Dear all, Regarding the AlphaFold2: while its results are impressive for properly folded proteins, I would caution against using it for IDPs: they do not have a stable 3D structure that can be predicted, instead they occupy an ensemble of structures, with a high degree of heterogeneity. While you will find models of IDPs predicted by AlphaFold, they only represent (at best) one of many local minima that these proteins occupy.
Again, many thanks for all your excellent contributions! Kind regards, Sorin On Tue, Aug 17, 2021 at 11:59 AM George Sheldrick <gshe...@uni-goettingen.de> 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> > <CCP4BB@JISCMAIL.AC.UK> on behalf of Roopa Thapar > <0000070a21fba45f-dmarc-requ...@jiscmail.ac.uk> > <0000070a21fba45f-dmarc-requ...@jiscmail.ac.uk> > *Sent:* Sunday, August 15, 2021 8:20 AM > *To:* CCP4BB@JISCMAIL.AC.UK <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> <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 > ######################################################################## 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/
