Basics of SAXS in structural analysis (of biomolecules) Karel Kubíček Motivation Screen Shot 2012-03-13 at 7.04.56 PM.png Terminology 1)X-ray – diffraction / scattering 2)XS - X-ray scattering 3)SAXS/WAXS - Small/Wide Angle X-ray Scattering 4)SANS - --------”---------- Neutron ---”--- A)Otto Kratky (1902, Vienna-1995, Graz) B)Günter Porod (1919 near Villach, 1984 Graz) C)Dmitri I. Svergun D) I)Scattering II)Scattering curve III)Guinier plot IV)PDF (Pair-distribution function) V) a)Bead model b)Bead model - / SAXS - envelope Experimental setup Screen Shot 2012-03-13 at 7.07.10 PM.png SAXS X-ray SAXS X-ray Crystalized Sample Sample in Solution (very tiny) bit of theory Screen Shot 2012-03-13 at 7.07.10 PM.png q=2k.sinq => q/2/k=sinq, k=2p/l => q=4p/l.sinq Often q is denoted as s k q Screen Shot 2012-03-13 at 7.13.29 PM.png => 1st step: scattering to scattering curve Screen Shot 2012-03-13 at 7.03.23 PM.png What can we learn from the scattering curve: 1)Shape of the studied molecule 2) 2)Fold 3) 3)Secondary structure 4) Screen Shot 2012-03-13 at 7.02.39 PM.png Is it really that easy? 2nd step: Guinier plot & Kratky plot (from the initial region of the scattering curve) Screen Shot 2012-03-13 at 7.13.29 PM.png Screen Shot 2014-03-25 at 08.52.46.png 2nd step: Guinier plot & Kratky plot (from the initial region of the scattering curve) Screen Shot 2012-03-13 at 7.02.39 PM.png 3rd step: PDF Pair-distribution function Screen Shot 2012-03-13 at 7.09.19 PM.png Screen Shot 2012-03-13 at 7.02.39 PM.png Screen Shot 2012-03-13 at 7.09.19 PM.png All information from the scattering curve together Screen Shot 2012-03-13 at 7.25.50 PM.png Bead model Spherical harmonics problem to be solved Screen Shot 2012-03-13 at 7.10.21 PM.png Solutions with similar “goodness” of fit may be obtained Screen Shot 2014-03-25 at 08.54.53.png Screen Shot 2012-03-13 at 7.08.49 PM.png Summary Screen Shot 2012-03-13 at 6.59.33 PM.png Summary (graphically) SAXS Hamburg Screen Shot 2012-03-13 at 6.41.34 PM.png IMG_0084.JPG IMG_0085.JPG IMG_0089.JPG IMG_0091.JPG IMG_0092.JPG IMG_0088.JPG IMG_0095.jpg IMG_0096.JPG IMG_0097.JPG IMG_0094.JPG IMG_0093.JPG IMG_0098.jpg IMG_0099.JPG Screen Shot 2012-02-15 at 4.19.01 PM.png SAXSvsNMR_Cid.png Evaluation of the experimental data Screen Shot 2012-03-14 at 2.23.47 PM.png References: 1)Koch, M.H.J., Vachette, P., Svergun, D.I. Quart Rev Biophys 2003 2)Jacques, D.A., Trewhella, J. Prot Sci 2010 3)Svergun, D.I., Petoukhov, M.V., Koch, M.H.J. Biophys J 2001 4)Wriggers, W. Biophys Rev 2010 5)Putnam, C.D., Hammel, M., Hura, G.L., Tainer, J.A. Quart Rev Biophys 2007 6)Madl, T. Gabel, F., Sattler, M. J Struct Biol 2010 Protein folding explained https://deepmind.com/blog/article/alphafold-a-solution-to-a-50-year-old-grand-challenge-in-biology AlphaFold: a solution to a 50-year-old grand challenge in biology em_spectrum.jpg NMR Confidence in structural features of proteins determined by X-ray crystallography (estimates are very rough and strongly depend on the quality of the data) Structural feature Resolution 5 Å 3 Å 2.5 Å 2 Å 1.5 Å Chain tracing - Fair Good Good Good Secondary structure Helices fair Fair Good Good Good Sidechain conformations - - Fair Good Good Orientation of peptide planes - - Fair Good Good Protein hydrogen atoms visible - - - - Good Screen Shot 2011-11-04 at 8.26.38 PM.png XAS = X-ray Absorption Spectroscopy XANES = X-ray Absorption Near Edge Structure EXAFS = Extended X-Ray Absorption Fine Structure SAXS/EXAFS Screen Shot 2011-11-04 at 8.30.51 PM.png Screen Shot 2011-11-04 at 8.27.57 PM.png Screen Shot 2011-11-04 at 8.27.00 PM.png Screen Shot 2011-11-04 at 8.26.38 PM.png EXAFS (Extended X-ray Absorption Fine Spectroscopy) poskytuje informace o nejbližších slupkách atomů sousedících