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] [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



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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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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"




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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


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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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
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



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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--
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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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

[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






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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




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






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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 

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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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 
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--
David Armstrong
Outreach and Training Lead
PDBe
European Bioinformatics Institute (EMBL-EBI)
European Molecular Biology Laboratory
Wellcome Trust Genome Campus
Hinxton
Cambridge CB10 1SD UK
Tel: +44 1223 492544



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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:
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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:
> 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

[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
> > 
> > > 
> 
> -
> 
> To unsubscribe from the CCP4BB list, click the following link:
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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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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:
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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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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 
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




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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
>
> 
>
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>
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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 

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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 =1 




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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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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
> 
>
> 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: 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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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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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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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
>
> --
>
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>



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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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>



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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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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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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
 
 
 


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 
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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 
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 
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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.
> 
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> 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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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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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
>
> 
>
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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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Re: [ccp4bb] Off topic: room temp FPLC column cooling

[John Newitt]

Bear in mind that if you have cold buffers going into a room
temperature (or warmer) pumps, you can get outgasing that will cause
inaccurate flow or loss of priming for the pumps. You may be able to
work around by using freshly degassed eluents, but the outgasing will
still be a problem for long runs.

On Fri, Jun 7, 2019 at 11:32 AM Gianluca Cioci  wrote:
>
> Hi,
>
> a cheap solution would be to put buffers, fraction collector, and the column 
> (if not too big)
> inside a cold cabinet, next to the FPLC.
> you probably need to use longer pipes but it works
>
> Best regards,
>
> GIA
>
>
>
> Le 07/06/2019 16:51, - - a écrit :
>
> Dear all,
>
> our cold cabinet FPLC is rotting away due to the high humidity in our lab. 
> Anybody having a room temperature setup, where only the buffers, column(s) 
> and fractions are being cooled? If so, by which means (cooling spiral, water 
> pump, peltier element?). Thank you for your suggestions! Michael
>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
> --
> 1st French Congress on Integrative Structural Biology
> Please check: http://bsi-2019.ipbs.fr
>
> Dr. Gianluca CIOCI
> BioCatalysis Team
> http://www.lisbp.fr/en/research/catalysis-and-molecular-enzymatic-engineering--ead1.html
> PICT - Plateforme Intégrée de Criblage de Toulouse
> http://cribligand.ipbs.fr/index.html
>
> Tel: +33 (0)5 61 55 97 68
> Tel: +33 (0)5 61 55 94 91
> E-mail: ci...@insa-toulouse.fr
>
> LISBP - INSA de Toulouse
> 135 avenue de Rangueil
> 31077 Toulouse CEDEX 04
> www.lisbp.fr
>
>
> 
>
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Re: [ccp4bb] Off topic: room temp FPLC column cooling

[Gianluca Cioci]

Hi,

a cheap solution would be to put buffers, fraction collector, and the 
column (if not too big)

inside a cold cabinet, next to the FPLC.
you probably need to use longer pipes but it works

Best regards,

GIA



Le 07/06/2019 16:51, - - a écrit :


Dear all,

our cold cabinet FPLC is rotting away due to the high humidity in our 
lab. Anybody having a room temperature setup, where only the buffers, 
column(s) and fractions are being cooled? If so, by which means 
(cooling spiral, water pump, peltier element?). Thank you for your 
suggestions! Michael





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--
1st French Congress on Integrative Structural Biology
Please check: http://bsi-2019.ipbs.fr

Dr. Gianluca CIOCI
BioCatalysis Team
http://www.lisbp.fr/en/research/catalysis-and-molecular-enzymatic-engineering--ead1.html
PICT - Plateforme Intégrée de Criblage de Toulouse
http://cribligand.ipbs.fr/index.html

Tel: +33 (0)5 61 55 97 68
Tel: +33 (0)5 61 55 94 91
E-mail: ci...@insa-toulouse.fr

LISBP - INSA de Toulouse
135 avenue de Rangueil
31077 Toulouse CEDEX 04
www.lisbp.fr




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Re: [ccp4bb] off topic: Membrane protein "into" soluble protein

[Bonsor, Daniel]

In theory yes, you can fuse the termini to restrain the protein. You could use 
T4 lysozyme and insert in the loop which they use for crystallizing GPCRs (many 
references, including https://www.ncbi.nlm.nih.gov/pubmed/25450769). The 
potential problems are that you have no idea where the termini are 
distance-wise to each other in your receptor. Maybe trying some modelling with 
Phyre or Rosetta) You would probably have to make several constructs with 
varying lengths of flexible linkers and then there is no guarantee the fusion 
protein will actually express or crystallize.

You say you have had no luck crystallize the construct you have at the moment. 
Is it His-tagged? Can you remove it? If C-terminal you could use 
Carboxypeptidase A (1:100 w/w ratio) or limited proteolysis in general? You 
said it is a receptor. Have you tried with the ligand/binding protein? Lysine 
methylation? Thermal Shift Assay to identify a storage buffer which increases 
the Tm of the protein?

You could make new constructs with trimmed termini. Surface entropy reduction 
of the protein (use the SER server, http://services.mbi.ucla.edu/SER/). You 
could try fusing it to MBP or surface entropy reduced MBP 
(https://www.ncbi.nlm.nih.gov/pubmed/20196072).

Hope something helps and it crystallizes.

Dan

Daniel A Bonsor PhD.
Sundberg Lab
Institute of Human Virology
University of Maryland, Baltimore
725 W Lombard Street N370
Baltimore
Maryland
MD 21201
Tel: (410) 706-7457


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrew 
Lovering
Sent: Wednesday, March 06, 2019 9:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off topic: Membrane protein "into" soluble protein

Dear ccp4bb members,

A quick off topic question. I have a protein whose domain structure runs 
N-TM-receptor-TM-enzyme-C, i.e. is a two transmembrane-helix containing 
signalling protein. One would suspect that the receptor domain has ends that 
cluster, and that the enzyme (via homology with known systems) is activated 
when the two helices self-interact in a dimeric protein as a four helix bundle.

So...my question - I have tried expressing the receptor domain as a soluble 
protein, it works well, folds, purifies, but doesn't crystallize. Does anyone 
know of an example of taking an exposed membrane protein loop/domain and making 
a chimera with a soluble 4 helix bundle such that the ends of the loop are 
tethered and restrained nicely?

I was thinking that this must've been attempted for transmembrane alpha-helical 
signalling proteins, perhaps even "PDB output" successfully!

Best
Andy



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Re: [ccp4bb] Off topic question

[Georg Hochberg]

Dear Reza,

CD hit will do exactly that.

Cheers,
Georg

Sent from my iPhone

On Jan 3, 2019, at 2:41 PM, Reza Khayat 
mailto:rkha...@ccny.cuny.edu>> wrote:


​Hi,


Happy new year to all!  A bit of an off topic question.  Does anyone know of a 
method/program to extract the most distinct "n" (n>2) sequences from a sequence 
alignment?  Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031



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Re: [ccp4bb] Off topic question

[Javier Gonzalez]

Hi Reza, happy new year!
The choice would depend on your alignment (aminoacid or nucleotides? are
the sequences closely or distantly related? is it a large alignment? are
there many gaps?)... Anyway, I think the safest, unbiased way to determine
a group of outliers might be to compute a phylogenetic tree and look for an
outgroup. But if the set of sequences is too large you might want (first)
to use a clustering algorithm, such as CD-HIT (
http://weizhongli-lab.org/cd-hit/).
HTH,
Javier

On Thu, Jan 3, 2019 at 6:08 PM Ethan A Merritt 
wrote:

> On Thursday, January 3, 2019 12:40:05 PM PST Reza Khayat wrote:
> > ?Hi,
> >
> >
> > Happy new year to all!  A bit of an off topic question.  Does anyone
> know of a method/program to extract the most distinct "n" (n>2) sequences
> from a sequence alignment?  Thanks.
>
> If these putative "most distinct" sequences are hypothesized to belong
> together,  then i suggest K-means clustering.  If they are hypothesized
> to be unrelated individual outliers then I think you would just take the
> worst scores using whatever metric you used to create original alignment.
>
> Ethan
>
> >
> >
> > Best wishes,
> > Reza
> >
> >
> > Reza Khayat, PhD
> > Assistant Professor
> > City College of New York
> > Department of Chemistry
> > New York, NY 10031
> >
> > 
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
> --
> Ethan A Merritt
> Biomolecular Structure Center,  K-428 Health Sciences Bldg
> MS 357742,   University of Washington, Seattle 98195-7742
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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>


-- 
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  LinkedIn




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Re: [ccp4bb] Off topic question

[Ethan A Merritt]

On Thursday, January 3, 2019 12:40:05 PM PST Reza Khayat wrote:
> ?Hi,
> 
> 
> Happy new year to all!  A bit of an off topic question.  Does anyone know of 
> a method/program to extract the most distinct "n" (n>2) sequences from a 
> sequence alignment?  Thanks.

If these putative "most distinct" sequences are hypothesized to belong
together,  then i suggest K-means clustering.  If they are hypothesized
to be unrelated individual outliers then I think you would just take the
worst scores using whatever metric you used to create original alignment.

Ethan

> 
> 
> Best wishes,
> Reza
> 
> 
> Reza Khayat, PhD
> Assistant Professor
> City College of New York
> Department of Chemistry
> New York, NY 10031
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1


-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
MS 357742,   University of Washington, Seattle 98195-7742



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Re: [ccp4bb] Off topic question

[Zhijie Li]

PCA?

