A Brief Introduction to Structural Biology and Protein Crystallography

Size: px
Start display at page:

Download "A Brief Introduction to Structural Biology and Protein Crystallography"

Transcription

1 A Brief Introduction to Structural Biology and Protein Crystallography

2 structural biology of H2O

3 Protein polymers fold up into well-defined structures 80 Å active sites ligand binding site

4 Protein Crystallography Pipeline cloning, protein expression and purification crystallization collect X-ray diffraction computer modeling and complemenary experiments extract functional insights 3-D structure phasing, modeling, refinement

5 Protein Crystals need lots of highly purified protein (10-50 mg) crystallization induced by evaporation of solvent crystals grow in days to weeks size: mm (bigger usually better) protein crystals are very fragile - 50 % by volume water frozen in liquid N2 prior to data collection

6 Typical experimental setup for X-ray diffraction From Crystallography 101, www-structure.llnl.gov/xray/101index.html

7 X-ray Diffraction Data diffraction is explained by Bragg s law diffraction spots are typically recorded as crystal is rotated atomic structure is encoded in diffraction pattern (Fourier transform) typical wavelength: 0.8 Å Å 5 x-ray diffraction pattern 6 State-of-the-art X-ray diffraction system in Schlundt Hall

8 Synchrotron X-Radiation Sources National Synchrotron Light Source Brookhaven National Lab, New York Advanced Light Source Lawrence Berkeley National Lab,California

9 Floor Plan of The Advanced Light Source

10 Advanced Light Source Beamline 4.2.2

11 Advanced Light Source Beamline 4.2.2

12 Advanced Photon Source Beamline 19ID

13 Typical Setup -170 C N2 X-rays goniometer

14 Advantages of Synchrotron X-Radiation Sources high brilliance results in higher resolution data in shorter time (1 hour vs. few days) tunable wavelength (0.8 Å Å) better signal/noise ratio

15 X-ray Crystallography Provides 3-D Structures of Proteins Protein crystals Atomic model of protein X-ray diffraction pattern More Computational analysis Computational analysis Electron density map

16 Interpret Electron Density Map Interpretation is a Pattern Recognition Problem

17

18

19 Phe Ala Ala Thr Asp Ser Ala Phe Phe GAFTAADSF Gly

20 the nitty gritty...the relationship between electron density (what we want) and the diffraction pattern (what we measure)

21 X-ray scatter off electrons (Rayleigh scattering )

22 spherical waves interfere constructively and destructively in phase: constructive out of phase: destructive

23 Bragg s Law the condition for constructive interference is given by Bragg s Law: n λ = 2dsinθ n = integer, order of diffraction λ = wavelength of X-rays θ = scattering angle

24 Consequences of Bragg s Law

25 Consequences of Bragg s Law Diffracted rays form a pattern of dots. The dots are called reflections. We think of this pattern as a three dimensional lattice with indices (h,k,l). The third dimension is revealed by rotating the crystal. This is the reciprocal lattice. We measure the intensity of each reflection, I (h,k,l). The value of I varies from reflection to reflection. to get the electron density, we use the square root of the intensity, which is called the structure factor amplitude, and given the symbol, F(h,k,l).

26 representation of F as a vector in the complex plane

27 from structure factors to electron density electron density at position (x,y,z) is related to the sum of all measured structure factors (nonlocal transformation) V is the volume of unit cell (known) x,y,z,h,k,l, are all known (independent variables) F(h,k,l) is a complex quantity. We directly measure the amplitude, F(h,k,l), but additional analysis is needed to the phase of each reflection α (h,k,l). This is the phase problem.

28 Crystallographic Resolution Resolution is a measure of the resolvability in the electron density map of a molecule. In X-ray crystallography, resolution is the highest angle resolvable peak in the diffraction pattern. resolution = (λ / sinθ)/2 high θ, high resolution low θ, low resolution

29 Effect of resolution on quality of electron density Resolution is a measure of the resolvability in the electron density map of a molecule. In X-ray crystallography, resolution is the highest angle resolvable peak in the diffraction pattern. resolution = (λ / sinθ)/2

30

31 from structure factors to electron density electron density at position (x,y,z) is related to the sum of all measured structure factors (nonlocal transformation) V is the volume of unit cell (known) x,y,z,h,k,l, are all known (independent variables) F(h,k,l) is a complex quantity. We directly measure the amplitude of F(h,k,l) but additional analysis is needed to get the phase

32