s absorbujícím atomem Fe N N N N His 108 Met 75 Met 86 Met 110 Cu(I) b4 b5’ H:\Personal\Thesis\Pictures\XAS.jpg X-Ray Absorption Spectroscopy Cu(I)DR1885 DE=-10.3 eV Ligand r(Å) 2s2.103(Å2) R-exafs e(fit index) Fit1 (1shell) 2S 2.299 4(1) 0.446 0.49 Fit2 (1shell) 3S 2.301 9(1) 0.403 0.41 Fit3 (2shells) 3S 2.300 8(1) 0.334 0.29 1N§ 1.982 4(1) Fit4 (2shells) 3S 2.303 8(1) 0.305 0.27 1N* 1.999 7(2) § no MS *His, MS A BLAST search over all non-redundant GenBank genomes M H M H M 75 86 108 110 C:\WINDOWS\Desktop\holo.tiff D:\Simone\Eudora\attach\apo_Fri04.jpg Conserved Met&His Other Met&His residues His 108 Met 73 Met 75 Met 88 Met 86 His 79 Met 83 Met 112 N C N C b1 b2 b3 b4 b5 b6 b7 b8 b9 Met 110 cyan – b-sheets grey – random coil Holo Apo Rentgenstrukturní analýza Krystalová mřížka působí na rentgenové záření jako optická mřížka na viditelné světlo. Nastávají ohybové jevy a na stínítku se objevuje difrakční obrazec. Tyto obrazce mohou být matematicky analyzovány, aby se získala informace o rozložení elektronů v molekulách tvořících krystal. Bragg‘s law 2d sinQ = nl 30/11/2018 Karel Kubíček 40 Bragg‘s law 2d sinQ = nl 30/11/2018 Karel Kubíček 41 Screen Shot 2011-11-02 at 10.10.21 AM.png Trojklonná Jednoklonná Kosočtverečná (ortorombická) Šesterečná Klencová Čtverečná Krychlová Krystalografická soustava Screen Shot 2011-11-02 at 10.11.11 AM.png Screen Shot 2011-11-02 at 10.10.43 AM.png Shape, rectangle Description automatically generated https://www.intechopen.com/books/x-ray-scattering/x-ray-diffraction-in-biology-how-can-we-see-dna-a nd-proteins-in-three-dimensions- A picture containing chart Description automatically generated Diagram Description automatically generated Diagram Description automatically generated https://doi.org/10.3390/cryst8110434 http://skuld.bmsc.washington.edu/~merritt/bc530/local_copies/phase_methods_files/vd_xtals.jpg Diagram Description automatically generated Diagram Description automatically generated Phase diagram of solubility of a protein in solutions as a function of the concentration of the precipitant. Screen Shot 2011-11-02 at 10.08.49 AM.png Screen Shot 2011-11-02 at 10.08.49 AM.png Screen Shot 2011-11-02 at 10.09.08 AM.png X-Ray Goniometer a device that permits the simultaneous recording of the direction of X rays diffracted by a specimen under study and of the position of the specimen at the time of diffraction. An X-ray goniometer can be an independent device recording the diffraction pattern on photographic film; in this case it is an X-ray camera. The term “X-ray goniometer” is also applied to goniometric devices that are components of X-ray diffractometers and are used for mounting the specimen and detector in positions corresponding to the conditions necessary for the occurrence of X-ray diffraction. https://encyclopedia2.thefreedictionary.com/X-Ray+Goniometer Screen Shot 2011-11-02 at 10.19.23 AM.png https://www.intechopen.com/books/x-ray-scattering/x-ray-diffraction-in-biology-how-can-we-see-dna-a nd-proteins-in-three-dimensions- Diagram Description automatically generated Map Description automatically generated Krystalogram B-DNA získaný v r. 1952 Rosalindou E. Franklinovou, na jehož základě Watson a Crick navrhli dvoušroubovicový model struktury DNA. C. & W. dostali v r.1962 společně s Mauricem Hugh Frederick Wilkinsem NC za fyziologii a medicínu „za jejich objevy týkající se molekulární struktury nukleových kyselin a jejich významu při přenosu informací v živých organizmech“ crick xrayhelix franklin watson F W C http://www.udel.edu/chem/fox/MassSpecLecture09.pdf Screen Shot 2015-04-14 at 11.18.55.png http://www.udel.edu/chem/fox/MassSpecLecture09.pdf Screen Shot 2015-04-14 at 11.19.46.png http://www.udel.edu/chem/fox/MassSpecLecture09.pdf Screen