Re: [ccp4bb] Co crystalization with less soluble ligand.
Fatty acids are very soluble in glycerol or the PEGs. Most proteins tolerate high concentrations (30-50%) glycerol and PEGs.� So, it is quite possible to make a solution that will allow the protein crsytal and the ligand to both be in the same solution. One of my former colleagues was in charge of dosing mice for drug testing.� He often had to get hydrophobic compounds into solution long enough to feed the compounds to the mice. If the key compound is not soluble in the current system, change the system. Some might complain that this is a lot of work (which it might be), but that might also be why no one else has done it yet. On 6/23/2021 5:53 AM, Tim Gruene wrote: Hi Frank, heme becomes near completely insoluble when it dimerizes. Fatty acids are also very insoluble. It is quite hard to get them into HSA, as far as I remember. Cheers, Tim On Wed, 23 Jun 2021 10:33:42 +0100 Frank von Delft wrote: And then of course, you need to decide whether you at all care to know anything about a compound that is so insoluble that it needs that kind of treatment :) On 23/06/2021 09:52, hoh wrote: Hi As total insolubility does not exist, I regularly use another method, which is to deposit a grain of the ligand directly into the drop. In this case, we no longer control the concentration. The goal is to regularly freeze crystals (1 hour, 2 days, 1 week ..). Depending on the results, it is possible to adjust the time. If after one hour, the crystal no longer diffracts, redo the experience by freezing at 10s, 30s, 2mn ..). If a blob appears, frozen after 1 week. This technique needs 2 things , time and several exploitable crystals in the drop. FH To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at 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=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] Co crystalization with less soluble ligand.
Hi Frank, heme becomes near completely insoluble when it dimerizes. Fatty acids are also very insoluble. It is quite hard to get them into HSA, as far as I remember. Cheers, Tim On Wed, 23 Jun 2021 10:33:42 +0100 Frank von Delft wrote: > And then of course, you need to decide whether you at all care to > know anything about a compound that is so insoluble that it needs > that kind of treatment :) > > On 23/06/2021 09:52, hoh wrote: > > Hi > > > > As total insolubility does not exist, I regularly use another > > method, which is to deposit a grain of the ligand directly into the > > drop. > > > > In this case, we no longer control the concentration. The goal is > > to regularly freeze crystals (1 hour, 2 days, 1 week ..). Depending > > on the results, it is possible to adjust the time. If after one > > hour, the crystal no longer diffracts, redo the experience by > > freezing at 10s, 30s, 2mn ..). If a blob appears, frozen after 1 > > week. This technique needs 2 things > > , time and several exploitable crystals in the drop. > > > > > > FH > > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a > mailing list hosted by www.jiscmail.ac.uk, terms & conditions are > available at https://www.jiscmail.ac.uk/policyandsecurity/ -- -- Tim Gruene Head of the Centre for X-ray Structure Analysis Faculty of Chemistry University of Vienna Phone: +43-1-4277-70202 GPG Key ID = A46BEE1A To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ pgpiKZin1MEqF.pgp Description: OpenPGP digital signature
Re: [ccp4bb] Co crystalization with less soluble ligand.
And then of course, you need to decide whether you at all care to know anything about a compound that is so insoluble that it needs that kind of treatment :) On 23/06/2021 09:52, hoh wrote: Hi As total insolubility does not exist, I regularly use another method, which is to deposit a grain of the ligand directly into the drop. In this case, we no longer control the concentration. The goal is to regularly freeze crystals (1 hour, 2 days, 1 week ..). Depending on the results, it is possible to adjust the time. If after one hour, the crystal no longer diffracts, redo the experience by freezing at 10s, 30s, 2mn ..). If a blob appears, frozen after 1 week. This technique needs 2 things , time and several exploitable crystals in the drop. FH To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Co crystalization with less soluble ligand.