On Jan 3, 2019, at 3:41 PM, Reza Khayat 
mailto:rkha...@ccny.cuny.edu>> wrote:


​Hi,


Happy new year to all!  A bit of an off topic question.  Does anyone know of a 
method/program to extract the most distinct "n" (n>2) sequences from a sequence 
alignment?  Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031



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Re: [ccp4bb] OFF TOPIC

[PULSARSTRIAN]

Hi Anamika,
 As far as I understood, the biotin in the elution
buffer is helping your protein to get stripped off from the Avidin column.
So, maybe you dialyze your purified protein (or run FPLC) and get rid of
biotin completely, before you load the protein on to strptavidin coated SPR
chip.
Hope this helps!!

Best,
Bhanu

On Tue, Nov 13, 2018 at 12:14 PM Anamika Singh 
wrote:

> Hi All,
>
> I am purifying the biotinylated protein (cloned into the pET28a vector)
> using Avidin beads. Since I need the protein for SPR but when I used the
> purified protein to interact with Streptavidin coated onto the SPR chip.
> There was no signal. Can anybody tell me why is it so or how can I make
> sure that the purified protein is biotinylated enough to interact and give
> the signal? Because when I ran the SDS-PAGE there was a band of purified
> protein.
>
> I have used the elution buffer (20mM Tris, 5mM Mgcl2, 500mM Nacl, and 2mM
> Biotin).
> PS: I have included the biotin during overexpression of the protein also.
>
> Please suggest.
>
> Thanks
> --
> Anamika
> Post-Doctoral Fellow
> Silberman Institute of Life Sciences
> Hebrew University of Jerusalem, Israel
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
B4U



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Re: [ccp4bb] OFF TOPIC

[Jonathan Elegheert]

Hi Anamika,

- Have you double-checked that the sequence of your cDNA is correct and 
includes the biotin acceptor peptide tag (BAP tag aka AviTag; GLNDIFEAQKIEWHE 
in single-letter amino acid code)?
- Are you using a dedicated bacterial strain that over-expresses BirA enzyme? 
This may not be strictly necessary because we often find that native E. coli 
BirA levels are sufficient to incorporate biotin, albeit with low efficiency.
- You could simply do a Western blot to confirm biotin incorporation. I like 
the high-sensitivity streptavidin-HRP conjugate from Pierce (cat. no. 21130).

Best wishes,
Jonathan

-
Jonathan Elegheert, PhD
Team Leader

Interdisciplinary Institute for NeuroScience (IINS) 
UMR5297 CNRS / UB
University of Bordeaux 
Centre Broca Nouvelle-Aquitaine
146 rue Léo Saignat
33076 Bordeaux Cedex
France

E-mail: jonathan.eleghe...@u-bordeaux.fr 

-






> On 13 Nov 2018, at 10:13, Anamika Singh  wrote:
> 
> Hi All,
> 
> I am purifying the biotinylated protein (cloned into the pET28a vector) using 
> Avidin beads. Since I need the protein for SPR but when I used the purified 
> protein to interact with Streptavidin coated onto the SPR chip. There was no 
> signal. Can anybody tell me why is it so or how can I make sure that the 
> purified protein is biotinylated enough to interact and give the signal? 
> Because when I ran the SDS-PAGE there was a band of purified protein. 
> 
> I have used the elution buffer (20mM Tris, 5mM Mgcl2, 500mM Nacl, and 2mM 
> Biotin). 
> PS: I have included the biotin during overexpression of the protein also. 
> 
> Please suggest. 
> 
> Thanks 
> -- 
> Anamika 
> Post-Doctoral Fellow
> Silberman Institute of Life Sciences 
> Hebrew University of Jerusalem, Israel
> 
> 
> 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

[Thomas Edwards]

You don’t say so, but one assumes that you have a BAP tag on the protein, and 
co-express a biotin ligase such as BirA?


Ed

T.A.Edwards Ph.D.
Associate Professor of Biochemistry
Deputy Head of School
_
Astbury Centre for Structural Molecular Biology
School of Molecular and Cellular Biology
Faculty of Biological Sciences
===
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
Faculty Staff 
Profile
Astbury Centre Web 
Page

Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka
[signature_1229172001]
Perturbation of Protein-Protein Interactions


From: CCP4 bulletin board  on behalf of Anamika Singh 

Reply-To: Anamika Singh 
Date: Tuesday, 13 November 2018 at 10:15
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] OFF TOPIC

Hi All,

I am purifying the biotinylated protein (cloned into the pET28a vector) using 
Avidin beads. Since I need the protein for SPR but when I used the purified 
protein to interact with Streptavidin coated onto the SPR chip. There was no 
signal. Can anybody tell me why is it so or how can I make sure that the 
purified protein is biotinylated enough to interact and give the signal? 
Because when I ran the SDS-PAGE there was a band of purified protein.

I have used the elution buffer (20mM Tris, 5mM Mgcl2, 500mM Nacl, and 2mM 
Biotin).
PS: I have included the biotin during overexpression of the protein also.

Please suggest.

Thanks
--
Anamika
Post-Doctoral Fellow
Silberman Institute of Life Sciences
Hebrew University of Jerusalem, Israel




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Re: [ccp4bb] OFF TOPIC

[Wim Burmeister]

  
  
Hello,
you probably purified a contaminant. Do a blot with an anti-biotin
antibody or get electro-spray mass spectrometry done in order to
confirm the identity of your protein.
Wim

On 13/11/2018 11:13, Anamika Singh
  wrote:


  
Hi All,

I am purifying the biotinylated protein (cloned into the pET28a vector) using Avidin beads. Since I need the protein for SPR but when I used the purified protein to interact with Streptavidin coated onto the SPR chip. There was no signal. Can anybody tell me why is it so or how can I make sure that the purified protein is biotinylated enough to interact and give the signal? Because when I ran the SDS-PAGE there was a band of purified protein. 


I have used the elution buffer (20mM Tris, 5mM Mgcl2, 500mM Nacl, and 2mM Biotin). 
PS: I have included the biotin during overexpression of the protein also. 
Please suggest. 
Thanks 
-- 


  

  

  Anamika 
Post-Doctoral Fellow
  Silberman Institute of Life Sciences 
  Hebrew University of Jerusalem, Israel
  
  

  

  

  
  
  
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following link:
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-- 
  
  
  


  Wim Burmeister
Professeur
  Institut de Biologie Structurale (IBS) CIBB
  71 avenue des Martyrs
  CS
  20192
  38044 Grenoble Cedex 9, FRANCE
  E-mail: wim.burmeis...@ibs.fr
Tel:    +33 (0) 457 42 87 41   Fax: +33 (0) 476 20
94 00
  website
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Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[Whitley, Matthew J]

Hi Andrew,


Thanks very much for your reply.


I have taken a look at the example data sets you referred me to, and I am most 
interested in example 7, the case with multiple lattices.  I will process this 
one myself and might include it during my tutorials.  This is for the Cold 
Spring Harbor x-ray course which takes place later this month.


Sincerely,

Matthew


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



From: Andrew Leslie 
Sent: Thursday, September 27, 2018 5:41 AM
To: Whitley, Matthew J
Cc: ccp4bb
Subject: Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Dear Matthew,

   I am also late in responding to this, but as part of a 
Nature Protocols paper on iMosflm (Supplementary Information for Nature 
Protocols 12, 1310-1325, 2017) I provided a number of examples of “problem 
datasets”. Some of these are just two images, to show issues in indexing, 
others are complete datasets showing a variety of pathologies.

All the images and a tutorial on how best to process them (with iMosflm) are 
available at the following URL:

www.mrc-lmb.cam.ac.uk/harry/imosflm/examples<https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.mrc-lmb.cam.ac.uk%2Fharry%2Fimosflm%2Fexamples=02%7C01%7Cmjw100%40PITT.EDU%7Cd146604484cb4dd13f5008d6245d717e%7C9ef9f489e0a04eeb87cc3a526112fd0d%7C1%7C0%7C636736381099125261=EzdbGDpEO126WIYz85Nj9QPvfuDXI31nj14OvyjjHzM%3D=0>


Best wishes,

Andrew


On 26 Sep 2018, at 03:15, Whitley, Matthew J 
mailto:mjw...@pitt.edu>> wrote:

For some reason, the September 19th ccp4bb digest got caught in my spam filter 
and didn't come through until a few minutes ago, so I didn't see several 
responses concerning interesting datasets for processing until just now.

Therefore, thanks also to Kay Diederichs, Eugene Osipov, and David Waterman for 
responding (and also to everyone else who responded if I am still overlooking 
anyone.)

As I mentioned before, I will be happy to compile a list of suggested datasets 
and make it available via this list.

Matthew


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



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Re: [ccp4bb] Off-topic, protein in dye-front (ion front?) on native-PAGE

[Xiao Lei]

Hi All,

Sorry to bring this old topic up again.  I planned to run tricine gels but
I found a possible error in table 2 (4% stacking gel formula) in Hermann
Schägger protocol (Nature Protocols volume 1, pages 16–22 (2006), the
author wrote 3ml 3X gel buffer in a total of 12 ml solution, it should be
4ml 3X gel buffer in a total of 12 ml solution right?

I tried to contact the author but unsuccessful, I do not know if anyone in
this forum has noticed if this is an error or not.

