BCM Lecture #3. Phase Improvement, Model Building & Refinement. Peter Pawelek & Mirek Cygler (slides 10-51(mostly))

Transcription

1 BCM Lecture #3 Phase Improvement, Model Building & Refinement Peter Pawelek & Mirek Cygler (slides 10-51(mostly))

2 Phase Improvement

3 Density Modification Methods as mentioned last week, initial phases will have some extent of inherent error a number of methods are commonly used to improve phases prior to the model building and refinement stage (and sometimes during): solvent flattening histogram matching NCS averaging Taylor,G., Acta Cryst. D59 (2003)

4 Solvent Flattening removal of electron density contribution from bulk solvent a mask is built around molecular density and ρ values outside of this mask are set to a flat value corresponding to solvent (typically 0.33 e Å -3 ) you need to have an initial electron density map in which the molecular boundary is clearly defined Taylor,G., Acta Cryst. D59 (2003)

5 Histogram Matching initial electron densities are modified to correspond to an expected distribution initial electron densities are expressed as a histogram on a grid and compared to a histogram corresponding to an ideal electron density map the histogram corresponding to the initial densities is modified to match the expected histogram Taylor,G., Acta Cryst. D59 (2003)

6 Non-Crystallographic Symmetry (NCS) Averaging NCS constraints can be applied (rigid or strict) such that electron densities of NCS-related subunits will be identical or highly similar (if weighting is used) a mask is built around a region of the asymmetric unit NCS operators are applied and density within mask is averaged the more subunits (N) within the asymmetric unit the better; noise is reduced by N 1/2 Taylor,G., Acta Cryst. D59 (2003)

7 Taylor,G., Acta Cryst. D59 (2003)

8 Model Building and Refinement

9 Model Building

10 Electron Density Maps - Electron density maps for model building are usually some combination of: - 2Fo-Fc (weighted towards experimental data) - Fo-Fc (difference map) - 3Fo-2Fc (intermediate between the above two) - Other maps can include: - 2mFo-DFc, mfo-dfc (likelihood-weighted maps: includes weighting for errors in experimental data and model) - omit maps (map around a portion of model in which model bias is removed) - Fo = observed structure factors (experimental data) - Fc = model structure factors (calculated from model phases)

11 Building Into a Difference Map - On the right we see a 2Fo-Fc map in blue and a Fo-Fc difference map superimposed - Differences densities from the Fo-Fc map are shown in green (positive difference) and red (negative difference) - Build into positive difference density and move atoms out of negative difference density

12 Slide courtesy Dr. Mirek Cygler General Protein Chemistry

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14 Helix Directionality Slide courtesy Dr. Mirek Cygler

15 Carbonyl Oxygens Point to C-Terminus Slide courtesy Dr. Mirek Cygler

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25 Model Building: Automated Approaches - Arp/wARP ( - RESOLVE ( Slide courtesy Dr. Mirek Cygler

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28 Model Refinement

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35 A Protein Molecule Divided into Two TLS Groups Macromolecular Crystallography, Volume 374 edited by Charles W. Carter, Robert M. Sweet

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48 The Over-Fitting Problem Slide courtesy Dr. Mirek Cygler

49 Data-To-Parameter Ratio as a Function of Resolution Figure from Biomolecular Crystallography (2010) B. Rupp. (Garland Science)

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52 Figure from Biomolecular Crystallography (2010) B. Rupp. (Garland Science

53 Adding Ordered Solvent Molecules ordered solvent (water) molecules are usually added towards the end of a rebuild/refinement cycle adding waters can signficantly decrease R, but be careful! (watch Rfree) only do this if your resolution is better than ~ 2.5 Å! water electron density should appear football shaped be careful not to build waters into density that may represent another molecule (sometimes this could even be a glycerol molecule from the cryoprotectant solution) Ordered waters observed in 1.2 Å dataset Figure from Biomolecular Crystallography (2010) B. Rupp. (Garland Science

54 Observable Ordered Solvents as a Function of Dataset Resolution Figure from Biomolecular Crystallography (2010) B. Rupp. (Garland Science)

55 Gradually Increase Parameters As You Converge on the Global Minimum

56 Slide courtesy Dr. Mirek Cygler