1. Protein Secondary Structures Assignment and prediction Pernille Haste Andersen 12.04.2005

2. Secondary Structure Elements ß-strand Helix Turn Bend

3. Use of secondary structure Classification of protein structures Definition of loops (active sites) Use in fold recognition methods Improvements of alignments Definition of domain boundaries

4. Classification of secondary structure Defining features –Dihedral angles –Hydrogen bonds –Geometry Assigned manually by crystallographers or Automatic –DSSP (Kabsch & Sander,1983) –STRIDE (Frishman & Argos, 1995) –DSSPcont (Andersen et al., 2002)

5. Dihedral Angles phi - dihedral angle about the N-Calpha bond psi - dihedral angle about the Calpha-C bond omega - dihedral angle about the C-N (peptide) bond From http://www.imb-jena.de

6. Helices phi(deg) psi(deg) H-bond pattern ------------------------------------------------------------------ right-handed alpha-helix -57.8 -47.0 i+4 pi-helix -57.1 -69.7 i+5 3 10 helix -74.0 -4.0 i+3 (omega is 180 deg in all cases) ----------------------------------------------------------------- From http://www.imb-jena.de

7. Beta Strands phi(deg) psi(deg) omega (deg) ------------------------------------------------------------------ beta strand -120 120 180 ----------------------------------------------------------------- Hydrogen bond patterns in beta sheets. Here a four-stranded beta sheet is drawn schematically which contains three antiparallel and one parallel strand. Hydrogen bonds are indicated with red lines (antiparallel strands) and green lines (parallel strands) connecting the hydrogen and receptor oxygen. From http://broccoli.mfn.ki.se/pps_course_96/

8. Secondary Structure Elements ß-strand Helix Turn Bend

9. Secondary Structure Type Descriptions *H = alpha helix *G = 3 10 - helix *I = 5 helix (pi helix) *E = extended strand, participates in beta ladder *B = residue in isolated beta-bridge *T = hydrogen bonded turn *S = bend *C = coil

