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How to Raise the Dead: The Nuts & Bolts of Ancestral Sequence Reconstruction Jeffrey Boucher Theobald Laboratory
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Talk Outline All the Pretty Corals: Convergent or Divergent Evolution How’d They Do That?
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45° 11-stranded β-barrel with chromophore positioned in center Green Fluorescent Protein (GFP)
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GFP Chromophore - Structure & Synthesis Ser 65 Tyr 66 Gly 67 Wachter 2006 Excitation - UV Blue Emission - Green Auto-catalyzation begins upon folding 1. Cyclization 3. Oxidation 2. Dehydation 3. Dehydation 2. Oxidation
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dendRFP clawGFP mc5 G5 2 scubGFP1 mc2 R2 mc3 mc4 mc1 R1 2 G1 2 Kaede Ugalde, Chang & Matz 2004 GFP Superfamily GFP-like Proteins from Coral
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Colors of the Rainbow Wachter 2006 2 Chemical Reactions ( Oxidation, Dehydration ) 3 Chemical Reactions ( Oxidation, Dehydration & 2 nd Oxidation extends π-system) Was complexity gained or lost?
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Convergent vs. Divergent Evolution Terrestrial Vertebrates Tree of Life (tolweb.org) - Slide courtesy of Kristine Mackin
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0.1 substitutions/site dendRFP clawGFP mc5 G5 2 scubGFP1 mc2 R2 mc3 mc4 mc1 R1 2 G1 2 Kaede Ugalde, Chang & Matz 2004 ALL RED/GREEN Pre-RED RED dendRFP clawGFP
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0.1 substitutions/site dendRFP clawGFP mc5 G5 2 scubGFP1 mc2 R2 mc3 mc4 mc1 R1 2 G1 2 Kaede 495 nm 505 nm 518 nm 578 nm ALL RED/GREEN Pre-RED RED Ugalde, Chang & Matz 2004
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mc5 G5 2 scubGFP1 mc2 R2 mc3 mc4 mc1 R1 2 G1 2 Kaede Ugalde, Chang & Matz 2004
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How to Resurrect a Protein 1) Acquire/Align Sequences 2) Construct Phylogeny (from Chang et al. 2002) 3) Infer Ancestral Nodes
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Acquiring Sequences BLAST (Basic Local Alignment Search Tool) How does alignment compare to alignment of random sequences? – E-value of 1 E -3 is a 1:1000 chance of alignment of random sequences
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Homology vs. Identity Significant BLAST hits inform us about evolutionary relationships Homologous - share a common ancestor – Homology is a hypothesis, identity is calculated – This is binary, not a percentile – Homology does not ensure common function
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Aligning Sequences Gap Penalty of -8 (heuristically determined) : 4 -8 5 4 0 6 2 4 6 5 4 0 3 4 -8 7 1 1 = 40 Orangutan Chimpanzee Multiple sequences aligned by similar method ClustalW MUSCLE (MUltiple Sequence Comparison by Log-Expectation) Faster than ClustalW, but methods similar
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How to Resurrect a Protein 1) Acquire/Align Sequences 2) Construct Phylogeny (from Chang et al. 2002) 3) Infer Ancestral Nodes
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Visual Depiction of Alignment Scores Suppose alignment of 3 sequences… Orangutan Chimpanzee Mouse O C M MCO 1919 4040 - 1818 -4040 -1818 1919 M O C Neighbor-Joining
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Phylogenetic Programs PHYLIP (PHYLogeny Inference Package) PAUP (Phylogeny Analysis Using Parsimony) – Now incorporates Maximum Likelihood PhyML (Phylogenetic Maximum Likelihood) MrBayes BAli-Phy (Bayesian Alignment and Phylogeny estimation)
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Maximum Likelihood (ML) Likelihood: – How surprised we should be by the data – Maximizing the likelihood, minimize your surprise Example: – Roll 20-sided die 9 times: Likelihood = Probability(Data|Model)
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Maximum Likelihood (ML) Fair Die Model: – 5% chance of rolling a 20 Trick Die Model: – 100% chance of rolling a 20 Likelihood = Probablity(Data|Model) Likelihood = (0.05) 9 = 2 E -11 Likelihood = (1) 9 = 1 Assuming trick model maximizes the likelihood
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From Dice to Trees – Data - Sequences/Alignment – Model - Tree topology, Branch lengths & Model of evolution Starting Tree Choose model that maximizes the likelihood Likelihood = Probablity(Data|Model) New Tree Likelihood Likelihood
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How to Resurrect a Protein 1) Acquire/Align Sequences 2) Construct Phylogeny (from Chang et al. 2002) 3) Infer Ancestral Nodes
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Reconstruction Methods Consensus Parsimony Maximum Likelihood
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Consensus Advantage: Easy & fast Disadvantages: Ignores phylogenetic relationships X X
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Parsimony Parsimony Principle – Best-supported evolutionary inference requires fewest changes – Assumes conservation as model Advantage over consensus: – Takes phylogenetic relationships into account
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Parsimony A B C D E F G H ABCDEFGHABCDEFGH
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Parsimony V V V I L L Example adapted from David Hillis I L {V} {L} {V, I} {V, I, L} Changes = 4 V L I I I V L
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Parsimony - Alternate Reconstructions Resolve ambiguous reconstructions Is conservation the best model?
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ML Improvements Over Parsimony PAML (Phylogeny Analysis by Maximum Likelihood) Includes evolutionary process & branch lengths – Reduction in ambiguous sites Fit of model included in calculation – Removes a priori choices – Use more complex models (when applicable) Confidence in reconstruction – Posterior probabilities
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Ugalde, Chang & Matz 2004 Ancestral GFP Reconstructions
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Ugalde, Chang & Matz 2004 Sites with PP<0.8 considered ambiguous Alt residues considered if PP>0.2 Alternative ancestors did not affect the conclusions PositionResiduePPResiduePPResiduePP 168P0.999 169K0.407R0.236S0.210 170V0.730I0.270 171I0.742V0.158R0.034
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Thanks for Your Attention Questions?
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