Experimental methods for 3D structure determination Josef Houser Autumn 2023 S1004 Methods for structural characterization of biomolecules Quick reminder-structure hierarchy Protein DNA Primary Sequence (aminoacids, N-term - C-term) Sequence (nucleotides, 5"- 3'end) Secondary a-helix, (3-sheet turns, loops (rotation along torsion angels MJ and 0) Watson-Crick base pairing (A-X C-G) Tertiary 3D organization of secondary motives A-form, B-form, Z-form Quarternary oligomerization nucleosomes 3D structure = tertiary. cally we also gain also primary, secondary and quarternary (not always) structure in one experiment Methods Nuclear magnetic resonance = NMR Cryo-electron microscopy = Cryo-EM Crystallography (diffraction methods) X-ray neutron electron Methods Nuclear magnetic resonance Cryo-electron microscopy Crystallography = NMR =Cryo-EM (diffraction methods) • High resolution up to individual atoms 1A ~ 0.1 nm ~ length of covalent bond • Results are x, y, z, coordinates of atoms position Require expensive instruments • Nontrivial principles and data analyses Methods NMR Cryo-EM Crystallography sample in solution in solution crystal sample concentration high (mM) low (pM) average (u.M) interact with nuclei electrons depends on radiation type size of molecule small (< 40 kDa) bis (> 100 kDa) both protein complexes dynamics no yes yes no with limits no resolution high (~ 1A) reasonable (2.5A) high (1A) duration of experiment days hours minutes (days for neutron crystallography) high throughput no no yes (X-ray crystallography) NMR https://youtu.be/RZLew6Ff-JE NMR Sample is placed inside a strong magnetic field. Nuclei in the sample are oriented along the magnetic field and start to spin = precession. Important! 1H, 13C, 15N, 19F isotopes needed in the sample NMR When activated by a radio frequency wave, the precession axis deviates and than aligns back. Allows to detect the frequency of precession and the time of return (= relaxation time) Depending on the nucleus surrounding the frequency of precession and relaxation time vary. NMR Ethanol H HC-C-OH i i H H I H 2 /ppm protein mm 11« -Dl MU 95 4 : Ba BII . , il 63 li 53 V3 4» ill U 30 2.» 2.'j 13 13 M C] 0.'. chemical shift > lkh ; ID experiment is sufficient for small organic molecules It is too crowded for biomacromolecules NMR For proteins (DNA) - more complex experiments and their combination needed • Series of activation pulses - signal generated by the same nucleus will corelate • Specific series of pulses enable the transfer of magnetization • To atoms bound with covalent bonds (J-coupling) • Sequence, the side chains orientation • To atoms in close vicinity without covalent bond (NOE = nuclear Overhauser effect) Tertiary structure NMR 10 9 8 7 5(1H)/ ppm Human hemoglobin PDB:2M6Z NMR Basic principle Measures precession of nuclei in strong magnetic field Sample requirements In solution at high concentration (mM) Pros Can detect dynamics of the molecule - sees also moving parts Sample in solution ^natural environment" Cons Only some isotopes are compatible - special sample preparation For smaller proteins (< 40 kDa) https://youtu.be/RZLew6Ff-JE G 9 https://youtu.be/Sn3dNMv-67k https://youtu.be/Enda859ftFQ http://dx.doi.Org/10.1016/j.ab.2016.05.006 CD Cryo-electron microscopy Recently very popular and developing method Nobel price in 2017 Produced in Brno-Thermofisher (Slatina) Tescan (Kohoutovice) Suitable for bigger molecules (> 100 kDa) and complexes Cryo-electron microscopy Electron beam interacts with sample molecules In vacuum Coils with magnetic field serve as lenses The image of the sample is bigger (magnification 1: 5 000 000) and inverted anode condenser lens 1 condenser lens 2 condenser diaphragm amorphous sample or not oriented objective lens objective diaphragm (back focal plane) selected area diaphragm intermediate lens projective lens final image screen Cryo-electron microscopy Sample preparation • Extraordinary sample purity is needed • Loading on the grid • Vitrification (flesh freeze in liquid etane -88 °C) • Needs to be thin - < 500 nm Embedded particles m mm**. 3mm square patch with regular holes in the carbon film carbon -1-3 pm thin super cooled vitreous ice layer DOI: 10.1007/978-l-4939-7033-9_28 Cryo-electron microscopy Data analysis: • Identification of the molecule of interest • Arranging into groups and averaging - 2D classification • Assessment of orientation • Combining to 3D Cryo-electron microscopy Result: • Electron density map in high resolution (around 3A, improving) Cryo-electron microscopy Basic principle Interaction of electrons with atoms of the sample. Sample requirements Low concentration, high purity, vitrification Pros Excelent method for protein complexes, viral particles Cons Smaller proteins (< 100 kDa) do not produce enough signal Demanding data analyses (months) Grant Jensen CALTECH youtube course https://youtu.be/ljTEG-B-kGc https://youtu.be/t4hhdgJADE8 https://doi.org/10.1007/978-l-4939-7033-9_28 https://doi.Org/10.1016/j.abb.2014.ll.011 CD Diffraction methods Diffraction happen, when the wavelenght of the radiation is at the similar range as the object (resolution) Not only diffraction: • Nothing - radiation goes through the sample • Absorbtion -> radiation damage • Inelastic scattering = change of the particle energy -> noise • Elastic scattering = the particle energy is conserved -> diffraction signal Diffraction methods Elastic scattering from single particle also happens, but the signal is very weak and noisy To increase the signal - CRYSTAL ■ Periodic arrangement of protein molecules with