©cmbi 2009 alignment & secondary structure you have learned about: data & databases tools...
TRANSCRIPT
©CMBI 2009
Alignment & Secondary Structure
You have learned about:
Data & databases
Tools
Amino Acids
Protein Structure
Today we will discuss: Aligning sequences
After this:
You know how to perform structural alignmentsYou are ready to apply this knowledge in your bioinformatics research project!
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Why align sequences?
The problem:
There a lots of sequences with unknown structure and/or functionThere are a few sequences with known structure and/or function
Alignment can help:
• If sequences align well, they are likely to be similar
• If they are similar, then they very likely share structural and/or functional aspects
• If one of them has known structure/function, then alignment gives us insight in structural and/or functional aspects of the aligned sequence(s)
TRANSFER OF INFORMATION!
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Sequence Alignment (1)
A sequence alignment is a representation of a whole series of evolutionary events, which left traces in the sequences.
Things that are more likely to happen during evolution should be most prominently observed in your alignment.
The purpose of a sequence alignment is to line up all residues in the sequence that were derived from the same residue position in the ancestral gene or protein.
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Sequence Alignment (2)
gap = insertion or deletion (indel)
A
B
B
A
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Structural alignment
To carry over information from a well studied protein sequence and its structure to a newly discovered protein sequence, we need a sequence alignment that represents the protein structures today, a structural alignment.
The implicit meaning of placing amino acid residues below each other in the same column of a protein (multiple) sequence alignment is that they are at the equivalent position in the 3D structures of the corresponding proteins!!
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Examples
1) the 3 active site residues H, D, S, of the serine protease we saw earlier
2) Cysteine bridges (disulfide bridges):STCTKGALKLPVCRKTSCTEG--RLPGCKR
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Transfer of information
Such information can be:
Phosphorylation sitesGlycosylation sitesStabilizing mutationsMembrane anchorsIon binding sitesLigand binding residuesCellular localization
Typically what one finds in the feature (FT) records of Swissprot!
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Significance of alignment
One can only transfer information if the similarity is significantly high between the two sequences.
Schneider (group of Sander) determined the “threshold curve” for transferring structural information from one known protein structure to another protein sequence:
If the sequences are > 80 aa long, then >25% sequence identity is enough to reliably transfer structural information.
If the sequences are smaller in length, a higher percentage of identity is needed.
Structure is much more conserved than sequence!
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Significance of alignment (2)
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Aligning sequences by hand
Most information that enters the alignment procedure comes from the physico-chemical properties of the amino acids.
Examples: which is the better alignment (left or right)?
1) CPISRTWASIFRCW CPISRTWASIFRCWCPISRT---LFRCW CPISRTL---FRCW
2) CPISRTRASEFRCW CPISRTRASEFRCWCPISRTK---FRCW CPISRT---KFRCW
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Aligning sequences by hand (2)
Procedure of aligning depends on information available:
1) Use “only” identity of amino acid and its physico-chemical properties. This is more or less what alignment programs do.
2) Also use explicitly the secondary structure preference of the amino acids.Example: aligning 2 helices when sequence identity is low
3) Use 3D information if one or more of the structures in the alignment are known.
In most cases you will start with a alignment program (e.g. CLUSTAL) and then use your knowledge of the amino acids to improve the alignment, for instance by correcting the position of gaps.
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Helix
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-4 -3 -2 -1 1 2 3 4 5 total
- - - - H H H H H
ASP 98 110 121 260 98 197 167 49 86 1186
Dataset of good helices from PDB files
Count all Asp residues in & before helices
Identify preferential positions for Asp residues
Positional preferences in helices (1)
Position 1 in helix
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Fill this table for all 20 amino acidsUse this information when aligning helices who have low percentage of sequence identity
-4 -3 -2 -1 1 2 3 4 5 total
- - - - H H H H H
ALA 143 148 99 58 189 205 187 241 268 1538
CYS 24 31 29 22 14 17 18 33 17 205
ASP 98 110 121 260 98 197 167 49 86 1186
GLU 91 100 71 71 152 287 269 70 147 1258
(…)
TRP 29 25 29 14 30 26 28 30 29 240
TYR 66 65 75 33 58 44 56 72 48 517
Positional preferences in helices (2)
Position 1 in helix
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Aligning 2 sequences when sequence identity is low
S G V S P D Q L A A L K L I L E L A L K
G T S L E T A L L M Q I A Q K L I A G
Helix 1:
Helix 2:
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Aligning 2 helices when sequence identity is low
S G V S P D Q L A A L K L I L E L A L K-1-4-4-1-4-1 3-2 1 1-2 2 -3-2 -3 2 5 1 2 2 1 5 4 -2 3 4 3 3 4 1 5 4 4 5 5 5
G T S L E T A L L M Q I A Q K L I A G-4-1-1-2 2-1 1-2-3 3 1 3 3 2 1 4 3 4 5 4 5 5
Final alignment:S G V S P D Q L A A L K L I L E L A L K
- G T S L E T A L L M Q I A Q K L I A G
Protein threading
The word threading implies that one drags the sequence (ACDEFG...) step by step through each location on the template
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Use of 3D structure info (1)
If you know that in structure 1 the Ala is pointing outside and the Ser is pointing inside:
Where does the Arg in structure 2 go?
