Advanced biochemistry and its methods Lecture 5 Lukáš Zídek lzidek@chemi.muni.cz Žídek: Strukturní biochemie (skripta k přednášce C9530), kapitoly 11-14 Lukáš Žídek C6215 1/69 COMPUTATIONAL: De novo structure calculation not reliable • Homology modeling using a similar known structure as a starting model o Prediction based on Machine Learning using sequence alignment and database including known structures (AlphaFold) using a similar known structure as a starting model • Structure determination based on experiment usually MD simulation with experimental restraints Methods of protein st mination EXPERIMENTAL: • Mass Spectrometry mostly analysis of cross-linked fragments a Scanning surface e.g. Atomic Force Microscopy (various modes) Interactions of electromagnetic (other) waves with molecules: • Spectroscopy how molecules change characteristics of the wave (intensity, phase, polarization, frequency) • diffraction methods (Microscopy) how molecules change direction of wave in space Lukáš Žídek C6215 3/69 A Photodiode Lukáš Žídek C6215 4/69 Scanning probe microscopy EEW508 KAI ST Imaging mode in atomic force microscopy Contact mode Feedback: lever deflection the feedback system adjusts the height of the cantilever base to keep this deflection constant as the tip moves over the surface (friction force microscopy, conductive probe AFM) Non-contact mode Feedback: oscillation amplitude the cantilever oscillates close to the sample surface, but without making contact with the surface. Electrostatic / magnetic force microscopy Intermittent contact Feedback: oscillation amplitude The cantilever oscillates andthetip makes repulsive contact with the surface of the sample at the lowest pointof the oscillation (Tapping modeAFM) Forco modulation mode Feedback: lever deflection the tip does not leave the surface at all during the oscillation cycle, (interfacial force microscopy) Lukáš Žídek C6215 5/69 image reconstruction "localization AFM" cf. super-resolution microscopy Localization AFM X-ray structure Lukáš Zídek C6215 7/69 Spectroscopy • UV/VIS spectrophotometry absorption, transition of electrons to higher orbitals concentration, content of aromatic amino acids, heme, prosthetic groups • IR spectroscopy absorption, transition of nuclei to higher vibration states Lukáš Zídek C6215 8/69 <□► 4 ^ > 4 = t 4 = > = ^0 q, o Lukáš Zídek C6215 9/69 <□► 4 ^ > 4 = t 4 = > = ^0 q, o Lukáš Zídek C6215 11/69 <□► 4 ^ > 4 = t 4 = > = ^0 q,o Lukáš Zídek C6215 12/69 • CD spectroscopy absorption differences of polarized light by chiral molecules y structure overall content of secondary structures Random coil a-helix /3-sheet 170 180 190 200 210 220 230 240 250 A / nm □ &\ Lukáš Zídek C6215 14/69 • NMR spectroscopy precession of magnetic moments of nuclei in magnetic field magnetic moments are slightly aligned in a static magnet axis of alignment is tilted by electromagnetic (radio) waves aligned magnetic moments precess about the static field resulting oscillating magnetic field is measured we do not observe the applied electromagnetic waves interactions of magnetic moments (mutual, with electrons) resolution, structural information atomic resolution structure determination, dynamics, interactions • molecule magnetic moment S 10"97 rads-1T"1 % e~ 1/2 -182.000 