Protein Structure Determination

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Presentation transcript:

Protein Structure Determination Part 1 -- X-ray Crystallography

The method FT-1 FT Crystals X-rays Atoms

microscope versus diffractometer wavelength ≈ size of cell transmitted light lenses 2D image measures relative absorbance image composed of pixels sample is thin section wavelength ≈ size of atom scattered light no lenses possible 0D images, 3D construction measures relative e-density composed of structure factors (waves) sample is thick crystal

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

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

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

Typical protein molecule: ~30Å = 0.000003 mm

Dimensions C-C bond distance: 1.52Å N O CH3 C C Wavelength of Cu K X-rays: 1.5418Å

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

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

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

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

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.

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

X-ray diffractometer

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

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

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

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

Supplementary reading Matrix algebra “An Introduction to Matrices, Sets and Groups for Science Students” by G. Stephenson ($7.95) Wave physics “Physics for Scientists and Engineers” by Paul A. Tipler 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

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