lDDT = Local Difference Distance Test, for reference... Harry -- Dr Harry Powell
> On 17 Aug 2021, at 20:21, Randy John Read <rj...@cam.ac.uk> wrote: > > What the DeepMind people say about this is that when part of a predicted > structure has a low predicted lDDT (a CASP measure of model quality), this is > a good indicator that it may be intrinsically disordered. > > Best wishes, > > Randy Read > >> On 17 Aug 2021, at 17:13, Sorin Draga <sor.dr...@gmail.com> wrote: >> >> 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> 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 >> >> >> 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 >> >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 >> > > ----- > Randy J. Read > Department of Haematology, University of Cambridge > Cambridge Institute for Medical Research Tel: +44 1223 336500 > The Keith Peters Building Fax: +44 1223 336827 > Hills Road E-mail: > rj...@cam.ac.uk > Cambridge CB2 0XY, U.K. > www-structmed.cimr.cam.ac.uk > > ######################################################################## > > 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/ ######################################################################## 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/
