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Manually Adjusting Multiple Alignments. Chris Wilton. Multiple Alignments. Reviewing multiple alignments what is a multiple alignment? Analyzing a multiple alignment what makes a ‘good’ multiple alignment? what can it tell us, why is it useful? Adjusting a multiple alignment

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multiple alignments
Multiple Alignments
  • Reviewing multiple alignments
    • what is a multiple alignment?
  • Analyzing a multiple alignment
    • what makes a ‘good’ multiple alignment?
    • what can it tell us, why is it useful?
  • Adjusting a multiple alignment
    • Alignment editors and HowTo
    • Demonstration and practice
what is a multiple alignment
What is a Multiple Alignment?
  • A comparison of sequences
    • “multiple sequence alignment”
  • A comparison of equivalents:
    • Structurally equivalent positions
    • Functionally equivalent residues
    • Secondary structure elements
    • Hydrophobic regions, polar residues
a good multiple alignment
A Good Multiple Alignment?
  • Difficult to define…
  • Good ones look pretty!
    • Aligned secondary structures
    • Strongly conserved residues / regions
    • Comparison with known structure helps
  • Bad ones look chaotic and random.
multiple alignment features
Multiple Alignment Features
  • Barton (1993)
    • “The position of insertions and deletions suggests regions where surface loops exist…
multiple alignment features2
Multiple Alignment Features
  • Barton (1993)
    • “The position of insertions and deletions suggests regions where surface loops exist…
    • Conserved glycine or proline suggests aβ-turn...
multiple alignment features4
Multiple Alignment Features
  • Barton (1993)
    • “The position of insertions and deletions suggests regions where surface loops exist…
    • Conserved glycine or proline suggests aβ-turn…
    • Residues with hydrophobic properties conserved at i, i+2, i+4 (etc) separated by unconserved or hydrophilic residues suggests a surface β-strand…
multiple alignment features6
Multiple Alignment Features
  • Barton (1993)
    • “The position of insertions and deletions suggests regions where surface loops exist…
    • Conserved glycine or proline suggests aβ-turn…
    • Residues with hydrophobic properties conserved at i, i+2, i+4 (etc) separated by unconserved or hydrophilic residues suggests a surfaceβ-strand…
    • A short run of hydrophobic amino acids (4 or 5 residues) suggests a buriedβ-strand…
multiple alignment features8
Multiple Alignment Features
  • Barton (1993)
    • Pairs of conserved hydrophobic amino acids separated by pairs of unconserved or hydrophilic residues suggests anα-helix with one face packed in the protein core. Similarly, an i, i+3, i+4, i+7 pattern of conserved residues.”
multiple alignment features10
Multiple Alignment Features
  • Barton (1993)
    • Pairs of conserved hydrophobic amino acids separated by pairs of unconserved or hydrophilic residues suggests anα-helix with one face packed in the protein core. Similarly, an i, i+3, i+4, i+7 pattern of conserved residues.”
  • Cysteine is a rare amino acid, and is often used in disulphide bonds ( pairs of conserved cysteines )
  • Charged residues ( histidine, aspartate, glutamate, lysine, arginine ) and other polar residues embedded in a conserved region indicate functional importance
quality assessment
Quality Assessment
  • Bad residues
    • Large distance from column consensus
  • Bad columns
    • Average distance from consensus is high – “entropy”
  • Bad regions
    • Profile scores
  • Bad quality doesn’t always mean badly aligned!

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quality assessment1
Quality Assessment
  • Profiles
    • A profile holds scores for each residue type (plus gaps) over every column of a multiple alignment
    • Concepts:
      • Consensus sequence
      • Amino acid similarity
    • Some multiple alignment programs use profiles to build or add to an alignment
    • Any alignment, or even one sequence, can be a profile (one sequence isn’t a very good one…)
what can we do with a ma
What can we do with a MA?
  • Identify subgroups (phylogeny)
    • Intra-group sequence conservation
    • Evolutionary relatedness (view tree)
  • Identify motifs (functionality)
    • Evolutionary signals
    • Highly conserved residues indicate functional or structural significance!
  • Widen search for related proteins
    • MA better than single sequence
    • Consensus sequence / profile useful