10. Automatic assignment programs DSSP ( http://www.cmbi.kun.nl/gv/dssp/ ) STRIDE ( http://www.hgmp.mrc.ac.uk/Registered/Option/stride.html ) DSSPcont ( http://cubic.bioc.columbia.edu/services/DSSPcont/ ) # RESIDUE AA STRUCTURE BP1 BP2 ACC N-H-->O O-->H-N N-H-->O O-->H-N TCO KAPPA ALPHA PHI PSI X-CA Y-CA Z-CA 1 4 A E 0 0 205 0, 0.0 2,-0.3 0, 0.0 0, 0.0 0.000 360.0 360.0 360.0 113.5 5.7 42.2 25.1 2 5 A H - 0 0 127 2, 0.0 2,-0.4 21, 0.0 21, 0.0 -0.987 360.0-152.8-149.1 154.0 9.4 41.3 24.7 3 6 A V - 0 0 66 -2,-0.3 21,-2.6 2, 0.0 2,-0.5 -0.995 4.6-170.2-134.3 126.3 11.5 38.4 23.5 4 7 A I E -A 23 0A 106 -2,-0.4 2,-0.4 19,-0.2 19,-0.2 -0.976 13.9-170.8-114.8 126.6 15.0 37.6 24.5 5 8 A I E -A 22 0A 74 17,-2.8 17,-2.8 -2,-0.5 2,-0.9 -0.972 20.8-158.4-125.4 129.1 16.6 34.9 22.4 6 9 A Q E -A 21 0A 86 -2,-0.4 2,-0.4 15,-0.2 15,-0.2 -0.910 29.5-170.4 -98.9 106.4 19.9 33.0 23.0 7 10 A A E +A 20 0A 18 13,-2.5 13,-2.5 -2,-0.9 2,-0.3 -0.852 11.5 172.8-108.1 141.7 20.7 31.8 19.5 8 11 A E E +A 19 0A 63 -2,-0.4 2,-0.3 11,-0.2 11,-0.2 -0.933 4.4 175.4-139.1 156.9 23.4 29.4 18.4 9 12 A F E -A 18 0A 31 9,-1.5 9,-1.8 -2,-0.3 2,-0.4 -0.967 13.3-160.9-160.6 151.3 24.4 27.6 15.3 10 13 A Y E -A 17 0A 36 -2,-0.3 2,-0.4 7,-0.2 7,-0.2 -0.994 16.5-156.0-136.8 132.1 27.2 25.3 14.1 11 14 A L E >> -A 16 0A 24 5,-3.2 4,-1.7 -2,-0.4 5,-1.3 -0.929 11.7-122.6-120.0 133.5 28.0 24.8 10.4 12 15 A N T 45S+ 0 0 54 -2,-0.4 -2, 0.0 2,-0.2 0, 0.0 -0.884 84.3 9.0-113.8 150.9 29.7 22.0 8.6 13 16 A P T 45S+ 0 0 114 0, 0.0 -1,-0.2 0, 0.0 -2, 0.0 -0.963 125.4 60.5 -86.5 8.5 32.0 21.6 6.8 14 17 A D T 45S- 0 0 66 2,-0.1 -2,-0.2 1,-0.1 3,-0.1 0.752 89.3-146.2 -64.6 -23.0 33.0 25.2 7.6 15 18 A Q T <5 + 0 0 132 -4,-1.7 2,-0.3 1,-0.2 -3,-0.2 0.936 51.1 134.1 52.9 50.0 33.3 24.2 11.2 16 19 A S E < +A 11 0A 44 -5,-1.3 -5,-3.2 2, 0.0 2,-0.3 -0.877 28.9 174.9-124.8 156.8 32.1 27.7 12.3 17 20 A G E -A 10 0A 28 -2,-0.3 2,-0.3 -7,-0.2 -7,-0.2 -0.893 15.9-146.5-151.0-178.9 29.6 28.7 14.8 18 21 A E E -A 9 0A 14 -9,-1.8 -9,-1.5 -2,-0.3 2,-0.4 -0.979 5.0-169.6-158.6 146.0 28.0 31.5 16.7 19 22 A F E +A 8 0A 3 12,-0.4 12,-2.3 -2,-0.3 2,-0.3 -0.982 27.8 149.2-139.1 120.3 26.5 32.2 20.1 20 23 A M E -AB 7 30A 0 -13,-2.5 -13,-2.5 -2,-0.4 2,-0.4 -0.983 39.7-127.8-152.1 161.6 24.5 35.4 20.6 21 24 A F E -AB 6 29A 45 8,-2.4 7,-2.9 -2,-0.3 8,-1.0 -0.934 23.9-164.1-112.5 137.7 21.7 37.0 22.6 22 25 A D E -AB 5 27A 6 -17,-2.8 -17,-2.8 -2,-0.4 2,-0.5 -0.948 6.9-165.0-123.7 138.3 18.9 38.9 20.8 23 26 A F E > S-AB 4 26A 76 3,-3.5 3,-2.1 -2,-0.4 -19,-0.2 -0.947 78.4 -27.2-127.3 111.5 16.4 41.3 22.3 24 27 A D T 3 S- 0 0 74 -21,-2.6 -20,-0.1 -2,-0.5 -1,-0.1 0.904 128.9 -46.6 50.4 45.0 13.4 42.1 20.2 25 28 A G T 3 S+ 0 0 20 -22,-0.3 2,-0.4 1,-0.2 -1,-0.3 0.291 118.8 109.3 84.7 -11.1 15.4 41.4 17.0 26 29 A D E < S-B 23 0A 114 -3,-2.1 -3,-3.5 109, 0.0 2,-0.3 -0.822 71.8-114.7-103.1 140.3 18.4 43.4 18.1 27 30 A E E -B 22 0A 8 -2,-0.4 -5,-0.3 -5,-0.2 3,-0.1 -0.525 24.9-177.7 -74.1 127.5 21.8 41.8 19.1

