitope in hand and would 
like to find out whether this also exists in other proteins. Most programs I 
accessed are based on sequence similarity but is there any program which 
searches a structure against a database of structures?

 

BRs

 

Sam

 

  _  

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Re: [ccp4bb] off-topic: structural motif / domain comparison

[orly avraham]

You might also find use in databases such as pfam, ecod, cath, scop.

Orly

On Fri, Aug 6, 2021, 09:43 Sam Tang  wrote:

> Dear all
>
> Sorry for an off-topic question here. I wonder if anyone may be aware of
> any search program which allows one to 'blast' a protein domain just like
> we 'blast' a protein sequence? For example I have an epitope in hand and
> would like to find out whether this also exists in other proteins. Most
> programs I accessed are based on sequence similarity but is there any
> program which searches a structure against a database of structures?
>
> BRs
>
> Sam
>
> --
>
> 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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Re: [ccp4bb] off-topic: structural motif / domain comparison

[Dalibor Košek]

Perhaps DALI can be of use
http://ekhidna2.biocenter.helsinki.fi/dali/

Dal.

pá 6. 8. 2021 v 8:42 odesílatel Sam Tang  napsal:

> Dear all
>
> Sorry for an off-topic question here. I wonder if anyone may be aware of
> any search program which allows one to 'blast' a protein domain just like
> we 'blast' a protein sequence? For example I have an epitope in hand and
> would like to find out whether this also exists in other proteins. Most
> programs I accessed are based on sequence similarity but is there any
> program which searches a structure against a database of structures?
>
> BRs
>
> Sam
>
> --
>
> 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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Re: [ccp4bb] off-topic: structural motif / domain comparison

[Chris Fage]

Hi Sam,

Perhaps the Modeller service is what you’re looking for?

https://salilab.org/modeller/

Best wishes,
Chris


On Fri, 6 Aug 2021 at 07:42 Sam Tang  wrote:

> Dear all
>
> Sorry for an off-topic question here. I wonder if anyone may be aware of
> any search program which allows one to 'blast' a protein domain just like
> we 'blast' a protein sequence? For example I have an epitope in hand and
> would like to find out whether this also exists in other proteins. Most
> programs I accessed are based on sequence similarity but is there any
> program which searches a structure against a database of structures?
>
> BRs
>
> Sam
>
> --
>
> 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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Re: [ccp4bb] off-topic: structural motif / domain comparison

[Jan Dohnalek]

Like PDBeFOLD search?
https://www.ebi.ac.uk/msd-srv/ssm/

Jan


On Fri, Aug 6, 2021 at 8:43 AM Sam Tang  wrote:

> Dear all
>
> Sorry for an off-topic question here. I wonder if anyone may be aware of
> any search program which allows one to 'blast' a protein domain just like
> we 'blast' a protein sequence? For example I have an epitope in hand and
> would like to find out whether this also exists in other proteins. Most
> programs I accessed are based on sequence similarity but is there any
> program which searches a structure against a database of structures?
>
> BRs
>
> Sam
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>


-- 
Jan Dohnalek, Ph.D
Institute of Biotechnology
Academy of Sciences of the Czech Republic
Biocev
Prumyslova 595
252 50 Vestec near Prague
Czech Republic

Tel. +420 325 873 758



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[ccp4bb] off-topic: structural motif / domain comparison

[Sam Tang]

Dear all

Sorry for an off-topic question here. I wonder if anyone may be aware of
any search program which allows one to 'blast' a protein domain just like
we 'blast' a protein sequence? For example I have an epitope in hand and
would like to find out whether this also exists in other proteins. Most
programs I accessed are based on sequence similarity but is there any
program which searches a structure against a database of structures?

BRs

Sam



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Re: [ccp4bb] off-topic: glycans

[Sam Tang]

Thanks David! This is exactly what I am looking for.

Sam


On Tue, 29 Jun 2021 at 19:35, David Briggs  wrote:

> Hi Sam,
>
> GlycoMod from Expasy sounds like it might do what you want to do.
>
> https://web.expasy.org/glycomod/
>
> D
>
> --
>
> *Dr David C. Briggs*
>
> Senior Laboratory Research Scientist
>
> Signalling and Structural Biology Lab
>
> The Francis Crick Institute
>
> London, UK
>
> ==
>
> Diamond User Committee (MX)
>
> CCP4 WG2
>
> ==
>
> about.me/david_briggs
> --
> *From:* CCP4 bulletin board  on behalf of Sam Tang
> 
> *Sent:* 29 June 2021 06:12
> *To:* CCP4BB@JISCMAIL.AC.UK 
> *Subject:* [ccp4bb] off-topic: glycans
>
>
> *External Sender:* Use caution.
>
> Dear community
>
> Sorry for an off-topic question here. I wonder if anyone may be aware of
> any glycan modification database where we can predict what is what. For
> example, if I got a mass difference of m/z X on LC-MS, and I would like to
> have a rough idea what it might be, where should I go for?
>
> Thanks!
>
> BRs
>
> Sam
>
> --
>
> 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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>
> The Francis Crick Institute Limited is a registered charity in England and
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> 06885462, with its registered office at 1 Midland Road London NW1 1AT
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Re: [ccp4bb] off-topic: glycans

[David Briggs]

Hi Sam,

GlycoMod from Expasy sounds like it might do what you want to do.

https://web.expasy.org/glycomod/

D


--

Dr David C. Briggs

Senior Laboratory Research Scientist

Signalling and Structural Biology Lab

The Francis Crick Institute

London, UK

==

Diamond User Committee (MX)

CCP4 WG2

==

about.me/david_briggs


From: CCP4 bulletin board  on behalf of Sam Tang 

Sent: 29 June 2021 06:12
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] off-topic: glycans


External Sender: Use caution.

Dear community

Sorry for an off-topic question here. I wonder if anyone may be aware of any 
glycan modification database where we can predict what is what. For example, if 
I got a mass difference of m/z X on LC-MS, and I would like to have a rough 
idea what it might be, where should I go for?

Thanks!

BRs

Sam



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The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 Midland Road London NW1 1AT



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[ccp4bb] off-topic: glycans

[Sam Tang]

Dear community

Sorry for an off-topic question here. I wonder if anyone may be aware of
any glycan modification database where we can predict what is what. For
example, if I got a mass difference of m/z X on LC-MS, and I would like to
have a rough idea what it might be, where should I go for?

Thanks!

BRs

Sam



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[ccp4bb] Off-topic: research associate job posting

[Artem Evdokimov]

Dear CCP4 friends

We are looking for fresh talent :) please see the posting below or directly
at MassBio.

https://careers.massbio.org/jobs/?keywords=Research+Associate+Silicon+Therapeutics=Boston%2C+MA%2C+United+States_completion=city%3DBoston%24state%3DMassachusetts%24country%3DUnited+States_type=city_text=Boston%2C+MA%2C+United+States_autocomplete=true=320


Artem


Silicon Therapeutics (“SITX”) is a privately held, physics-driven
integrated drug discovery company. SITX’s discovery efforts as well as
physics-based platform are all completely in-house, with a full wet lab as
well as a team of experienced R professionals spanning biology,
biochemistry, chemistry, biophysics (NMR + X-ray) & preclinical sciences.
SITX’s engine leverages quantum mechanics and molecular dynamics, which are
deployed on its own internal supercomputer composed of over 400 GPU’s and
FPGA’s and allows SITX to accurately simulate the physical motion and
properties of biological targets at an atomistic level resolution. We are
the only company that owns the entire spectrum of physics-driven drug
discovery from chip-to-clinic with a team of over 60 individuals in Boston.