Xiao


On Thu, Jan 19, 2017 at 8:00 AM Didier Spittler 
wrote:

> Yes Tris-Tricine gel !
>
> Try to obtain this article from nature protocol.
>
> Best,
>
> Didier
>
>
> 2017-01-19 14:47 GMT+01:00 zeyaul islam :
>
>> Try Tricine gel. It is particularly suited for low molecular wt proteins
>> and it will give you very good resolution. Even you can run it overnight at
>> 30 V (16-18 hours).
>>
>> On Thu, Jan 19, 2017 at 9:33 AM, Walt  wrote:
>>
>>> Hi,
>>>
>>> I have a small protein (~9 kDa) with acidic pI (~4).
>>> When I run 18% native-PAGE, it appears my protein is in the dye front.
>>> How can I fix this problem? Changing the pH of separating gel
>>> might help? How about gradient native-PAGE? Thank you!
>>>
>>> Walt
>>>
>>
>>
>
>
> --
> Didier Spittler, PhD
> Phone number : +33658576481
>



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Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[Gloria Borgstahl]

Hi Matthew,  I am also a bit late in responding, I have a few
incommensurately modulated protein crystal datasets that you would be
welcome to use in your course.  It would be neat for students to at
least know that this type of diffraction exists.  As far as I know,
they can only be processed with Eval software.  Please let me know if
you are interested and we can transfer images to you.  Thanks, Gloria


On Thu, Sep 27, 2018 at 4:42 AM Andrew Leslie  wrote:
>
> Dear Matthew,
>
>I am also late in responding to this, but as part of a 
> Nature Protocols paper on iMosflm (Supplementary Information for Nature 
> Protocols 12, 1310-1325, 2017) I provided a number of examples of “problem 
> datasets”. Some of these are just two images, to show issues in indexing, 
> others are complete datasets showing a variety of pathologies.
>
> All the images and a tutorial on how best to process them (with iMosflm) are 
> available at the following URL:
>
> www.mrc-lmb.cam.ac.uk/harry/imosflm/examples
>
>
> Best wishes,
>
> Andrew
>
>
> On 26 Sep 2018, at 03:15, Whitley, Matthew J <> wrote:
>
> For some reason, the September 19th ccp4bb digest got caught in my spam 
> filter and didn't come through until a few minutes ago, so I didn't see 
> several responses concerning interesting datasets for processing until just 
> now.
>
> Therefore, thanks also to Kay Diederichs, Eugene Osipov, and David Waterman 
> for responding (and also to everyone else who responded if I am still 
> overlooking anyone.)
>
> As I mentioned before, I will be happy to compile a list of suggested 
> datasets and make it available via this list.
>
> 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:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
>
> 
>
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> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



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Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[Andrew Leslie]

Dear Matthew,

   I am also late in responding to this, but as part of a 
Nature Protocols paper on iMosflm (Supplementary Information for Nature 
Protocols 12, 1310-1325, 2017) I provided a number of examples of “problem 
datasets”. Some of these are just two images, to show issues in indexing, 
others are complete datasets showing a variety of pathologies.

All the images and a tutorial on how best to process them (with iMosflm) are 
available at the following URL:

www.mrc-lmb.cam.ac.uk/harry/imosflm/examples 



Best wishes,

Andrew


> On 26 Sep 2018, at 03:15, Whitley, Matthew J  wrote:
> 
> For some reason, the September 19th ccp4bb digest got caught in my spam 
> filter and didn't come through until a few minutes ago, so I didn't see 
> several responses concerning interesting datasets for processing until just 
> now.
> 
> Therefore, thanks also to Kay Diederichs, Eugene Osipov, and David Waterman 
> for responding (and also to everyone else who responded if I am still 
> overlooking anyone.)
> 
> As I mentioned before, I will be happy to compile a list of suggested 
> datasets and make it available via this list.
> 
> 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:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 



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Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[Whitley, Matthew J]

For some reason, the September 19th ccp4bb digest got caught in my spam filter 
and didn't come through until a few minutes ago, so I didn't see several 
responses concerning interesting datasets for processing until just now.

Therefore, thanks also to Kay Diederichs, Eugene Osipov, and David Waterman for 
responding (and also to everyone else who responded if I am still overlooking 
anyone.)

As I mentioned before, I will be happy to compile a list of suggested datasets 
and make it available via this list.

Matthew


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



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Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[David Waterman]

Hi Matthew,

I'm a little late to the thread, but I thought I would still like to add
DPF3b, kindly provided by Wolfram Tempel. This dataset is available on
zenodo and forms the basis of a tutorial for using DIALS:
https://dials.github.io/documentation/tutorials/correcting_poor_initial_geometry_tutorial.html.
As the tutorial states,

This is a challenging dataset to process. There are a combination of
problems, including:

   - A ‘reversed’ rotation axis
   - Incorrect beam centre recorded in the image headers
   - Split spots
   - Multiple lattices
   - Systematically weak spots that may correspond to pseudocentring

Cheers
-- David


On Tue, 25 Sep 2018 at 18:27, Whitley, Matthew J  wrote:

> Dear colleagues,
>
> I want to thank the following people for providing suggestions and
> comments about ‘difficult’ datasets suitable for teaching data processing:
>
> Tim Craig
> Jacob Keller
> Graeme Winter
> Aleksandar Bijelic
> Clemens Vonrhein
> Loes Kroon-Batenburg
> James Holton
>
> If anyone else has suggestions for good datasets for teaching processing,
> I would still be happy to hear them.
>
> Finally, several people asked me to make available a list of all the
> dataset suggestions I receive.  I am happy to do so, and I will post a
> message to this list when the information is up and available, probably
> later in the fall.
>
>
> Sincerely,
> Matthew
>
>
>
> ---
> Matthew J. Whitley, Ph.D.
> Research Instructor
> Department of Pharmacology & Chemical Biology
> University of Pittsburgh School of Medicine
>
>
>
>
> On 9/19/2018 5:15 PM, Whitley, Matthew J wrote:
>
> Dear colleagues,
>
> For teaching purposes, I am looking for a small number (< 5) of
> macromolecular diffraction datasets (raw images) that might be
> considered 'difficult' for a beginning crystallography student to
> process.  By 'difficult' I generally mean not able to be processed
> automatically by a common processing package (XDS, Mosflm, DIALS, etc)
> using default settings, i.e., no black box "click and done" processing.
> The datasets I am looking for would have some stumbling block such as
> incorrect experimental parameters recorded in the image headers,
> multiple lattices that cause indexing to fail, datasets for which
> determining the correct space group is tricky, datasets for experiments
> in which the crystal slipped or moved in the beam, or anything else you
> can think of.  The idea is for these beginning students to examine
> several datasets that highlight various phenomena that can lead one
> astray during processing.
>
> A good candidate dataset would also ideally comprise a modest number of
> images so as to keep integration time to a minimum.  Factors that are
> mostly irrelevant for my purpose: resolution (as long as better than
> ~3.5 Å), source (home vs synchrotron), presence/absence of anomalous
> scattering,  presence/absence of ligands, monomeric vs oligomeric
> structures, etc.  Also, to be clear, I am not looking for datasets that
> have so many pathologies that they would require many long hours of work
> for an expert to process correctly.
>
> I have checked public repositories such as proteindiffraction.org and
> SBGrid databank, but all of the datasets I acquired from these sources
> process satisfactorily with little effort, and in any event I know of no
> way to search for 'challenging' datasets.  (I also wonder whether
> anybody is in the habit of depositing, shall we say, less-than-pristine
> images to public repositories?)
>
> If you know of such a dataset that is already publicly available, or if
> you have such a dataset that you are willing to share for solely
> educational purposes, I would appreciate hearing from you, either on- or
> off-list.
>
> Thank you in advance for your suggestions.
>
> Matthew
>
>
>
>
> --
>
> 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: 'Difficult' Datasets for Processing Practice

[Whitley, Matthew J]

Dear colleagues,

I want to thank the following people for providing suggestions and comments 
about ‘difficult’ datasets suitable for teaching data processing:

Tim Craig
Jacob Keller
Graeme Winter
Aleksandar Bijelic
Clemens Vonrhein
Loes Kroon-Batenburg
James Holton

If anyone else has suggestions for good datasets for teaching processing, I 
would still be happy to hear them.

Finally, several people asked me to make available a list of all the dataset 
suggestions I receive.  I am happy to do so, and I will post a message to this 
list when the information is up and available, probably later in the fall.


Sincerely,
Matthew



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




On 9/19/2018 5:15 PM, Whitley, Matthew J wrote:
Dear colleagues,

For teaching purposes, I am looking for a small number (< 5) of
macromolecular diffraction datasets (raw images) that might be
considered 'difficult' for a beginning crystallography student to
process.  By 'difficult' I generally mean not able to be processed
automatically by a common processing package (XDS, Mosflm, DIALS, etc)
using default settings, i.e., no black box "click and done" processing.
The datasets I am looking for would have some stumbling block such as
incorrect experimental parameters recorded in the image headers,
multiple lattices that cause indexing to fail, datasets for which
determining the correct space group is tricky, datasets for experiments
in which the crystal slipped or moved in the beam, or anything else you
can think of.  The idea is for these beginning students to examine
several datasets that highlight various phenomena that can lead one
astray during processing.

A good candidate dataset would also ideally comprise a modest number of
images so as to keep integration time to a minimum.  Factors that are
mostly irrelevant for my purpose: resolution (as long as better than
~3.5 Å), source (home vs synchrotron), presence/absence of anomalous
scattering,  presence/absence of ligands, monomeric vs oligomeric
structures, etc.  Also, to be clear, I am not looking for datasets that
have so many pathologies that they would require many long hours of work
for an expert to process correctly.

I have checked public repositories such as 
proteindiffraction.org and
SBGrid databank, but all of the datasets I acquired from these sources
process satisfactorily with little effort, and in any event I know of no
way to search for 'challenging' datasets.  (I also wonder whether
anybody is in the habit of depositing, shall we say, less-than-pristine
images to public repositories?)

If you know of such a dataset that is already publicly available, or if
you have such a dataset that you are willing to share for solely
educational purposes, I would appreciate hearing from you, either on- or
off-list.

