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Protein Structure Determination

Protein Structure Determination. Part 2 -- X-ray Crystallography. The method. FT -1. FT. Crystals. X-rays. Atoms. EM versus x-ray. electron microcope resolution ≈ 1nm de Broglie wavelength of e- ≈ size of atom transmitted light lensing possible, 10 6 x mag. 2D image w/tilt

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Protein Structure Determination

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  1. Protein Structure Determination Part 2 -- X-ray Crystallography

  2. The method FT-1 FT Crystals X-rays Atoms

  3. EM versus x-ray • electron microcope • resolution ≈ 1nm • de Broglie wavelength of e- ≈ size of atom • transmitted light • lensing possible, 106x mag. • 2D image w/tilt • measures density. • sample is thin section • diffractometer • resolution up to 0.1nm = 1Å • wavelength ≈ size of atom • scattered light • no lens possible • 3D reconstruction • measures relative e-density • sample is single crystal

  4. X-ray diffractometer

  5. Experimental setup X-ray detector X-ray source beam stop

  6. Dimensions X-ray detector X-ray beam Beam width: ~0.20 mm Crystal thickness: 0.10-1.00 mm

  7. X-ray detector Unit cell: ~100Å = 0.00001mm

  8. Typical protein molecule: ~30Å = 0.000003 mm

  9. N CH3 Cα O C Dimensions C-C bond distance: 1.52Å Wavelength of Cu Kα X-rays: 1.5418Å

  10. N CH3 Cα O C Dimensions Angle of incidence=θ: 0-90° Bragg plane separation distance (resolution): 0.7-50Å

  11. C C N Dimensions Carbon atom amount an electron moves in one xray cycle X-rays see e- as if they were standing still.

  12. Electromagnetic spectrum Wavelength of X-rays used in crystallography: 1Å - 3Å (Å = 10-10m) most commonly 1.54Å (Cu ) Frequency = c/λ =(3x108m/s)/(1.54x10-10m) ≈ 2x1018 s-1

  13. oscillating e- scatter X-rays …in all direction. oscillation e- emission

  14. Reflection planes • The “amplitude” of scattering is measured. • The amplitude is proportional to the differences of e- density in the direction of “reflection planes” • The orientation and separation of reflection planes is determined by the directions of the incoming and scattered rays.

  15. 10K+ reflections • Moving the X-rays and the detector gives a new set of planes. • Changing the angle of reflection changes the spacing (resolution).

  16. Reconstruction of e- density The density at every point in the crystal is calculated by summing over all of the density waves.

  17. Topics covering in this course • Crystal growth • Diffraction theory • Symmetry • Experimental methods • Interpretation of data • Software

  18. Equations you will need to know Euler's theorem Bragg's law Reciprocol space Symmetry Fourier transform Inverse Fourier transform

  19. How to know that you know • all terms defined • physical/geometric interpretation

  20. “Physics for Scientists and Engineers” by Paul A. Tipler Supplementary reading Matrix algebra “An Introduction to Matrices, Sets and Groups for Science Students” by G. Stephenson ($7.95) Wave physics Protein structure “Introduction to Protein Structure”-- by Carl-Ivar Branden and John Tooze “Introduction to Protein Architecture : The Structural Biology of Proteins” -- by Arthur M. Lesk

  21. Materials Gale Rhodes “Crystallography Made Crystal Clear” 3rd Ed. Academic Press graph paper straight edge protractor compass calculator w/trig functions http://www.bioinfo.rpi.edu/bystrc/courses/bcbp4870/bcbp4870.html

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