Our diversity and our cross-functional, multi-dimensional teams make us
strong. We foster an open-minded culture where we come to work without
preconceptions about how people think. With all channels open for
communication, we can rapidly fuse information across disciplines and teams.

Job Summary

Silicon Therapeutics is seeking an energetic, passionate practitioner of
the laboratory arts to join the Biochemistry/Biophysics/Structural Biology
platform as a Research Associate . This is a foundational support role for
someone who enjoys technical challenges and is not afraid to get their
hands dirty by helping with core lab functions from cell culture, protein
purification, or biochemical experiments all the way to laboratory
automation, instrument maintenance, etc. The qualified candidate will have
basic laboratory practices under their belt and would be passionate to
learn additional techniques while supporting a growing group dedicated to
Discovery research. The successful candidate will work as part of a
collaborative team of scientists with expertise in protein production,
biochemistry, structure, and biophysics supporting hit-ID, hit-to-lead, and
lead-ID phases of our innate immuno-oncology drug discovery programs and
will have opportunities to learn cutting edge techniques.

Job Responsibilities

Responsible for bacterial and eukaryotic cell culture for the purpose of
recombinant protein expression.
Basic protein purification under supervision of expert practitioners.
Preparation of common biochemical reagents, maintenance of lab readiness.
Biochemical/biophysical characterization of recombinant proteins
Work as part of a cross-functional project team(s) to provide timely,
robust, high-impact results.
Carefully document research workflows, data and protocols using an
electronic laboratory notebook.
Capture experimental results in database.
Organize and present data as needed
Other duties as assigned


Requirements

A minimum of B.Sc. with 2+ years of research experience
Documented experience with recombinant protein production
Propensity towards tinkering with complex equipment
Ability to multitask on the job, combining diligence and tenacity to thrive
in a highly malleable and fluid research environment
Demonstrated ability to execute experiments independently and under the
supervision of experts.
Passion for supporting teams and learning on the job.
Excellent communication skills.
Silicon Therapeutics provides equal employment opportunities to all
employees and applicants for employment and prohibits discrimination and
harassment of any type without regard to race, color, religion, age, sex,
national origin, disability status, genetics, protected veteran status,
sexual orientation, gender identity or expression, or any other
characteristic protected by federal, state or local laws.

Job Information

Job ID: 55846631
Location:
Boston, Massachusetts, United States
Position Title: Research Associate, Biochemistry/Biophysics/Structural
Biology
Company Name: Silicon Therapeutics
Job Function: Scientist
Job Type: Full-Time
Job Duration: Indefinite
Min Education: BA/BS/Undergraduate
Min Experience: 2-3 Years
Required Travel: None


Silicon Therapeutics (“SITX”) is a privately-held, physics-driven
integrated drug discovery company in the preclinical IND stage targeting
innate immunity to start, but with the capability to move into many other
franchise opportunities. The Company’s initial pipeline is focused on
modulation of the innate immune system to “light the spark” within
immunologically cold tumors by using its physics-enabled drug discovery
engine.



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This 

Re: [ccp4bb] OFF TOPIC question

[Peat, Tom (Manufacturing, Parkville)]

Hello Anamika,

>From the information you gave, you have two different promoters- araC and the 
>lac promoter. LacI is the lac inhibitor. The lac promoter is a two part 
>system- when lactose is not present, the inhibitor sits near the promoter and 
>blocks transcription of genes downstream.
Almost any E. coli system will work the expression of proteins from these 
promoters, excepting those that have been modified in some way to block 
arabinose or lactose use. The BL21 (DE3) system has the T7 system implemented, 
but as you are not using the T7 promoter, there is no particular reason to use 
this version of E. coli.
Best of luck, tom

Tom Peat, PhD
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au


From: CCP4 bulletin board  on behalf of Anamika Singh 

Sent: Thursday, November 26, 2020 8:59 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] OFF TOPIC question

Hi all,

I have two constructs having different ori, p15ori and M13 ori, different 
promoters araC and LacI, and different antibiotic resistance chloramphenicol 
and Ampicillin respectively. I would like to know which expressing E. coli host 
cells will be good for the co-transformation of these constructs?

Thanks

Anamika




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Re: [ccp4bb] OFF TOPIC question

[Crissy L Tarver]

BL21(DE3) pLysS using 5X KCM and heat shock transformation worked for me.

Crissy L Tarver
Postdoctoral Researcher
Department of Structural Biology
Stanford University School of Medicine

From: CCP4 bulletin board  on behalf of Hughes, Jonathan 

Sent: Thursday, November 26, 2020 2:48:24 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] AW: [ccp4bb] OFF TOPIC question


Bl21Pro

j



Von: CCP4 bulletin board  Im Auftrag von Anamika Singh
Gesendet: Donnerstag, 26. November 2020 11:07
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] OFF TOPIC question



One correction to the previous question: I have two constructs having different 
ori, p15ori and M13 ori, different promoters araBAD promoter and LacI, and 
different antibiotic resistance chloramphenicol and Ampicillin respectively. I 
would like to know which E. coli host cells will be good for the 
co-transformation of these constructs?



On Thu, 26 Nov 2020 at 11:59, Anamika Singh 
mailto:anamika.ii...@gmail.com>> wrote:

Hi all,



I have two constructs having different ori, p15ori and M13 ori, different 
promoters araC and LacI, and different antibiotic resistance chloramphenicol 
and Ampicillin respectively. I would like to know which expressing E. coli host 
cells will be good for the co-transformation of these constructs?



Thanks



Anamika






--

Dr. Anamika Singh
Post-Doctoral Fellow

Silberman Institute of Life Sciences

Hebrew University of Jerusalem, Israel

No: 054-294-8036





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[ccp4bb] AW: [ccp4bb] OFF TOPIC question

[Hughes, Jonathan]

Bl21Pro
j

Von: CCP4 bulletin board  Im Auftrag von Anamika Singh
Gesendet: Donnerstag, 26. November 2020 11:07
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] OFF TOPIC question

One correction to the previous question: I have two constructs having different 
ori, p15ori and M13 ori, different promoters araBAD promoter and LacI, and 
different antibiotic resistance chloramphenicol and Ampicillin respectively. I 
would like to know which E. coli host cells will be good for the 
co-transformation of these constructs?

On Thu, 26 Nov 2020 at 11:59, Anamika Singh 
mailto:anamika.ii...@gmail.com>> wrote:
Hi all,

I have two constructs having different ori, p15ori and M13 ori, different 
promoters araC and LacI, and different antibiotic resistance chloramphenicol 
and Ampicillin respectively. I would like to know which expressing E. coli host 
cells will be good for the co-transformation of these constructs?

Thanks

Anamika



--
Dr. Anamika Singh
Post-Doctoral Fellow
Silberman Institute of Life Sciences
Hebrew University of Jerusalem, Israel
No: 054-294-8036



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Re: [ccp4bb] OFF TOPIC question

[Anamika Singh]

*One correction to the previous question:* I have two constructs having
different ori, p15ori and M13 ori, different promoters *araBAD promoter*
and LacI, and different antibiotic resistance chloramphenicol and
Ampicillin respectively. I would like to know which E. coli host cells will
be good for the co-transformation of these constructs?

