doc. Mgr. Karel Kubíček, Ph.D., Ústav fyziky kondenzovaných látek, CEITEC MU, e: karelk77@gmail.com 20563@mail.muni.cz t: +420 549 49 3253 17/10/2023 Karel Kubíček 1 em_spectrum.jpg NMR 17/10/2023 Karel Kubíček 2 NMR 1)Jaderný spin ¹ 0 (1H, 13C, 15N, 31P) - počet neutronů a počet protonů jsou sudá čísla (12C=6p+6n) Þ nulový spin - počet neutronů plus počet protonů je liché číslo (1H=p, 13C=6p+7n) Þ neceločíselný spin (i.e. ½, 3/2, 5/2) - počet neutronů a počet protonů jsou lichá čísla (2H=p+n) Þ celočíselný spin (i.e. 1, 2, 3) 1) n=g*B (1) – pokud vložíme do magnetického pole intezity B, jádro mající nenulový spin může absorbovat foton frekvence n. Frekvence n závisí na gyromagnetickém poměru g jader 2)Z kvantové mechaniky víme, že spin I může nabývat 2I +1 orientací Þ jádro se spinem ½ může mít dvě orientace v externím magnetickém poli– nižší / vyšší energie N S N S N S E=h n (2) 17/10/2023 Karel Kubíček 3 Electron Proton Neutron Atom = In the planetary model of the atom, an electron orbits a nucleus, forming a closed-current loop and producing a magnetic field with a north pole and a south pole. Molecule is hence a group of small magnetic fields and each atom within the molecule experiences different local magnetic field. A picture containing game Description automatically generated 17/10/2023 Karel Kubíček 4 earth-magfield.jpg magnetic field = 0 magnetic field > 0 For NMR, nuclear spin is needed!!! Spin analogy to a compass needle 17/10/2023 Karel Kubíček 5 Nuclear Magnetic Resonance Stručně Z (1) a (2): E=h g B N S N S 17/10/2023 Karel Kubíček 6 N S N S 17/10/2023 Karel Kubíček 7 Magnet •supravodivé solenoidy na bázi •Nb a Sn ponořené do heliové a dusíkové láznĕ •He-lázeň ~4 K dále snížena J-T •pumpou na ~2.1 K •v současnosti až 22 Tesla •magnetické pole země ~50mT - y:\Presentations\080430_UFKL_NMRSeminar\26542.jpg y:\Presentations\080430_UFKL_NMRSeminar\wirestrain.gif y:\Presentations\080430_UFKL_NMRSeminar\n4a02f3.jpg (Nb, Ta)3Sn supravodič o šírce 0.81 mm s 271 vlákny vnořenými do OFHC mĕdĕné matrice 17/10/2023 Karel Kubíček 8 y:\Presentations\080430_UFKL_NMRSeminar\fig6.jpg y:\Presentations\080430_UFKL_NMRSeminar\c-mag-4.gif Díra cca 55mm He-plnění N2-plnění 17/10/2023 Karel Kubíček 9 17/10/2023 Karel Kubíček 10 17/10/2023 Karel Kubíček 11 y:\Presentations\080430_UFKL_NMRSeminar\6914430-0-large.jpg y:\Presentations\080430_UFKL_NMRSeminar\probe.jpeg y:\Presentations\080430_UFKL_NMRSeminar\probe4.jpeg y:\Presentations\080430_UFKL_NMRSeminar\probe5.jpeg NMR měřicí sonda y:\Presentations\080430_UFKL_NMRSeminar\LRC.jpg 17/10/2023 Karel Kubíček 12 Spektrometr CBU Control board unit FGU Frequency gen. u. Shimms Temperature Unit AcquisitionControler Transmitter 17/10/2023 Karel Kubíček 13 Size Relaxation FID NMR line(width) after FT slow (i.e. long t2 time) medium fast A close up of a glove Description automatically generated A picture containing ball, game Description automatically generated A yellow ball Description automatically generated A close up of a logo Description automatically generated Time Time Hz Hz Hz 17/10/2023 Karel Kubíček 14 Size Relaxation FID NMR line(width) after FT slow (i.e. long t2 time) medium fast A close up of a logo Description automatically generated A close up of a logo Description automatically generated Time Time Hz Hz Hz e.g. Cholesterol Biomolecules 5-30 kDa Large molecules 50+ kDa 17/10/2023 Karel Kubíček 15 NMR as a tool for study structure, dynamics and interactions of biomolecules 1)Structure determination of NAs and proteins 2)Protein – metal interaction 3)Protein – ligand interaction For most of the modern applications, enrichment by 13C, 15N and often 2H needed! 1) Isotope Ground state spin Natural abundance [%] Rel. Sensitivity 1H ½ ~100__ 1.00x10+0 13C ½ 1.10 1.59x10-2 15N ½ 0.37 1.04x10-3 19F ½ 100__ 8.30x10-1 31P ½ ~100__ 6.63x10-2 12C 0 98.90 - 16O 0 ~100__ - 17/10/2023 Karel Kubíček 16 http://www.fbreagents.com/basics_nmr/9proteins.htm Structure calculation Iterative procedure of structure determination by NMR N C Nrd1 CID PDB ID: 2LO6 Uncertainty of the final structure represented as a family of 10-20 structures with deviation among individual members indicated by RMSD (typically <1.5 Å2) Final structure 17/10/2023 Karel Kubíček 17 17/10/2023 Karel Kubíček 18 Diagram Description automatically generated Optical microscope Electron microscope 17/10/2023 