Re: [ccp4bb] cavities volume calculations
Dear Nathalie, try CASTp ( http://sts.bioe.uic.edu/castp/index.html?2cpk ) Cheers, Flavio. Il giorno lun 6 lug 2020 alle ore 15:41 Nathalie Colloc'h < coll...@cyceron.fr> ha scritto: > Hi everybody, > > I am aware of programs which compute the volume of cavities and pockets. > > However, I am looking for a program which can compute the volume around > a given point (given by its coordinates), and return also the > information about if it is an open surface pocket or an internal cavity. > > Thanks a lot > > Nathalie > > > -- > Dr. Nathalie Colloc'h > équipe CERVOxy > ISTCT UMR 6030 - CNRS - Université Caen-Normandie - CEA > Centre Cyceron > bd Becquerel > 14074 Caen cedex > France > 33.2.31.47.01.32 > coll...@cyceron.fr > http://www.istct.cyceron.fr/ > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a > mailing list hosted by www.jiscmail.ac.uk, terms & conditions are > available at https://www.jiscmail.ac.uk/policyandsecurity/ > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter
Dear community, I'm looking for a commercial expression vector for E.Coli with a cleavable his-tag at the c-ter. Anyone can help me? Thank you in advance, Flavio. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter
Dear all, thank you for your valuable suggestions. Have a good day, Flavio. Il 16/02/24 15:36, Karla J. F. Satchell ha scritto: Sorry did not mean to go off topic. Original question was requests for suggestions of cleavable c-term tag vectors. I only meant to provide info on one type recommended by another. Others may have further suggestions for Flavio. *From: *CCP4 bulletin board on behalf of Karla J. F. Satchell *Date: *Friday, February 16, 2024 at 7:13 AM *To: *CCP4BB@JISCMAIL.AC.UK *Subject: *Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter Hi everyone— Thought I might chime in. This technology was developed in my lab based on our studies of toxins and we have methods papers and two patent. The CPD-tag works great for production of protein with minimal residual residues on the protein. Our studies indicate that it can help solubilize protein during expression. Protein is purified with the C-terminal CPD tag, and the entire CPD and 6xHis-tag are released by addition of InsP6 (phytic acid) which is very inexpensive. The only restriction of the recognition sequence is small-Leu-small so a small residue plus Leucine does remain on your protein. There does also need to be a little flexibility at the end of your protein for access to the protease so our vectors add extra Gly-Ala so the residual is GAAL. CPD is particularly useful for small proteins if the additional residues do not interfere with your assays. https://pubmed.ncbi.nlm.nih.gov/28056928/ <https://urldefense.com/v3/__https:/pubmed.ncbi.nlm.nih.gov/28056928/__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HimFkBThs$> https://pubmed.ncbi.nlm.nih.gov/31773580/ <https://urldefense.com/v3/__https:/pubmed.ncbi.nlm.nih.gov/31773580/__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HijC4KPFp$> https://patents.google.com/patent/US8257946B2/en <https://urldefense.com/v3/__https:/patents.google.com/patent/US8257946B2/en__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HirGMggut$> https://patents.google.com/patent/US8383400B2/en <https://urldefense.com/v3/__https:/patents.google.com/patent/US8383400B2/en__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HiqMuRtc4$> Here is a link to the original paper on mechanism of action of CPD https://pubmed.ncbi.nlm.nih.gov/19620709/ <https://urldefense.com/v3/__https:/pubmed.ncbi.nlm.nih.gov/19620709/__;!!Dq0X2DkFhyF93HkjWTBQKhk!WmwLxh4JzUmaD1lNY20_ID-cV87BW0TsBDnbHKRlFiLvNtDYLZXp5v_vR6P8_ReHlWVV8DU7ZEy69-OGnYVPAhVTl75HinAiY8dQ$> These are not commercially available as despite multiple marketing attempts, there was little interest mid-2010s from biotech for licensing new cloning technologies and we abandoned advanced development If this technology is interesting to you to add to your commercial biotech portfolio, please feel free to reach out to me. Flavio, we can share our vector, but my institution does require an MTA as technology is patented. Most people just use synthetic DNA to generate the exact clone they want so we rarely get requests but happy to share sequences if you need for your synthetic design. Karla Satchell *From: *CCP4 bulletin board on behalf of Srivastava, Dhiraj <bda2be8a675a-dmarc-requ...@jiscmail.ac.uk> *Date: *Friday, February 16, 2024 at 6:42 AM *To: *CCP4BB@JISCMAIL.AC.UK *Subject: *Re: [ccp4bb] [External] [ccp4bb] Off topic : plasmid expression vector for E.Coli with a cleavable his-tag at the c-ter Hi Flavio While i am not aware of any commercial vector with cleavable c terminal tag, you can easily make your own by introducing protease cleavage site of your choice. However this strategy will results in quite a few extra residues from protease site. An alternative, which is not commercial but available through addgene, is cpd-his tag. It’s a bigger tag (~25 kda) but for me, it helped in solubility and expression. https://www.addgene.org/38251/ <https://urldefense.com/v3/__https:/www.addgene.org/38251/__;!!Dq0X2DkFhyF93HkjWTBQKhk!X4zQW9pYePXhpOWDRmDhx8F9CS1fcLsydhbQbLz2By2Pl6vW6Rnzq4kJp8HcdKp1EvVk5oIaXe__iBvZYFJul3k-1u1NgbhjZdL2xUs9Cg$> There are other variants of this vector available on addgene that you can choose from. Depending on your c terminal sequence, it may leave either no extra residues or only one or two residues. Dhiraj *From:*CCP4 bulletin board on behalf of Flavio Di Pisa *Sent:* Friday, February 16, 2024 3:33 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [External] [ccp4bb] Off topic : plasmid expression vector fo
Re: [ccp4bb] Question about metal behavior in microbatch under oil vs sitting drop crystallization setup
Thank you all for your helpful discussion.
