Workshop in X-ray structure analysis

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1 10th International Symposium and Summer School on Bioanalysis Workshop in X-ray structure analysis Practical parts II & III Ivica Đilović & Dalibor Milić Zagreb, 7 July 010

2 Aims and objectives You will: solvethe crystal (and molecular) structure of a metal complex from the corresponding single-crystal X-ray diffraction data learn how to visualize and interpret crystal structures learn how to retrieve structural information from the databases of crystal structures You will not: learn how to solve structures of biological macromolecules (proteins, nucleic acids, macromolecular complexes) it is more complicated! learn how to solve structures from powder (polycrystalline) diffraction data it is even more complicated!!! become an expert in X-ray structural analysis (just by attending this workshop)

3 Workshop materials Create a new folder in D:\ - it is going to be your working folder Download files X.hkl and X.ins in your working folder 3

4 10th International Symposium and Summer School on Bioanalysis Workshop in X-ray structure analysis Practical part II solving the structure Dalibor Milić Zagreb, 7 July 010

5 Problem What is the crystal structure of chemical compound X? 5

6 Chemical synthesis 1) Copper(II) hydroxide was dissolved in warm 10 % water solution of acetic acid. ) The obtained clear solution was cooled off until bluishgreen crystalline product X appeared. 3) Crystals X were filtered off and dried in air. T Cu(OH) (s) + CH 3 COOH(aq) X(s) Crystals of X 6

$90 mm 3 Oxford Diffraction Xcalibur (Mo-K) = 0.$

7 Single-crystal X-ray data collection Crystal size: mm 3 Oxford Diffraction Xcalibur (Mo-K) = Å Sapphire 3 CCD-detector T = 95 K ω- and φ-scans by 1 steps exposure time:1.5 s per step 7

8 443 diffraction images (frames) 8

9 Crystallographic unit cell Crystallographic unit cell Crystal lattice

10 For crystal X: Basic crystallographic data Crystal system: monoclinic a b c = = 90 o 90 o Unit-cell parameters: a = (6) Å b = (3) Å c = (7) Å = = 90 o = (6) o 10

11 Known unit-cell parameters Miller indices (hkl) can be assigned to each diffraction maximum 11

$Data reduction Integration of diffraction maxima intensities Ω I$ Miller indices Icorr F(hkl ) Corrected intensities I corr Standard

12 Data reduction Integration of diffraction maxima intensities Ω I ov3 int ( hkl) k I LPTE F( hkl) Applying of various corrections Miller indices Icorr F(hkl ) Corrected intensities I corr Standard uncertainty of I corr (hkl) h k l I corr (hkl) [I corr (hkl)]

13 Data reduction For crystal X (have a look at X.hkl): 438 measured diffraction maxima ( reflections ) 164 unique diffraction maxima (because of the crystal symmetry) 13

14 hkl : h + k = n +1 h0l : h, l = n +1 0kl : k = n +1 hk0 : h + k = n +1 0k0 : k = n +1 h00 : h = n +1 00l : l = n +1 Crystal space group Systematic absences for X (not obseved diffraction maxima): n +1 = any odd number space groups with such systematic absences: Cc (no. 9) C/c (no. 15) non-centrosymmetric centrosymmetric 14

15 E-statistics Normalized structure factor: Crystal space group E hkl F j hkl f j T Centrosymmetric random structure: E Non-centrosymmetric random structure: E For X: E Crystal structure of X is most probably centrosymmetric! 15

16 Crystal space group Cc (no. 9) C/c (no. 15) non-centrosymmetric centrosymmetric 16

17 C/c Crystal space group Asymmetric unit the smallest part of the unit cell from which, by application of all symmetry operations of the space group, the whole space is filled For crystallographic analysis it is enough to use just an asymmetric unit an asymmetic unit can be made of: a) just one whole chemical entity (e.g. a molecule) b) more (different or same) chemical entities c) the part of a chemical entity (e.g. one half of a molecule)

18 WinGX a software package for X-ray structure analysis free download from: 1. Start a new project in WinGX (X.ins)!. Check the space group of X in WinGX! 3. Open the INS file and have a look at it! 18