identical orientation ■ Difficult to obtain ■ Fragile Diffraction methods Signal is enhanced on crystal by a constructive addition of elastically diffracted waves Waves need to stay in the same phase The extra path equals to integer multiple of A nA = 2dsin9 Bragg's law Diffraction methods • If we want atomic resolution, we need to choose the radiation with wavelength at the same range The radiation can be: photons (X-ray) neutrons electrons particles obey quantum physics Diffraction methods Photons (X-ray) Neutrons Electrons Scattered by Scattering factor of elements Speed Rest mass Energy Wavelength Crystal size electrons dependent on Z c = 299 792 458 m.s" none 7-17 keV 0.07-0.17 nm Medium (pirn) nuclei independent on Z ~ 2600 m.s1 1.675 x 1027kg 0.1meV-0.5 eV 0.01-3 nm Big (mm) both dependent on Z and atomic charge ~ 6 000 000 m.s 1 9.1091 xlO31 kg 100 - 300 keV 2-4 pm Small (nm) X-ray diffraction The oldest and best established diffraction method • Uses synchrotron radiation • Quick data collection (minutes) • High throughput X-ray diffraction X-ray = photons Elastic scattering Diffraction pattern Synchrotron Crystal To prevent radiation damage - measurement at cryogenic temperatures (-196 °C) Oscilation during data collection typically 0.1° X-ray diffraction Diffraction pattern E|ectron densjty mgp X-ray diffraction X-ray diffraction Basic principle Elastic scattering of x-ray photons from electron cloud of the sample arranged in crystal Sample requirements Crystal (medium size, pirn) Typically cryogenic temperatures Pros Quick data collection and analyses, high throughput, automation Cons Crystals are sometimes difficult to obtain H atoms are not visible even at high resolution Costly instrumentation (synchrotron) https://youtu.be/QuCRBxjk3fg https://doi.org/10.3390/molecules25051030 https://doi.org/10.1007/978-l-60327-159-2_3 CD Neutron diffraction The source of neutrons is nuclear reaction Slow data collection (days) Requires huge crystals Can visualise hydrogens Neutron diffraction How to produce free neutrons? 1. Radioactive decay 2. Fission 3. Spallation 4. Fusion Particles are typically protons, targets include Ta, W, U, Hg 235U + nslow -> 2 fission fragments + 2.5 n,^ 180 MeV Sp*IUUon Spallation production: - 60 n.proton J neutron leu'.ron ^ • fission product lj neutron nucleus fission product J neutron Reactor production: - 1 n.decay -i Neutron diffraction Scattering factor independent on Z (proton number of element) HDCNOPS Fe Akwn Nu. o4 ein trum Coherent u flMrfM A »74 V7 N0» I • u » Fe ti I* «♦,*-» I **'4 ♦♦■»' •*•*« «$H .?(» .10 1 O O O ooO X N X-ray do not see H Neutrons can see H Neutron diffraction Scattering factor independent on Z (proton number of element) 1.0 0.8 _ 0.6 oi Ü 0.4 c !a 0.2 u -0.2 - -0.4 □ Neutrons X-rays Ü c 0 01 2 I E - D n c o i— TO U c a o Ol >^ x O o One catch - H has negative scattering lenght CH2 group cancel each other out ^ Sampl e deuteration needed Deuteration also to improve signal noise ratio (H has large incoherent scattering) Neutron diffraction Low energy No radiation damage Measurement at room temperature for very long time (neutron flux is low, so long expositions is needed to have reasonable signal intensity) Neutron diffraction Laue diffraction Compensation for a long exposition time Use neutrons of multiple wavelenghts at the same time Intersection of the detector with more Ewalds spheres on the same picture Allows to used higher angles of oscilation Requires special data processing Neutron diffraction Neutrons Elastic scattering Diffraction pattern Nuclear reactor Crystal Radiation damage is low - measurent at room temperature Oscilation during data collection up to 7° Neutron diffraction Basic principle Elastic scattering of neutrons from nuclei of the sample arranged in crystal Sample requirements Crystal (big, mm) Typically at room temperature Pros Visible hydrogens - exact study of hydrogen bonds, protonation states Cons Huge crystals are needed - even more difficult to obtain Requires deuteration of the sample - expensive Very limited access to radiation sources https://youtu.be/Ep8qWJhS894 https://doi.org/10.1002/9780470015902.a0003045.pub2 CO Electron diffraction The youngest of diffraction methods Performed at electron microscopes Needs only tiny crystals (nm) Incident beams Specimen Objective lens Electron diffraction Why to use electrons? They strongly interacts with matter - elastic scattering represents 25 % of scattered electrons n Needs only sub-micrometer crystals - perfect for systems that do not form bigger crystals (membrane proteins) Electron diffraction Why not to use electrons? They strongly interacts with matter - problem of multiple scattering of 1 electron inside the sample \7 Difficult to take into account in data processing - introduces errors Increases with sample thickness - ideal size 100-200 nm Electron diffraction Electrons Elastic scattering Diffraction Oscilation during data collection usually 0.1° Sample holder allows to rotate only 80° of the crystal Electron diffraction Basic principle Elastic scattering of electrons from the sample arranged in crystal Sample requirements Crystal (tiny, nm) Performed in vacuum Pros Better accesibility of cryo-electron microscopes Microcrystals are easier to produce Cons https://youtu.be/s5lWzflFZB0 Secondary scattering of electrons Still in development ♦V.j//. https://doi.org/10.1107/s2059798320016368 Biomolecular I nteractions and Crystallography Core Facility bic@ceitec.cz bic.ceitec.cz CF Head Josef Houser • +420 549 492 527 josef.houser@ceitec.cz MUNI 0<=^iti=c: