Re: [ccp4bb] Teaching models and cognition w/ xtallography as example, high school lvl
Thanks for the help all, I ended up using Protopedia for the list of Nobel Prizes but I really like the Oxygen We Breathe tutorial and I've bookmarked it for future use. In the end I had them read Ed Yong's article in The Atlantic about the inevitable evolution of bad science. We talked about the transfer of models (evolution model transferred from biology to social science) and the problems and solutions for science listed in the article. I then used those as a segue to talk about how one field, structural biology, actually had already implemented several of those solutions and how successful the field was, e.g. Nobel Prizes. The solutions were sharing models, sharing data, cross-validation. And we also talked a bit about how there are too many degrees of freedom compared to observations but we can add information about order/connectivity of atoms, bond angles, bond lengths, etc. Even something as simple as the rule that two atoms can't be on top of each other is a lot of information. Unfortunately, I ended up with too much information for 45 minutes. I could have taken out the stuff about destructive and constructive interference and why we chose x-ray over visible light without losing the points I wanted to make. And I could have to sharpened up the lesson by spending more time thinking about the take home message. Cheers, Morten On 19 September 2016 at 15:09, Joel Sussman <joel.suss...@weizmann.ac.il> wrote: > 19-Sep-2016 > Dear Morten > Please consider looking at *Proteopedia*: http://proteopedia.org, e.g. > see: > * 3D molecular models: an introduction > *http://www.proteopedia.org/w/3D_Molecular_Models > <http://www.proteopedia.org/w/3D_Molecular_Models>* > * HIV-1 *protease http://proteopedia.org/w/HIV-1_protease > <http://proteopedia.org/w/HIV-1_protease>* > * Group:SMART:A Physical Model of the β2-Adrenergic Receptor > *http://www.proteopedia.org/w/Group:SMART:A_Physical_Model_of_the_%CE%B22-Adrenergic_Receptor > <http://www.proteopedia.org/w/Group:SMART:A_Physical_Model_of_the_%CE%B22-Adrenergic_Receptor>* > * Tutorial:How do we get the oxygen we breathe > *http://proteopedia.org/w/Tutorial:How_do_we_get_the_oxygen_we_breathe > <http://proteopedia.org/w/Tutorial:How_do_we_get_the_oxygen_we_breathe>* > best regards, > Joel > > > Prof. Joel L. Sussman > joel.suss...@weizmann.ac.il *www.weizmann.ac.il/~joel > <http://www.weizmann.ac.il/~joel>* > Dept. of Structural Biology tel: +972 (8) 934 6309 > *www.weizmann.ac.il/ISPC > <http://www.weizmann.ac.il/ISPC>* > Weizmann Institute of Science fax: +972 (8) 934 6312 *www.proteopedia.org > <http://www.weizmann.ac.il/~joel>* > Rehovot 76100 ISRAEL mob: +972 (50) 510 9600 > > - > > On 19Sep, 2016, at 13:53, Morten Grøftehauge < > mortengroftehauge.w...@gmail.com <mortengroftehauge.w...@gmail.com>> > wrote: > > Hi everybody, > > I am teaching a single 45 minute lesson about models in natural science in > a week long module on models and cognition. The students are in a science > high school, age approx. 17. I thought xtallography would be a good example > because it's very model-oriented, there's some stuff about validation and > model precision indicators (e.g. r-values), models that build on other > models (e.g. bond angles and lengths), data sharing vs not sharing etc. > They can open PyMol and see some electron density, and I can automate a lot > with scripts. > > Now I've googled a bit and looked at the teaching resources at RCSB PDB > 101 but I can't seem to find anything that helps with what I want to show > them. The guide to understanding PDB data looks like it has some useful > things but it's very practically oriented (http://pdb101.rcsb.org/learn/ > guide-to-understanding-pdb-data/introduction). What I need to teach is > more meta. > *Does anyone know of any teaching resources that uses x-ray > crystallography models as a basis for talking about scientific models in > general?* > If anyone has any great examples, specific structure-wise then please > mention them. But I may just use some of my own as examples. > > Sincerely, > Morten > > >
Re: [ccp4bb] Your top choice of crystal screens!