On Thu, 26 Nov 2020 at 11:59, Anamika Singh  wrote:

> Hi all,
>
> I have two constructs having different ori, p15ori and M13 ori, different
> promoters araC and LacI, and different antibiotic resistance
> chloramphenicol and Ampicillin respectively. I would like to know which 
> *expressing
> E. coli host cells* will be good for the co-transformation of these
> constructs?
>
> Thanks
>
> Anamika
>
>

-- 
Dr. Anamika Singh
Post-Doctoral Fellow
Silberman Institute of Life Sciences
Hebrew University of Jerusalem, Israel
No: 054-294-8036



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[ccp4bb] OFF TOPIC question

[Anamika Singh]

Hi all,

I have two constructs having different ori, p15ori and M13 ori, different
promoters araC and LacI, and different antibiotic resistance
chloramphenicol and Ampicillin respectively. I would like to know
which *expressing
E. coli host cells* will be good for the co-transformation of these
constructs?

Thanks

Anamika



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Re: [ccp4bb] (off-topic) beamline for XAFS

[Jan Kern]

Dear Banu,
I would recommend looking also at SSRL beam line 7-3 and 9-3 as they are
well set up for protein EXAFS.
Greetings,
Jan

On Tue, Nov 10, 2020 at 11:03 AM PULSARSTRIAN 
wrote:

> Dear all,
>   Sorry for the off topic.
> Looking for suggestions on beamlines for XAFS on proteins with [4Fe-4S]
> clusters:
> I have two options, one at BNL and another at APS@ANL.
> At BNL, I think 6-BM (BMM) seems to be more for biological samples on
> metalloproteins on the other hand, ANL has several beamlines for XAFS, and
> not sure which one is the best for [4Fe-4S] proteins.
> Any suggestions on these beamlines for XAFS on [4Fe-4S] proteins, would be
> a great help.
>
> Regards,
> Bhanu
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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[ccp4bb] (off-topic) beamline for XAFS

[PULSARSTRIAN]

Dear all,
  Sorry for the off topic.
Looking for suggestions on beamlines for XAFS on proteins with [4Fe-4S]
clusters:
I have two options, one at BNL and another at APS@ANL.
At BNL, I think 6-BM (BMM) seems to be more for biological samples on
metalloproteins on the other hand, ANL has several beamlines for XAFS, and
not sure which one is the best for [4Fe-4S] proteins.
Any suggestions on these beamlines for XAFS on [4Fe-4S] proteins, would be
a great help.

Regards,
Bhanu



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Re: [ccp4bb] Off-topic: Mild cross-linking protocol

[Artem Evdokimov]

If you have hopes for cysteine residues in reasonable proximity, then
bis-iodoacetamide (with a suitable spacer, commercially available is the
ethylenediamine spacer). Any reasonable chemist can make you other spacer
lengths to order.

Homobifunctional PEG with maleimide, N-hydroxy succinimide esters, etc
might work also.

Homobifunctional PEG with amines on ends can be used with a water soluble
coupler e.g. EDC NBT mix.

Notably if you are lucky just treating protein as a dimer with EDC NBT mix
can promote clinking.

Bis aldehydes are an old standby but they are usually quite harsh.

Feel free to write directly for/with additional details.

Artem

On Mon, Oct 19, 2020, 3:18 AM Chiara Bruckmann 
wrote:

> Dear all,
>
> Sorry for the off-topic question. I need to prepare an heterodimeric
> protein complex for an immunisation, and I would like to make sure that the
> dimer will be stable and it won't dissociate after injection.
>
> I am wondering if any of you has a mild cross-linking protocol (and
> suggestions of a suitable reagent) to share.
>
> Thanks and regards,
> Chiara
>
> 
>
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>
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[ccp4bb] Off-topic: Mild cross-linking protocol

[Chiara Bruckmann]

Dear all,

Sorry for the off-topic question. I need to prepare an heterodimeric protein 
complex for an immunisation, and I would like to make sure that the dimer will 
be stable and it won't dissociate after injection. 

I am wondering if any of you has a mild cross-linking protocol (and suggestions 
of a suitable reagent) to share.

Thanks and regards,
Chiara



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[ccp4bb] Off-topic: amphipathic polymer NV-10

[Uma Gabale]

Dear all,Sorry for an extremely off-topic question. We are looking for an 
amphipathic polymer, NV-10. It used to be made by Expedeon and has been used 
for assisting proper protein folding as latest as in 2019 as gathered from the 
literature. Now in the year 2020, it seems to have disappeared from the face of 
the Earth. Expedeon was taken over by Abcam and Abcam no longer manufactures it.
Real trouble is that I couldn’t really find any more details about the polymer, 
which would have assisted me to find a substitute. My question is: does anyone 
have any suggestion as which other amphipathic polymers (A8-35, PMALs, NAPol or 
any others) could be used as a substitute for NV-10? Given that these amphipols 
are very expensive, and we would need considerable amounts of them, I would 
certainly like to narrow down.

I will highly appreciate any input.

Thanks and regards,
Uma.

--Uma Gabale, PhDResearch AssociateMolecular and Cellular Biochemistry
Indiana University Bloomington  



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[ccp4bb] [OFF TOPIC] Cell biology bb

[Rafael Marques]

Hi Folks, how are you doing? Hope everyone is safe!

I was wondering if there is a bulletin board email group like this one 
concerning Cell Biology, where we could find not only discussions about related 
topics but also post-doc opportunities. I would be very glad if you guys know 
any and could share it with me.

Kind Regards

__

Rafael Marques da Silva
Mestrando em Física Biomolecular
Universidade de São Paulo

Bacharel em Ciências Biológicas
Universidade Federal de São Carlos

phone: +55 16 99766-0021

   "A sorte acompanha uma mente bem treinada"





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[ccp4bb] off-topic: coronavirus test summaries

[Artem Evdokimov]

Just FYI, here are two useful links for those who would like to see what's
being used to test for coronavirus:

https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf


https://www.finddx.org/covid-19/pipeline/

May be this saves someone twenty minutes of searching online.

Artem

- Cosmic Cats approve of this message



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[ccp4bb] off-topic: work opportunities at Enko

[Artem Evdokimov]

Hello!

Sorry (not sorry) for the off-topic post. We have a few jobs posted and I'd
like to re-post them here.

Please see the link for full descriptions >>>
https://www.enkochem.com/careers-enko

-Scientist, Structural Biology & Protein Chemistry
-Senior Scientist, Protein Target Discovery
-Scientist, Enzymology & Protein Chemistry
-Enzymology Research Associate

(may be less relevant to this crowd - below)

-Regulatory Toxicologist
-Analytical Scientist, Biology
-Greenhouse Technician, Biology
-Greenhouse Manager, Biology

Key points:

(1) if you'd like to learn more, informally - please feel free to write me
directly
(2) if you'd like to apply, please write to *care...@enkochem.com*


Artem

VP of Biochemistry/Molecular Biology
Enko



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Re: [ccp4bb] Off-topic: (micro)array image analysis software

[Darren Hart]

Hello,

You could run Image Quant TL in a VM (parallels, vmware or virtual box).

https://bmi.cchmc.org/resources/software/imagequant-tl

For this appliation (arrays), we use an old program called VisualGrid 
that is no longer available and run it an isolated XP VM via virtual box 
(in linux).