Shot 2015-04-14 at 11.18.35.png http://www.udel.edu/chem/fox/MassSpecLecture09.pdf Screen Shot 2015-04-14 at 11.20.13.png http://www.udel.edu/chem/fox/MassSpecLecture09.pdf Screen Shot 2015-04-14 at 11.20.34.png http://www.udel.edu/chem/fox/MassSpecLecture09.pdf Screen Shot 2015-04-14 at 11.20.57.png http://www.udel.edu/chem/fox/MassSpecLecture09.pdf Screen Shot 2015-04-14 at 11.23.12.png http://www.udel.edu/chem/fox/MassSpecLecture09.pdf Screen Shot 2015-04-14 at 11.23.23.png Screen Shot 2015-04-14 at 11.27.03.png Screen Shot 2015-04-14 at 11.28.19.png Screen Shot 2015-04-14 at 11.30.27.png Screen Shot 2015-04-14 at 11.44.34.png 2D_electrophoresis.png Screen Shot 2015-04-14 at 12.42.12.png metox.1 Amino Acid Residue Composition Residue Monoisotopic Mass Delta Mass Methionine C5H9NOS 131.0405 0 Methionine Sulfoxide C5H9NO2S 147.0354 15.9949 Methionine Sulfone C5H9NO3S 163.0303 31.9898 Sulfur S 31.9721 - Y:\Presentations\071123-PhD_Def\DR1885_digerite.png Y:\Presentations\071123-PhD_Def\PepMapMALDI.png Mass Spectrometry ESI - electron spray ionization – high voltage applied to a liquid jet producing highly charged droplets MALDI - matrix-assisted laser desorption/ionization - three step process: 1) sample mix with matrix and deposition on a metal plate, 2) laser pulse desorbs the sample with matrix, 3) analyte molecules are ionized and analyzed (TOF – time of flight MS technique) Paul_Patent_2939952_Fig5.gif Quadrupole mass analyzer Screen Shot 2015-04-27 at 22.48.22.png Screen Shot 2015-04-27 at 22.39.13.png HDX MS - Hydrogen Deuterium Exchange with Mass Spec HDX-MS_Nature2013_GroeL_Fig2.jpg ARDD - average relative D-uptake difference Scientific Reports 3, Article number: 1247, 2013 Screen Shot 2015-05-12 at 14.08.56.png http://www.biopharminternational.com/performing-hydrogendeuterium-exchange-mass-spectrometry ITC – isothermal titration calorimetry -1760s – Black measured the heat capacity and latent heat of water -1780s – Lavoisier designed an ice calorimeter and used this instrument to measure the metabolic heat produced by a guinea pig confined in the measurement chamber => Calorimeter was one of the earliest scientific instruments & first calorimetric experiment was a biologically relevant measurement Screen Shot 2013-05-09 at 11.51.35 AM.png Why ITC? 1)ITC is a quantitative technique 2)can determine: I.binding affinity (Ka), II.enthalpy changes (ΔH), III.binding stoichiometry (n) of the interaction between two or more molecules in solution. IV. From these initial measurements: 3)Gibbs energy changes (ΔG) 4)entropy changes (ΔS) 5) 5) ΔG = -RTlnKa = ΔH-TΔS Screen Shot 2013-05-09 at 2.38.12 PM.png Screen Shot 2013-05-09 at 2.38.22 PM.png Screen Shot 2013-05-09 at 9.48.43 AM.png Screen Shot 2013-05-09 at 9.49.02 AM.png Screen Shot 2013-05-09 at 9.49.25 AM.png Screen Shot 2013-05-09 at 9.49.35 AM.png Screen Shot 2013-05-09 at 9.53.37 AM.png Screen Shot 2013-05-09 at 11.52.37 AM.png Three possibilities of calorimetric measurement: 1)temperature change (either adiabatic or isoperibol) [°C/time] 2)power compensation (often called isothermal) [mcal/time] 3)heat conduction (Lavoisier design) 4) Screen Shot 2013-05-09 at 9.45.58 AM.png Screen Shot 2013-05-09 at 9.46.12 AM.png Screen Shot 2013-05-09 at 9.46.30 AM.png Screen Shot 2013-05-09 at 9.46.43 AM.png Screen Shot 2013-05-09 at 9.47.05 AM.png Screen Shot 2013-05-09 at 9.47.18 AM.png Screen Shot 2013-05-09 at 9.47.52 AM.png Screen Shot 2013-05-09 at 9.48.09 AM.png Screen Shot 2013-05-09 at 10.54.53 AM.png Screen Shot 2013-05-09 at 9.48.09 AM.png Screen Shot 2013-05-09 at 9.50.39 AM.png Screen Shot 2013-05-09 at 9.50.03 AM.png Screen Shot 2013-05-09 at 2.38.47 PM.png The experimental binding isotherm can be characterized by the unitless value c