Fabrice Gorrec, at Cambridge, made the MORPHEUS screen and I really, really like it http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3246824/ It's easy to mix yourself and if you make extra of the stocks it is also very easy to mix a follow-up grid screen. The conditions are cryoprotected from the outset (which I very much agree with - if your initial hits from other screens freeze with ice rings then you should attempt to optimize towards cryoconditions rather than cryoprotect crystals after they have grown, in my opinion). I disagree with the choice to include calcium + magnesium since I think you should have screened your protein to find out if it binds any metal ions before you start crystallization screening (since it will also influence your optimal purification protocol). If you have stabilizing salts or other factors you can try setting up the same screen multiple times but with different protein buffer. pH is restricted to 6.5, 7.5, and 8.5 but that's where the majority of hits occur anyway. And the grid optimization goes both higher and lower than those max / min values. I've had great success with Morpheus. Morten On 17 October 2014 00:15, xaravich ivan xaravich.i...@gmail.com wrote: Hi everyone, Several of you wanted to know what kind of suggestions I received. Attached you will find all the replies. Thanks On Thu, Oct 16, 2014 at 9:57 AM, xaravich ivan xaravich.i...@gmail.com wrote: Dear Xtallographers, As the budget becomes tighter it is difficult to get hold of all the crystal screens one would want to try, to crystallize a protein. What in your opinion/experience is/are the first few commercial crystal screens you would try? ( or what are the ones you routinely start with and have got more success with?) With a very limited budget and without crystal robot access ( which means larger volumes of screens/ well) I would like to know your personal favorites. Thanks Ivan -- Morten K Grøftehauge, PhD Pohl Group Durham University
Re: [ccp4bb] Thermofluor assay
I've tried to post my reply to this quite a few times but as best I can tell (and my friends can tell from their CCP4bb subscriptions) I have been foiled by jiscmail every time. Hopefully this will go through. And hopefully I'm not reposting stuff I already sent. Here we go: Okay, the consensus here seems to be that you don't have a thermal shift assay up and running with your membrane protein or membrane proteins in general. Like a couple of people mentioned the Alexandrov 2008 is the standard reference for membrane proteins (CPM reacts with a thiol). You have options though: Thiol-reaction activated fluorophore (e.g. CPM) High-throughput static light scattering (Harbinger Stargazer, Avacta Optim) High-throughput intrinsic fluorescence (Avacta Optim) High-throughput fluorescence lifetime (NovaFluor PR Fluorescence Lifetime Plate Reader) Western blotting (CETSA, FASTpp) Environmental rigidity sensitive dyes (e.g. DCVJ) Environmental dielecticity / hydrophobicity sensitive dyes (e.g. SYPRO Orange, bis-ANS) Possibly differential scanning calorimetry (DSC) Thiol-reaction activated fluorophore Cysteine side chains are typically buried in the core of a protein. When the protein is denatured they become solvent accessible. A dye like CPM can then react with it and only the thiol-reacted compound is fluorescent. CPM has maximum excitation/emission of ~385/470 nm which is a slight problem. Most qPCR machines have excitation filters that begin at ~450 nm and emission filters that begin at ~500 nm. I have however seen somewhere in the literature someone excite CPM with about ~405 nm and reading fluorescence above 500 nm. I haven't tried it but I have tried bis-ANS where I excited at 455-485 nm but without getting a useful signal (bis-ANS has the same excitation maximum as CPM). Some qPCR machines can be fitted with excitation filters that start at 350 nm (Stratagene MX3000/3005, Qiagen Rotor Gene, possibly more). More fluorescence plate readers have these wavelengths but then often don't have temperature control or the temperature is capped at 42 or 65 C. 65 is probably fine for membrane proteins but I would want to start with a test protein and I can't think of any that would melt at that temperature. But actually, you could just add Gu-HCl to ß-lactoglobulin - ß-LG melts in the 70-80 degrees interval but with enough guanidinium it should be fine. Without temp. control you could incubate outside the plate reader but you should be quick about it. I've been told not to incubate with CPM since it does degrade. CPM is of course incompatible with ß-mercaptoethanol, DTT, and tris (tris because of the primary amine unless you are at pH where it is well protonated and has no buffering capacity). pH is restricted to ~neutral. http://www.ncbi.nlm.nih.gov/pubmed/18334210 I tried what Artem suggested with having a friendly chemist cook me up one of those Korean / Chinese compounds that work like CPM but fluoresces in the visible spectrum. It didn't work for me but perhaps I didn't try hard enough or it wasn't pure enough or something. There are a lot of them but keep in mind that a lot of them are designed to react with both the thiol and the amine of cysteine. In a protein that amine is usually an amide and therefore no go. Update: this is the one Artem used http://www.ncbi.nlm.nih.gov/pubmed/19343759 There's a paper where they used CPM with membrane protein in LCP. They had to centrifuge it after each heating step though because the LCP became cloudy. High-throughput static light scattering Using a specialised machine to read the aggregation state of many wells in parallel using static light scattering while heating. I know of two machines, Harbinger Stargazer and Avacta Optim, but unless you can find one to borrow this might be a bit over budget. High-throughput intrinsic fluorescence The Avacta Optim also reads intrinsic fluorescence at the same time. I don't know if this adds any useful information not already supplied by the light scattering, I haven't tried it. High-throughput intrinsic fluorescence lifetime The lifetime of tryptophan fluorescence differs between folded and unfolded protein and by measuring the lifetime of UV-excited fluorescence at temperature intervals you can get a measurement of the melting temperature of your protein. NovaFluor PR Fluorescence Lifetime Plate Reader is the only machine for this that I know of. Western blotting This is pretty interesting. As far as I can see CETSA is a simplified version of FASTpp but with broader applicability. FASTpp is Fast Parallel Protealysis. Crude lysate + thermolysin. Heat but take out aliquots at intervals. Thermolysin is specific for bulky hydrophobic residues and since most of them are buried the digestion is greatly accelerated when proteins denature (this is true of all proteases though, regardless of their specificity). The aliquots are run on an SDS gel and Western blotting is performed to determine at what temperature the protein of
[ccp4bb] Coot set-refine-max-residues
Hi ccp4bb, I've been having trouble with Coot. Specifically the scripting to change the maximum number of residues to refine. So if I try to refine more than 20 residues I get this neat little warning message in the terminal: WARNING:: Hit heuristic fencepost! Too many residues to refine FYI: 23 20 (which is your current maximum). Use (set-refine-max-residues 40) to increase limit but when I try set-refine-max-residues 40 or set-refine-max-residues 30 in either the terminal or the scheme scripting command window, it doesn't actually change anything. I've run the Python version of the hydrogen bond restraints script before all this. Hope someone can point me in the right direction. Cheers, Morten -- Morten K Grøftehauge, PhD Pohl Group Durham University