Thank you in advance for your suggestions.

Matthew






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Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[James Holton]

It was brought to my attention that the link to the preprint I provided 
below doesn't work, but this one does:


https://www.biorxiv.org/content/early/2018/08/18/394965

Thanks to Folmer Fredslund for pointing this out to me!

-James Holton
MAD Scientist

On 9/21/2018 3:50 PM, James Holton wrote:
For teaching purposes I have found that controlled pairs of data sets 
are most instructive.  You are right that an easy one-button-push 
processing run tells you nothing, but so does a 
bang-it-crashed-now-what data set.  Most useful are two data sets that 
are identical in every respect but one, and that one thing is the 
point you are trying to get across.  It's hard to collect such 
perfectly paired data sets, so I ended up just simulating them. I 
deliberately chose a high-symmetry space group to keep the download 
size small. You can download them from here:


http://bl831.als.lbl.gov/~jamesh/workshop/

These five datasets represent the four biggest problems I see users 
have when trying to solve structures: 1) poor anomalous signal, 2) 
overlaps from a bad crystal orientation, 3) hidden radiation damage to 
sites, and 4) ice rings.  The 5th "goodsignal" dataset is the positive 
control.


The web page contains everything from images to processed MTZ files, 
maps and the "right answer" in pdb and mtz format.  A slightly more 
"realistic" version with a bigger download size is here:


http://bl831.als.lbl.gov/~jamesh/workshop2/

This is the one I used for my "weak anomalous challenge" a few years 
back. The teaching advantage is that you can use the image-mixer 
script to modulate the severity of problems like ice rings and 
anomalous signal.  If you make a competition of it, people tend to get 
more interested.


When it comes to beam centers, it is not all that hard to take a data 
set with a "correct" beam center and just edit the headers. How you do 
this depends on the file format, but I have some instructions for 
editing images in general here:


http://bl831.als.lbl.gov/~jamesh/bin_stuff/

In general, you can usually separate the header from the data with the 
unix command "head" or "dd", edit the header with your favorite text 
editor, and then put the two parts back together with "cat". As for 
which beam center is "correct", it is important to tell your students 
that that depends on which software you are using.  I wrote all this 
down in the last paragraph on page 7 of this doc:


https://submit.biorxiv.org/submission/pdf?msid=BIORXIV/2018/394965

This doc also describes another simulated data set that demonstrates 
the challenges of combining lots of short wedges together.  May or may 
not be too advanced a topic for your students?  Or maybe not. As you 
can guess I'm experimenting with biorxiv.  So far, no comments.


Good luck with your class!

-James Holton
MAD Scientist


On 9/19/2018 5:15 PM, Whitley, Matthew J wrote:

Dear colleagues,

For teaching purposes, I am looking for a small number (< 5) of
macromolecular diffraction datasets (raw images) that might be
considered 'difficult' for a beginning crystallography student to
process.  By 'difficult' I generally mean not able to be processed
automatically by a common processing package (XDS, Mosflm, DIALS, etc)
using default settings, i.e., no black box "click and done" processing.
The datasets I am looking for would have some stumbling block such as
incorrect experimental parameters recorded in the image headers,
multiple lattices that cause indexing to fail, datasets for which
determining the correct space group is tricky, datasets for experiments
in which the crystal slipped or moved in the beam, or anything else you
can think of.  The idea is for these beginning students to examine
several datasets that highlight various phenomena that can lead one
astray during processing.

A good candidate dataset would also ideally comprise a modest number of
images so as to keep integration time to a minimum.  Factors that are
mostly irrelevant for my purpose: resolution (as long as better than
~3.5 Å), source (home vs synchrotron), presence/absence of anomalous
scattering,  presence/absence of ligands, monomeric vs oligomeric
structures, etc.  Also, to be clear, I am not looking for datasets that
have so many pathologies that they would require many long hours of work
for an expert to process correctly.

I have checked public repositories such as proteindiffraction.org and
SBGrid databank, but all of the datasets I acquired from these sources
process satisfactorily with little effort, and in any event I know of no
way to search for 'challenging' datasets.  (I also wonder whether
anybody is in the habit of depositing, shall we say, less-than-pristine
images to public repositories?)

If you know of such a dataset that is already publicly available, or if
you have such a dataset that you are willing to share for solely
educational purposes, I would appreciate hearing from you, either on- or
off-list.

Thank you in advance for your suggestions.

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[Andreas Förster]

Hi James,

you’re probably aware of this but you can edit CBF headers in place with
sed. That’s what I do when I make the detector on my diffractometer go
closer than the hardware limit.

All best - Andreas
On Sat, 22 Sep 2018 at 00:51, James Holton 
wrote:

> For teaching purposes I have found that controlled pairs of data sets
> are most instructive.  You are right that an easy one-button-push
> processing run tells you nothing, but so does a bang-it-crashed-now-what
> data set.  Most useful are two data sets that are identical in every
> respect but one, and that one thing is the point you are trying to get
> across.  It's hard to collect such perfectly paired data sets, so I
> ended up just simulating them. I deliberately chose a high-symmetry
> space group to keep the download size small. You can download them from
> here:
>
> http://bl831.als.lbl.gov/~jamesh/workshop/
>
> These five datasets represent the four biggest problems I see users have
> when trying to solve structures: 1) poor anomalous signal, 2) overlaps
> from a bad crystal orientation, 3) hidden radiation damage to sites, and
> 4) ice rings.  The 5th "goodsignal" dataset is the positive control.
>
> The web page contains everything from images to processed MTZ files,
> maps and the "right answer" in pdb and mtz format.  A slightly more
> "realistic" version with a bigger download size is here:
>
> http://bl831.als.lbl.gov/~jamesh/workshop2/
>
> This is the one I used for my "weak anomalous challenge" a few years
> back. The teaching advantage is that you can use the image-mixer script
> to modulate the severity of problems like ice rings and anomalous
> signal.  If you make a competition of it, people tend to get more
> interested.
>
> When it comes to beam centers, it is not all that hard to take a data
> set with a "correct" beam center and just edit the headers. How you do
> this depends on the file format, but I have some instructions for
> editing images in general here:
>
> http://bl831.als.lbl.gov/~jamesh/bin_stuff/
>
> In general, you can usually separate the header from the data with the
> unix command "head" or "dd", edit the header with your favorite text
> editor, and then put the two parts back together with "cat". As for
> which beam center is "correct", it is important to tell your students
> that that depends on which software you are using.  I wrote all this
> down in the last paragraph on page 7 of this doc:
>
> https://submit.biorxiv.org/submission/pdf?msid=BIORXIV/2018/394965
>
> This doc also describes another simulated data set that demonstrates the
> challenges of combining lots of short wedges together.  May or may not
> be too advanced a topic for your students?  Or maybe not. As you can
> guess I'm experimenting with biorxiv.  So far, no comments.
>
> Good luck with your class!
>
> -James Holton
> MAD Scientist
>
>
> On 9/19/2018 5:15 PM, Whitley, Matthew J wrote:
> > Dear colleagues,
> >
> > For teaching purposes, I am looking for a small number (< 5) of
> > macromolecular diffraction datasets (raw images) that might be
> > considered 'difficult' for a beginning crystallography student to
> > process.  By 'difficult' I generally mean not able to be processed
> > automatically by a common processing package (XDS, Mosflm, DIALS, etc)
> > using default settings, i.e., no black box "click and done" processing.
> > The datasets I am looking for would have some stumbling block such as
> > incorrect experimental parameters recorded in the image headers,
> > multiple lattices that cause indexing to fail, datasets for which
> > determining the correct space group is tricky, datasets for experiments
> > in which the crystal slipped or moved in the beam, or anything else you
> > can think of.  The idea is for these beginning students to examine
> > several datasets that highlight various phenomena that can lead one
> > astray during processing.
> >
> > A good candidate dataset would also ideally comprise a modest number of
> > images so as to keep integration time to a minimum.  Factors that are
> > mostly irrelevant for my purpose: resolution (as long as better than
> > ~3.5 Å), source (home vs synchrotron), presence/absence of anomalous
> > scattering,  presence/absence of ligands, monomeric vs oligomeric
> > structures, etc.  Also, to be clear, I am not looking for datasets that
> > have so many pathologies that they would require many long hours of work
> > for an expert to process correctly.
> >
> > I have checked public repositories such as proteindiffraction.org and
> > SBGrid databank, but all of the datasets I acquired from these sources
> > process satisfactorily with little effort, and in any event I know of no
> > way to search for 'challenging' datasets.  (I also wonder whether
> > anybody is in the habit of depositing, shall we say, less-than-pristine
> > images to public repositories?)
> >
> > If you know of such a dataset that is already publicly available, or if
> > you have such a dataset that you are 

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[James Holton]

For teaching purposes I have found that controlled pairs of data sets 
are most instructive.  You are right that an easy one-button-push 
processing run tells you nothing, but so does a bang-it-crashed-now-what 
data set.  Most useful are two data sets that are identical in every 
respect but one, and that one thing is the point you are trying to get 
across.  It's hard to collect such perfectly paired data sets, so I 
ended up just simulating them. I deliberately chose a high-symmetry 
space group to keep the download size small. You can download them from 
here:


http://bl831.als.lbl.gov/~jamesh/workshop/

These five datasets represent the four biggest problems I see users have 
when trying to solve structures: 1) poor anomalous signal, 2) overlaps 
from a bad crystal orientation, 3) hidden radiation damage to sites, and 
4) ice rings.  The 5th "goodsignal" dataset is the positive control.


The web page contains everything from images to processed MTZ files, 
maps and the "right answer" in pdb and mtz format.  A slightly more 
"realistic" version with a bigger download size is here:


http://bl831.als.lbl.gov/~jamesh/workshop2/

This is the one I used for my "weak anomalous challenge" a few years 
back. The teaching advantage is that you can use the image-mixer script 
to modulate the severity of problems like ice rings and anomalous 
signal.  If you make a competition of it, people tend to get more 
interested.


When it comes to beam centers, it is not all that hard to take a data 
set with a "correct" beam center and just edit the headers. How you do 
this depends on the file format, but I have some instructions for 
editing images in general here:


http://bl831.als.lbl.gov/~jamesh/bin_stuff/

In general, you can usually separate the header from the data with the 
unix command "head" or "dd", edit the header with your favorite text 
editor, and then put the two parts back together with "cat". As for 
which beam center is "correct", it is important to tell your students 
that that depends on which software you are using.  I wrote all this 
down in the last paragraph on page 7 of this doc:


https://submit.biorxiv.org/submission/pdf?msid=BIORXIV/2018/394965

This doc also describes another simulated data set that demonstrates the 
challenges of combining lots of short wedges together.  May or may not 
be too advanced a topic for your students?  Or maybe not. As you can 
guess I'm experimenting with biorxiv.  So far, no comments.


Good luck with your class!

-James Holton
MAD Scientist


On 9/19/2018 5:15 PM, Whitley, Matthew J wrote:

Dear colleagues,

For teaching purposes, I am looking for a small number (< 5) of
macromolecular diffraction datasets (raw images) that might be
considered 'difficult' for a beginning crystallography student to
process.  By 'difficult' I generally mean not able to be processed
automatically by a common processing package (XDS, Mosflm, DIALS, etc)
using default settings, i.e., no black box "click and done" processing.
The datasets I am looking for would have some stumbling block such as
incorrect experimental parameters recorded in the image headers,
multiple lattices that cause indexing to fail, datasets for which
determining the correct space group is tricky, datasets for experiments
in which the crystal slipped or moved in the beam, or anything else you
can think of.  The idea is for these beginning students to examine
several datasets that highlight various phenomena that can lead one
astray during processing.

A good candidate dataset would also ideally comprise a modest number of
images so as to keep integration time to a minimum.  Factors that are
mostly irrelevant for my purpose: resolution (as long as better than
~3.5 Å), source (home vs synchrotron), presence/absence of anomalous
scattering,  presence/absence of ligands, monomeric vs oligomeric
structures, etc.  Also, to be clear, I am not looking for datasets that
have so many pathologies that they would require many long hours of work
for an expert to process correctly.

I have checked public repositories such as proteindiffraction.org and
SBGrid databank, but all of the datasets I acquired from these sources
process satisfactorily with little effort, and in any event I know of no
way to search for 'challenging' datasets.  (I also wonder whether
anybody is in the habit of depositing, shall we say, less-than-pristine
images to public repositories?)

If you know of such a dataset that is already publicly available, or if
you have such a dataset that you are willing to share for solely
educational purposes, I would appreciate hearing from you, either on- or
off-list.

Thank you in advance for your suggestions.

Matthew





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Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[Loes Kroon-Batenburg]

Dear Matthew,

In my search for the validity of meta data, I went through several data 
sets in SBGrid and proteindiffraction.org (IRRMC), especially those 
where automatic processing did not succeed are gave different results 
with different processing software. We reprocessed those data sets with 
EVAL and located some issues with the meta data. Most of the problems 
are related to wrong or absent primary beam positions.
SBGrid is very useful to find problematic data sets, since it lists four 
attempts to re-process the data.


Some interesting cases could be:
SBGrid:
373 automatic processing failed to see the crystal is C (or I)-centered 
monoclinic
254 unit cell no so easy to index and inaccurate (and therefore space 
group wrong) because of presence of a second and third lattice

426 beam center very wrong (has to be manually set)
415 another beam center problem; caused large errors in unit cell 
dimensions in automatic processing (only xia2-3dii has it right)
383 is a very challenging case; might be too complicated; at least two 
lattices amd C-centered orthorombic (which none of the automatic 
processing software found)


IRRMC
3m8t: apparent monoclinic C2 symmetry, but is in fact triclinic. Also a 
second smaller fragment present.

4i08: wrong beam center. Rotation direction may have to be reversed.

Store.Synchrotron:
public data set https://store.synchrotron.org.au/experiment/view/1037/  
: one long unit cell axis: quite a challenge combined with inaccurate 
beam center.


Let me add: several initiatives exist or are on the way to publish and 
describe raw data of which the structure could not be solved due to all 
kinds of reasons: indexing fails, phase problem can not be solved, lots 
of additional reflections present, diffuse scattering.  See progress 
report and references in there 
(http://forums.iucr.org/viewtopic.php?f=21=396) of the IUCr working 
group DDDWG, now continued as COMMDAT committee.


Best wishes,
Loes
On 09/20/18 02:15, Whitley, Matthew J wrote:

Dear colleagues,

For teaching purposes, I am looking for a small number (< 5) of
macromolecular diffraction datasets (raw images) that might be
considered 'difficult' for a beginning crystallography student to
process.  By 'difficult' I generally mean not able to be processed
automatically by a common processing package (XDS, Mosflm, DIALS, etc)
using default settings, i.e., no black box "click and done" processing.
The datasets I am looking for would have some stumbling block such as
incorrect experimental parameters recorded in the image headers,
multiple lattices that cause indexing to fail, datasets for which
determining the correct space group is tricky, datasets for experiments
in which the crystal slipped or moved in the beam, or anything else you
can think of.  The idea is for these beginning students to examine
several datasets that highlight various phenomena that can lead one
astray during processing.

A good candidate dataset would also ideally comprise a modest number of
images so as to keep integration time to a minimum.  Factors that are
mostly irrelevant for my purpose: resolution (as long as better than
~3.5 Å), source (home vs synchrotron), presence/absence of anomalous
scattering,  presence/absence of ligands, monomeric vs oligomeric
structures, etc.  Also, to be clear, I am not looking for datasets that
have so many pathologies that they would require many long hours of work
for an expert to process correctly.

I have checked public repositories such as proteindiffraction.org and
SBGrid databank, but all of the datasets I acquired from these sources
process satisfactorily with little effort, and in any event I know of no
way to search for 'challenging' datasets.  (I also wonder whether
anybody is in the habit of depositing, shall we say, less-than-pristine
images to public repositories?)

If you know of such a dataset that is already publicly available, or if
you have such a dataset that you are willing to share for solely
educational purposes, I would appreciate hearing from you, either on- or
off-list.

Thank you in advance for your suggestions.

Matthew




--

__

Dr. Loes Kroon-Batenburg
Dept. of Crystal and Structural Chemistry
Bijvoet Center for Biomolecular Research
Utrecht University
Padualaan 8, 3584 CH Utrecht
The Netherlands

E-mail : l.m.j.kroon-batenb...@uu.nl
phone  : +31-30-2532865
fax: +31-30-2533940
__



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Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[Kay Diederichs]

Hi Matthew,

I have some notes which indicate that SBgrid data sets 5, 62, 78, 117, 218 
posed problems for "automatic" processing using generate_XDS.INP / XDS when I 
saw them for the first time. 
Some of these problems (mainly the conversion of header values to ORGX ORGY) 
are taken care of in current generate_XDS.INP; others remain (defaults for 
MAXIMUM_ERROR_OF_SPOT_POSITION, MAXIMUM_ERROR_OF_SPINDLE_POSITION too low for 
some data sets) or cannot be fixed (radiation damage or detector defects? in 
data set 68). 
This reflects of course the general progress  - what was difficult some time 
ago may be almost trivial now.

My advice would be to go all the way from data processing to refinement. 
Otherwise you end up with a heap of numbers that might tell you something about 
the precision of the data, but nothing about their accuracy. 
And if you do that, keep in mind that the RCSB-deposited PDBs may not be 
optimal interpretations of the data. Also consider the re-refined PDBs from the 
PDB_REDO website.

best,

Kay



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Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[graeme.win...@diamond.ac.uk]

Matthew,

One SBGrid example I used for a workshop was

https://data.sbgrid.org/dataset/218/

This has the “wrong” beam centre as understood by (me/dials/xia2) which causes 
a certain amount of fun, nice example for use of the reciprocal lattice viewer 
and image viewer in DIALS to make sensible choices

Best wishes Graeme




On 20 Sep 2018, at 01:15, Whitley, Matthew J 
mailto:mjw...@pitt.edu>> wrote:

Dear colleagues,

For teaching purposes, I am looking for a small number (< 5) of
macromolecular diffraction datasets (raw images) that might be
considered 'difficult' for a beginning crystallography student to
process.  By 'difficult' I generally mean not able to be processed
automatically by a common processing package (XDS, Mosflm, DIALS, etc)
using default settings, i.e., no black box "click and done" processing.
The datasets I am looking for would have some stumbling block such as
incorrect experimental parameters recorded in the image headers,
multiple lattices that cause indexing to fail, datasets for which
determining the correct space group is tricky, datasets for experiments
in which the crystal slipped or moved in the beam, or anything else you
can think of.  The idea is for these beginning students to examine
several datasets that highlight various phenomena that can lead one
astray during processing.

A good candidate dataset would also ideally comprise a modest number of
images so as to keep integration time to a minimum.  Factors that are
mostly irrelevant for my purpose: resolution (as long as better than
~3.5 Å), source (home vs synchrotron), presence/absence of anomalous
scattering,  presence/absence of ligands, monomeric vs oligomeric
structures, etc.  Also, to be clear, I am not looking for datasets that
have so many pathologies that they would require many long hours of work
for an expert to process correctly.