100 1H 1/2 0.277 99.98 13C 1/2 0.067 1.1 14N 1 0.019 99.6 15N 1/2 -0.027 0.4 170 5/2 -0.036 0.04 19p 1/2 0.252 100 31 p 1/2 0.108 100 129Xe 1/2 -0.075 24.4 quadrupolar (relax fast) rare isotopes (enrichment) in equilibrium (spherical symmetry) Lukáš Žídek C6215 17/69 jl-B Boltzmann distribution: P(9) oc e kzT = e^7" Precession (angular momentum in afield): ú u - njIb_ - V3kBT -7B Lukáš Zídek C6215 18/69 x reproduced from M. H. Levitt: Spin Dynamics Lukáš Žídek C6215 21/69 nd electrons Lukáš Žídek C6215 22/69 NOESY spectra Nuclear Overhauser effect: result of dipole-dipole interactions Peak intensities are proportional to nuclear Overhauser effect 9.0 0.5H 1.0H E 1-5H Q. Q. 2.(H 2.5^ 3.0H 8 5 O Ji Ô 8.0 7.5 7.0 6.5 0 o O o o 9.0 8.5 0 o ň 8 0 o Q h0.5 M .o H .5 h2.0 h2.5 h3.0 8.0 7.5 C02 - 1H (ppm) 7.0 6.5 □ ť3? - = Lukáš Zídek C6215 23/69 user effect Nuclear Overhauser effect proportional to 1 /d6 {d = distance) Lukáš Zídek C6215 24/69 lation Model of the protein built from known distances lation Model of the protein built from known distances lation Model of the protein built from known distances lation Model of the protein built from known distances <□► 4 ^ > 4 = t 4 = > = ^0 q, o Lukáš Zídek C6215 29/69 <□► 4 ^ > 4 = t 4 = > = ^0 q, o Lukáš Zídek C6215 30/69 Lukáš Zídek C6215 31/69 <□► 4 ^ > 4 = t 4 = > = ^0 q, o Lukáš Zídek C6215 32/69 Lukáš Žídek C6215 33/69 Lukáš Žídek C6215 34/69 stals Lukáš Žídek C6215 38/69 Lukáš Žídek C6215 39/69 Lukáš Žídek C6215 40/69 Lukáš Žídek C6215 41/69 Lukáš Žídek C6215 42/69 Lukáš Žídek C6215 44/69 Lukáš Žídek C6215 45/69 Real space cat Circular rainbow scale of phases i i i Fourier transform Linear intensity scale of amplitude size Fourier amplitudes and phases F(hkl) = V J J j p (x y z) exp[2m (/zx + ky + h)] d x dy d z x=0y=0 z=0 Observed amplitudes Lukáš Zídek C6215 46/69 Electron density equation & PHASE PROBLEM Lukáš Žídek C6215 47/69 • Direct interpretation of amplitudes mutual positions of atoms calculated from amplitudes for simple molecules (Patterson function/map) a Using heavy atoms • Molecular replacement Diffraction back-calculated from a known structure similar to the studied proteins Orientation and position of the molecule in the crystal obtained by searching for the match of diffraction patterns (measured vs. back-calculated) Calculated phases used for the unknown molecule Lukáš Žídek C6215 48/69 o o o o o o Lukáš Žídek C6215 49/69 Known structure *1 Manx cat Fourier transforn, try different orientations Observed amplitudes Phases unknown! Fourier cat Calculated amplitudes and phases FT of Manx cat Wrong orientation! Lukáš Zídek C6215 50/69 Observed amplitudes (tailed cat), calculated pha es (Manx cat) Inverted Fourier transform Even the tail becomes visible! Lukáš Zídek C6215 51/69 Model building & resolution 3.OÄ 4.0Ä Lukáš Žídek C6215 52/69 Model building & refinement Lukáš Žídek C6215 53/69 R-factor, Rf factor Film X-ray beam X-ray tube Diffracted x-rays Crystal Observed reflections Model Calculated diffraction pattern R-factor R = hkl \f^\ hkl Rfree faCt0r ^ 11 foto I -*|fcalcll fifree = hklcT [fobs hklcT Lukáš Zídek C6215 54/69 <□► 4 ^ > 4 = t 4 = > = ^0 q,o Lukáš Zídek C6215 55/69 Lukáš Žídek C6215 56/69 Lukáš Žídek C6215 57/69 Lukáš Žídek C6215 58/69 5» 2« • 2 •• •• I ••• i m • •••• • »• • ! • Lukáš Zídek C6215 < □ ► 4 ^ > 4 = t 4 = > = ^0 q, o 59/69 <□► 4 ^ > 4 = t 4 = > = ^0 q, o Lukáš Žídek C6215 60/69 Lukáš Žídek C6215 63/69 AXS) crystal diffraction: l(q) = Jv Jv Ap(r)Ap(r + Ar)e^r6rd(r + Ar) SAXS: l(q) = /fvJv Ap{r)Ap{r + Ar)e^AFdrd(r + Ar) = 4tt / ŕ o Ap(T)Ap(r + AF)dAr ) ^^dr 0r,4>r P(r)=r*Vp^0(r) Guinier law: l(q) « l(0)e~R^q2/3 for q 0 0: scattering angle g: scattering vector q = |q| = 47rsin(6>/2)/A: momentum transfer /(g): scattering intensity in direction given by q 70: probability of finding a point at r from a given point RQ\ radius of gyration Svergun and Koch, Rep. Prog. Phys. 66 (2003) 1735, stacks.iop.org/RoPP/66/1735 Lukáš Zídek C6215 66/69 iAXS) I tesolulion (rm-2 00 1.00 0.67 u£0 oas -!-1-1-1-1— I-1-1-1— s (nm-1) from Wikipedia Lukáš Žídek C6215 67/69 Lukáš Žídek C6215 68/69 iAXS) A ■E 03 Q IE 04 o 1C-06H B j.OE-06 2.0E-CG 1 OF-Ofi H O.GE+03 000 0 05 D 10 O'S 0?0 C 75 0 30 0 35 04C qd/A) í: CM 4 Ľ - -Od D" Q" 2 C £-05 0.0Ľ+00 <= Í.13 2E-oa 03 v—J 11=—U 5 □.00 D.OE 0.10 0.15 G .20 C.2E 0.30 3 C.4C Exp. Homo(. Mode 10 70 30 10 S3 GO 70 80 ľ(A) o ft o o o ■J* 0.300 P ULXJ3 qd/A] q4 Evangelista, D., New Biotechnology 2017, DOI: 10.1016/j.nbt.2017.10.001 □ i5P - = 5 ^) o Lukáš Zídek C6215 69/69 <□► 4 ^ > 4 = t 4 = > = ^0 q, o Lukáš Žídek C6215 70/69 Lukáš Žídek C6215 72/69 • a Perfect lens in focus provide contrast in intensity of scattered vs. original wave if the scattered wave is absorbed by the sample o b Perfect lens in focus provide no contrast in intensity of scattered vs. original wave if the scattered wave is not absorbed by the sample, but only phase-shifted by 90° • c Imperfect and defocused lens provide contrast in intensity of scattered vs. original wave if the scattered wave is absorbed by the sample • d Defocus and spherical aberration of lens provide contrast in intensity of scattered vs. original wave if the scattered wave is not absorbed by the sample, but only phase-shifted by 90°. Defocus and spherical aberration introduce another phase shift scattered have oposite phase and cancel each other. Waves Lukáš Žídek C6215 74/69 Photons scatter as waves =4> limited resolution resolution < 0.61 X/n (A = wavelegth, n = refractive index =4> A « 0.1 nm (distances of atoms in molecules), X-rays Lens are not available for X-rays (A ^ 0.1 nm) no material has sufficient refractive index SOLUTIONS: • Analysis of diffraction patterns intensity enhanced if molecules are aligned in crystals =4> X-ray crystallography applicable also to electron and neutron waves • Microscopy with electrically charged waves electrons scatter as waves electron beams are bent in electromagnetic field =4> Electron microscopy Lukáš Žídek C6215 75/69 Transmission Electron Microscope Vacuum! Electron source: Thermal emission from heated cathode Focussing: Electro-magnetic Lenses Detection: Phosphor screen or CCD camera (former times: negative) Lukáš Žídek C6215 76/69 Proteins in vitrous ice, can reach atomic resolution Lukáš Žídek C6215 77/69 Amplitude vs. phase objects Macromolecules in water / vitreous ice are phase objects im«».»!. Wavelength Refractive Amplitude * index(RI) = n Undisturbed Wave ♦ rn mmw'n, Light\ WW c Rl = n Amplitude Specimen [ Rl> n Phase men • Amnliti [ A Ä ■ Rl>n Amplitude