Hi As total insolubility does not exist, I regularly use another method, which is to deposit a grain of the ligand directly into the drop. In this case, we no longer control the concentration. The goal is to regularly freeze crystals (1 hour, 2 days, 1 week ..). Depending on the results, it is possible to adjust the time. If after one hour, the crystal no longer diffracts, redo the experience by freezing at 10s, 30s, 2mn ..). If a blob appears, frozen after 1 week. This technique needs 2 things , time and several exploitable crystals in the drop. FH -- François Hoh Centre de Biochimie Structurale, UMR 5048 CNRS, UMR 1054 INSERM 29, rue de navacelles 34090 Montpellier Cedex, France. Phone: +33 467 417 706 Fax: +33 467 417 913 -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Co crystalization with less soluble ligand.
Dear Gourab,
Sorry for the delayed response, I missed your message earlier.
Can you grow the protein crystals easily (do you have lots of them)?
Is your ligand soluble in PEG, MPD, or Solutol?
Often, you can exchange the crystal solution without destroying the
crystal. But, you have to match the water activity of the original
growth solution with the new solution. This way you could get your
ligand into one of these non-salt solutions and then move a crystal from
your grow solution into the new solution with your ligand. This would
expose your crystal to the ligand.
Obviously, you will probably burn through several crystals to see what
PEG, MPD, of Solutol concentration your crystals will tolerate. To some
degree, this is no different than hunting for the best cryo-condition.
Thus, I would see which solution your ligand prefers first, so you can
limit your crystal trials.
I worked on an enzyme that required calcium for activity, but we could
only grow the crystals in ammonium sulfate. I was able to exchange the
crystal growth solution for a PEG solution. The protein has some
affinity for the sulfate ions, so the exchange needed to be done over a
~4 hour period of time. If I went faster, the sulfate would remain in
the crystal channels and would precipitate when exposed to calcium. It
was tedious working this out, but it was essential for everything else
that came later.
Steven Herron [sherron_...@yahoo.com]
On 4/23/2021 11:40 PM, Gourab Basu Choudhury wrote:
I tried sokaing ,high concentration of DMSO is effecting crystal, so I
tried other contidtion where 10%DMSO is there. Got 1.9 A data with
protein co crystalization with 10mM ligand. But ligand occupancy is
not visble.
I got the KD value from ITC, with reverse titration. With 10uM ligand
in cell and 150uM protein in syringe. .
Can anybody suggest some views.please feel free to share your experiences.
On Sat, Apr 24, 2021, 10:49 AM Peat, Tom (Manufacturing, Parkville)
wrote:
Hello Gourab,
DMSO is not the only possible solvent- there are others to try.
I don't want to sound negative, but 40 micromolar is not a very
tight binding compound. It would also depend on how that binding
constant was measured- how did someone get enough in solution to
measure that?
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 mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Gourab Basu Choudhury
mailto:goura...@csiriicb.res.in>>
*Sent:* Saturday, April 24, 2021 12:46 PM
*To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
mailto:CCP4BB@JISCMAIL.AC.UK>>
*Subject:* [ccp4bb] Co crystalization with less soluble ligand.
Hello everyone,
I am finding it difficult for getting a ligand density inside the
protein as the ligand is very much insoluble. It's only soluble in
100% DMSO. I tried for co crystalization. Kd value of the ligand
is near 40um. Any suggestion what to do?
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Re: [ccp4bb] Co crystalization with less soluble ligand.