Darren


On 21/01/2020 15:48, Bärbel Blaum wrote:


Hello,

clearly off-topic but maybe someone here is experienced in the 
analysis of array scans and can offer some advice? That would be 
beautiful. Here’s the problem: I want to test an ELISA-based HT array 
for phosphorylation profiling of a whole pathway, with almost 200 
antibodies at once. The array company offers free scanning of the 
arrays, i.e. we do the experiment, sent the slides back, and receive 
the scans as raw images in tiff format. Can anyone suggest a suitable 
program to analyse such images of array scans on a Mac? There are six 
replicates per antibody per slide so in theory a good basis for at 
least semi-quantitative analysis - but finding a program to analyse 
these data is a real pain. The scans are obtained with a GenePix 
scanner I am being told but the instrument’s software does not run on 
a Mac. I tried ImageJ, which is open source and runs but needs some 
plugin for arrays that does not work for me (I suspect it worked for a 
previous version of ImageJ). I also tried TIGR Spotfinder, which in 
theory seems the perfect program (plenty of documentation), is also 
open source and Mac compatible - but when I try to compile it several 
files seem missing from the sourceforge package (and I cannot find 
another source).


Does anyone use either ImageJ with the array plugin or the Spotfinder 
and can assure me that these options are still being developed or 
maybe knows for sure they are dead and not worth investing any more 
time? Or, even better, could point me to a program that runs on a Mac 
and is suitable for array analysis (it does not actually have to be 
free as long as it works for users who do not write code). I do have a 
GAL file for the images.


Many thanks for your help and sorry for the spam!

Bärbel

--

Bärbel Blaum, PhD

Inthera Bioscience AG

Einsiedlerstrasse 34

CH-8820 Waedenswil

Switzerland

E-Mail: baerbel.bl...@intherabio.com

Phone: +41 43 477 94 72--




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--

**

Dr. Darren J. Hart,

CNRS Research Director, Institut de Biologie Structurale (IBS)
Unité Mixte de Recherche UMR5075 (CEA-CNRS-UGA)

Director, Integrated Structural Biology Grenoble (ISBG)
Unité Mixte de Service UMS3518 (CNRS-UGA-CEA-EMBL)

**

Email: darren.h...@ibs.fr

Tel: +33 4 57 42 85 86

Physical address: IBS/ISBG, 71 avenue des Martyrs, 38000 Grenoble, France

Postal address: IBS/ISBG, 71 avenue des Martyrs, CS 20192, 38042 
Grenoble, Cedex 9, France


**




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[ccp4bb] Off-topic: (micro)array image analysis software

[Bärbel Blaum]

Hello,

 

clearly off-topic but maybe someone here is experienced in the analysis of 
array scans and can offer some advice? That would be beautiful. Here’s the 
problem: I want to test an ELISA-based HT array for phosphorylation profiling 
of a whole pathway, with almost 200 antibodies at once. The array company 
offers free scanning of the arrays, i.e. we do the experiment, sent the slides 
back, and receive the scans as raw images in tiff format. Can anyone suggest a 
suitable program to analyse such images of array scans on a Mac? There are six 
replicates per antibody per slide so in theory a good basis for at least 
semi-quantitative analysis - but finding a program to analyse these data is a 
real pain. The scans are obtained with a GenePix scanner I am being told but 
the instrument’s software does not run on a Mac. I tried ImageJ, which is open 
source and runs but needs some plugin for arrays that does not work for me (I 
suspect it worked for a previous version of ImageJ). I also tried TIGR 
Spotfinder, which in theory seems the perfect program (plenty of 
documentation), is also open source and Mac compatible - but when I try to 
compile it several files seem missing from the sourceforge package (and I 
cannot find another source). 

 

Does anyone use either ImageJ with the array plugin or the Spotfinder and can 
assure me that these options are still being developed or maybe knows for sure 
they are dead and not worth investing any more time? Or, even better, could 
point me to a program that runs on a Mac and is suitable for array analysis (it 
does not actually have to be free as long as it works for users who do not 
write code). I do have a GAL file for the images.

 

Many thanks for your help and sorry for the spam!

 

Bärbel 

 

-- 

Bärbel Blaum, PhD

Inthera Bioscience AG

Einsiedlerstrasse 34

CH-8820 Waedenswil

Switzerland

E-Mail: baerbel.bl...@intherabio.com

Phone: +41 43 477 94 72--

 

 




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Re: [ccp4bb] Off-topic (somewhat): Call for papers on structure-guided kinase drug discovery

[John R Helliwell]

Dear Isabel,
I strongly support and **heartily thankyou** for your email below in support of 
IUCr Journals. A wide range of readerships are covered, now also including 
IUCrJ with its articles describing results aimed at highly diverse readerships 
ie well beyond crystallography. I would also add that the open access fees or 
journal subscription costs are the most competitive ie lowest available. The 
small surpluses from the IUCr Journals that are made are ploughed back into the 
journals further development or support for student conference bursaries and 
community workshops. Contrast that with typically 40 % or more profits made by 
the commercial publishers of research. It is not a given that our community 
IUCr journals will simply just survive. We all need to take responsibility for 
ensuring that they do.
Yours sincerely,
John
Emeritus Professor John R Helliwell DSc
Editor in Chief Acta Cryst and Chairman of the IUCr Journals Commission 1996 to 
2005




> On 22 Nov 2019, at 13:58, Isabel Uson  wrote:
> 
> Dear Julie and Mathew,
> 
> I feel advertisement on behalf of professional publishers is not appropriate 
> for the bulletin board. MDPI should pay for its advertisements, rather than 
> get them for free. (Being a for-profit firm, they should also pay for, rather 
> than invite editing, but this is of course personal). They stand in direct 
> competition with IUCr journals, which it should be in our best interest to 
> protect. We all profit from constant -rather than occasional- scientific 
> editing and the IUCr support to our community (meetings, fellowships, awards).
> I know it is not the first time such a call is posted in the bb and that it 
> is not for me to say what is appropriate or not but I will really miss 
> journals from our scientific societies the day they become extinct.
> Best wishes,
> 
> Isabel
> 
> 
>> On Thu, Nov 21, 2019 at 1:07 AM CCP4BB automatic digest system 
>>  wrote:
>> There are 2 messages totaling 363 lines in this issue.
>> 
>> --
>> 
>> Date:Wed, 20 Nov 2019 17:24:15 +
>> From:Julie Tucker 
>> Subject: Off-topic (somewhat): Call for papers on structure-guided kinase 
>> drug discovery
>> 
>> Dear colleagues,
>> 
>> Mathew Martin and I would very much appreciate your contributions on the
>> topic of "Recent Advances in Structure-Guided Kinase Drug Discovery" for a
>> special issue of the International Journal of Molecular Sciences
>>  (IJMS, ISSN 1422-0067, Current Impact
>> Factor: 4.183). We encourage submission of both *original research articles
>> and topical reviews* on all aspects of *structure-guided drug discovery
>> targeting the phosphotransferase enzyme family*. We welcome accounts of
>> your experiences of applying structure-guided methods of all types to the
>> discovery and development of inhibitors of protein, lipid and small
>> molecule kinases from bacteria to man. More information can be found here:
>> https://www.mdpi.com/journal/ijms/special_issues/structure_guided_kinase.
>> 
>> Due to its open access policy, the journal charges publication fees,
>> however, please contact us directly for the chance to secure a discount on
>> the usual fee. The deadline for submissions is nominally 20th April 2020,
>> however, articles will be peer-reviewed and published on an ongoing basis.
>> 
>> Our apologies to those of you with no interest in kinase drug discovery,
>> and our heartfelt thanks to any of you who feel moved to contact us to
>> discuss potential contributions. Please also feel free to pass this
>> invitation on to any interested colleagues.
>> 
>> Best wishes,
>> Julie Tucker and Mathew Martin
>> -- 
>> Julie Tucker
>> York Biomedical Research Institute
>> Department of Biology and HYMS
>> University of York
>> Heslington
>> YORK
>> YO10 5DD
>> 
>> Tel. 01904 328912
>> 
>> 
>> Co-guest editor of "Recent Advances in Structure-Guided Kinase Drug
>> Discovery
>> "
>> 
>> A special issue of *International Journal of Molecular Sciences*
>>  (IF 4.183) (ISSN 1422-0067).
>> 
>> Email disclaimer
>> 
>> 
>> 
>> 
> 
> 
> -- 
> ICREA Res. Prof. Isabel Usón
> Crystallographic Methods
> Department of Structural Biology (“Maria de Maeztu” Unit of Excellence), 
> Molecular Biology Institute of Barcelona, Spanish Research Council; 
> Barcelona Science Park, Helix Building, 08028 Barcelona (Spain)
> http://chango.ibmb.csic.es/ARCIMBOLDO
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



