5. A typical crystallography experiment Pure protein Grow crystal Characterize crystals Collect diffraction data Solve phase problem Calculate electron density map Build/rebuild model Refine model Analyze structure
13. Remove cover slip and fish out crystal with a small nylon loop Mount loop on goniostat in a stream of nitrogen gas Surface tension of the liquid in the loop holds crystal in place Mounting crystals
19. Waves & the phase problem The amplitudes of the diffracted X-rays can be experimentally measured, but the phases cannot = phase problem . i.e. we don’t know the phase of each diffracted ray relative to the others! X ? A Z X Y
26. Molecular Replacement By determining the correct orientation and position of a molecule in the unit cell using a previously solved structure as a ‘search model’. This model can then be used to calculate phases
31. Resolution 6 Å : Outline of the model, feature such as helices can be identified. 3Å : Can trace polypeptide chain using sequence data, establish folding topology. Assign side chains. 2Å : Accurately establish mainchain conformation, assign sidechains without sequence data, I.d water molecules. 1.5Å : Individual atoms are almost resolved, detailed discription of water structure. 1.2Å : Hydrogen atoms may become visible.
35. Deviation of bond lengths & angles from ideal. All based on the geometry of small molecules. Rms deviation for bond lengths should be less than 0.02 Å and less than 4º for bond angles Determined using a Ramachandran plot . Rms deviation of bond length & bond angle
50. Fluorescence ENERGY S 0 S 1 S 2 T 2 T 1 ABS FL I.C. ABS - Absorbance S 0.1.2 - Singlet Electronic Energy Levels FL - Fluorescence T 1,2 - Corresponding Triplet States I.C.- Nonradiative Internal Conversion IsC - Intersystem Crossing PH - Phosphorescence IsC IsC PH [Vibrational sublevels] Jablonski Diagram Vibrational energy levels Rotational energy levels Electronic energy levels Singlet States Triplet States
54. Corrected excitation spectra (corrected for source output and monochromator throughput) can be obtained by using a reference channel equipped with a "quantum counter". This is a concentrated dye solution (typically 3 mg/mL rhodamine B in ethylene glycol). A tiny fraction of the excitation beam is diverted to the reference detector. The quantum counter absorbs all of this light, and converts it (with 100% efficiency to fluorescence), the intensity of which is independent of wavelength between 220 and 580 nm. Any changes in lamp output or monochromator throughput will cause corresponding alterations in the output of the reference channel. By dividing the fluorescence signal by the reference signal, these wavelength-dependent variations are cancelled out. Unfortunately, the quantum counter will not entirely correct the emission spectrum. However, instrument manufacturers supply correction factors for their monochromators. Application of these will give an approximately correct spectrum. If more accuracy is needed, the spectrum of a known standard compound (fluorescing in the region of interest) can be compared to published standards. j. Biological fluorophores 1) Intrinsic fluorophores a) Proteins Tryptophan dominates protein fluorescence spectra - high molar absorptivity - moderate quantum yield - ability to quench tyrosine and phenylalanine emission by energy transfer. Free tyrosine has a relatively high fluorescent output, but is strongly quenched by trptophan in native proteins. Unless tyrosine and tryptophan are absent, emission from phenylalanine is not observed in protein fluorescent spectra.
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57. Effect of Ca 2+ on Intrinsic Trp-fluorescence and on Fluorescence Anisotropy ▼ Wild type • Dome loop mutant Blue shift and intensity enhancement upon addition of Ca 2+ Change in anisotropy upon titration in the wild type, but not in the mutant