These are my go-to review articles for ideas for getting a ligand into a crystal. https://scripts.iucr.org/cgi-bin/paper?S0907444906047020 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297911/ I have also seen a talk where someone successfully dissolved their water-insoluble ligand in Octan-1-ol, and layered that solution over the drop after crystals had grown. They incubated for X period of time (I don't recall) froze the crystals and found the ligand had diffused into the crystals and was in the active site. Maybe someone else knows of the/a paper describing this? Hope this helps, D -- Dr David C. Briggs Senior Laboratory Research Scientist Signalling and Structural Biology Lab The Francis Crick Institute London, UK == Diamond User Committee MX representative == about.me/david_briggs From: CCP4 bulletin board on behalf of Gourab Basu Choudhury Sent: 24 April 2021 03:46 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Co crystalization with less soluble ligand. External Sender: Use caution. Hello everyone, I am finding it difficult for getting a ligand density inside the protein as the ligand is very much insoluble. It's only soluble in 100% DMSO. I tried for co crystalization. Kd value of the ligand is near 40um. Any suggestion what to do? To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1<https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1=04%7C01%7C%7Ce31fd41ae0d94d54f04108d906cc8c65%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C637548297877580067%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C2000=dSgnMbz9Zrqhw3oPKKs51HY2Zbr7ZAr4rmk1KIqFCJE%3D=0> 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 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Co crystalization with less soluble ligand.
Dear Gourab, You might just want to add the ligand in pure, solid form to the edge of the crystallization drop. This worked in our case beautifully (see Angew. Chem. Int. Ed. 2012, 51, 3354). We had to soak it for 4 weeks. Good luck! Best regards Bernhard To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Sv: [ccp4bb] Co crystalization with less soluble ligand.
Some ligands/ small molecules solubility is pH- dependent. (E.g. biotin, EDTA) Try adjusting the pH (it doesn't have to be accurate, just use a pH paper if you have very small volumes). It might help you out to improve ligand solubility (and binding with your protein). Also, not to ruin the party, but have you done a base line ITC measurement (protein to buffer, no ligand) and substracted it from the protein - ligand measurement? It might improve your calculated kd slightly, or, alas, you'll realise the measured affinity was due to protein's denaturing upon interaction with the solvent. Good luck, Amit Quoting Jon Cooper <488a26d62010-dmarc-requ...@jiscmail.ac.uk>: I realised afterwards that this might be an experimental example of desperately trying to turn a negative result into a positive one, which is something that has been discussed here recently (sorry, Dale) ;-0 ;-0 Sent from ProtonMail mobile Original Message On 24 Apr 2021, 14:33, Jon Cooper wrote: If you want to try co-crystallisation again, if you dissolve your ligand in say DMSO or anything that works, e.g. isopropanol, then add it to your protein up to its maximum tolerable level (i.e. the level up to which the protein is not denatured/inactivated by the solvent - you need to test this), the ligand will start to precipitate out on a grand scale but don't worry, stir the mix at 4°C for a couple of days so that the ligand can slowly partition into the protein binding sites, hopefully. Then you can dialyse out the organic solvent and/or use one of the spin