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Re: [ccp4bb] Off-topic (somewhat): Call for papers on structure-guided kinase drug discovery

[Aaron Finke]

Yes, this issue will definitely compete with IUCr's many journals dedicated to 
kinase drug discovery. /sarcasm

On a more serious note: nobody should be "loyal" to a journal or scientific 
society; both are servants of the scientific community, not the other way 
around. If they stop supporting scientists with their roles and policies, those 
scientists will move elsewhere (note, for example, the American 
Crystallographic Association and the diaspora of structural biologists/protein 
crystallographers from the ACA). If I submit to an IUCr journal, it is because 
my work is geared toward the community of IUCr journal readers. IUCr journals 
are not always going to attract the right audience for a particular study, and 
that's fine. Not everyone here is a capital-C Crystallographer, and if someone 
in this community wants to bring to our attention an opportunity that may 
benefit its users, that's great! I take issue with MDPI's policies as a 
publishing house, but guest editors posting about a special issue on behalf of 
them is more beneifical than harmful.

In that vein, advertistments for job postings are professionally-related and 
also not relevant to CCP4 but I think everyone, especially young scientists, 
benefit from those as well.

Aaron
--
Aaron Finke
Staff Scientist, MacCHESS
Cornell University
e-mail: af...@cornell.edu

On Nov 22, 2019, at 8:58 AM, Isabel Uson 
mailto:iuf...@ibmb.csic.es>> wrote:

Dear Julie and Mathew,

I feel advertisement on behalf of professional publishers is not appropriate 
for the bulletin board. MDPI should pay for its advertisements, rather than get 
them for free. (Being a for-profit firm, they should also pay for, rather than 
invite editing, but this is of course personal). They stand in direct 
competition with IUCr journals, which it should be in our best interest to 
protect. We all profit from constant -rather than occasional- scientific 
editing and the IUCr support to our community (meetings, fellowships, awards).
I know it is not the first time such a call is posted in the bb and that it is 
not for me to say what is appropriate or not but I will really miss journals 
from our scientific societies the day they become extinct.
Best wishes,

Isabel


On Thu, Nov 21, 2019 at 1:07 AM CCP4BB automatic digest system 
mailto:lists...@jiscmail.ac.uk>> wrote:
There are 2 messages totaling 363 lines in this issue.

--

Date:Wed, 20 Nov 2019 17:24:15 +
From:Julie Tucker mailto:julie.tuc...@york.ac.uk>>
Subject: Off-topic (somewhat): Call for papers on structure-guided kinase drug 
discovery

Dear colleagues,

Mathew Martin and I would very much appreciate your contributions on the
topic of "Recent Advances in Structure-Guided Kinase Drug Discovery" for a
special issue of the International Journal of Molecular Sciences
 (IJMS, ISSN 1422-0067, Current Impact
Factor: 4.183). We encourage submission of both *original research articles
and topical reviews* on all aspects of *structure-guided drug discovery
targeting the phosphotransferase enzyme family*. We welcome accounts of
your experiences of applying structure-guided methods of all types to the
discovery and development of inhibitors of protein, lipid and small
molecule kinases from bacteria to man. More information can be found here:
https://www.mdpi.com/journal/ijms/special_issues/structure_guided_kinase.

Due to its open access policy, the journal charges publication fees,
however, please contact us directly for the chance to secure a discount on
the usual fee. The deadline for submissions is nominally 20th April 2020,
however, articles will be peer-reviewed and published on an ongoing basis.

Our apologies to those of you with no interest in kinase drug discovery,
and our heartfelt thanks to any of you who feel moved to contact us to
discuss potential contributions. Please also feel free to pass this
invitation on to any interested colleagues.

Best wishes,
Julie Tucker and Mathew Martin
--
Julie Tucker
York Biomedical Research Institute
Department of Biology and HYMS
University of York
Heslington
YORK
YO10 5DD

Tel. 01904 328912


Co-guest editor of "Recent Advances in Structure-Guided Kinase Drug
Discovery
"

A special issue of *International Journal of Molecular Sciences*
 (IF 4.183) (ISSN 1422-0067).

Email disclaimer





--
ICREA Res. Prof. Isabel Usón
Crystallographic Methods
Department of Structural Biology (“Maria de Maeztu” Unit of Excellence),
Molecular Biology Institute of Barcelona, Spanish Research Council;
Barcelona Science Park, Helix Building, 08028 Barcelona (Spain)

Re: [ccp4bb] Off-topic (somewhat): Call for papers on structure-guided kinase drug discovery

[Isabel Uson]

Dear Julie and Mathew,

I feel advertisement on behalf of professional publishers is not
appropriate for the bulletin board. MDPI should pay for its advertisements,
rather than get them for free. (Being a for-profit firm, they should also
pay for, rather than invite editing, but this is of course personal). They
stand in direct competition with IUCr journals, which it should be in our
best interest to protect. We all profit from constant -rather than
occasional- scientific editing and the IUCr support to our community
(meetings, fellowships, awards).
I know it is not the first time such a call is posted in the bb and that it
is not for me to say what is appropriate or not but I will really miss
journals from our scientific societies the day they become extinct.
Best wishes,

Isabel


On Thu, Nov 21, 2019 at 1:07 AM CCP4BB automatic digest system <
lists...@jiscmail.ac.uk> wrote:

> There are 2 messages totaling 363 lines in this issue.
>
> --
>
> Date:Wed, 20 Nov 2019 17:24:15 +
> From:Julie Tucker 
> Subject: Off-topic (somewhat): Call for papers on structure-guided kinase
> drug discovery
>
> Dear colleagues,
>
> Mathew Martin and I would very much appreciate your contributions on the
> topic of "Recent Advances in Structure-Guided Kinase Drug Discovery" for a
> special issue of the International Journal of Molecular Sciences
>  (IJMS, ISSN 1422-0067, Current Impact
> Factor: 4.183). We encourage submission of both *original research articles
> and topical reviews* on all aspects of *structure-guided drug discovery
> targeting the phosphotransferase enzyme family*. We welcome accounts of
> your experiences of applying structure-guided methods of all types to the
> discovery and development of inhibitors of protein, lipid and small
> molecule kinases from bacteria to man. More information can be found here:
> https://www.mdpi.com/journal/ijms/special_issues/structure_guided_kinase.
>
> Due to its open access policy, the journal charges publication fees,
> however, please contact us directly for the chance to secure a discount on
> the usual fee. The deadline for submissions is nominally 20th April 2020,
> however, articles will be peer-reviewed and published on an ongoing basis.
>
> Our apologies to those of you with no interest in kinase drug discovery,
> and our heartfelt thanks to any of you who feel moved to contact us to
> discuss potential contributions. Please also feel free to pass this
> invitation on to any interested colleagues.
>
> Best wishes,
> Julie Tucker and Mathew Martin
> --
> Julie Tucker
> York Biomedical Research Institute
> Department of Biology and HYMS
> University of York
> Heslington
> YORK
> YO10 5DD
>
> Tel. 01904 328912
>
>
> Co-guest editor of "Recent Advances in Structure-Guided Kinase Drug
> Discovery
>  >"
>
> A special issue of *International Journal of Molecular Sciences*
>  (IF 4.183) (ISSN 1422-0067).
>
> Email disclaimer
> 
>
> 
>
>
-- 
ICREA Res. Prof. Isabel Usón
Crystallographic Methods
Department of Structural Biology (“Maria de Maeztu” Unit of Excellence),
Molecular Biology Institute of Barcelona, Spanish Research Council;
Barcelona Science Park, Helix Building, 08028 Barcelona (Spain)
http://chango.ibmb.csic.es/ARCIMBOLDO



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[ccp4bb] Off-topic (somewhat): Call for papers on structure-guided kinase drug discovery

[Julie Tucker]

Dear colleagues,

Mathew Martin and I would very much appreciate your contributions on the
topic of "Recent Advances in Structure-Guided Kinase Drug Discovery" for a
special issue of the International Journal of Molecular Sciences
 (IJMS, ISSN 1422-0067, Current Impact
Factor: 4.183). We encourage submission of both *original research articles
and topical reviews* on all aspects of *structure-guided drug discovery
targeting the phosphotransferase enzyme family*. We welcome accounts of
your experiences of applying structure-guided methods of all types to the
discovery and development of inhibitors of protein, lipid and small
molecule kinases from bacteria to man. More information can be found here:
https://www.mdpi.com/journal/ijms/special_issues/structure_guided_kinase.

Due to its open access policy, the journal charges publication fees,
however, please contact us directly for the chance to secure a discount on
the usual fee. The deadline for submissions is nominally 20th April 2020,
however, articles will be peer-reviewed and published on an ongoing basis.

Our apologies to those of you with no interest in kinase drug discovery,
and our heartfelt thanks to any of you who feel moved to contact us to
discuss potential contributions. Please also feel free to pass this
invitation on to any interested colleagues.

Best wishes,
Julie Tucker and Mathew Martin
-- 
Julie Tucker
York Biomedical Research Institute
Department of Biology and HYMS
University of York
Heslington
YORK
YO10 5DD

Tel. 01904 328912


Co-guest editor of "Recent Advances in Structure-Guided Kinase Drug
Discovery
"

A special issue of *International Journal of Molecular Sciences*
 (IF 4.183) (ISSN 1422-0067).

Email disclaimer




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[ccp4bb] (off-topic) fundraising campaign - EMERGENCY VENICE

[Benini Stefano]

Dear All,

On behalf of Elisa Moretti (in Cc.) I share this fundraising initiative for 
Venice that was recently damaged by flooding:

Dear all,

I am sharing with you the news about the initiatives launched by Ca' Foscari 
University of Venice (Italy) in support of Venice and its citizens, hit hard by 
the adverse weather conditions of the past few days.

Ca' Foscari has had its own damage in some of its sites, but these days it is 
the whole city which is at its knees, with homes, businesses, libraries, 
bookshops severely damaged, monuments soaked in salty waters, boarding stations 
destroyed, boats sunk. . .

We have always been in Venice with our community, and on this emergency we 
thought it is important to be proactive in supporting the city and its 
residents.
We have thus decided to launch a fundraising campaign - EMERGENCY VENICE - 
through our fundraising platform.  It takes just a few minutes to donate, at 
the following link:
https://supportacafoscari.unive.it/emergenzavenezia/~my-donation.
If you deem it appropriate, please contribute to the campaign and help us share 
it with your contacts.

Thanks to all for what you will be able to do, even a small gesture means a lot!

Kindest regards,
Elisa Moretti









Stefano Benini, Ph.D. Assistant Professor
Guest editor of "Carbohydrate-Active Enzymes: Structure, Activity and Reaction 
Products"
A special issue of International Journal of Molecular 
Sciences (IF 4.183) (ISSN 1422-0067). This 
special issue belongs to the section "Molecular 
Biophysics". 
https://www.mdpi.com/journal/ijms/special_issues/carbohydrate-active_enzymes

[https://s3.amazonaws.com/zapnito/uploads/f9805f649da7ceb38a2059ab1ce4f9bc/SciReports_EBM_Branded_Sig_v2.jpg]

https://sbenini.people.unibz.it/
"And money wasn't what I had in mind. Oh God, no, what I wanted was to do good. 
I was dying to do something good." Saul Bellow

"I don't like anything that's fake and I hate pretenders!" Stefano Benini

"articolo 21 della Costituzione Italiana:  Tutti hanno diritto di manifestare 
liberamente il proprio pensiero con la parola, lo scritto e ogni altro mezzo di 
diffusione."
*
Bioorganic chemistry and Bio-Crystallography laboratory (B2Cl)
Faculty of Science and Technology, Libera Università di Bolzano
Piazza Università, 5
39100 Bolzano, Italy
Office (room K2.14):  +39 0471 017128
Laboratory (room E.021): +39 0471 017910
Fax: +39 0471 017009
https://sbenini.people.unibz.it/
orcid.org/-0001-6299-888X

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[ccp4bb] Off topic: 384 well stamper

[Artem Evdokimov]

Colleagues

I have a quick question for you: we are in need of a small footprint 384
well plate stamper with dilution (optional but desired) capability. We are
already evaluating Integra, Jena Analytic, and Apricot models but we are
curious if there are other companies who sell this sort of machinery (any
personal impressions of these and any other similar LH devices are very
welcome!). I would be happy to post a condensed summary of replies.

Thank you

Artem



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Re: [ccp4bb] [Off-Topic] 3D Vision stereo with Ubuntu 18.04

[Chris Richardson]

Thanks to everyone who gave helpful suggestions; I now have stereo working on 
Ubuntu 18.04.

To help anyone who comes across this in the CCP4 archives in the future, it was 
necessary to:

1) Install lightdm and set it as the default display manager.  Other display 
managers that don't do compositing may also work.

2) Install a suitable desktop environment (I used gnome flashback metacity in 
the end, but XFCE also works).

3) Edit the Xorg configuration (which is now a series of files in 
/usr/share/X11/xorg.conf.d) to include "Stereo" "10", and "Composite" "Disable".

The step I was missing was the first one.  In Ubuntu 16.04, stereo worked 
without having to do this.

Thanks again,

Chris
-- 
Dr Chris Richardson :: Sysadmin, structural biology, icr.ac.uk
 
 
 

On 08/11/2019, 12:19, "CCP4 bulletin board on behalf of Chris Richardson" 
 wrote:

Apologies for the only slightly relevant question.

Does anyone know the correct incantations to get nVidia 3D Vision glasses 
and emitter working with Ubuntu 18.04?

None of the tricks that work with 16.04 are helping with the new release.  
In particular, disabling composite in the extensions makes the display blank 
while X11 restarts itself every few seconds.

