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! 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