concentrators to remove it and concentrate the protein for crystallisation. There will be tons of precipitated ligand which you can centrifuge out whenever necessary. Sent from ProtonMail mobile Original Message On 24 Apr 2021, 13:08, abhimanyu singh wrote: You may soak the crystals with ligands in maximum tolerable DMSO percentage and harvest crystals at different time points. In my experience working with some poor affinity, highly hydrophobic compounds, it may take up-to several days for the ligand to bind with good occupancy (in one instance it was four days). This may also depend on the crystal soaking/growth temperature. Ethylene glycol is a good alternative to DMSO in some cases, which might particularly be helpful in co-crystallisation experiments. Greetings, Abhi On Sat, 24 Apr 2021 at 09:11, Casper Wilkens <5d34ab0aef35-dmarc-requ...@jiscmail.ac.uk> wrote: How long did you wait before freezing the crystal? Sometimes I have to wait days before the ligand finds it way into the binding site. Casper Wilkens Asst. Prof. Structural Enzymology & Biorefining DTU Bioengineering Technical University of Denmark Department of Biotechnology and Biomedicine Søltofts Plads Building 224 Room 028 2800 Kgs. Lyngby [c...@dtu.dk](mailto:c...@bio.dtu.dk) www.bioengineering.dtu.dk/ https://www.cazypedia.org/index.php/User:Casper_Wilkens https://twitter.com/protein_artist https://www.researchgate.net/profile/Casper_Wilkens https://www.cazypedia.org/index.php/User:Casper_Wilkens --- Fra: CCP4 bulletin board på vegne af Gourab Basu Choudhury Sendt: 24. april 2021 07:40:20 Til: CCP4BB@JISCMAIL.AC.UK Emne: Re: [ccp4bb] Co crystalization with less soluble ligand. I tried sokaing ,high concentration of DMSO is effecting crystal, so I tried other contidtion where 10%DMSO is there. Got 1.9 A data with protein co crystalization with 10mM ligand. But ligand occupancy is not visble. I got the KD value from ITC, with reverse titration. With 10uM ligand in cell and 150uM protein in syringe. . Can anybody suggest some views.please feel free to share your experiences. On Sat, Apr 24, 2021, 10:49 AM Peat, Tom (Manufacturing, Parkville) wrote: Hello Gourab, DMSO is not the only possible solvent- there are others to try. I don't want to sound negative, but 40 micromolar is not a very tight binding compound. It would also depend on how that binding constant was measured- how did someone get enough in solution to measure that? Best of luck, tom Tom Peat, PhD Proteins Group Biomedical Program, CSIRO [343 Royal Parade](https://www.google.com/maps/search/343+Royal+Parade+%0D%0AParkville,+VIC,+3052?entry=gmail=g) [Parkville, VIC, 3052](https://www.google.com/maps/search/343+Royal+Parade+%0D%0AParkville,+VIC,+3052?entry=gmail=g) +613 9662 7304 +614 57 539 419 tom.p...@csiro.au --- From: CCP4 bulletin board on behalf of Gourab Basu Choudhury Sent: Saturday, April 24, 2021 12:46 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Co crystalization with less soluble ligand. Hello everyone, I am finding it difficult for getting a ligand density inside the protein as the ligand is very much insoluble. It's only soluble in 100% DMSO. I tried for co crystalization. Kd value of the ligand is near 40um. Any sugge
Re: [ccp4bb] Sv: [ccp4bb] Co crystalization with less soluble ligand.
I realised afterwards that this might be an experimental example of desperately trying to turn a negative result into a positive one, which is something that has been discussed here recently (sorry, Dale) ;-0 ;-0 Sent from ProtonMail mobile Original Message On 24 Apr 2021, 14:33, Jon Cooper wrote: > If you want to try co-crystallisation again, if you dissolve your ligand in > say DMSO or anything that works, e.g. isopropanol, then add it to your > protein up to its maximum tolerable level (i.e. the level up to which the > protein is not denatured/inactivated by the solvent - you need to test this), > the ligand will start to precipitate out on a grand scale but don't worry, > stir the mix at 4°C for a couple of days so that the ligand can slowly > partition into the protein binding sites, hopefully. Then you can dialyse out > the organic solvent and/or use one of the spin concentrators to remove it and > concentrate the protein for crystallisation. There will be tons of > precipitated ligand which you can centrifuge out whenever necessary. > > Sent from ProtonMail mobile > > Original Message > On 24 Apr 2021, 13:08, abhimanyu singh wrote: > >> You may soak the crystals with ligands in maximum tolerable DMSO percentage >> and harvest crystals at different time points. In my experience working with >> some poor affinity, highly hydrophobic compounds, it may take up-to several >> days for the ligand to bind with good occupancy (in one instance it was four >> days). This may also depend on the crystal soaking/growth temperature. >> Ethylene glycol is a good alternative to DMSO in some cases, which might >> particularly be helpful in co-crystallisation experiments. >> >> Greetings, >> Abhi >> >> On Sat, 24 Apr 2021 at 09:11, Casper Wilkens >> <5d34ab0aef35-dmarc-requ...@jiscmail.ac.uk> wrote: >> >>> How long did you wait before freezing the crystal? Sometimes I have to wait >>> days before the ligand finds it way into the binding site. >>> >>> Casper Wilkens >>> Asst. Prof. >>> Structural Enzymology & Biorefining >>> DTU Bioengineering >>> >>> Technical University of Denmark >>> >>> Department of Biotechnology and Biomedicine >>> Søltofts Plads >>> Building 224 >>> Room 028 >>> 2800 Kgs. Lyngby >>> [c...@dtu.dk](mailto:c...@bio.dtu.dk) >>> www.bioengineering.dtu.dk/ >>> https://www.cazypedia.org/index.php/User:Casper_Wilkens >>> https://twitter.com/protein_artist >>> https://www.researchgate.net/profile/Casper_Wilkens >>> https://www.cazypedia.org/index.php/User:Casper_Wilkens >>> >>> --- >>> >>> Fra: CCP4 bulletin board på vegne af Gourab Basu >>> Choudhury >>> Sendt: 24. april 2021 07:40:20 >>> Til: CCP4BB@JISCMAIL.AC.UK >>> Emne: Re: [ccp4bb] Co crystalization with less soluble ligand. >>> >>> I tried sokaing ,high concentration of DMSO is effecting crystal, so I >>> tried other contidtion where 10%DMSO is there. Got 1.9 A data with protein >>> co crystalization with 10mM ligand. But ligand occupancy is not visble. >>> I got the KD value from ITC, with reverse titration. With 10uM ligand in >>> cell and 150uM protein in syringe. . >>> >>> Can anybody suggest some views.please feel free to share your experiences. >>> >>> On Sat, Apr 24, 2021, 10:49 AM Peat, Tom (Manufacturing, Parkville) >>> wrote: >>> >>>> Hello Gourab, >>>> >>>> DMSO is not the only possible solvent- there are others to try. >>>> I don't want to sound negative, but 40 micromolar is not a very tight >>>> binding compound. It would also depend on how that binding constant was >>>> measured- how did someone get enough in solution to measure that? >>>> Best of luck, tom >>>> >>>> Tom Peat, PhD >>>> Proteins Group >>>> Biomedical Program, CSIRO >>>> [343 Royal >>>> Parade](https://www.google.com/maps/search/343+Royal+Parade+%0D%0AParkville,+VIC,+3052?entry=gmail=g) >>>> [Parkville, VIC, >>>> 3052](https://www.google.com/maps/search/343+Royal+Parade+%0D%0AParkville,+VIC,+3052?entry=gmail=g) >>>> +613 9662 7304 >>>> +614 57 539 419 >>>> tom.p...@csiro.au >>>> >>>> ---
Re: [ccp4bb] Sv: [ccp4bb] Co crystalization with less soluble ligand.