11. Secondary Structure Prediction What to predict? –All 8 types or pool types into groups H E C DSSP Q3 *H = alpha helix *G = 3 10 -helix *I = 5 helix (pi helix) *E = extended strand *B = beta-bridge *T = hydrogen bonded turn *S = bend *C = coil

12. Straight HEC Secondary Structure Prediction What to predict? –All 8 types or pool types into groups H E C Q3 *H = alpha helix *E = extended strand *T = hydrogen bonded turn *S = bend *C = coil *G = 3 10 -helix *I = 5 helix (pi helix) *B = beta-bridge

13. Secondary Structure Prediction Simple alignments Align to a close homolog for which the structure has been experimentally solved. Heuristic Methods (e.g., Chou-Fasman, 1974) Apply scores for each amino acid an sum up over a window. Neural Networks (different inputs) Raw Sequence (late 80’s) Blosum matrix (e.g., PhD, early 90’s) Position specific alignment profiles (e.g., PsiPred, late 90’s) Multiple networks balloting, probability conversion, output expansion (Petersen et al., 2000).

14. Improvement of accuracy 1974 Chou & Fasman~50-53% 1978 Garnier63% 1987 Zvelebil66% 1988 Quian & Sejnowski64.3% 1993 Rost & Sander70.8-72.0% 1997 Frishman & Argos<75% 1999 Cuff & Barton72.9% 1999 Jones76.5% 2000 Petersen et al.77.9%

15. Simple Alignments Solved structure of a homolog to query is needed Homologous proteins have ~88% identical (3 state) secondary structure If no close homologue can be identified alignments will give almost random results

16. Amino acid preferences in  - Helix

17. Amino acid preferences in  - Strand

18. Amino acid preferences in coil

19. Chou-Fasman NameP(a)P(b)P(turn)f(i)f(i+1)f(i+2)f(i+3) Ala 14283660.060.0760.0350.058 Arg 9893950.0700.1060.0990.085 Asp 101541460.1470.1100.1790.081 Asn 67891560.1610.0830.1910.091 Cys 701191190.1490.0500.1170.128 Glu 15137740.0560.0600.0770.064 Gln 111110980.0740.0980.0370.098 Gly 57751560.1020.0850.1900.152 His 10087950.1400.0470.0930.054 Ile 108160470.0430.0340.0130.056 Leu 121130590.0610.0250.0360.070 Lys 114741010.0550.1150.0720.095 Met 145105600.0680.0820.0140.055 Phe 113138600.0590.0410.0650.065 Pro 57551520.1020.3010.0340.068 Ser 77751430.1200.1390.1250.106 Thr 83119960.0860.1080.0650.079 Trp 108137960.0770.0130.0640.167 Tyr 691471140.0820.0650.1140.125 Val 106170500.0620.0480.0280.053

20. Chou-Fasman 1. Assign all of the residues in the peptide the appropriate set of parameters. 2. 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) P(b-sheet) for that segment, the segment can be assigned as a helix. 3. Repeat this procedure to locate all of the helical regions in the sequence. 4. 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) 105 and the average P(b-sheet) > P(a-helix) for that region. 5. 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. 6. 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 tetra-peptide; and (3) the averages for the tetra-peptide obey the inequality P(a-helix) P(b-sheet), then a beta-turn is predicted at that location.

21. Chou-Fasman General applicable Works for sequences with no solved homologs But the accuracy is low!

22. Neural Networks Benefits –General applicable –Can capture higher order correlations –Inputs other than sequence information Drawbacks –Needs many data (different solved structures). However, theese does exist today (nearly 2500 solved structures with low sequence identity/high resolution.) –Complex method with several pitfalls.

