Jürgen Sühnel jsuehnel@fli-leibniz.de

1. 3D Structures of Biological Macromolecules Part 4: Protein Structure Prediction Jürgen Sühnel jsuehnel@fli-leibniz.de Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena Centre for Bioinformatics Jena / Germany Supplementary Material: http://www.fli-leibniz.de/www_bioc/3D/

2. Protein Structure D. W. Mount: Bioinformatics, Cold Spring Harbor Laboratory Press, 2001.

3. Protein Structure Sequence Protein-protein or protein-nucleic acid complexes Secondary structure elements Domains/Folds Chains

4. Protein Structure Prediction http://speedy.embl-heidelberg.de/gtsp/flowchart2.html

5. A Good Protein Structure • Minimizes disallowed torsion angles • Maximizes number of hydrogen bonds • Minimizes interstitial cavities or spaces • Minimizes number of “bad” contacts • Minimizes number of buried charges

6. Protein Structure Prediction – CAFASP Contest http://www.cs.bgu.ac.il/~dfischer/CAFASP3/

7. Protein Structure Prediction – CASP Contest http://predictioncenter.llnl.gov/

8. Secondary structure • 3D structure • Modeling by homology (Comparative modeling) • Fold recognition (Threading) • Ab initio prediction • Rule-based approaches • Lattice models • Simulating the time dependence of folding • Refinement • Exploring the effect of single amino acid substitutions • Ligand effects on protein structure and dynamics (induced fit) Protein Structure Prediction

9. Lysozyme

10. Lysozyme – 5lyz

11. Lysozyme – 5lyz: Information from the JenaLib Atlas Page

12. Lysozyme – 5lyz: Information from the JenaLib Atlas Page

13. Lysozyme – 5lyz: Information from the JenaLib Atlas Page

14. Lysozyme – 5lyz: Information from the JenaLib Atlas Page

15. Lysozyme – 5lyz: PROSITE Signature

16. Lysozyme – 5lyz: PROSITE Signature

17. PROMOTIF Secondary Structure Analysis – 5lyz . .

18. Protein Backbone Torsion Angles D. W. Mount: Bioinformatics, Cold Spring Harbor Laboratory Press, 2001.

19. PROMOTIF Secondary Structure Analysis – 5lyz

20. PROMOTIF Secondary Structure Analysis – 5lyz

21. PROMOTIF Secondary Structure Analysis – 5lyz

22. Chou-Fasman Secondary Structure Prediction

23. Amino Acid Propensities From a database of experimental 3D structures, calculate the propensity for a given amino acid to adopt a certain type of secondary structure • Example: N(Ala)=2,000; N(tot)=20,000; N(Ala, helix)=500;N(helix)=4,000. P(Ala,helix) = [N(Ala,helix)/N(helix)] / [N(Ala)/N(tot)] P(Ala,helix) = [500/4,000]/[2,000/20,000] = 1.25 Used in Chou-Fasman algorithm

24. Chou-Fasman Secondary Structure Prediction • Assign all of the residues in the peptide the appropriate set of parameters. • Scan through the peptide and identify regions where 4 out of 6 contiguous residues have P(a-helix) > 100. • That region is declared an alpha-helix. Extend the helix in both directions until a set of four contiguous • residues that have an average P(a-helix) < 100 is reached. That is declared the end of the helix. • If the segment defined by this procedure is longer than 5 residues and the average • P(a-helix) > P(b-sheet) for that segment, the segment can be assigned as a helix. • Repeat this procedure to locate all of the helical regions in the sequence. • Scan through the peptide and identify a region where 3 out of 5 of the residues have a value of • P(b-sheet) > 100. That region is declared as a beta-sheet. Extend the sheet in both directions • until a set of four contiguous residues that have an average P(b-sheet) < 100 is reached. • That is declared the end of the beta-sheet. Any segment of the region located by this procedure • is assigned as a beta-sheet if the average P(b-sheet) > 105 and the average P(b-sheet) > P(a-helix) • for that region. • Any region containing overlapping alpha-helical and beta-sheet assignments are taken to be helical if the • average P(a-helix) > P(b-sheet) for that region. It is a beta sheet if the average • P(b-sheet) > P(a-helix) for that region. • To identify a bend at residue number j, calculate the following value • p(t) = f(j)f(j+1)f(j+2)f(j+3) • where the f(j+1) value for the j+1 residue is used, the f(j+2) value for the j+2 residue is used and • the f(j+3) value for the j+3 residue is used. If: (1) p(t) > 0.000075; (2) the average value for • P(turn) > 1.00 in the tetrapeptide; and (3) the averages for the tetrapeptide obey the inequality • P(a-helix) < P(turn) > P(b-sheet), then a beta-turn is predicted at that location.