If you want to try co-crystallisation again, if you dissolve your ligand in say DMSO or anything that works, e.g. isopropanol, then add it to your protein up to its maximum tolerable level (i.e. the level up to which the protein is not denatured/inactivated by the solvent - you need to test this), the ligand will start to precipitate out on a grand scale but don't worry, stir the mix at 4°C for a couple of days so that the ligand can slowly partition into the protein binding sites, hopefully. Then you can dialyse out the organic solvent and/or use one of the spin concentrators to remove it and concentrate the protein for crystallisation. There will be tons of precipitated ligand which you can centrifuge out whenever necessary. Sent from ProtonMail mobile Original Message On 24 Apr 2021, 13:08, abhimanyu singh wrote: > You may soak the crystals with ligands in maximum tolerable DMSO percentage > and harvest crystals at different time points. In my experience working with > some poor affinity, highly hydrophobic compounds, it may take up-to several > days for the ligand to bind with good occupancy (in one instance it was four > days). This may also depend on the crystal soaking/growth temperature. > Ethylene glycol is a good alternative to DMSO in some cases, which might > particularly be helpful in co-crystallisation experiments. > > Greetings, > Abhi > > On Sat, 24 Apr 2021 at 09:11, Casper Wilkens > <5d34ab0aef35-dmarc-requ...@jiscmail.ac.uk> wrote: > >> How long did you wait before freezing the crystal? Sometimes I have to wait >> days before the ligand finds it way into the binding site. >> >> Casper Wilkens >> Asst. Prof. >> Structural Enzymology & Biorefining >> DTU Bioengineering >> >> Technical University of Denmark >> >> Department of Biotechnology and Biomedicine >> Søltofts Plads >> Building 224 >> Room 028 >> 2800 Kgs. Lyngby >> [c...@dtu.dk](mailto:c...@bio.dtu.dk) >> www.bioengineering.dtu.dk/ >> https://www.cazypedia.org/index.php/User:Casper_Wilkens >> https://twitter.com/protein_artist >> https://www.researchgate.net/profile/Casper_Wilkens >> https://www.cazypedia.org/index.php/User:Casper_Wilkens >> >> --------------- >> >> Fra: CCP4 bulletin board på vegne af Gourab Basu >> Choudhury >> Sendt: 24. april 2021 07:40:20 >> Til: CCP4BB@JISCMAIL.AC.UK >> Emne: Re: [ccp4bb] Co crystalization with less soluble ligand. >> >> I tried sokaing ,high concentration of DMSO is effecting crystal, so I tried >> other contidtion where 10%DMSO is there. Got 1.9 A data with protein co >> crystalization with 10mM ligand. But ligand occupancy is not visble. >> I got the KD value from ITC, with reverse titration. With 10uM ligand in >> cell and 150uM protein in syringe. . >> >> Can anybody suggest some views.please feel free to share your experiences. >> >> On Sat, Apr 24, 2021, 10:49 AM Peat, Tom (Manufacturing, Parkville) >> wrote: >> >>> Hello Gourab, >>> >>> DMSO is not the only possible solvent- there are others to try. >>> I don't want to sound negative, but 40 micromolar is not a very tight >>> binding compound. It would also depend on how that binding constant was >>> measured- how did someone get enough in solution to measure that? >>> Best of luck, tom >>> >>> Tom Peat, PhD >>> Proteins Group >>> Biomedical Program, CSIRO >>> [343 Royal >>> Parade](https://www.google.com/maps/search/343+Royal+Parade+%0D%0AParkville,+VIC,+3052?entry=gmail=g) >>> [Parkville, VIC, >>> 3052](https://www.google.com/maps/search/343+Royal+Parade+%0D%0AParkville,+VIC,+3052?entry=gmail=g) >>> +613 9662 7304 >>> +614 57 539 419 >>> tom.p...@csiro.au >>> >>> --- >>> >>> From: CCP4 bulletin board on behalf of Gourab Basu >>> Choudhury >>> Sent: Saturday, April 24, 2021 12:46 PM >>> To: CCP4BB@JISCMAIL.AC.UK >>> Subject: [ccp4bb] Co crystalization with less soluble ligand. >>> >>> Hello everyone, >>> >>> I am finding it difficult for getting a ligand density inside the protein >>> as the ligand is very much insoluble. It's only soluble in 100% DMSO. I >>> tried for co crystalization. Kd value of the ligand is near 40um. Any >>> suggestion what to do? >>> >>> --- >>> >>> To
Re: [ccp4bb] Sv: [ccp4bb] Co crystalization with less soluble ligand.