Cheers,
Flavio
Il 22/04/25 19:27, Patrick Shaw Stewart ha scritto:
We used to do a lot of microbatch, but I've never come across
something like this before!
I agree with Pat that Cd++ ions can't dissolve in oil because they're
far too polar.
I think there is a subtle effect related to protein concentration
and/or water activity.
Do you have a (protein) skin on the vapor diffusion drops? If so, the
protein concentration in the vapor diffusion setup may be much lower.
I would not use Al's Oil for this because it complicates things - I
would try to find the microbatch conditions with paraffin oil that are
equivalent to the vapor diffusion condition. This depends a bit on
whether the main precipitant is a salt, which causes significant
dehydration during equilibration, or, for example, PEG, where little
dehydration occurs, or if it does, it's a slow process. What was the
main precipitant, and how long did the crystals take to grow in vapor
diffusion?
I would try to establish seeding conditions, which would give you much
more control. You may need to dilute the seedstock significantly to
get the crystal size that you want.
A mini phase diagram might be helpful - say 6 microbatch wells (with
paraffin) where you vary the ratio of protein to crystallization
cocktail. Then another 6 wells with a small volume of diluent eg
water added. Ideally, you would do it with and without seed to
establish the metastable zone of the phase diagram. We're working on
a couple of scripts to do similar phase diagrams automatically!
Good luck
Patrick
On Tue, Apr 22, 2025 at 2:41 PM Patrick Loll wrote:
I’m skeptical that the oil is acting as a reservoir for the metal,
as shouldn’t the metal be too hydrophilic to partition into the
oil phase? This is testable, at least.
Another (to me, more plausible) explanation is that there are
subtle differences in water activity in your two crystallization
setups. Many years ago (doi: 10.1021/bi00013a021) I saw a metal
change position owing to crystal dehydration and concomitant
subtle shifts in atomic positions; perhaps this is what’s going on?
FWIW,
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
> On Apr 22, 2025, at 5:03 AM, Flavio Di Pisa
wrote:
>
> Dear community,
> I’ve observed differences when crystallizing the same protein
using two different setups: microbatch under (Al’s) oil and vapor
diffusion sitting drop.
> The protein crystallizes in a condition containing 50 mM cadmium
as one of the precipitating agents. In the sitting drop setup, I
observe 2 well-defined cadmium ions at the so-called
mineralization site (please see PDB entry 5lg8 and the related
paper: https://www.pnas.org/doi/10.1073/pnas.1614302114), with
occupancies close to 1, as confirmed by the anomalous signal, plus
other "anomalous blob" near to this site.
> However, in the microbatch under oil setup, I never observe
these cadmium ions. Instead, I consistently detect only one
cadmium ion with high occupancy, and occasionally a second one
with lower occupancy.
> In summary, crystallizing the same protein using these two
setups results in different metal-binding behavior.
> My question is: could it be that in microbatch under oil, ions
might diffuse away from the mineralization site? Could this
account for the reduced number of cadmium ions observed?
Additionally, and more importantly, could the crystallization
setup influence the soaking efficiency of other metals, such as
iron (the natural substrate of this protein)?
> I’ve attached two screenshots:
> • One (orange blob) represents the protein crystallized via
vapor diffusion, showing two well-defined anomalous peaks.
> • The other shows the same site in a crystal grown via
microbatch, where only one anomalous signal (in white) is visible.
> I hope I’ve been clear. Thank you in advance, and I wish you all
a great day!
> Best regards,
> Flavio
>
>
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Patrick Loll
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