Thank you all for the very helpful information and suggestions! Kind regards,
Sorin On Sun, Aug 15, 2021 at 12:48 PM Jon Cooper < 0000488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote: > As Joel has suggested before, alphafold on an IDP would be interesting and > would seem like a zero-cost starting point - perhaps one you have tried > already. > > > Sent from ProtonMail mobile > > > > -------- Original Message -------- > On 15 Aug 2021, 15:53, Scott Horowitz < scott.horow...@du.edu> wrote: > > > Hi Sorin, > > I hate to say it, but this is a really tough and expensive one. Solving a > true conformational ensemble of one IDP of decent size (~>70 residues) at > something like decent resolution is hard, and not that many labs actually > do it (it's usually a different set of NMR techniques than solving folding > proteins, and that knowledge is even somewhat specialized even within the > NMR community). Solving a co-structural ensemble of two IDPs that bind is > even harder, and I'm hard pressed to remember a single case right now where > it's been done (probably has, but very rarely). Assuming they express > really well and produce decent spectra, it is in theory doable, but I'd > assume multiple years of work by a very good student or postdoc from a lab > that specializes in this and many thousands of dollars (I'd very roughly > assume ~$10k in materials costs alone) would be required for that > co-structure. > > The SAXS route is certainly less expensive and faster if it works and gets > you the info you need, but it certainly will be low-res. I'm not as > familiar with it, but if you can differentially label the proteins, the > neutron equivalent of SAXS might also help with the co-structural ensemble > to differentiate which protein is where in the resulting blob. > > Scott > > Scott Horowitz, Ph.D. > > Assistant Professor > > Department of Chemistry & Biochemistry > > Knoebel Institute for Healthy Aging > > University of Denver > > > > ECS Building > > 2155 E. Wesley Ave > > Denver, CO 80208 > > Phone: 303-871-4326 > > Fax: 303-871-7915 > > Zoom Room: https://udenver.zoom.us/my/scotthorowitz > > Email: scott.horow...@du.edu > > Office: Room 561 Lab: Room 505 > > ------------------------------ > *From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Roopa > Thapar <0000070a21fba45f-dmarc-requ...@jiscmail.ac.uk> > *Sent:* Sunday, August 15, 2021 8:20 AM > *To:* CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK> > *Subject:* [EXTERNAL] Re: [ccp4bb] biomolecular NMR for IDPs > > [External Email From]: *owner-ccp...@jiscmail.ac.uk* > > Hello Sorin, > > > 1. The cost of getting NMR data on the IDPs you propose depends upon the > expression levels of the protein/s as you will need to label with 15N and > 13C - and depending upon your overall yields per liter of E.coli culture, > this can add up. In addition you will need to run triple resonance > experiments - so you should look into the hourly charge to access the NMR > spectrometers where you are located. Moreover, you need to account for > time required for optimization of solution conditions to collect the NMR > data as the sample needs to be homogenous (as in no aggregation) at > millimolar or hundreds of micromolar concentration. As Ethan Merritt > suggested, it would be a good idea to use SAXS first as it requires very > little sample, no isotope labeling, and you can try to narrow down the > solution conditions that would be best suited for NMR. The Kratky plots, > Rg values under different solution conditions can give very useful > information about conformational states and ensembles populated by IDPs. > However, although NMR tends to be more expensive than other techniques > but is perfect for IDPs as you point out you can get residue specific > information. A combined NMR/SAXS approach has proven to be very useful to > validate computational models. > > 2. In general, CROs are much more expensive particularly for generating > isotopically labeled samples - it is cost-prohibitive for academic labs. > Genscript is one CRO that will express proteins, but I am not sure if they > will make isotopically labeled proteins for NMR. > > 3. The amount of protein needed depends upon the size of the molecule. > You will need at least 2-3 samples that are differentially labeled with > 15N, 13C (also since you want data on the free and bound forms of the > complex) at 0.5 - 1 mM depending upon the size of the molecule which > relates to the complexity of the NMR spectrum due to number of resonances > and the relaxation times. The total volume required for each sample is > between 280 ul - 600 ul, depending upon which type of instrumentation and > NMR probes you have access to. > > Hope this helps! > > Best regards, > Roopa > > On Saturday, August 14, 2021, 04:12:58 PM CDT, Sorin Draga < > sor.dr...@gmail.com> wrote: > > > Hello everyone, > > I do realize that this is not a NMR focused group, but I do hope that > there are a few spectroscopists lurking around that could possibly answer a > few questions (I am more of a modeler/computationalist): > > The problem: I have two intrinsically disordered proteins that are known > to interact (let's call them 1 and 2). I would like to get structural > information (a conformational ensemble) for 1 and for the "complex" (1+2). > Further down the line (depending on whether this is possible) I would also > like to evaluate potential small molecule inhibitors for the said complex. > Both 1 and 2 are <200 aminoacids long. > > The questions: > > 1. Could the cost of determining the "structure" for 1 and 1+2 be > estimated? To be more precise, I am looking for a ball-park figure on how > much a NMR measurement would cost in this case. > 2. Could anyone recommend a good group/CRO that could provide such a > service and not have an astronomical cost? > 3. Any other suggestions/thoughts that you think might be worth mentioning > (minimum quantity of protein necessary, purity, type of NMR etc) > > Many thanks for your help and time! > > Cheers! > > Sorin > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1__;!!NCZxaNi9jForCP_SxBKJCA!DbtqkRPBtMNlqc7ScfGFuxp3RZP6f9Omit6_JrUQlHKBIgVjntXF_s7XNuXVXZqqeLwS$> > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1__;!!NCZxaNi9jForCP_SxBKJCA!DbtqkRPBtMNlqc7ScfGFuxp3RZP6f9Omit6_JrUQlHKBIgVjntXF_s7XNuXVXZqqeLwS$> > > ------------------------------ > > 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/
