4.1.pairwise Intro

Comparing Two Protein
Sequences
Cdric Notredame
Cdric Notredame (12/01/16)
Our Scope
Look once Under the Hood

Pairwise Alignment methods are POWERFUL
Pairwise Alignment methods are LIMITED
If You Understand the LIMITS

they Become VERY POWERFUL
Outline
-WHY Does It Make Sense To Compare Sequences

-HOW Can we Compare Two Sequences ?
-HOW Can we Align Two Sequences ?
-HOW can I Search a Database ?
Why Does It Make Sense

To Compare Sequences ?
Sequence Evolution
Why Do We Want To Compare Sequences
wheat
?????
--DPNKPKRAMTSFVFFMSEFRSEFKQKHSKLKSIVEMVKAAGER
| | |||||||| || | |||
||| | |||| ||||
KKDSNAPKRAMTSFMFFSSDFRS----KHSDL-SIVEMSKAAGAA
EXTRAPOLATE
Homology?
??????
SwissProt
Why Do We Want To Compare Sequences
Why Does It Make Sense To Align

Sequences ?
-Evolution is our Real Tool.

-Nature is LAZY and Keeps re-using Stuff.
-Evolution is mostly DIVERGEANT
Same Sequence Same Ancestor
Why Does It Make Sense To Align

Sequences ?
Same
Sequence
Same
Function
Same
Origin
Same
3D Fold
Many
Counter-examples!
Comparing Is Reconstructing Evolution
An Alignment is a STORY
ADKPKRPLSAYMLWLN
ADKPKRPLSAYMLWLN
ADKPKRPLSAYMLWLN
Mutations
+
Selection
ADKPKRPKPRLSAYMLWLN
ADKPRRPLS-YMLWLN
An Alignment is a STORY
ADKPKRPLSAYMLWLN
ADKPKRPLSAYMLWLN
ADKPKRPLSAYMLWLN
Mutations
+
Selection
ADKPKRPKPRLSAYMLWLN
ADKPRRPLS-YMLWLN
Insertion
Deletion
ADKPRRP---LS-YMLWLN
ADKPKRPKPRLSAYMLWLN
Mutation
Evolution is NOT Always Divergent

Chen et al, 97, PNAS, 94, 3811-16
AFGP with (ThrAlaAla)n

Similar To Trypsynogen
S
NOT
Similar to
Trypsinogen
Evolution is NOT Always Divergent

Similar To Trypsynogen
N
S
NOT
Similar to Trypsinogen
SIMILAR Sequences
BUT
DIFFERENT origin
Evolution is NOT always Divergent

But in MOST cases, you may assume it is
Similar Function
DOES NOT REQUIRE
Similar Sequence
Same
Sequence
Same
Origin
Same
Function
Same
3D Fold
Similar Sequence
Historical Legacy
How Do Sequences Evolve
Each Portion of a Genome has its own Agenda.
How Do Sequences Evolve ?

CONSTRAINED Genome Positions Evolve SLOWLY
EVERY Protein Family Has its Own Level Of Constraint
Family
KS
KA
Histone3
Insulin
Interleukin I
Globin
Apolipoprot. AI
Interferon G
6.4
4.0
4.6
5.1
4.5
8.6
0
0.1
1.4
0.6
1.6
2.8
Rates in Substitutions/site/Billion Years as measured on Mouse Vs Human (80 Million years)

Ks Synonymous Mutations, Ka Non-Neutral.
Differentmolecularclocksfordifferentproteinsanotherprediction

The amino Acids Venn Diagram
To Make Things Worse, Every Residue has its Own
Personality
L V
I
Aliphatic
Aromatic
P
AG G
T C S
D
N
Y HKE
Q
W R
Hydrophobic
Polar
Small
In a structure, each Amino Acid plays a Special Role
+
On the surface,
CHARGE MATTERS
OmpR, Cter Domain
In the core,
SIZE MATTERS

Accepted Mutations Depend on the Structure
Big -> Big
Small ->Small
NO DELETION
+
Charged -> Charged
Small <-> Big or Small
DELETIONS
How Can We Compare

Sequences ?
Substitution Matrices
How Can We Compare Sequences ?

To Compare Two Sequences, We need:
Their Structure
We Do Not
Have Them !!!
Their Function

We will Need To Replace Structural Information With
Sequence Information.
Same
Sequence
Same
Origin
Same
Function
Same
3D Fold
It CANNOT Work ALL THE TIME !!!

To Compare Sequences, We need to Compare Residues
We Need to Know How Much it COSTS to SUBSTITUTE
an Alanine into an Isoleucine
a Tryptophan into a Glycine
The table that contains the costs for all the possible
substitutions is called the SUBSTITUTION MATRIX
How to derive that matrix?

Using Knowledge Could Work
C
Aliphatic
L V
I
Aromatic
Y
W
Small
A G
G
CC
D
K E
S
N
Q
Hydrophobic
Polar
But we do not know enough about Evolution and

Structure.
Using Data works better.