23. Architecture I K E E H V I I Q A E H E C IKEEHVIIQAEFYLNPDQSGEF….. Window Input Layer Hidden Layer Output Layer Weights

24. Sparse encoding Inp Neuron 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 AAcid A 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 R 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 N 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 D 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 C 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Q 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 E 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0

25. Input Layer I K E E H V I I Q A E 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0

26. BLOSUM 62 A R N D C Q E G H I L K M F P S T W Y V B Z X * A 4 -1 -2 -2 0 -1 -1 0 -2 -1 -1 -1 -1 -2 -1 1 0 -3 -2 0 -2 -1 0 -4 R -1 5 0 -2 -3 1 0 -2 0 -3 -2 2 -1 -3 -2 -1 -1 -3 -2 -3 -1 0 -1 -4 N -2 0 6 1 -3 0 0 0 1 -3 -3 0 -2 -3 -2 1 0 -4 -2 -3 3 0 -1 -4 D -2 -2 1 6 -3 0 2 -1 -1 -3 -4 -1 -3 -3 -1 0 -1 -4 -3 -3 4 1 -1 -4 C 0 -3 -3 -3 9 -3 -4 -3 -3 -1 -1 -3 -1 -2 -3 -1 -1 -2 -2 -1 -3 -3 -2 -4 Q -1 1 0 0 -3 5 2 -2 0 -3 -2 1 0 -3 -1 0 -1 -2 -1 -2 0 3 -1 -4 E -1 0 0 2 -4 2 5 -2 0 -3 -3 1 -2 -3 -1 0 -1 -3 -2 -2 1 4 -1 -4 G 0 -2 0 -1 -3 -2 -2 6 -2 -4 -4 -2 -3 -3 -2 0 -2 -2 -3 -3 -1 -2 -1 -4 H -2 0 1 -1 -3 0 0 -2 8 -3 -3 -1 -2 -1 -2 -1 -2 -2 2 -3 0 0 -1 -4 I -1 -3 -3 -3 -1 -3 -3 -4 -3 4 2 -3 1 0 -3 -2 -1 -3 -1 3 -3 -3 -1 -4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 -2 2 0 -3 -2 -1 -2 -1 1 -4 -3 -1 -4 K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5 -1 -3 -1 0 -1 -3 -2 -2 0 1 -1 -4 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 0 -2 -1 -1 -1 -1 1 -3 -1 -1 -4 F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6 -4 -2 -2 1 3 -1 -3 -3 -1 -4 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 -1 -1 -4 -3 -2 -2 -1 -2 -4 S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 1 -3 -2 -2 0 0 0 -4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5 -2 -2 0 -1 -1 0 -4 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11 2 -3 -4 -3 -2 -4 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 -1 -3 -2 -1 -4 V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4 -3 -2 -1 -4

27. Input Layer I K E E H V I I Q A E -1 0 0 2 -4-4 2 5 -2-2 0 -3-3 -3-3 1 -2-2 -3-3 -1 0 -1 -3-3 -2-2 -2-2

28. Secondary networks (Structure-to-Structure) H E C H E C H E C H E C IKEEHVIIQAEFYLNPDQSGEF….. Window Input Layer Hidden Layer Output Layer Weights

29. PHD method (Rost and Sander) Combine neural networks with sequence profiles –6-8 Percentage points increase in prediction accuracy over standard neural networks Use second layer “Structure to structure” network to filter predictions Jury of predictors Set up as mail server

30. PSI-Pred (Jones) Use alignments from iterative sequence searches (PSI-Blast) as input to a neural network Better predictions due to better sequence profiles Available as stand alone program and via the web

