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Using X-ray structures for bioinformatics Robbie P. Joosten Netherlands Cancer Institute Autumnschool 2013.

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Presentation on theme: "Using X-ray structures for bioinformatics Robbie P. Joosten Netherlands Cancer Institute Autumnschool 2013."— Presentation transcript:

1 Using X-ray structures for bioinformatics Robbie P. Joosten Netherlands Cancer Institute Autumnschool 2013

2 Structures in bioinformatics Understand biology – Direct interpretation – Data mining – Homology modeling Drug design Molecular dynamics Basic rule: Better structures → Better results Introduction

3 Right structure(s) for the job 1.Selection: find (a number of) PDB entries 2.Validation: check the quality of your selection 3.Optimisation: maximise the quality of your selection Focus on X-ray structures Introduction

4 X-ray structures have a history 1.Protein expression 2.Crystallisation 3.X-ray diffraction experiment 4.Model building and refinement 5.Deposition at the PDB All these steps affect the final PDB file Selection

5 Protein expression A ‘construct’ is made Partial proteins – E.g. only extracellular domain of membrane protein Frankenstein proteins – Fusion proteins or chimeras Mutants are introduced – Some by accident! Poly-histidine tags added for purification Altered glycosylation state – Large sugars hamper crystallisation History

6 Crystallisation The protein stacks regularly to form a crystal Protein still functional in the crystal Much solvent in the crystal (~40%) Some residues can move – Disorder: missing loops/side chains – Alternate conformation History

7 Crystallisation Beware of crystal packing One copy of the protein can influence the next History

8 Crystallisation Chemicals are used for crystallisation Buffers to stabilise the pH Precipitants – Change solubility of the protein – Neutralise local charges – Bind water – High concentrations are used Compounds compete with natural ligands Examples: – Polyethylene glycol (PEG) – Ammonium sulphate History

9 Crystallisation Beware of the crystallisation conditions History

10 Crystallisation Beware of the crystallisation conditions History

11 X-ray diffraction Typical experiment History X-ray source Detector

12 X-ray diffraction X-rays interact with electrons – Atoms with few electrons (H, Li) do not diffract well X-rays cause damage to the protein – Acidic groups (ASP en GLU) can be destroyed – Disulphide bridges are broken – Hydrogens are stripped – Cooling crystals in liquid nitrogen helps Glycerol added to the crystal! History

13 X-ray diffraction We are not using a microscope We don’t measure everything we need History X-ray diffraction gives an indirect and incomplete measurement Measured Missing: phase

14 Model building and refinement Iterative process History Phases + calculated X-ray data Electron density maps Structure model Measured X-ray diffraction data Initial phases FT Model building

15 History Two types of maps 1.Regular electron density map (2mFo-DFc) 2.Difference map (mFo-DFc) Model building and refinement

16 Fitting atoms to the ED map and trying to remove difference density peaks History Model building and refinement

17 Requires skill and experience Requires time and patience Requires good software Lack of any of these can be seen in the final PDB file History Model building and refinement

18 Both coordinates and experimental X-ray data are deposited PDB standardises files and adds annotation Sometimes things go wrong History Deposition at the PDB

19 LINKs between alternate conformations History Deposition at the PDB

20 History Deposition at the PDB Un-biological LINKs (in 1a1a) LINK C ACE C 100 N PTH C 101 LINK C PTH C 101 N GLU C 102 LINK CF PTH C 101 OG SER A 188 LINK N DIP C 103 C GLU C 102 LINK C ACE D 100 N PTH D 101 LINK C PTH D 101 N GLU D 102 LINK N DIP D 103 C GLU D 102

21 Think of what happened to the structure before you downloaded it

22 Use the experimental data Resolution says very little about the structure (free) R-factor gives the overall fit of the structure to the experimental data For biological interpretation more detail is needed Use the maps Validation X-ray specific validation

23 Which is the better structure of berenil bound to DNA? Validation X-ray specific validation PDB idResolutionR 268d2.00.160 1d632.00.183

24 Validation X-ray specific validation The real-space R-factor (RSR) A per-residue score of how well the atoms fit the map Works like the R-factor (lower is better)

25 Maps can help distinguish the good and bad bits of a structure Validation X-ray specific validation

26 Poorly fitted side-chains Evil peptides Validation Things you can find in maps

27 The wrong drug Validation Things you can find in maps

28 Sequence error K -> R Accidental mutant Also a missing sulfate Validation Things you can find in maps

29 Missing water Missing alternate conformation Validation Things you can find in maps

30 Visualisation in Coot – http://www2.mrc- Get maps and real-space R values from the Electron Density Server – – Direct interface with Coot Get maps and updated models from PDB_REDO Practical session Validation Checking maps

31 Maps show things you cannot see otherwise

32 Solved by a diverse group of scientists – People make errors & gain experience Since 1976 – Structures are not updated Solved with the methods of their era – Methods improve over time Structures in the PDB do not represent the best we can do NOW Optimisation Structures in the PDB

33 Take structure + experimental data Use latest X-ray crystallography methods – Decision making: use case-specific methods – Create new methods when needed Improve model quality – Fit with experimental data – Geometric quality Fix errors PDB_REDO Optimisation Improve structures in PDB

34 Step 1: prepare data Clean-up structure and X-ray data Data mining Step 2: establish baseline Fit with experimental data (R-factors) Geometric quality – Validation with WHAT_CHECK Optimisation PDB_REDO method

35 Step 3: re-refine structure (with Refmac) Improve fit with experimental data – Use restraints to improve geometric quality Improve description of protein dynamics – Concerted movement of groups of atoms (TLS) – Anisotropic movement of individual atoms Optimisation PDB_REDO method

36 Step 4: rebuild structure Delete nonsense waters Flip peptide planes Rebuild side-chains – Add missing ones – Optimise H-bonding Step 5: validate structure Geometry Density map fit Ligand interactions Optimisation PDB_REDO method

37 – > 72,000 structures (98%) – Detailed methods & reprints Directly in molecular graphics software – YASARA – CCP4mg – Coot (needs plugin) – PyMOL (needs plugin) Linked via PDBe & RCSB Availability PDB_REDO databank

38 Improved fit with the data Better geometry Optimisation Does it work? (12,000 structures)

39 MolProbity validation (1eoi) PDBPDB_REDO Optimisation

40 Electrostatics calculations ‘Missing’ positive lysine atoms distort electrostatics calculations Adding missing atoms correctly describes C-terminus interaction with side chains

41 Wrong peptide plane in peptide ligand Fixed by PDB_REDO Better understanding of H-bonds in the interaction Optimisation Protein-ligand interaction

42 Optimisation Protein-protein interaction Packing interface with poor ionic interactions Rebuilt interface properly describes ionic dimerisation interactions

43 Optimised structures give a better view of the biology of the protein

44 PDB_REDOers Amsterdam: R Joosten K Joosten A Perrakis Key contributors: Eleanor Dodson, Ian Tickle, Paul Emsley, Ethan Merritt, Elmar Krieger, Thomas Lütteke, Rachel Kramer Green, Sanchayita Sen Nijmegen: T te Beek M Hekkelman G Vriend Cambridge: G Murshudov F Long

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