25. Lysozyme – 5lyz: Chou-Fasman Secondary Structure Prediction http://fasta.bioch.virginia.edu/fasta_www/chofas.htm

26. Lysozyme – 5lyz: Chou-Fasman Secondary Structure Prediction GRCE (0.57|0.98|0.70|1.39) 0.91 RCEL (0.98|0.70|1.39|1.41)1.12 CELA (0.70|1.39|1.41|1.42)1.23 ELAA (1.39|1.41|1.42|1.42) 1.41 http://fasta.bioch.virginia.edu/fasta_www/chofas.htm

27. Lysozyme – 5lyz: PhD/PROF Structure Prediction PROF_sec: PROF predicted secondary structure: H=helix, E=extended (sheet), blank=other (loop) PROF = PROF: Profile network prediction Heidelberg Rel_sec reliability index for PROF_sec prediction (0=low to 9=high) SUB_sec subset of the PROFsec prediction, for all residues with an expected average accuracy > 82% (tables in header) NOTE: for this subset the following symbols are used: L: is loop (for which above ' ' is used) .: means that no prediction is made for this residue, as the reliability is: Rel < 5 O3_acc observed relative solvent accessibility (acc) in 3 states: b = 0-9%, i = 9-36%, e = 36-100%. P3_acc PROF predicted relative solvent accessibility (acc) in 3 states: b = 0-9%, i = 9-36%, e = 36-100%. Rel_acc reliability index for PROFacc prediction (0=low to 9=high) SUB_acc subset of the PROFacc prediction, for all residues with an expected average correlation > 0.69 (tables in header) NOTE: for this subset the following symbols are used: I: is intermediate (for which above ' ' is used) .: means that no prediction is made for this residue, as the reliability is: Rel < 4 http://cubic.bioc.columbia.edu/predictprotein/submit_def.html#top

28. Lysozyme – 5lyz: PhD/PROF Structure Prediction, BLAST http://cubic.bioc.columbia.edu/predictprotein/submit_def.html#top

29. Lysozyme – 5lyz: PhD/PROF Structure Prediction, BLAST http://cubic.bioc.columbia.edu/predictprotein/submit_def.html#top

30. Lysozyme – 5lyz: PhD/PROF Structure Prediction • Perform BLAST search to find local alignments • Remove alignments that are “too close” • Perform multiple alignments of sequences • Construct a profile (PSSM) of amino-acid frequencies at each residue • Use this profile as input to the neural network • A second network performs “smoothing” • The third level computes jury decision of several different instantiations of the first two levels. http://cubic.bioc.columbia.edu/predictprotein/submit_def.html#top

31. Lysozyme – 5lyz: PsiPred Structure Prediction http://bioinf.cs.ucl.ac.uk/psipred/psiform.html

32. PsiPred PSIPRED is a  simple and reliable secondary structure prediction method, incorporating two feed-forward neural networks which perform an analysis on output obtained from PSI-BLAST (Position Specific Iterated - BLAST). Version 2.0 of PSIPRED includes a new algorithm which averages the output from up to 4 separate neural networks in the prediction process to further increase prediction accuracy. Using a very stringent cross validation method to evaluate the method's performance, PSIPRED 2.0 is capable of achieving an average Q3 score of nearly 78%. Predictions produced by PSIPRED were also submitted to the CASP4 server and assessed during the CASP4 meeting, which took place in December 2000 at Asilomar. PSIPRED 2.0 achieved an average Q3 score of 80.6% across all 40 submitted target domains with no obvious sequence similarity to structures present in PDB, which placed PSIPRED in first place out of 20 evaluated methods (an earlier version of PSIPRED was also ranked first in CASP3 held in 1998). http://bioinf.cs.ucl.ac.uk/psipred/psiform.html

33. PSI-BLAST Position specific iterative BLAST (PSI-BLAST) refers to a feature of BLAST 2.0 in which a profile (or position specific scoring matrix, PSSM) is constructed (automatically) from a multiple alignment of the highest scoring hits in an initial BLAST search. The PSSM is generated by calculating position-specific scores for each position in the alignment. Highly conserved positions receive high scores and weakly conserved positions receive scores near zero. The profile is used to perform a second (etc.) BLAST search and the results of each "iteration" are used to refine the profile. This iterative searching strategy results in increased sensitivity.

