Dear Amit I did not follow the full thread, just in case this was not covered already some additional considerations: - 10% DMSO (if I understand your CoX setup correctly) is on the high side, we had cases where this was tolerated but more where not - depending on the Kd of your ligand you want to consider going down to 1% / 100µM ligand - with 15mg/ml protein, where are you in terms of mM protein? Figuring this out and considering law of mass action will tell you if your setup leaves you with sufficient occupancy (% ligand bound) - for poorly soluble ligands (and to reduce DMSO) you can use an alternative CoX protocol: incubate diluted protein and diluted ligand (I often use 5-10µM protein and 10x molar excess ligand) and reconcentrate after incubation (eg overnight) - try to use the available apo crystals as seeds! - the above will probably nor work if you have a ligand that is weak (Kd> 1-10µM) AND poorly soluble. In this case the already mentioned strategy of adding solid material to crystal drops is probably your only chance.
Good luck! Alex Alexander Pautsch Global NCE Boehringer Ingelheim Pharma GmbH & Co. KG Birkendorfer Str. 65 | 88397 Biberach T +49 (7351) 54-4683<tel:+49%20(7351)%2054-4683> M +49 (151) 15022743<tel:+49%20(151)%2015022743> E alexander.paut...@boehringer-ingelheim.com<mailto:alexander.paut...@boehringer-ingelheim.com> [cid:image001.png@01DB1590.DC556720]<https://www.boehringer-ingelheim.com/de/> Pflichtangaben finden Sie unter: hier<https://www.boehringer-ingelheim.com/de/unser-unternehmen/gesellschaften-in-deutschland> Mandatory information can be found at: here<https://www.boehringer-ingelheim.com/de/unser-unternehmen/gesellschaften-in-deutschland> Datenschutzhinweis: Klicken Sie hier<https://www.boehringer-ingelheim.com/de/datenschutz>, um weitere Informationen auf der lokalen Unternehmensinternetseite des betreffenden Landes über Datenschutz bei Boehringer Ingelheim und zu Ihren Rechten zu erhalten. Privacy Notice: Click here<https://www.boehringer-ingelheim.com/de/datenschutz> for more information on the local company website of the respective country about data protection at Boehringer Ingelheim and your rights. Diese E-Mail ist vertraulich zu behandeln. Sie kann besonderem rechtlichem Schutz unterliegen. Wenn Sie nicht der richtige Adressat sind, senden Sie bitte diese E-Mail an den Absender zurück, löschen die eingegangene E-Mail und geben den Inhalt der E-Mail nicht weiter. Jegliche unbefugte Bearbeitung, Nutzung, Vervielfältigung oder Verbreitung ist verboten. / This e-mail is confidential and may also be legally privileged. If you are not the intended recipient please reply to sender, delete the e-mail and do not disclose its contents to any person. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited. Von: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> Im Auftrag von Tom Peat Gesendet: Mittwoch, 2. Oktober 2024 23:23 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Co-Crystallization with drug molecule CAUTION: Do not click links, scan QR codes, or open attachments unless you trust the sender; this email is from an external sender (outside Boehringer Ingelheim). Hello Amit, In addition to what others have written, if you have some of your compound dry (not in DMSO), then adding this directly to your preformed crystals has worked for us on several occasions. In this instance, one would take a small/ fine pipette tip and dip this into your compound and then touch this to your crystallisation drop. Even if the compound is mostly insoluble, one still gets a little in solution and if this small amount binds to your protein, it is taken out of solution, and more goes into solution (mass action). It is very manual, so you don't want to do a high throughput screen this way, but if you get can get apo crystals and you don't have too many compounds, it can work. Best regards, tom ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Artem Evdokimov <artem.evdoki...@gmail.com<mailto:artem.evdoki...@gmail.com>> Sent: Thursday, October 3, 2024 5:42 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> Subject: Re: [ccp4bb] Co-Crystallization with drug molecule Dear Amit As David already pointed out, all proteins are different and it's hard to say in advance what amount of DMSO may work (or not). An additional concern is that DMSO can also interfere with ligand binding (cases from my personal past history), especially if these inhibitors/ligands are on the weaker side. Solutions: Despite its very high boiling point (189C) DMSO can in fact be evaporated from a small sample of your inhibitor, resulting in more or less solid inhibitor sample that can be re-dissolved in the same DMSO (but higher concentration), some other solvent, or perhaps directly in the protein solution. The latter is sometimes the only way to do this - I used to set up drops of DMSO solutions, then evaporate the DMSO in high vacuum (heating helps) with a cryofinger, then set up protein drops on top. This of course requires access to a lyophilizer or something similar. If you have a vial of your solution you can freeze-dry DMSO with water, by first diluting the sample then freeze-drying it. Also water can sometimes crash the substance out (if not water, then perhaps Ether or another solvent where your inhibitor does not dissolve) which makes it easier to redissolve (but there will be a loss of course). Find a friendly chemist nearby and ask then to put your sample in a speedvac on 'high BP' setting Notably, if you're "blessed" with an inhibitor that has the general solubility of a Sony Walkman, once you get rid of the DMSO, you may find out that the damned thing does not want to dissolve in anything else, including your protein solution. This happens a lot during early discovery phases when compounds are not very active (micromolar) and also poorly soluble (also micromolar). This is by far the most frequent cause for failing to co-crystallize (or soak) a ligand of interest. Very frustrating. Some success can be achieved using high DMSO or DMF (DMA also can be good) in your crystallization, or by phase transfer catalysts like Cyclodextrin(s) or appropriately formulated micelles. All of which can also mess up crystallization, needless to say. Best of luck in your endeavors! Artem - Cosmic Cats approve of this message On Wed, Oct 2, 2024 at 2:51 PM amit gaur <cdriamitg...@gmail.com<mailto:cdriamitg...@gmail.com>> wrote: Hi everyone, I am trying to crystallize a protein with a drug molecule. The protein concentration is 15.5 mg/ml, the drug stock concentration is 10 mM, and the drug is dissolved in DMSO. I am adding the drug to a final concentration of 1 mM in 100 ul of protein, and the DMSO volume is 10 ul for Co-crystallization. I want to know how much DMSO is permissible during co-crystallization with the drug and if DMSO can poison crystal formation. I have not been successful in getting crystals with inhibitors till now, but I obtained crystals of protein without DMSO, and those diffracted to 2.5A. Thanks, Dr. Amit Gaur, Research Scientist Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 1623 15th Street, Troy, NY, 12180 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## 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/