Making a Substitution Matrix
-Take 100 nice pairs of Protein Sequences,
easy to align (80% identical).
-Align them
-Count each mutations in the alignments
-25 Tryptophans into phenylalanine
-30 Isoleucine into Leucine
-For each mutation, set the substitution score to the log odd ratio:
Log
Observed
Expected by chance
Youre kidding! I was struck by a lightning twice too!!

Garry Larson, The Far Side

-Take 100 nice pairs of Protein Sequences,
easy to align (80% identical).
-Align them
-Count each mutations in the alignments
-25 Tryptophans into phenylalanine
-30 Isoleucine into Leucine
-For each mutation, set the substitution score to the log odd ratio:
Log
Observed
Expected by chance

The Diagonal Indicates How

Conserved a residue tends to be.
W is VERY Conserved
Some Residues are Easier To
mutate into other similar
Cysteins that make disulfide
bridges and those that do not
get averaged


Using Substitution Matrix
Given two Sequences and a substitution Matrix,

We must Compute the CHEAPEST Alignment
Insertion
Deletion
ADKPRRP---LS-YMLWLN
ADKPKRPKPRLSAYMLWLN
Mutation
Scoring an Alignment
Most popular Subsitution Matrices
PAM250
Blosum62 (Most widely used)
Raw Score
TPEA
TPEA
|
| ||
APGA
APGA
Score =1 + 6 + 0 + 2 = 9
Question: Is it possible to get such a good alignment

by chance only?
Insertions and Deletions

Gap Penalties
Gap Opening Penalty
Gap Extension Penalty
gap
Seq
Seq
Seq
Seq
AAGARFIELDTHE----CAT
GARFIELDTHE----CAT
|||||||||||
|||
|||||||||||
|||
BBGARFIELDTHELASTCAT
GARFIELDTHELASTCAT
Opening a gap is more expensive than extending

it

Limits of the substitution Matrices
They ignore non-local interactions and Assume that
identical residues are equal
ADKPKRPLSAYMLWLN
They assume evolution rate

to be constant
ADKPKRPLSAYMLWLN
ADKPKRPLSAYMLWLN
Mutations
+
Selection
ADKPKRPKPRLSAYMLWLN
ADKPRRPLS-YMLWLN

Substitution Matrices Cannot Work !!!

I know But at least, could I get some idea of

when they are likely to do all right

The Twilight Zone
%Sequence Identity
Similar Sequence
Similar Structure
Different Sequence
Structure ????
Same 3D Fold
30%
30
Twilight Zone
Length
100

The Twilight Zone
Substitution Matrices Work Reasonably Well on

Sequences that have more than 30 % identity over
more than 100 residues

Which Matrix Shall I used
The Initial PAM matrix was computed on 80%
similar Proteins
It been extrapolated to more distantly
related sequences.
Pam 250
Pam 350
Other Matrices Exist:
BLOSUM 42
BLOSUM 62
BLOSUM 62

Which Matrix Shall I use
PAM: Distant Proteins High Index (PAM 350)

BLOSUM: Distant Proteins Low Index (Blosum30)
Choosing The Right Matrix may be Tricky
GONNET 250> BLOSUM62>PAM 250.
But This will depend on:
The Family.
The Program Used and Its Tuning.
Insertions, Deletions?
HOW Can we Align Two

Sequences ?
Dot Matrices
Global Alignments
Local Alignment
Dot Matrices
QUESTION
What are the elements shared by
two sequences ?
Dot Matrices
>Seq1
THEFATCAT
>Seq2
THELASTCAT
T H E F A T C A T
Window
Stringency
T
H
E
F
A
S
T
C
A
T
Dot Matrices
Sequences
Window size
Stringency
Dot Matrices
Strigency
Window=1
Stringency=1
Window=11
Stringency=7
Window=25
Stringency=15
Dot Matrices
x
y
x
y
Dot Matrices
http://myhits.isb-sib.ch/cgi-bin/dotlet
Dot Matrices
Dot Matrices
Dot Matrices
Dot Matrices
Limits
-Visual aid
-Best Way to EXPLORE the Sequence Organisation
-Does NOT provide us with an ALIGNMENT
wheat
?????
--DPNKPKRAMTSFVFFMSEFRSEFKQKHSKLKSIVEMVKAAGER
| | |||||||| || | |||
||| | |||| ||||
KKDSNAPKRAMTSFMFFSSDFRS----KHSDL-SIVEMSKAAGAA
Global Alignments
-Take 2 Nice Protein Sequences
-A good Substitution Matrix (blosum)
-A Gap opening Penalty (GOP)
-A Gap extension Penalty (GEP)
Cost
GOP
GOP
GEP
GOP
GOP
L
Afine Gap Penalty

Parsimony:
Evolution takes the simplest path
(So We Think)
Insertions and Deletions