31. Position specific scoring matrices (PSI-BLAST profiles) A R N D C Q E G H I L K M F P S T W Y V 1 I -2 -4 -5 -5 -2 -4 -4 -5 -5 6 0 -4 0 -2 -4 -4 -2 -4 -3 4 2 K -1 -1 -2 -2 -3 -1 3 -3 -2 -2 -3 4 -2 -4 -3 1 1 -4 -3 2 3 E 5 -3 -3 -3 -3 3 1 -2 -3 -3 -3 -2 -2 -4 -3 -1 -2 -4 -3 1 4 E -4 -3 2 5 -6 1 5 -4 -3 -6 -6 -2 -5 -6 -4 -2 -3 -6 -5 -5 5 H -4 2 1 1 -5 1 -2 -4 9 -5 -2 -3 -4 -4 -5 -3 -4 -5 1 -5 6 V -3 0 -4 -5 -4 -4 -2 -3 -5 1 -2 1 0 1 -4 -3 3 -5 -3 5 7 I 0 -2 -4 1 -4 -2 -4 -4 -5 1 0 -2 0 2 -5 1 -1 -5 -3 4 8 I -3 0 -5 -5 -4 -2 -5 -6 1 2 4 -4 -1 0 -5 -2 0 -3 5 -1 9 Q -2 -3 -2 -3 -5 4 -1 3 5 -5 -3 -3 -4 -2 -4 2 -1 -4 2 -2 10 A 2 -4 -4 -3 2 -3 -1 -4 -2 1 -1 -4 -3 -4 1 2 3 -5 -1 1 11 E -1 3 1 1 -1 0 1 -4 -3 -1 -3 0 3 -5 4 -1 -3 -6 -3 -1 12 F -3 -5 -5 -5 -4 -4 -4 -1 -1 1 1 -5 2 5 -1 -4 -4 -3 5 2 13 Y 3 -5 -5 -6 3 -4 -5 -2 -1 0 -4 -5 -3 3 -5 -2 -2 -2 7 1 14 L -1 -3 -4 -2 1 5 1 -1 -1 -1 1 -3 -3 1 -5 -1 -1 -2 3 -2 15 N -1 -4 4 1 5 -3 -4 2 -4 -4 -4 -3 -2 -4 -5 2 0 -5 0 0 16 P -2 4 -4 -4 -5 0 -3 3 2 -5 -4 0 -4 -3 0 1 -2 -1 5 -3 17 D -3 -2 1 5 -6 -2 2 2 -1 -2 -2 -3 -5 -4 -5 -1 2 -6 -3 -4

32. Sequence-to-structure –Window sizes15,17,19 and 21 –Hidden units50 and 75 –10-fold cross validation => 80 predictions Structure-to-structure –Window size17 –Hidden units40 –10-fold cross validation => 800 predictions Several different architectures Output: C C H H C C C Output: C C C C C C C

33. Activities to probabilities 0.050.10.15…1.0 0.050.99 0.10 0.150.90.830.75. 1.0 Helix activities (output) Strand activities (output) Coil probabilities! (calculated) Coil conversion

34. Confidence of a per residue prediction –P(Highest) – P(second highest) –H: 0.80 E: 0.05 C:0.15 => conf.=0.65 Mean per chain confidence for all 800 predictions –Calculate Mean and Standard deviation –Averaging of per chain predictions with  Balloting procedure

35. Benchmarking secondary structure predictions CASP –Critical Assessment of Structure Predictions –Sequences from about-to-be-deposited-structures are given to groups who submit their predictions before the structure is published –Every 2. year EVA –Newly solved structures are send to prediction servers. –Every week

36. EVA results (Rost et al., 2001) PROFphd77.0% PSIPRED76.8% SAM-T99sec76.1% SSpro76.0% Jpred275.5% PHD71.7% –Cubic.columbia.edu/eva

37. Links to servers Database of links http://mmtsb.scripps.edu/cgi bin/renderrelres?protmodel ProfPHD http://www.predictprotein.org/ PSIPRED http://bioinf.cs.ucl.ac.uk/psipred/ JPred http://www.compbio.dundee.ac.uk/~www-jpred/

38. Practical Conclusion If you need a secondary structure prediction use one of the newer ones such as : –ProfPHD, –PSIPRED, and –JPred And not one of the older ones such as : –Chou-Fasman –Garnier