19 Phase problem Reciprocal space F( hkl) N j 1 f j ( hkl)exp[i( hx j ky j lz j )] FT 1 FT Real space 1 ( xyz) F( hkl)exp[ i( hx V h k l ky lz)] Do not forget: F( hkl) F( hkl) expi ( hkl) 1 ( xyz) F( hkl) exp[ i( hx ky lz) i( hkl)] V h k l I F(hkl) PHASE PROBLEM 19

20 Solving the phase problem Patterson method Direct methods Charge flipping... 0

21 SHELX a group of programs for solving and refinement of crystal structures freely available at COMPUTER.cif.ins CRYSTALLOGRAPHER.res Model building 1

$recycling diffraction experiment$ FT calculation phases FT 1

22 Kevin Cowtan's Book of Fourier REAL STRUCTURE Fourier s recycling diffraction experiment STRUCTURAL AMPLITUDES amplitudes FT calculation phases FT 1 calculation MODEL STRUCTURAL AMPLITUDES & PHASES

23 Structure solving Solve the structure of X by direct methods! Vizualize the structure solution by SXGRAPH! Build the atomic model from Q-peaks (maxima in difference electron density maps)! 3

24 Minimization of function: Y is usually F Refinement w( Yo Yc ) Y o is observed value and Y c is calculated value based on the actual model w is weighting parameter which is different for different hkl least-squares method - the most common refinement method in small molecule crystallography atomic coordinates (3 per atom), atomic displacement parameters (1 per atom, if it is isotropic; 6 per atom, if it is anisotropic), global scale factor between measured and calculated intensities sometimes additional restraints are added in the refinement w( Yo Yc ) wr ( rt rc ) Refine the structural model of X by SHELXL! 4

5 isotropic sin 8 exp ) (iso; U j hkl T anisotropic ) aniso; ( hkl T a c b )] 3 13 1

25 5 isotropic sin 8 exp ) (iso; U j hkl T anisotropic ) aniso; ( hkl T a c b )] c klb U c hla U b hka U ( exp[ c l U b k U a h U Atomic displacement parameter

26 INS file atomic coordinates section atom name type x fractional coordinates y z site occupancy factor anisotropic atomic displacement parameters

27 ORTEP Software for visualization of displacement ellipsoids Free download for Windows from: Visualize ADPs for the structural model of X by ORTEP! Grow fragments! crystallographic inversion center (center of symmetry) in the middle of the dinuclear complex 7

28 R Fo F Structure evaluation structure has to be chemically sound!!! some parameters for evaluation they measure fitting of structural model to experimental data o F c F o observed structure factor F c calculated structure factor R-factor is usually lower than 5% for correctly determined structures Weighted R-factor: wr w( Y o Yc ) o wy 1/ Goodnes of fit: S ( w Y N P o Yc ) 1/ It is expected to be S 1 for correct structural models No. of data No. of refined parameters 8

29 Hydrogen atoms X-ray diffraction gives information about electron density Positions of hydrogen atom nuclei do not correspond to maxima in electron density Hydrogen atoms (just 1 e ) are poor X-ray scatterers H atoms are frequently built in ideal calculated positions or treated with restraints during refinement For X use: HFIX 137 generation of idealised methyl group, torsion angle is found by fitting to the electron density DFIX O5 H51 O5 H5 DANG H51 H5 Restraints for a water molecule 9

30 CIF file Crystallographic Information File standard text format for exchange of crystallographic data for generation of CIF file after refinement just write in INS: OMIT (instead of: OMIT ) ACTA Reference That s it! - the first structural report for X: J. N. van Niekerk & F. R. L. Schoening, Acta Crystallogr. 6 (1953) 7 3.

Key crystallographic concepts: Theory of diffraction. (Crystallography y without tears, Part 1)

$Key crystallographic concepts: Theory of diffraction. (Crystallography y without tears, Part 1)$ Protein Crystallography (3) Key crystallographic concepts: Theory of diffraction. (Crystallography y without tears, Part 1) Cele Abad-Zapatero University of Illinois at Chicago Center for Pharmaceutical