2Why X-rays? Optics: Limit of resolution ~ l/2 Distance between atoms: 0.1 – 0.3 nmWavelenght of X-rays: 0.1 – 10 nmAllows structure determinationat atomic resolutionProblem:No lenses available for X-rays(as for visible light in microscope)Remember:Wavelength, Amplitude, Phase
3Overview of steps Growing a Crystal Collecting X-ray diffraction patternSolving of Phase ProblemCalculate Electron Density MapConstructing a Structural ModelRefining the Structural Model
4Growth of Crystals - Background Why? Diffraction pattern of single molecule too weak for detection=> sum of several diffraction patterns of identically orientedmolecules with identical conformationPorcine ElastaseSupersaturated Solution: Concentration higher than intrinsic solubility S0
5Growth of Crystals - Approaches Hanging Drop Method:Empirical Approach:Test hundreds of conditionsParameters:pHsalt type & concentrationprecipitant(ammonium sulfate, polyethylene glycol, 2-methyl-2,4-dimethyl-pentane diol (MPD), propanol)temperature (2 – 30°C)protein concentration(1 – 100 mg/ml)additional substances(substrates, inhibitors, cofactors etc.)Alternative: Sitting DropMicrodialysis
6Description of Crystals bγUnit cell:characterized by edge length and anglescan contain several moleculesis repeated multiple times along the axis of the crystalAssymetric Unit: minimal part of the unit cell which is related to other parts by defined symmetry operations
7Space Groups Crystal can be described by shape of unit cells (called lattice type, see figure) and symmetry type of asymmetric unit65 different space groups possible for natural biomoleculesExample:P212121
8Diffraction and Interference Huygen’s principle of diffraction:Each point in front of a wavefront acts as a point of progapation for a new wavelet.b) Constructive interference of scattered waves=> Amplitude = 2Ec) Destructive interference of scattered waves=> Amplitude = 0 (wave 180° out of phase)
9Bragg’s Law Requirement for constructive interference: 2 d sin θ = n l n: integer values (0, 1, 2, ...)If angle θ and wavelenght l are known, the distance d between planes can be determined.Simplification: reflection at planes (one dimension: distance d of planes)!
10Diffraction in 3D: Miller Indices Van Laue Conditions replace Bragg’s Law(too much to explain in Bchm 4000)Instead of “n”, three Miller Indices (h, k, l) define the integer number of wavelenght that result in an observed reflection froma 3D crystal.Example: 2 points in space1D reflection2D reflection
11More Diffraction Patterns 2D example:Each diffraction spot corresponds to one set of Miller indices (h, k, l).Friedel’s law:I(h,k,l) = I(-h,-k,-l)
12Diffraction Photograph Each spot contains information from all atoms!“Pattern of spots” (diffraction pattern) allows determination of crystal parameters (size of unit cell, symmetry within unit cell).Structural information has to de derived (see below) from difference in spot intensities.To obtain enough information, diffraction photographs are taken at various different angles of the crystal in the X-ray beam.X-ray diffraction photographof Sperm Whale Myoglobin crystal
13The Structure Factor: F(S) How to get to an electron density map from the intensities of reflections?Intrinsic amplitude of scattered X-ray from each type of atom depends on its electron density r(xyz) (described by atomic scattering factor f)f is used to describe scattering vector for each atom for one reflection (with Miller indices h,k,l)Sum of atomic scattering vectors is molecular scattering factor F, also called structure factor (can be calculated for a given structure):F(hkl) = ∫r(xyz) e-2pi(hx+ky+lz) Fourier Transformation
14The Phase ProblemContinued: How to get to an electron density map from the intensities of reflections?Fourier Transformation (other way around)r(xyz) = ∫F(hkl) e2pi(hx+ky+lz)Magnitude of F is proportional to square root of intensity:|F(hkl)| ~ √ I(hkl)Thus: r(xyz) = 1/NV ∑ ∑ ∑ √I(hkl) e-i(hkl) e2pi(hx+ky+lz)h k lElectron DensityIntensityPhaseMissing Information to get electron density!
15Molecular Replacement Method to obtain Phase informationOnly possible if structure of a very similar molecule is known (native vs. Mutant, close homolog), at least 25% sequence identityCalculate structure factors Fcalc for known moleculeCompare with data set to estimate phase for known structure (acalc)Use this Phase information together with observed structure factors (Fobs) to calculate first electron density mapRefine Structure (see below)
16Multiple Isomorphous Replacement 2. Method to obtain Phase informationUse crystals containing heavy atoms at specific positions (isomorphous crystals) as well as crystal of native proteinHeavy atoms strongly diffractDifference data set of heavy atom derivative and native crystals gives structure factors of heavy atoms only = limited data setCan be used to determine position and phase of heavy atoms (as for small molecule crystals, Patterson function)Needs to be done for at least 2 heavy atom derivatives to obtain enough information to estimate phase of native data set and to solve the structure
17Multiple Anomalous Dispersion 3. Method to obtain Phase informationUse one crystal containing heavy atoms at specific positions as well as crystal of native proteinHeavy atoms not only diffract X-rays, but also absorbs X-rays of certain wavelengthUsing synchroton X-ray source, diffraction data are generated at different wavelenghts of the heavy-atom crystalDifferent data sets can be used to obtain phase information similar to multiple isomorphous replacement
18Electron Density MapComputed electron density map can be represented by “chicken wire” on computer.Knowing the primary sequence of a protein (or nucleic acid) the atoms can be placed by hand into the electron density map.Rough, inaccurate model of protein structureWhat about hydrogen atoms?
19Structural Refinement Iterative Process:model is subtly changedStructure Factors are calculated (Fcalc) and compared by Rcrys factor with measured Fobs to see how well a model fits the data:differences are minimized (minimal R)good structure: Rcrys < 20% (0.2)Problem:R factor can be artifically lowered by adding more solventbetter Rfree: as Rcrys, but calculated with 10% reflections which have not been used for refinement (unbiased data set)
20Resolution Limited by: Quality of crystal (Conformational inhomogenities, Mobility of individual atoms)Number of unique reflections used for structure determination (at least four times more than atoms required)Typically not by wavelength5Å - a helices roughly visible3Å - peptide chain visible, side chains if sequence known2Å - conformations of side chains visible
21Temperature Factor (B factor) Describes local imprecision in electron densityCalculated during structure refinementMeasured in Å²The higher the B factor, the more imprecise the atom position.Good protein structure: B < Å²Reasons for higher B factor:Thermal motion of atomDifferent conformations of side chain, i.e. lower occupancy at specific positionProtein disordered, e.g. In surface exposed loops
22Other Important Parameters Protein backbone should have only allowed conformations (as described in Ramachandran plot)Completenes of structure (should be >90%)
23Parameters in Publication Rasmussen et al., Science 2007
24Protein Data Basestructure data (x, y, z coordinates of each atom) are submitted to Protein Data Basefreely accessible, structure can be downloaded and visualized with free programs such as RasMol or Swiss PDB Viewer
25Native Conformation?Is protein structure determined from crystal the same asin vivo solution structure?Most likely:Crystals contain 40 – 60 % solvent, i.e. the protein is in simialr environment as in cellOften proteins crystallize in different space groups with different crystal contacts between proteins, but the determined structure is the same, i.e. It is independent of crystal packing.Often X-ray structures and solution NMR structures superimpose well.Many enzymes are active in crystalline stateBUT: You never know! Exceptions are possible!