You may soak the crystals with ligands in maximum tolerable DMSO percentage and harvest crystals at different time points. In my experience working with some poor affinity, highly hydrophobic compounds, it may take up-to several days for the ligand to bind with good occupancy (in one instance it was four days). This may also depend on the crystal soaking/growth temperature. Ethylene glycol is a good alternative to DMSO in some cases, which might particularly be helpful in co-crystallisation experiments. Greetings, Abhi On Sat, 24 Apr 2021 at 09:11, Casper Wilkens < 5d34ab0aef35-dmarc-requ...@jiscmail.ac.uk> wrote: > How long did you wait before freezing the crystal? Sometimes I have to > wait days before the ligand finds it way into the binding site. > > > > Casper Wilkens > Asst. Prof. > Structural Enzymology & Biorefining > DTU Bioengineering > > > > Technical University of Denmark > Department of Biotechnology and Biomedicine > Søltofts Plads > Building 224 > Room 028 > 2800 Kgs. Lyngby > c...@dtu.dk > www.bioengineering.dtu.dk/ > https://www.cazypedia.org/index.php/User:Casper_Wilkens > https://twitter.com/protein_artist > https://www.researchgate.net/profile/Casper_Wilkens > <https://www.cazypedia.org/index.php/User:Casper_Wilkens> > > -- > *Fra:* CCP4 bulletin board på vegne af Gourab > Basu Choudhury > *Sendt:* 24. april 2021 07:40:20 > *Til:* CCP4BB@JISCMAIL.AC.UK > *Emne:* Re: [ccp4bb] Co crystalization with less soluble ligand. > > I tried sokaing ,high concentration of DMSO is effecting crystal, so I > tried other contidtion where 10%DMSO is there. Got 1.9 A data with protein > co crystalization with 10mM ligand. But ligand occupancy is not visble. > I got the KD value from ITC, with reverse titration. With 10uM ligand in > cell and 150uM protein in syringe. . > > Can anybody suggest some views.please feel free to share your experiences. > > On Sat, Apr 24, 2021, 10:49 AM Peat, Tom (Manufacturing, Parkville) > wrote: > >> Hello Gourab, >> >> DMSO is not the only possible solvent- there are others to try. >> I don't want to sound negative, but 40 micromolar is not a very tight >> binding compound. It would also depend on how that binding constant was >> measured- how did someone get enough in solution to measure that? >> Best of luck, tom >> >> Tom Peat, PhD >> Proteins Group >> Biomedical Program, CSIRO >> 343 Royal Parade >> <https://www.google.com/maps/search/343+Royal+Parade+%0D%0AParkville,+VIC,+3052?entry=gmail=g> >> Parkville, VIC, 3052 >> <https://www.google.com/maps/search/343+Royal+Parade+%0D%0AParkville,+VIC,+3052?entry=gmail=g> >> +613 9662 7304 >> +614 57 539 419 >> tom.p...@csiro.au >> >> -- >> *From:* CCP4 bulletin board on behalf of Gourab >> Basu Choudhury >> *Sent:* Saturday, April 24, 2021 12:46 PM >> *To:* CCP4BB@JISCMAIL.AC.UK >> *Subject:* [ccp4bb] Co crystalization with less soluble ligand. >> >> Hello everyone, >> >> I am finding it difficult for getting a ligand density inside the protein >> as the ligand is very much insoluble. It's only soluble in 100% DMSO. I >> tried for co crystalization. Kd value of the ligand is near 40um. Any >> suggestion what to do? >> >> -- >> >> 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: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > -- *Abhimanyu Kumar Singh, PhD* Post-doctoral Research Fellow Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research Department of Microbiology and Immunology, KU Leuven Herestraat 49, box 1030, 3000 Leuven Belgium. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Sv: [ccp4bb] Co crystalization with less soluble ligand.