I have checked public repositories such as 
proteindiffraction.org and
SBGrid databank, but all of the datasets I acquired from these sources
process satisfactorily with little effort, and in any event I know of no
way to search for 'challenging' datasets.  (I also wonder whether
anybody is in the habit of depositing, shall we say, less-than-pristine
images to public repositories?)

If you know of such a dataset that is already publicly available, or if
you have such a dataset that you are willing to share for solely
educational purposes, I would appreciate hearing from you, either on- or
off-list.

Thank you in advance for your suggestions.

Matthew

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




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Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

[Keller, Jacob]

I deposited a dataset on SBGrid from a calmodulin-peptide complex which has 
some "nice" features: merohedral twinning (with variable twin fraction in the 
same dataset) and unavoidable detector cutoffs. It's relatively easy to 
integrate, but solving is somewhat harder. There's a paper on it too which 
gives the "answers" I came up with.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
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its deletion, so that we can ensure such a mistake does not occur in the future.

-Original Message-
From: CCP4 bulletin board  On Behalf Of Whitley, Matthew 
J
Sent: Wednesday, September 19, 2018 8:16 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Dear colleagues,

For teaching purposes, I am looking for a small number (< 5) of macromolecular 
diffraction datasets (raw images) that might be considered 'difficult' for a 
beginning crystallography student to process.  By 'difficult' I generally mean 
not able to be processed automatically by a common processing package (XDS, 
Mosflm, DIALS, etc) using default settings, i.e., no black box "click and done" 
processing. The datasets I am looking for would have some stumbling block such 
as incorrect experimental parameters recorded in the image headers, multiple 
lattices that cause indexing to fail, datasets for which determining the 
correct space group is tricky, datasets for experiments in which the crystal 
slipped or moved in the beam, or anything else you can think of.  The idea is 
for these beginning students to examine several datasets that highlight various 
phenomena that can lead one astray during processing.

A good candidate dataset would also ideally comprise a modest number of images 
so as to keep integration time to a minimum.  Factors that are mostly 
irrelevant for my purpose: resolution (as long as better than
~3.5 Å), source (home vs synchrotron), presence/absence of anomalous 
scattering,  presence/absence of ligands, monomeric vs oligomeric structures, 
etc.  Also, to be clear, I am not looking for datasets that have so many 
pathologies that they would require many long hours of work for an expert to 
process correctly.

I have checked public repositories such as proteindiffraction.org and SBGrid 
databank, but all of the datasets I acquired from these sources process 
satisfactorily with little effort, and in any event I know of no way to search 
for 'challenging' datasets.  (I also wonder whether anybody is in the habit of 
depositing, shall we say, less-than-pristine images to public repositories?)

If you know of such a dataset that is already publicly available, or if you 
have such a dataset that you are willing to share for solely educational 
purposes, I would appreciate hearing from you, either on- or off-list.

Thank you in advance for your suggestions.

Matthew

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




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Re: [ccp4bb] Off-topic: protein 2d diagrams

[Gert Vriend]

 Batch mode generation of residue-based diagrams of proteins

Fabien CampagneEmmanuel BettlerGert VriendHarel Weinstein
/Bioinformatics/, Volume 19, Issue 14, 22 September 2003, Pages 
1854–1855,https://doi.org/10.1093/bioinformatics/btg236

Published:
22 September 2003

On 4-8-2018 10:38, Joana Pereira wrote:

Dear CCP4 community,

I have seen many papers with detailed protein 2d diagrams like this one:

However, I can never find a reference for any tool that can compute 
such diagrams from a 3D structure. Do you know of any?


Many thanks and enjoy the warm weather!

Joana


—
Dr. Joana Pereira
Postdoctoral Researcher
Department of Protein Evolution

Max Planck Institute for Developmental Biology
Max-Planck-Ring 5
72076 Tübingen
GERMANY



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Re: [ccp4bb] Off-topic: protein 2d diagrams

[Schertler Gebhard (PSI)]

Try

http://gpcrdb.org/

They have a tool for snake plots of GPCRs

Best
Gebhard



Prof. Gebhard F.X. Schertler
Structural Biology ETH Zürich D-BIOL

Head of Biology and Chemistry
Division
Paul Scherrer Institut
Laboratory of Biomolecular Research,
LBR
OFLC 109
CH-5232 Villigen PSI
gebhard.schert...@psi.ch
phone +41 56 310 4265

Am 04.08.2018 um 10:38 schrieb Joana Pereira 
mailto:joana.pere...@tuebingen.mpg.de>>:

Dear CCP4 community,

I have seen many papers with detailed protein 2d diagrams like this one:

<2D-transmembrane-protein-diagram-A-convenient-representation-of-the-receptor-sequence.jpeg>
However, I can never find a reference for any tool that can compute such 
diagrams from a 3D structure. Do you know of any?

Many thanks and enjoy the warm weather!

Joana


—
Dr. Joana Pereira
Postdoctoral Researcher
Department of Protein Evolution

Max Planck Institute for Developmental Biology
Max-Planck-Ring 5
72076 Tübingen
GERMANY



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Re: [ccp4bb] Off-topic question

[Debanu Das]

Hi,
I was going to suggest the same but since it has already been said, here’s a 
cheeky suggestion: you could try determining the crystal structure of the 
complex and get a direct sequence readout for the nuclei acid.
Best,
Debanu 

> On Mar 5, 2018, at 9:34 PM, Philippe BENAS 
> <0d88e888355a-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> Hi Jobi,
> 
> Phenol/CHCl3 extraction, iPrOH precipitation and then nucleic acid sequencing.
> 
> Best regards,
> Philippe
>  
> Philippe BENAS, Ph.D.
> 
> Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS
> Faculté de Pharmacie, Université Paris Descartes
> Case 48
> Av, de l'Observatoire
> F-75270 PARIS cedex 06
> +33.1.5373.1599
> E-mails: philippe.be...@parisdescartes.fr, philippe_be...@yahoo.fr
> URLs: http://lcrbw.pharmacie.univ-paris5.fr/ , 
> http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18
> 
> 
> 
> De : Jobichen Chacko 
> À : CCP4BB@JISCMAIL.AC.UK 
> Envoyé le : Mardi 6 mars 2018 5h11
> Objet : [ccp4bb] Off-topic question
> 
> Dear All,
> We are trying to identify/sequence a DNA/RNA fragment (around 100bp) which 
> was co-purified along with our protein.
> The expression was done in E.coli.
> Any suggestions on how to do this.
> Thank you.
> Jobi
> 
> 

Re: [ccp4bb] Off-topic question

[Philippe BENAS]

Hi Jobi,
Phenol/CHCl3 extraction, iPrOH precipitation and then nucleic acid sequencing.

Best regards,Philippe Philippe BENAS, Ph.D.

Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS
Faculté de Pharmacie, Université Paris Descartes
Case 48
Av, de l'Observatoire
F-75270 PARIS cedex 06
+33.1.5373.1599
E-mails: philippe.be...@parisdescartes.fr, philippe_be...@yahoo.fr
URLs: http://lcrbw.pharmacie.univ-paris5.fr/ , 
http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18



  De : Jobichen Chacko 
 À : CCP4BB@JISCMAIL.AC.UK 
 Envoyé le : Mardi 6 mars 2018 5h11
 Objet : [ccp4bb] Off-topic question
   
Dear All,
We are trying to identify/sequence a DNA/RNA fragment (around 100bp) which was 
co-purified along with our protein.
The expression was done in E.coli.
Any suggestions on how to do this.
Thank you.
Jobi


   

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

[Pavel Afonine]

Perhaps this can be automated:

https://www.phenix-online.org/papers/wd5073_reprint.pdf

Software doesn't get tired or bored, and thus potentially can try more and
produce more plausible interretations. Then one can hire a number of people
of various expertise to choose "best" result according to their subjective
interpretations.

Pavel

On Mon, Nov 20, 2017 at 12:25 PM, Shintaro Aibara  wrote:

> Dear All,
>
> Apologies for the slightly off-topic, but I was wondering if anybody knew
> of a paper/textbook where a protein model was built by multiple people
> (ranging from novice to experienced builders) and compared. I believe the
> conclusion was that while the overall trace was broadly correct,
> experienced builders maintained better geometry compared to beginners.
>
> I distinctly remember reading it a couple of years ago but cannot seem to
> find the figure. If anybody knows of this figure/paper/textbook it would be
> much appreciated if you could point me in the direction.
>
> Yours faithfully,
> Shintaro
>

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

[Marcin Wojdyr]

It sounds a bit like this paper from a year ago:
https://www.nature.com/articles/ncomms12549
apart from the conclusion: here the point is that amateurs build
higher quality models than crystallographers.

On 20 November 2017 at 20:25, Shintaro Aibara  wrote:
> Dear All,
>
> Apologies for the slightly off-topic, but I was wondering if anybody knew of
> a paper/textbook where a protein model was built by multiple people (ranging
> from novice to experienced builders) and compared. I believe the conclusion
> was that while the overall trace was broadly correct, experienced builders
> maintained better geometry compared to beginners.
>
> I distinctly remember reading it a couple of years ago but cannot seem to
> find the figure. If anybody knows of this figure/paper/textbook it would be
> much appreciated if you could point me in the direction.
>
> Yours faithfully,
> Shintaro

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

[Shintaro Aibara]

Thanks Mark for the paper, it kind of catches the essence of what I was
looking for. I do however remember it was more people and I think I'm
looking for a figure where all the builds (which if I remember correctly
was in the 10's rather than 3) were superimposed over each other to make
what looked similar to an NMR ensemble.