34. Comparing Secondary Structure Prediction Results PsiPred Chou-Fasman Phd/PROF

35. Comparing Secondary Structure Prediction Results

36. Protein Secondary Structure Prediction - Summary • 1st Generation - 1970s • Chou & Fasman, Q3 = 50-55% • 2nd Generation -1980s • Qian & Sejnowski, Q3 = 60-65% • 3rd Generation - 1990s • PHD, PSI-PRED, Q3 = 70-80% • Features of the new methods: • Taking into account evolutionary information • Neural networks • Failures: • Nonlocal sequence interactions • Wrong prediction at the ends of H/E Q3 – Percentage of correctly assigned amino acids in a test set

37. Protein Structure Prediction http://speedy.embl-heidelberg.de/gtsp/flowchart2.html

38. Modeling by Homology (Comparative Modeling) http://salilab.org/modeller/

39. Modeling by Homology (Comparative Modeling) http://modbase.compbio.ucsf.edu/modbase-cgi-new/search_form.cgi

40. Modeling by Homology (Comparative Modeling) http://modbase.compbio.ucsf.edu/modbase-cgi-new/search_form.cgi

41. Modeling by Homology (Comparative Modeling) http://modbase.compbio.ucsf.edu/modbase-cgi-new/search_form.cgi

42. Modeling by Homology (Comparative Modeling) http://swissmodel.expasy.org/

43. Modeling by Homology (Comparative Modeling) • Comparative modeling predicts the three-dimensional structure of a given • protein sequence (target) based primarily on its alignment to one or more proteins • of known structure (templates). • The prediction process consists of • fold assignment, • target template alignment, • model building, and • model evaluation and refinement. • The number of protein sequences that can be modeled and the accuracy of • the predictions are increasing steadily because of the growth in the number of • known protein structures and because of the improvements in the modeling • software. • Further advances are necessary in recognizing weak sequence structure • similarities, aligning sequences with structures, modeling of rigid body shifts, • distortions, loops and side chains, as well as detecting errors in a model. • Despite these problems, it is currently possible to model with useful accuracy • significant parts of approximately one third of all known protein sequences. http://salilab.org/modeller/

44. Fold Recognition (Threading) Methods of protein fold recognition attempt to detect similarities between protein 3D structure that are not accompanied by any significant sequence similarity. The unifying theme of these appraoches is to try and find folds that are compatible with a particular sequence. Unlike sequence-only comparison, these methods take advantage of the extra information made available by 3D structure information. Rather than predicting how a sequence will fold, they predict how well a fold will fit a sequence.

45. Fold Recognition (Threading) – Why ? • Secondary structure is more conserved than primary structure • Tertiary structure is more conserved than secondary structure • Therefore very remote relationships can be better detected through 2o or 3o structural homology instead of sequence homology

46. Fold Recognition (Threading)

47. Fold Recognition (Threading) – 2 Kinds • 2D Threading or Prediction Based Methods (PBM) • Predict secondary structure (SS) or ASA of query • Evaluate on basis of SS and/or ASA matches • 3D Threading or Distance Based Methods (DBM) • Create a 3D model of the structure • Evaluate using a distance-based “hydrophobicity” or pseudo-thermodynamic potential

48. Fold Recognition • Database of 3D structures and sequences • Protein Data Bank (or non-redundant subset) • Query sequence • Sequence < 25% identity to known structures • Alignment protocol • Dynamic programming • Evaluation protocol • Distance-based potential or secondary structure • Ranking protocol

49. Fold Recognition http://www.sbg.bio.ic.ac.uk/~3dpssm/index2.html

50. Ab Initio Prediction • Predicting the 3D structure without any “prior knowledge” • Used when homology modelling or threading have failed (no homologues are evident) • Equivalent to solving the “Protein Folding Problem” • Still a research problem