Protein functions prediction
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Introduction
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Signal peptides
Transmembrane regions
and topology
PTM (post-translational
modifications)
Low complexity and
biased regions
Repeats
Coils
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Secondary structure
Antigenic peptides
Domain/Motifs
Tools
The EMBOSS package
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Different techniques
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Algorithms
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Sliding window, Nearest Neighbor
Patterns, regular expression
Weight matrices
HMM, profiles
Neural Networks
Rules
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Sliding window
THISISATESTSEQVENCETHATDISPLAYSTHESLIDINGWINDQW
Score1
Score2
Scoren
Width or Size=11, Step=5
Results are usually displayed
as a graph, see example ->
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Patterns / regular expression
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Pattern: <A-x-[ST](2)-x(0,1)-{V}
Regexp: ^A.[ST]{2}.?[^V]
Text: The sequence must start with an
alanine, followed by any amino acid, followed
by a serine or a threonine, two times,
followed by any amino acid or nothing,
followed by any amino acid except a valine.
Simply the syntax differ…
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Weight matrices (PSSM)
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
HMM / profiles
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Neural Networks
General principle:
Example:
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Signals found in proteins
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N-ter
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exportation - secretion
mitochondria
chloroplast
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internal
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NLS (nuclear localization
signal)
C-ter
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GPI-anchor (Glycosyl
Phosphatidyl Inositol)
other membrane
anchors (see PTM)
other unknown ?
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Signals detection tools
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SignalP
MitoProt
ChloroP
Predotar
PSort
TargetP
Sigcleave (EMBOSS)
Phobius
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Big-PI
DGPI
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Transmembrane regions
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Detection (signal peptide, hydropathy, helices)
Organisation (topology)
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Transmembrane detection tools
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TMHMM
TMPred
TopPred2
DAS
HMMTop
Tmap (EMBOSS)
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Mixture of tools
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Phobius
ConPred II
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Post translational modifications
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Phosphorylation
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S - T - (HO)K
Acetylation, methylation
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N
O-glycosylation
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S-T-Y
N-glycosylation
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D-E-K
Sulfation
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Y
Farnesylation, myristylation,
palmitoylation,
geranylgeranylation, GPIanchor
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Ubiquitination and family
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C - Nter - Cter
K - Nter
Inteins (protein splicing)
Pre-translational
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Selenoprotein
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C
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
PTM detection
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Pattern prediction
(PROSITE)
Short or weak signal
Frequent hit producer
Best method is experimental
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MS/MS detection
Most method use « rules »
joining pattern detection and
knowledge to predict sites.
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NetOGlyc - Prediction of type Oglycosylation sites in mammalian
proteins
DictyOGlyc - Prediction of GlcNAc
O-glycosylation sites in
Dictyostelium
YinOYang - O-beta-GlcNAc
attachment sites in eukaryotic
protein sequences
NetPhos - Prediction of Ser, Thr
and Tyr phosphorylation sites in
eukaryotic proteins
NMT - Prediction of N-terminal Nmyristoylation
Sulfinator - Prediction of tyrosine
sulfation sites
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Low complexity regions
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repeats
compositional bias
PEST
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Low complexity / Repeats
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DUST (DNA) / SEG
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search collection
search collection
REPRO, Radar
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REP
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de novo detection
EMBOSS (DNA)
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RepeatMasker (DNA)
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de novo detection
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einverted
equicktandem
etandem
palindrome
EMBOSS (protein)
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oddcomp
PEST, PESTFind
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de novo detection
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Coils
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Helix of helix
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coiled-coil
Leu-zipper
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Coils detection
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COILS
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Paircoil, Multicoil
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Pairwise correlation
Marcoil
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Weight matrices
HMM
Pepcoil (EMBOSS)
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Weight matrices
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Secondary structure
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Structure to predict
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Alpha-helices
Beta-sheets
Turns
Random coil
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Garnier (EMBOSS)
PHD
DSC
PREDATOR
NNSSP
Jpred
Jnet
Many others
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Antigenic peptide
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Peptides binding to MHC
class I
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Use of experimental
knowledge
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8, 9, 10 mers
class II
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15 mers (3+9+3)
Depend highly on MHC type
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Databases of known
peptides
SYFPEITHI
HLA_Bind (BIMAS)
MAPPP combined expert
Antigenic (EMBOSS)
Many more
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Prediction of proteasome
cleavage sites
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NetChop
PaProc
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Domain / Motif
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All the protein domain
descriptors
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PROSITE
PFAM
SMART
PRODOM
BLOCKS
PRINTS
TIGRfam
…
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Federation: InterPro
Many techniques
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Patterns, Regexp
PSSM (PSI-BLAST)
Profiles
HMM
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Other Tools
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You can find some of them on our servers
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Or on ExPASy server
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www.ch.embnet.org
www.expasy.org/tools
Or ask Google!!