On Mon, Nov 20, 2017 at 10:04 PM, Mark J. van Raaij 
wrote:

>
> This one?
> http://scripts.iucr.org/cgi-bin/paper?SE0260
>
>
> Mark J van Raaij
> CNB-CSIC
> wwwuser.csic.es/~mjvanraaij
>
>  Original message 
> From: Shintaro Aibara 
> Date: 20/11/2017 21:25 (GMT+01:00)
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] [Off-topic] Comparison of the same structure built by
> many people
>
> Dear All,
>
> Apologies for the slightly off-topic, but I was wondering if anybody knew
> of a paper/textbook where a protein model was built by multiple people
> (ranging from novice to experienced builders) and compared. I believe the
> conclusion was that while the overall trace was broadly correct,
> experienced builders maintained better geometry compared to beginners.
>
> I distinctly remember reading it a couple of years ago but cannot seem to
> find the figure. If anybody knows of this figure/paper/textbook it would be
> much appreciated if you could point me in the direction.
>
> Yours faithfully,
> Shintaro
>



-- 
Yours Sincerely,
Shintaro Aibara

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

[Mark J. van Raaij]

This one?http://scripts.iucr.org/cgi-bin/paper?SE0260

Mark J van RaaijCNB-CSICwwwuser.csic.es/~mjvanraaij
 Original message From: Shintaro Aibara 
 Date: 20/11/2017  21:25  (GMT+01:00) To: 
CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] [Off-topic] Comparison of the same 
structure built by many people 
Dear All,
Apologies for the slightly off-topic, but I was wondering if anybody knew of a 
paper/textbook where a protein model was built by multiple people (ranging from 
novice to experienced builders) and compared. I believe the conclusion was that 
while the overall trace was broadly correct, experienced builders maintained 
better geometry compared to beginners.
I distinctly remember reading it a couple of years ago but cannot seem to find 
the figure. If anybody knows of this figure/paper/textbook it would be much 
appreciated if you could point me in the direction.
Yours faithfully,
Shintaro

Re: [ccp4bb] OFF topic (Protein degrades during Dailysis)

[Éverton D'Andréa]

   Hi Anamika,

As it was said before, a cocktail of protease inhibitors should be
fine. But let`s say you can not overcome the problem. It is not specified
if you are doing this dialysis to cleave or not your His-tagged protein but
even this you could try getting samples in 1 hour intervals then you could
monitor the stability (and/or cleavage) of your protein. Another thing is
if you are doing this at room temperature or at 4C.

Best regards,

Everton.

On Thu, Oct 26, 2017 at 5:11 PM, Smith Liu  wrote:

> suppose protease is the issue, then avoid overnight dialysis
>
>
> 发自网易邮箱大师
>
> 在2017年10月26日 22:18，Anamika Singh  写道：
> Dear All,
>
> I am purifying the His-tagged proteins ( 21Kda) using buffer 20mM Tris,
> 150mM Nacl, and 5mM MgCl2 with 500mM imidazole. Earlier I was facing the
> precipitation problem during dialysis but with the addition of 50mM L-Arg
> somehow managed to overcome the precipitation issue. But this time I have
> seen after overnight dialysis there are degradation products on SDS page. I
> have used protease inhibitor (PMSF) in my sonication buffer.
>
> Please suggest me to overcome this problem.
>
> Thanks
> --
> Anamika
>
>
>
> 【网易自营】好吃到爆！鲜香弹滑加热即食，经典13香/麻辣小龙虾仅75元3斤>>
> 
>
>



-- 
Éverton Dias D'Andréa

Re: [ccp4bb] OFF topic (Protein degrades during Dailysis)

[Smith Liu]

suppose protease is the issue, then avoid overnight dialysis


发自网易邮箱大师


在2017年10月26日 22:18，Anamika Singh 写道：
Dear All,


I am purifying the His-tagged proteins ( 21Kda) using buffer 20mM Tris, 150mM 
Nacl, and 5mM MgCl2 with 500mM imidazole. Earlier I was facing the 
precipitation problem during dialysis but with the addition of 50mM L-Arg 
somehow managed to overcome the precipitation issue. But this time I have seen 
after overnight dialysis there are degradation products on SDS page. I have 
used protease inhibitor (PMSF) in my sonication buffer. 


Please suggest me to overcome this problem. 



Thanks
--

Anamika

Re: [ccp4bb] OFF topic (Protein degrades during Dailysis)

[Ruud Hovius]

Hello,

PMSF inhibits only 1 class of proteinases

You can try to add other inhibitors :
e.g. for purification from yeast I used the following in addition to PMSF:

1000 x stock individual inhibitors

pepstatin5 mg/ml methanol

chymostatin1 mg/mlDMSO

antipain3 mg/mlmethanol (sonicate to dissolve)

aprotinin2 mg/mlwater

leupeptin1 mg/ml water

E-642 mg/ml50 % ethanol

bestatin5 mg/mlmethanol

as well as EDTA for metallo-proteases

Some suppliers like Roche sell pre-made mixtures like protease inhibitor 
pills.



Also be as fast as possible with all steps and keep on ice


Best of luck,


Ruud



On 26/10/17 16:18, Anamika Singh wrote:

Dear All,

I am purifying the His-tagged proteins ( 21Kda) using buffer 20mM 
Tris, 150mM Nacl, and 5mM MgCl2 with 500mM imidazole. Earlier I was 
facing the precipitation problem during dialysis but with the addition 
of 50mM L-Arg somehow managed to overcome the precipitation issue. But 
this time I have seen after overnight dialysis there are degradation 
products on SDS page. I have used protease inhibitor (PMSF) in my 
sonication buffer.


Please suggest me to overcome this problem.

Thanks
--
Anamika


--

Ruud Hovius
EPFL SB ISIC LIP
BCH 4209
CH-1015 Lausanne
+41-21-693-9442
http://lip.epfl.ch

Re: [ccp4bb] Off topic: denaturing urea gels

[Mohammad Khan]

Dear all,

Thanks for all the replies. I adjusted my volumes and stopped reactions
with 8 M urea and 25 mM EDTA. The gels now run fine :)

-Mohammad

On 05-Oct-2017 7:29 PM, "Phoebe A. Rice" <pr...@uchicago.edu> wrote:

> Even though the protein should be denatured by all that urea, we find such
> gels sometimes look nicer if stop the reaction with SDS and protease K,
> and/or phenol extract the remains of the protein before loading the
> high-urea gel.
>
> ++
>
> Phoebe A. Rice
> Dept. of Biochemistry & Molecular Biology
> The University of Chicago
> pr...@uchicago.edu
>
>
> 
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Opher
> Gileadi [opher.gile...@sgc.ox.ac.uk]
> Sent: Saturday, September 30, 2017 3:44 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Off topic: denaturing urea gels
>
> In addition to the previous suggestions:
>
> With very small gels, the sample composition and depth (in the well) have
> a strong effect on the resolution.
> Rinse the wells with TBE buffer just before loading, as urea from the gel
> diffuses into the well and may prevent the sample from settling at the
> bottom. Minimize the amount of salt in the samples; try to load very small
> volumes (1-3 ul), even if this means longer exposures later.

Re: [ccp4bb] Off topic: denaturing urea gels

[Phoebe A. Rice]

Even though the protein should be denatured by all that urea, we find such gels 
sometimes look nicer if stop the reaction with SDS and protease K, and/or 
phenol extract the remains of the protein before loading the high-urea gel.

++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
pr...@uchicago.edu



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Opher Gileadi 
[opher.gile...@sgc.ox.ac.uk]
Sent: Saturday, September 30, 2017 3:44 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic: denaturing urea gels

In addition to the previous suggestions:

With very small gels, the sample composition and depth (in the well) have a 
strong effect on the resolution.
Rinse the wells with TBE buffer just before loading, as urea from the gel 
diffuses into the well and may prevent the sample from settling at the bottom. 
Minimize the amount of salt in the samples; try to load very small volumes (1-3 
ul), even if this means longer exposures later.

Re: [ccp4bb] Off topic: denaturing urea gels

[Opher Gileadi]

In addition to the previous suggestions:

With very small gels, the sample composition and depth (in the well) have a 
strong effect on the resolution.
Rinse the wells with TBE buffer just before loading, as urea from the gel 
diffuses into the well and may prevent the sample from settling at the bottom. 
Minimize the amount of salt in the samples; try to load very small volumes (1-3 
ul), even if this means longer exposures later.

Re: [ccp4bb] Off topic: denaturing urea gels

[Zhijie Li]

Smith:
One should expect to see ladders each separated by 1 nt in such cases.

Mohammed:

My suggestions:
1) running at 70V seems low. I do not see a reason for not using higher 
voltages. 200Vx45min should be OK. IT is better to not let the BPB front run 
out of the gel - so that you know whether the DNA had all been chopped into 
single nts. (how do you visualize the bands? Is the oligo end-labelled?  EtBr 
should not work well for small fragments. But UV shadowing may work.)

2) boil the sample before loading (6-8 M urea should always be included in 
sample buffers). This is important because the enzyme might bind the DNA and 
slowly release them causing the smearing.

3) How does the uncleaved DNA alone (no enzyme at all) look on the same gel? If 
it is a sharp single band then you might need to pay extra attention to 
denaturing the samples before loading. If it is also smeary then it might be 
the gel system needs optimizing.

4) use 0.75mm gel. Thinner gels give better resolution. 

5) I found that DNA ran just fine in the discontinuous tris-glycine 
system(Laemmli). In other words,  the PAGE system routinely used for proteins 
(without SDS) works for DNA too, with the benefit of sharpening the bands.
 You may also consider experimenting with gel percentages(25%-30 % for example) 
so that you can see even single nts. The acry:bis ratio is not that important 
as long as you stick to one.