Thanks in advance,

Chris
-- 
Dr Chris Richardson :: Sysadmin, structural biology, icr.ac.uk
 
 
 


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The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company 
Limited by Guarantee, Registered in England under Company No. 534147 with its 
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Re: [ccp4bb] [Off-Topic] 3D Vision stereo with Ubuntu 18.04

[Xiao Lei]

I used to use Ubuntu Mate OS to get 3D work for coot and pymol. I am not
sure if the latest version still works.

Regards

Xiao

On Fri, Nov 8, 2019, 7:19 AM Chris Richardson 
wrote:

> Apologies for the only slightly relevant question.
>
> Does anyone know the correct incantations to get nVidia 3D Vision glasses
> and emitter working with Ubuntu 18.04?
>
> None of the tricks that work with 16.04 are helping with the new release.
> In particular, disabling composite in the extensions makes the display
> blank while X11 restarts itself every few seconds.
>
> Thanks in advance,
>
> Chris
> --
> Dr Chris Richardson :: Sysadmin, structural biology, icr.ac.uk
>
>
>
>
>
> The Institute of Cancer Research: Royal Cancer Hospital, a charitable
> Company Limited by Guarantee, Registered in England under Company No.
> 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP.
>
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Re: [ccp4bb] [Off-Topic] 3D Vision stereo with Ubuntu 18.04

[Christine Gee]

Hi Chris
We have it working with the Mate desktop. Composite disabled. Let me know if 
you would like more details
Regards
Christine. 

Sent from my iPad

> On Nov 8, 2019, at 4:18 AM, Chris Richardson  
> wrote:
> 
> Apologies for the only slightly relevant question.
> 
> Does anyone know the correct incantations to get nVidia 3D Vision glasses and 
> emitter working with Ubuntu 18.04?
> 
> None of the tricks that work with 16.04 are helping with the new release.  In 
> particular, disabling composite in the extensions makes the display blank 
> while X11 restarts itself every few seconds.
> 
> Thanks in advance,
> 
> Chris
> -- 
> Dr Chris Richardson :: Sysadmin, structural biology, icr.ac.uk
> 
> 
> 
> 
> 
> The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company 
> Limited by Guarantee, Registered in England under Company No. 534147 with its 
> Registered Office at 123 Old Brompton Road, London SW7 3RP.
> 
> This e-mail message is confidential and for use by the addressee only.  If 
> the message is received by anyone other than the addressee, please return the 
> message to the sender by replying to it and then delete the message from your 
> computer and network.
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> 
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Re: [ccp4bb] [Off-Topic] 3D Vision stereo with Ubuntu 18.04

[Wim Burmeister]

Hello,
The desktop changed in the passage from Ubuntu16 to Ubuntu18.
I think Nvidia stereo now works only with a xfce desktop.
The passage from debian 8 to debian 9 was not a problem as long as xfce is kept.
Best regards
Wim 

- Mail original -
De: "Chris Richardson" 
À: "CCP4BB" 
Envoyé: Vendredi 8 Novembre 2019 13:18:44
Objet: [ccp4bb] [Off-Topic] 3D Vision stereo with Ubuntu 18.04

Apologies for the only slightly relevant question.

Does anyone know the correct incantations to get nVidia 3D Vision glasses and 
emitter working with Ubuntu 18.04?

None of the tricks that work with 16.04 are helping with the new release.  In 
particular, disabling composite in the extensions makes the display blank while 
X11 restarts itself every few seconds.

Thanks in advance,

Chris
-- 
Dr Chris Richardson :: Sysadmin, structural biology, icr.ac.uk
 
 
 


The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company 
Limited by Guarantee, Registered in England under Company No. 534147 with its 
Registered Office at 123 Old Brompton Road, London SW7 3RP.

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[ccp4bb] [Off-Topic] 3D Vision stereo with Ubuntu 18.04

[Chris Richardson]

Apologies for the only slightly relevant question.

Does anyone know the correct incantations to get nVidia 3D Vision glasses and 
emitter working with Ubuntu 18.04?

None of the tricks that work with 16.04 are helping with the new release.  In 
particular, disabling composite in the extensions makes the display blank while 
X11 restarts itself every few seconds.

Thanks in advance,

Chris
-- 
Dr Chris Richardson :: Sysadmin, structural biology, icr.ac.uk
 
 
 


The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company 
Limited by Guarantee, Registered in England under Company No. 534147 with its 
Registered Office at 123 Old Brompton Road, London SW7 3RP.

This e-mail message is confidential and for use by the addressee only.  If the 
message is received by anyone other than the addressee, please return the 
message to the sender by replying to it and then delete the message from your 
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[ccp4bb] Summary: [ccp4bb] off topic: microscope in glove box

[Guenter Fritz]

Dear Artem, Marta, Arwen, Robin,

thank you all for the detailed info. We had a try today and it worked 
smoothly.

Thanks again and best regards,
Guenter
Short version: It should be OK, especially since the vacuum is 
transient and not particularly 'strong*' :)


Long version: if this is an older microscope there may be further 
delamination of optically bonded components if air is already admitted 
between glass planes (i.e. the optical cement is worn and old). Also 
some of the fancier models may have pneumatic balance elements for 
gross motion of the optical column - those may experience pressure 
differentials above their maximum tolerances. Finally, and very 
unlikely you may have a situation where multiple optical elements are 
sealed together in a single tube with air trapped between them - if 
the 'vent' is blocked (by e.g. old grease or something) then these may 
pop.


https://www.olympus-lifescience.com/en/microscope-resource/primer/anatomy/oculars/ 



But the short version is right 99% of the time.

Artem

*"Professor Hubert Farnsworth 
: Well, it's a space 
ship, so I'd say anywhere between zero and one."

https://www.imdb.com/title/tt0584455/characters/nm0921942

- Cosmic Cats approve of this message


On Mon, Oct 21, 2019 at 10:00 AM Guenter Fritz 
> wrote:


Dear all,

I want to put one of our microscopes into the glove box. Does anybody
know whether some parts of the microscope optics do not like
vacuum in
the air lock ?

Thanks and best regards, Guenter



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Re: [ccp4bb] off topic: microscope in glove box

[Artem Evdokimov]

Short version: It should be OK, especially since the vacuum is transient
and not particularly 'strong*' :)

Long version: if this is an older microscope there may be further
delamination of optically bonded components if air is already admitted
between glass planes (i.e. the optical cement is worn and old). Also some
of the fancier models may have pneumatic balance elements for gross motion
of the optical column - those may experience pressure differentials above
their maximum tolerances. Finally, and very unlikely you may have a
situation where multiple optical elements are sealed together in a single
tube with air trapped between them - if the 'vent' is blocked (by e.g. old
grease or something) then these may pop.

https://www.olympus-lifescience.com/en/microscope-resource/primer/anatomy/oculars/


But the short version is right 99% of the time.

Artem

*"Professor Hubert Farnsworth
: Well, it's a space ship,
so I'd say anywhere between zero and one."
https://www.imdb.com/title/tt0584455/characters/nm0921942

- Cosmic Cats approve of this message


On Mon, Oct 21, 2019 at 10:00 AM Guenter Fritz <
guenter.fritz.phenix.c...@gmail.com> wrote:

> Dear all,
>
> I want to put one of our microscopes into the glove box. Does anybody
> know whether some parts of the microscope optics do not like vacuum in
> the air lock ?
>
> Thanks and best regards, Guenter
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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[ccp4bb] off topic: microscope in glove box

[Guenter Fritz]

Dear all,

I want to put one of our microscopes into the glove box. Does anybody 
know whether some parts of the microscope optics do not like vacuum in 
the air lock ?