RPDDWHLHLR

GGIDTHVHFI

GFTLTHEHIC

PFVEPHIHLD

PKVELHVHLD

what do we want to do
What do we want to do?
  • Build a homology model?
    • Accuracy
  • Perform phylogenetic analysis?
    • Completeness
  • Functional analysis of a protein family?
    • Diversity
building the initial alignment
Building the initial alignment
  • Fetch related sequences and run alignment
    • Clustal, Dialign, TCoffee, Muscle …
  • Fetch a multiple alignment from a database and add sequences of interest
    • Pfam, ProDom, ADDA …
  • Start from a motif-finding procedure
    • MEME, Pratt, Gibbs Sampler …
adjusting the alignment
Adjusting the alignment
  • Filter alignment:
    • Remove any redundancy
    • Remove unrelated sequences
    • Remove unwanted domains
    • Recalculate alignment if necessary
  • Look for conserved motifs, adjust any misalignments. Try different colour schemes and thresholds.
  • One step at a time…
jalview alignment editor
Jalview Alignment Editor

Clamp, M., Cuff, J., Searle, S. M. and Barton, G. J. (2004), "The Jalview Java Alignment Editor", Bioinformatics, 20, 426-7.

colouring your alignment

HYDROPHOBIC

/ POLAR

hydrophobic

polar

BURIED INDEX

buried

surface

β-STRAND

LIKELIHOOD

probable

unlikely

HELIX LIKELIHOOD

probable

unlikely

Colouring your alignment
colouring your alignment1
Colouring your alignment
  • By conservation thresholds:
colouring your alignment2
Colouring your alignment
  • Conservation index

Amino Acid Property Classification Schema, eg: Livingstone & Barton 1993

check pdb structures
Check PDB Structures
  • Load MA with sequence(s) for known PDB structure
    • View >> Feature Settings >> Fetch DAS Features (wait...) OR
    • Right-click >> Associate Structure with Sequence >> Discover PDB ids (quicker)
  • Right-click sequence name >> View PDB Entry
  • Structure opens in new window – residues acquire MA colours
  • Highlight residues by hovering mouse over alignment or structure
  • Label residues by clicking on structure
compare alignment to structure1
Compare Alignment to Structure
  • Crucial way of checking alignment!
  • Where are gaps / insertions /deletions ?
    • In secondary structures: bad
    • In surface loops: okay
  • Where are our key / functional residues?
    • Are they in probable active site?
    • Check they are clustered
    • Check they are accessible, not buried
demonstration and practice
Demonstration and Practice
  • Start Jalview (click here)
  • Tools >> Preferences >>

Visual

select Maximise Window, unselect Quality, set Font Size to 8 or 9, Colour >> Clustal, uncheck Open File

Editing

check Pad Gaps When Editing

  • File >> Input Alignment >> from URL (use this one)
  • Get used to the controls – selecting and deselecting sequences/groups (drag mouse), dragging sequences/groups (use shift/ctrl), selecting sequence regions, hiding sequences/groups, removing columns and regions… Then explore menus and tools.
  • Now load this alignment – I’ve messed up a good alignment, and now I’d like you to correct it! There are two groups of sequences and one single sequence to adjust.
demonstration and practice1
Demonstration and Practice
  • View >> Feature Settings >> DAS Settings
    • select Uniprot, dssp, cath, Pfam, PDBsum_ligands, PDBsum_DNAbinding, then click ‘Save as default’
    • click Fetch DAS Features (then click yes at prompt) ...
    • Move mouse over alignment and read information about features
    • Move mouse over sequence names to check for PDB ids
  • Open a PDB structure (choose any)
  • View >> uncheck Show All Chains, then use up-arrow key to increase structure size.
  • Hover mouse over structure (see how residues are highlighted in the sequence), then do same for sequence. Select residues in the structure by clicking them – a label will appear. Click again to remove label.
  • Check position of insertions & deletions using this method.
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