Zhijie



> On Sep 30, 2017, at 7:54 AM, Smith Liu  wrote:
> 
> your enzyme cannot give definite fragment. thus smear
> 
> 
> 发自网易邮箱大师
> 
> 在2017年09月30日 19:28，Mohammad Khan 写道：
> Dear all,
> 
> I am working with an exonuclease and I run the digested DNA on a 
> 8Murea-20%acrylamide gel in TBE buffer. I use the Mini-Protean BioRad system 
> and cast gels of about 8.6x6.5 cm dimensions with 1.5 mm thickness. I use a 
> 15 well comb. I run my gels at 70 V for as long as 4 hours till my undigested 
> DNA reaches half the gel distance. I use 20-30 nt long susbtrates.
> 
> I am mostly not able to get distinct bands of the digested products but 
> rather get a smear. Is there any way to make sure that I get distinct 
> digested products rather than a smear?
> 
> I am looking forward for suggestions from all!
> 
> Thank you.
> 
> Ciao!

Re: [ccp4bb] Off topic: denaturing urea gels

[Smith Liu]

your enzyme cannot give definite fragment. thus smear


发自网易邮箱大师


在2017年09月30日 19:28，Mohammad Khan 写道：
Dear all,


I am working with an exonuclease and I run the digested DNA on a 
8Murea-20%acrylamide gel in TBE buffer. I use the Mini-Protean BioRad system 
and cast gels of about 8.6x6.5 cm dimensions with 1.5 mm thickness. I use a 15 
well comb. I run my gels at 70 V for as long as 4 hours till my undigested DNA 
reaches half the gel distance. I use 20-30 nt long susbtrates.


I am mostly not able to get distinct bands of the digested products but rather 
get a smear. Is there any way to make sure that I get distinct digested 
products rather than a smear?


I am looking forward for suggestions from all!


Thank you.


Ciao!

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Mohammad Khan]

Dear Thomas,

I do all my experiments in a 384 well, non-binding plates. I have a salt
concentration of 50 mM NaCl, with BSA. i will try the other suggestions as
well.

>From a couple of experiments, I could determine the Kd to be approx. 10 nM.

Thanks for all the suggestions!



On Fri, Jul 21, 2017 at 4:11 PM, Thomas Edwards 
wrote:

> A few tips, some/all of which may be relevant. Or not…
>
>
>
> If you are in 96 or 384 well plates, they vary. We tried quite a few
> batches and brands before settling on one.
>
>
>
> Keep your salt concentration as low as possible.
>
>
>
> You should be able to measure Kd in a range of about 1nM to low uM.
> Outside that range is harder, or possibly impossible.
>
>
>
> Buffers matter.
>
> RNA binding proteins have preferred phosphates, PPIs Tris.
>
> Some triton and/or some BSA can help.
>
>
>
> All proteins are different, and each likes some or all or none of the
> above…..
>
>
>
> Ideally you should convert polarization to anisotropy. Simple enough – but
> some referees can get picky…
>
>
>
> *Ed*
>
>
>
> *T.A.Edwards Ph.D.*
>
> *Deputy Director* Astbury Centre for Structural Molecular Biology
>
> Ass. Professor, School of Molecular and Cellular Biology
>
> Garstang 8.53d
>
> University of Leeds, Leeds, LS2 9JT  Telephone: 0113 343 3031
>
> http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
>
> Invention, my dear friends, is 93% perspiration, 6% electricity, 4%
> evaporation, and 2% butterscotch ripple. ~Willy Wonka
>
>
>
> *From: *CCP4 bulletin board  on behalf of Mohammad
> Khan 
> *Reply-To: *Mohammad Khan 
> *Date: *Friday, 21 July 2017 at 14:32
> *To: *"CCP4BB@JISCMAIL.AC.UK" 
> *Subject: *[ccp4bb] Off topic: Flourescence anisotropy measurement
>
>
>
> Dear all,
>
>
>
> I am trying to measure the difference in polarization upon the binding of
> the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying
> dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in
> difference of polarization with decrease in protein concentration. However,
> the results are difficult to reproduce and also vary greatly within
> triplicates of an experiment.
>
>
>
> Similar observations have been observed by my colleagues with their
> proteins.
>
>
>
> Are there any tips or precautions to keep in mind while setting up these
> reactions?
>
>
>
> Looking forward for suggestions.
>
>
>
> Thank you.
>

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Mohammad Khan]

Dear Didier and Opher,

The difference in polarities can reach upto 60 units. I have tried
concentrations between 1-5 nM DNA. Maybe I will give higher concentrations
a shot.

Thanks!

On Fri, Jul 21, 2017 at 4:02 PM, Didier Spittler 
wrote:

> Hello,
>
> What is your difference between the maximum and minimum value ? Have you
> try to change the probe concentration?
>
> Best,
>
> Didier
>
> Le 21 juil. 2017 15:34, "Mohammad Khan"  a écrit :
>
> Dear all,
>
> I am trying to measure the difference in polarization upon the binding of
> the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying
> dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in
> difference of polarization with decrease in protein concentration. However,
> the results are difficult to reproduce and also vary greatly within
> triplicates of an experiment.
>
> Similar observations have been observed by my colleagues with their
> proteins.
>
> Are there any tips or precautions to keep in mind while setting up these
> reactions?
>
> Looking forward for suggestions.
>
> Thank you.
>
>
>

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Mohammad Khan]

Dear Julius,

That is a good suggestion. I will definitely try this.

Thanks!


On Fri, Jul 21, 2017 at 3:41 PM, Rabl, Julius  wrote:

> Dear Mohammad,
> your buffer is key to success in biophysical measurements. While your
> protein may be totally fine in standard buffer, the large and hydrophobic
> fluorophores used in FP, MST and other assays are prone to adsorbing to
> plasticware etc. Also, at 1nM concentration, you are losing much more of
> your protein to adsorption, so you will have far less active protein at
> those concentrations than calculated. I would try to add detergent and, if
> necessary, BSA to your buffer. For my assay development projects, Tween20
> or Brij35 (0.03%) have worked well and I have used 0.5% BSA. Once you get
> the buffer right, measurements will be far more reproducible. Also, try to
> include a good control, e.g. DNA with Cy3, but sequence that does not bind
> to test for nonspecific, concentration dependent effects.
> I hope this helps, if you have any questions, feel free to email me!
> Best,
> Julius
>
>
> > On 21 Jul 2017, at 15:32, Mohammad Khan  wrote:
> >
> > Dear all,
> >
> > I am trying to measure the difference in polarization upon the binding
> of the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying
> dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in
> difference of polarization with decrease in protein concentration. However,
> the results are difficult to reproduce and also vary greatly within
> triplicates of an experiment.
> >
> > Similar observations have been observed by my colleagues with their
> proteins.
> >
> > Are there any tips or precautions to keep in mind while setting up these
> reactions?
> >
> > Looking forward for suggestions.
> >
> > Thank you.
>
>

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Smith Liu]

May i ask, whether the fluoresnce anisotropy method was reliable enough to 
determine the stoichiometry of a protein complex?


发自网易邮箱大师


在2017年07月22日 03:44，Phoebe A. Rice 写道：
You might also be getting aggregation.
If you do an old-fashioned EMSA ("gel shift") assay, does it hang up in the 
well?




++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago

pr...@uchicago.edu


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Morgan Milton 
[eilise.mil...@gmail.com]
Sent: Friday, July 21, 2017 9:44 AM
To:CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic: Flourescence anisotropy measurement


Completely agree, you need a higher DNA concentration. We have had luck with 10 
nM DNA.


Also, bubbles have a HUGE impact on how the fluorescent signal is measured. 
Make sure you spin your plates down (assuming you are using them) to remove any 
bubbles. We just had an undergrad read his anisotropy assay and the data looked 
horrible. He then realized he had not spun his plate down, after doing so the 
data was much more consistent.


Are you doing technical replicates? We do at least triplicates per plate.


All the best,

Morgan





Morgan E. Milton, PhD




On Fri, Jul 21, 2017 at 9:48 AM, Opher Gileadi <opher.gile...@sgc.ox.ac.uk> 
wrote:
FP is the ratio between two fluorescence measurements; if the fluorescence 
signal is too low, you will still get a ratio but it will be essentially noise. 
Try to perform the measurements at 10-50 nM DNA. If your binding affinity is in 
the low nM range, you may have to use other methods to measure KD.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Phoebe A. Rice]

You might also be getting aggregation.
If you do an old-fashioned EMSA ("gel shift") assay, does it hang up in the 
well?


++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago

pr...@uchicago.edu<mailto:pr...@uchicago.edu>


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Morgan Milton 
[eilise.mil...@gmail.com]
Sent: Friday, July 21, 2017 9:44 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

Completely agree, you need a higher DNA concentration. We have had luck with 10 
nM DNA.

Also, bubbles have a HUGE impact on how the fluorescent signal is measured. 
Make sure you spin your plates down (assuming you are using them) to remove any 
bubbles. We just had an undergrad read his anisotropy assay and the data looked 
horrible. He then realized he had not spun his plate down, after doing so the 
data was much more consistent.

Are you doing technical replicates? We do at least triplicates per plate.

All the best,
Morgan


Morgan E. Milton, PhD


On Fri, Jul 21, 2017 at 9:48 AM, Opher Gileadi 
<opher.gile...@sgc.ox.ac.uk<mailto:opher.gile...@sgc.ox.ac.uk>> wrote:
FP is the ratio between two fluorescence measurements; if the fluorescence 
signal is too low, you will still get a ratio but it will be essentially noise. 
Try to perform the measurements at 10-50 nM DNA. If your binding affinity is in 
the low nM range, you may have to use other methods to measure KD.