How long did you wait before freezing the crystal? Sometimes I have to wait days before the ligand finds it way into the binding site. Casper Wilkens Asst. Prof. Structural Enzymology & Biorefining DTU Bioengineering Technical University of Denmark [http://www.dtu.dk/%7E/media/DTU_Generelt/Andet/DTU_email_logo_01.gif] Department of Biotechnology and Biomedicine Søltofts Plads Building 224 Room 028 2800 Kgs. Lyngby c...@dtu.dk<mailto:c...@bio.dtu.dk> www.bioengineering.dtu.dk/<http://www.bioengineering.dtu.dk/> https://www.cazypedia.org/index.php/User:Casper_Wilkens https://twitter.com/protein_artist https://www.researchgate.net/profile/Casper_Wilkens <https://www.cazypedia.org/index.php/User:Casper_Wilkens> Fra: CCP4 bulletin board på vegne af Gourab Basu Choudhury Sendt: 24. april 2021 07:40:20 Til: CCP4BB@JISCMAIL.AC.UK Emne: Re: [ccp4bb] Co crystalization with less soluble ligand. I tried sokaing ,high concentration of DMSO is effecting crystal, so I tried other contidtion where 10%DMSO is there. Got 1.9 A data with protein co crystalization with 10mM ligand. But ligand occupancy is not visble. I got the KD value from ITC, with reverse titration. With 10uM ligand in cell and 150uM protein in syringe. . Can anybody suggest some views.please feel free to share your experiences. On Sat, Apr 24, 2021, 10:49 AM Peat, Tom (Manufacturing, Parkville) wrote: Hello Gourab, DMSO is not the only possible solvent- there are others to try. I don't want to sound negative, but 40 micromolar is not a very tight binding compound. It would also depend on how that binding constant was measured- how did someone get enough in solution to measure that? 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 mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Gourab Basu Choudhury mailto:goura...@csiriicb.res.in>> Sent: Saturday, April 24, 2021 12:46 PM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: [ccp4bb] Co crystalization with less soluble ligand. Hello everyone, I am finding it difficult for getting a ligand density inside the protein as the ligand is very much insoluble. It's only soluble in 100% DMSO. I tried for co crystalization. Kd value of the ligand is near 40um. Any suggestion what to do? 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: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Co crystalization with less soluble ligand.
I tried sokaing ,high concentration of DMSO is effecting crystal, so I tried other contidtion where 10%DMSO is there. Got 1.9 A data with protein co crystalization with 10mM ligand. But ligand occupancy is not visble. I got the KD value from ITC, with reverse titration. With 10uM ligand in cell and 150uM protein in syringe. . Can anybody suggest some views.please feel free to share your experiences. On Sat, Apr 24, 2021, 10:49 AM Peat, Tom (Manufacturing, Parkville) wrote: > Hello Gourab, > > DMSO is not the only possible solvent- there are others to try. > I don't want to sound negative, but 40 micromolar is not a very tight > binding compound. It would also depend on how that binding constant was > measured- how did someone get enough in solution to measure that? > 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 Gourab > Basu Choudhury > *Sent:* Saturday, April 24, 2021 12:46 PM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Co crystalization with less soluble ligand. > > Hello everyone, > > I am finding it difficult for getting a ligand density inside the protein > as the ligand is very much insoluble. It's only soluble in 100% DMSO. I > tried for co crystalization. Kd value of the ligand is near 40um. Any > suggestion what to do? > > -- > > 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: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Co crystalization with less soluble ligand.
Hello Gourab, DMSO is not the only possible solvent- there are others to try. I don't want to sound negative, but 40 micromolar is not a very tight binding compound. It would also depend on how that binding constant was measured- how did someone get enough in solution to measure that? 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 Gourab Basu Choudhury Sent: Saturday, April 24, 2021 12:46 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Co crystalization with less soluble ligand. Hello everyone, I am finding it difficult for getting a ligand density inside the protein as the ligand is very much insoluble. It's only soluble in 100% DMSO. I tried for co crystalization. Kd value of the ligand is near 40um. Any suggestion what to do? 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: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Co crystalization with less soluble ligand.
Hello everyone, I am finding it difficult for getting a ligand density inside the protein as the ligand is very much insoluble. It's only soluble in 100% DMSO. I tried for co crystalization. Kd value of the ligand is near 40um. Any suggestion what to do? To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