Gap Penalties
Gap Opening Penalty
Gap Extension Penalty
gap
Seq
Seq
Seq
Seq
AAGARFIELDTHE----CAT
GARFIELDTHE----CAT
|||||||||||
|||
|||||||||||
|||
BBGARFIELDTHELASTCAT
GARFIELDTHELASTCAT
Opening a gap is more expensive than extending

it
Global Alignments
-Take 2 Nice Protein Sequences
-A good Substitution Matrix (blosum)
-A Gap opening Penalty (GOP)
-A Gap extension Penalty (GEP)
-DYNAMIC PROGRAMMING
>Seq1
THEFATCAT
>Seq2
THEFASTCAT
THEFA-TCAT
THEFASTCAT
DYNAMIC
PROGRAMMING
Global Alignments
DYNAMIC PROGRAMMING
Brute Force Enumeration

F A S T
F A T
----FAT
FAST-----FATFAST----F-ATFAST---
(L1+l2)!
(L1)!*(L2)!
Global Alignments
DYNAMIC PROGRAMMING
Dynamic Programming (Needlman and Wunsch)

Match=1
MisMatch=-1
Gap=-1
F A S T
0
F -1
A -2
T -3
-1 -2 -3 -4
1
F A S T
0
-1 -2 -3 -4
-1 1 0 -1 0
F
A -2
T -3
-1 -1
F A S T
F A - T
F A S T
0
F
A
T
-1 -2 -3 -4
1
2
1
2
Global Alignments
DYNAMIC PROGRAMMING
Global Alignments are very sensitive to gap

Penalties
GOP
GEP
Global Alignments
DYNAMIC PROGRAMMING
Global Alignments are very sensitive to gap

Penalties
Global Alignments do not take into account the
MODULAR nature of Proteins
C: K vitamin dep. Ca Binding
K: Kringle Domain
G: Growth Factor module
F: Finger Module
Local Alignments
GLOBAL Alignment
LOCAL Alignment
Smith And Waterman (SW)=LOCAL Alignment
Local Alignments
We now have a PairWise Comparison Algorithm,
We are ready to search Databases
Database Search
Q
SW
1.10e-20
10
1.10e-100
1.10e-2
1.10e-1
10
QUERRY
Comparison Engine
Database
3
1
3
6
1.10e-2
1
20
15
E-values
How many time do we expect such an
Alignment by chance?
13
CONCLUSION
Sequence Comparison
-Thanks to evolution, We CAN compare

Sequences
-There is a relation between Sequence and

Structure.
-Substitution matrices only work well with

similar Sequences (More than 30% id).
The Easiest way to Compare Two Sequences is

a dotplot.
A few Addresses

4.1.pairwise Intro

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4.1.pairwise Intro

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Comparing Two Protein

Cdric Notredame (12/01/16)

Look once Under the Hood

If You Understand the LIMITS

-WHY Does It Make Sense To Compare Sequences

Cdric Notredame (12/01/16)

Why Does It Make Sense

Cdric Notredame (12/01/16)

Why Do We Want To Compare Sequences

Cdric Notredame (12/01/16)

Why Do We Want To Compare Sequences

Cdric Notredame (12/01/16)

Why Does It Make Sense To Align

-Evolution is our Real Tool.

Cdric Notredame (12/01/16)

Why Does It Make Sense To Align

Cdric Notredame (12/01/16)

Comparing Is Reconstructing Evolution

Cdric Notredame (12/01/16)

Cdric Notredame (12/01/16)

Cdric Notredame (12/01/16)

Evolution is NOT Always Divergent

AFGP with (ThrAlaAla)n

Evolution is NOT Always Divergent

Evolution is NOT always Divergent

Cdric Notredame (12/01/16)

How Do Sequences Evolve

Each Portion of a Genome has its own Agenda.

Cdric Notredame (12/01/16)

How Do Sequences Evolve ?

Rates in Substitutions/site/Billion Years as measured on Mouse Vs Human (80 Million years)

Cdric Notredame (12/01/16)

Cdric Notredame (12/01/16)

How Do Sequences Evolve ?

How Do Sequences Evolve ?

In a structure, each Amino Acid plays a Special Role

How Do Sequences Evolve ?

Cdric Notredame (12/01/16)

How Can We Compare

Cdric Notredame (12/01/16)

How Can We Compare Sequences ?

Cdric Notredame (12/01/16)

How Can We Compare Sequences ?

It CANNOT Work ALL THE TIME !!!

Cdric Notredame (12/01/16)

How Can We Compare Sequences ?

Cdric Notredame (12/01/16)

How Can We Compare Sequences ?

But we do not know enough about Evolution and

How Can We Compare Sequences ?

Cdric Notredame (12/01/16)

Youre kidding! I was struck by a lightning twice too!!

Garry Larson, The Far Side

How Can We Compare Sequences ?

Cdric Notredame (12/01/16)

How Can We Compare Sequences ?

The Diagonal Indicates How

Cdric Notredame (12/01/16)

How Can We Compare Sequences ?

Cdric Notredame (12/01/16)

Cdric Notredame (12/01/16)

How Can We Compare Sequences ?

Given two Sequences and a substitution Matrix,