Karel Kubíček 19 Diagram, engineering drawing Description automatically generated FEG - Field Emission Gun Diagram Description automatically generated SEM – skenovací EM, TEM – transmisní/prozařovací EM 17/10/2023 Karel Kubíček 20 17/10/2023 Karel Kubíček 21 Diagram Description automatically generated Focused Ion Beam (FIB) + SEM 17/10/2023 Karel Kubíček 22 Villa et al. Current Opinion in Structural Biology, 23(5), 2013, 771-7 Diagram Description automatically generated 17/10/2023 Karel Kubíček 23 Villa et al. Current Opinion in Structural Biology, 23(5), 2013, 771-7 Diagram Description automatically generated Diagram Description automatically generated 17/10/2023 Karel Kubíček 24 A picture containing text, outdoor Description automatically generated 17/10/2023 Karel Kubíček 25 Villa et al. Current Opinion in Structural Biology, 23(5), 2013, 771-7 Graphical user interface Description automatically generated with low confidence 17/10/2023 Karel Kubíček 26 A picture containing graphical user interface Description automatically generated TEM – Sample preparation 17/10/2023 Karel Kubíček 27 A picture containing text Description automatically generated e.g. uranyl acetate Diagram Description automatically generated Sample vitrification 17/10/2023 28 Karel Kubíček 17/10/2023 Karel Kubíček 29 A picture containing key Description automatically generated ThermoFisherScientific Grid 17/10/2023 Karel Kubíček 30 Text Description automatically generated A picture containing indoor, person, hand, disk brake Description automatically generated A picture containing indoor Description automatically generated 17/10/2023 Karel Kubíček 31 A picture containing text, electronics Description automatically generated cryoem101.org Autogrid: grid + C-shape spring (C-clip) + clip ring Auto-loader: up to 12 autogrids 17/10/2023 Karel Kubíček 32 A picture containing indoor, tableware Description automatically generated ThermoFisherScientific; MiTeGen 17/10/2023 Karel Kubíček 33 A picture containing text Description automatically generated A picture containing text Description automatically generated Note the ice thickness Grids get damaged 17/10/2023 Karel Kubíček 34 A picture containing text Description automatically generated A picture containing text Description automatically generated single particles 17/10/2023 Karel Kubíček 35 https://doi.org/10.1016/j.cell.2019.04.006 Diagram Description automatically generated A picture containing several Description automatically generated Structure determination using single particle cryoEM 1) Sample preparation – 2) vitrification – 3) measurement – 4) particle picking – 5) 2D classification – 6) 3D model reconstruction 17/10/2023 Karel Kubíček 36 A collage of images of human brain Description automatically generated 17/10/2023 Karel Kubíček 37 A collage of images of a surface Description automatically generated 17/10/2023 Karel Kubíček 38 A collage of images of lines and lines Description automatically generated bioRxiv preprint doi: https://doi.org/10.1101/2023.09.13.557623 A collage of images of different types of cells Description automatically generated A collage of images of a dna molecule Description automatically generated A diagram of a dna structure Description automatically generated with medium confidence Yang et al.,Science375, 167–172 (2022) Cryo-EM structures of amyloid-b 42 filaments from human brains 17/10/2023 Karel Kubíček 39 17/10/2023 Karel Kubíček 40 Text Description automatically generated 17/10/2023 Karel Kubíček 41 Graphical user interface, application, website Description automatically generated https://time.com/collection/best-inventions-2022/6229912/deepmind-alphafold/ 17/10/2023 Karel Kubíček 42 Graphical user interface Description automatically generated 43 A screenshot of a cellphone Description automatically generated with low confidence 17/10/2023 Karel Kubíček 17/10/2023 Karel Kubíček 44 Text Description automatically generated 17/10/2023 Karel Kubíček 45 A picture containing text, indoor, items, cluttered Description automatically generated 17/10/2023 Karel Kubíček 46 A map of a city Description automatically generated with low confidence 17/10/2023 Karel Kubíček 47 A picture containing chart Description automatically generated 17/10/2023 48 A picture containing text Description automatically generated Shape Description automatically generated Karel Kubíček