(and what will CLUSTAL choose?)
1
2
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Use of 3D structure info (2)
1 2 3 4 5 6 7 8 9 10 11 A ILE CYS ARG LEU PRO GLY SER ALA GLU ALA VALB1 VAL CYS ARG THR PRO --- --- --- GLU ALA ILEB2 VAL CYS ARG --- --- --- THR PRO GLU ALA ILE
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An even more real example
1 2 3 4 5 6 7 8 9 10 11 A ILE CYS ARG LEU PRO GLY SER ALA GLU ALA VALB1 VAL CYS ARG THR PRO --- --- --- GLU ALA ILEB2 VAL CYS ARG --- --- --- THR PRO GLU ALA ILE
IVV CCC
RRR
LT-
PP- G- -
S-T
A-P
EEE
AAAV I I
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1)Are crucial for being able to transfer information
2)Can be optimized by using secondary structure preferences (e.g. helix positioning)
3)Can be optimized by using 3D structure info
We have seen that alignments ….
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If we have more than two related sequences aligned, the alignment is called a multiple sequence alignment (MSA)
MSA’s can:
1)reveal structural information (e.g. cys-bridges, calcium binding sites)
2)validate PROSITE search results
3)confirm or improve pair-wise sequence alignments (Course Day 6)
Multiple sequence alignments
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MSA and cysteine bridges
Multiple sequence alignments can reveal structural information:
1 2 3 4ASCTRGCIKLPTCKKMGRCTGYSTCTKGALKLPVCRKMGKSSAYATSTHGCMKLPCSRRFGKCSSYTSCTEGCLRLPGCKRFGRCTSYTTCTKGLLKLPGCKRFGKSSAYASSTKGCMKLPVSRRFGRCTAY
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MSA to validate PROSITE results (1)
You have seen the PROSITE database of protein patterns before.
PROSITE glycosylation pattern:
N-{P}-[ST]-{P} where N is the glycosylation site.
PROSITE Syntax:A-[BC]-X-D(2,5)-{EFG}-HMeans:
AB or CAnything2-5 D’sNot E,F or GH
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MSA to validate PROSITE results (2)
The chance of finding N-{P}-[ST]-{P} is rather high.
So how can you be sure? Look at the MSA of related sequences!
ASLRNASTVVTIGDTITGNLTLASYHWGSIKNGSSVITLPGTMEGNLSTTTYHYATLRNASTVMEINGTITGDLTLASFHW
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MSA to validate PROSITE results (3)
The chance of finding N-{P}-[ST]-{P} is rather high.
So how can you be sure? Look at the MSA of related sequences!
ASLRNASTVVTIGDTITGNLTLASYHWGSIKNGSSVITLPGTMEGNLSTTTYHYATLRNASTVMEINGTITGDLTLASFHW
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What you have learned today
• A good sequence alignment is necessary to carrying over information between proteins.
•Putting amino acids below each other in a sequence alignment implies that you predict that they are on equivalent positions in both proteins.
• If the aligned sequences are > 80 aa long, then >25% sequence identity is enough to reliably transfer structural information.
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You are now capable of…
• Applying these lessons to the practical exercises
• Performing your own bioinformatics research project!
Take home lesson:
Please remember to always use all structural information available to you to optimize a sequence alignment. This can be real 3D data, but can also be “just” your own knowledge about the properties and preferences of the amino acids.
©CMBI 2009
CWPVAASYGRCWPT---YGRCWPTA-SYGRCWPTLGLFGR
MSA for improvement of pair-wise alignments