Thanks and best regards, Guenter



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[ccp4bb] off topic: job posting

[Artem Evdokimov]

Good morning,

We have two relevant job postingings that I would like to share.

Thank you for your attention :)

Artem

https://careers.massbio.org/job/sr-scientistprincipal-scientist-computational-chemistry/49455467/


and


https://careers.massbio.org/job/scientist-enzymology-protein-chemistry/49690257/

Sr Scientist/Principal Scientist - Computational Chemistry

Enko discovers and develops sustainable solutions for farmers to protect
their crops from pests and disease. It’s proprietary discovery platform
represents a revolutionary approach to rapidly discover novel, safe and
economical solutions that are needed to ensure efficient food production.
Led by a team of proven scientists, entrepreneurs and ag-industry veterans,
Enko uses innovative science and agile design principles to rapidly develop
field-ready crop protection solutions that overcome the rising resistance
barriers traditional pesticides face.

Enko team members will be entitled to participate in Health, Dental,
Vision, Life and Disability insurance, 401(k) plan, and other similar
fringe benefit plans as may be provided by the Company.

We are looking to add a Computational Chemist to join the Chemistry team.
This person will enable the discovery of small molecules for the modulation
of novel biological targets for use in the control of agriculturally
important weeds and pests.

Major responsibilities will include:

Build the computational chemistry project strategy to enhance the
advancement of targets from DNA-encoded library hits to candidate compound
selection
Build the requisite infrastructure to conduct this research, including
identifying tools and technologies needed to support and accelerate
pipeline development and champion their acquisition or the establishment of
collaborations
Lead the structure-based drug design (SBDD) efforts utilizing the suite of
computational chemistry tools including docking/scoring, molecular dynamics
simulations, homology modeling, watermap, and FEP+
Collaborate with medicinal chemists and biologists to create a rich and
diverse pipeline that will address the company’s goals
The ideal candidate will have demonstrated an ability to apply their
expertise in computational chemistry to advance multiple, small molecule
discovery campaigns from screening to development candidates. They will
have demonstrated the ability to thrive in a dynamic, fast-paced,
innovative environment. Excellent written and oral communication skills are
required, as is the desire and ability to excel in a multidisciplinary
environment both as a leader and member across multiple project teams. As
an early addition to the scientific team, this person will have an
opportunity to help us establish a culture that nurtures scientific
excellence, a sense of urgency to achieve project success, and success
built on collaboration and personal accountability.

Qualifications and Skills

D. in Chemistry or a related discipline with 3-5 years of experience in
application of computational chemistry to drug or crop protection chemistry
discovery
Demonstrated expertise in all aspects of modern computational chemistry
including receptor- and structure-based design, conformational analysis,
molecular dynamics, QSAR, binding free energy calculations and
cheminformatics
Track record of accomplishment in small molecule discovery and/or molecular
modelling fields
Demonstration of project leadership and data-driven decision making
Excellent written and oral communication
Strong background in medicinal chemistry, biology, statistics and computer
programming is a plus
No Employment Agencies please, the Company is not responsible for any fees
related to candidates that are unsolicited.

Job Code – PGP13 (Use Job Code in subject line of all communications)

Company: Enko Chem

Contact Name: John Gilroy

Email: john.gil...@enkochem.com

Position Location: Boston, MA

Minimum Required Education: Doctorate

Minimum Years’ Experience: 3-5 years

Scientist, Enzymology & Protein Chemistry

Enko discovers and develops sustainable solutions for farmers to protect
their crops from pests and disease. It’s proprietary discovery platform
represents a revolutionary approach to rapidly discover novel, safe and
economical solutions that are needed to ensure efficient food production.
Led by a team of proven scientists, entrepreneurs and ag-industry veterans,
Enko uses innovative science and agile design principles to rapidly develop
field-ready crop protection solutions that overcome the rising resistance
barriers traditional pesticides face.

Enko team members will be entitled to participate in Health, Dental,
Vision, Life and Disability Insurance, 401(k) Plan and other similar fringe
benefit plans.

We are looking for a creative and highly skilled protein
chemist/enzymologist to join our multidisciplinary team. This scientist
will be responsible for designing, validating, and conducting
enzymatic/biochemical assays and for supporting the overall activities of
the team 

Re: [ccp4bb] Off-topic question

[Jan Stransky]

Hi,
there is a guide here, but you should get the proper script for Pymol
from the Consurf server.
http://www.protein.osaka-u.ac.jp/rcsfp/supracryst/suzuki/jpxtal/Katsutani/en/consurf.php
Jan

On 6/22/19 10:24 PM, khaja faisal tarique wrote:
> Hi everyone,
> 
> I was wondering can anyone suggest me how to project the primary
> sequence conservation calculated through the Consurf server.  Any help
> or suggestion will be appreciated. I have pasted a figure from an
> article just for reference.
> 
> 
> Best
> 
> Khaja
> 
> image.png
> 
> 
> 
> 
> 
> 
> 
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[ccp4bb] Off-topic question

[khaja faisal tarique]

Hi everyone,

I was wondering can anyone suggest me how to project the primary sequence
conservation calculated through the Consurf server.  Any help or suggestion
will be appreciated. I have pasted a figure from an article just
for reference.


Best

Khaja

[image: image.png]



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[ccp4bb] Off topic: room temp FPLC column cooling

[marco.mazzor...@diamond.ac.uk]

Hi Michael,
besides the very valuable suggestion of Gianluca, which worked well in my PhD 
lab, more recently I had very good results using silica gel.

You can find desiccant beads of different diameter and pore size (for absorbing 
water normally around 2.5 nm). Self-indicating ones help you identify when the 
beads need changing. The resin is inexpensive and can be bought from well known 
lab chemicals suppliers as well as from Amazon and eBay. For 5 kilos resin 
2.5-6.0 nm pores, self indicating (usually orange), which will last you ages 
you might be looking in the range of £200.

We placed a couple of 40x30 cm pyrex oven trays, filled with beads, in 
proximity of the FPLC and fluorescence detector. 
RH in the double-glass door cabinet was monitored with a £10 humidity sensor. 
Every week/fortnight we swapped the wet beads with dry ones and regenerated the 
resin by leaving the trays overnight in the glassware drying oven (luckily we 
had synthetic chemists in the department who let us use it).
After cooling down, the resin was stored in a sealed vessel until next use.

A possibility we ruled out is to use a dehumidifier. This would require extra 
space to be located outside the FLPC cabinet, which is noisy, and needs 
adapting connectors which might not be H compliant or invalidate warranties. 
Putting the dehumidifier inside the cabinet would waste useful cold-storage 
space and create vibrations which interfered with the sensitive equipment.

Hope this helps.

Best

Marco


--
Marco Mazzorana, Ph.D.
MX Senior Support Scientist

Diamond Light Source, Ltd.
Harwell Science and Innovation Campus
OX11 0DE Didcot (United Kingdom)

Tel +44 (0)1235 778643

--
Marco Mazzorana, Ph.D.
MX Senior Support Scientist

Diamond Light Source, Ltd.
Harwell Science and Innovation Campus
OX11 0DE Didcot (United Kingdom)

Tel +44 (0)1235 778643


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