Manually adjusting multiple alignments
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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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Manually Adjusting Multiple Alignments

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Manually adjusting multiple alignments

Manually Adjusting Multiple Alignments

Chris Wilton


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.


A good multiple alignment1

conservation

quality

consensus

?

A Good Multiple Alignment?


Multiple alignment features

Multiple Alignment Features

  • Barton (1993)

    • “The position of insertions and deletions suggests regions where surface loops exist…


Multiple alignment features1

Multiple Alignment Features


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 features3

Multiple Alignment Features


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 features5

Multiple Alignment Features


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 features7

Multiple Alignment Features


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 features9

Multiple Alignment Features


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


Multiple alignment features11

Multiple Alignment Features


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


Sequence features

Sequence Features


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 structure

Compare Alignment to 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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