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www.google.com
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
European Molecular Biology Open Software Suite
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
How to use EMBOSS/Jemboss at SIB
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
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Free Open Source (for most Unix plateforms)
GCG successor (compatible with GCG file format)
More than 150 programs (ver. 2.9.0)
Easy to install locally
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Interfaces
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but no interface, requires local databases
Unix command-line only
Jemboss, www2gcg, w2h, wemboss… (with account)
Pise, EMBOSS-GUI, SRSWWW (no account)
Staden, Kaptain, CoLiMate, Jemboss (local)
Access: www.emboss.org or emboss.sourceforge.net
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Some details
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Format USA
'asis'
Format
Format
Format
Format
Format
Format
Format
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::
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::
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::
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::
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Sequence [start : end : reverse]
'@' ListFile [start : end : reverse]
'list' : ListFile [start : end : reverse]
Database : Entry [start : end : reverse]
Database - SearchField : Word [start : end : reverse]
File : Entry [start : end : reverse]
File : SearchField : Word [start : end : reverse]
Program Program-parameters '|' [start : end : reverse]
Example: fasta::Swissprot:UBP5_HUMAN[200:300]
Databases
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Any can be added, use showdb to display the available databases
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
databases
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showdb
Displays information on the currently available databases
# Name
Type ID Qry All Comment
# ====
==== == === === =======
ipr_fetch
P
OK OK OK InterPro current by fetch
ipi_fetch
P
OK OK OK IPI current by fetch
refseq_fetch P
OK OK OK refseq current by fetch
repbase_fetch P
OK OK OK repbase current by fetch
swiss_fetch
P
OK OK OK SwissProt current by fetch
swissprot
P
OK OK OK SWISSPROT sequences
trembl
P
OK OK OK TREMBL sequences
trembl_fetch P
OK OK OK trembl current by fetch
tremblnew
P
OK OK OK TREMBL New sequences
ug_fetch
P
OK OK OK Unigene by fetch
embl
N
OK OK OK EMBL release
emhum
N
OK OK OK EMBL release, Human section by emboss index
emrod
N
OK OK OK EMBL release, Rodent section by emboss index
emvrt
N
OK OK OK EMBL release, Vertebrate (nonhuman, nonrodent)
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seqret (seqretall, seqretset, seqretsplit)
entret (for complete untouched entry, e.g., for unigene, interpro, swissprot…)
Possible to define your own « .embossrc » file
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
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Some tools for DNA
redata
remap
restover
restrict
showseq
silent
cirdna
lindna
revseq
…
Search REBASE for enzyme name, references, suppliers etc
Display a sequence with restriction cut sites, translation etc
Finds restriction enzymes that produce a specific overhang
Finds restriction enzyme cleavage sites
Display a sequence with features, translation etc
Silent mutation restriction enzyme scan
Draws circular maps of DNA constructs
Draws linear maps of DNA constructs
Reverse and complement a sequence
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Example: remap
ECLAC
E.coli lactose operon with lacI, lacZ, lacY and lacA genes.
Hin6I
TaqI
| HhaI
| Bsc4I
| Bsu6I
| |
Hin6I
| BssKI
| |
| HhaI
AciI
| | BsiSI
\ \
\ \
\
\ \ \
GACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGT
10
20
30
40
50
60
----:----|----:----|----:----|----:----|----:----|----:----|
CTGTGGTAGCTTACCGCGTTTTGGAAAGCGCCATACCGTACTATCGCGGGCCTTCTCTCA
/ /
/ /
/
/ / ///
| TaqI | Hin6I
AciI
| | ||BssKI
Bsc4I HhaI
| | |BsiSI
| | Bsu6I
| Hin6I
HhaI
# Enzymes that cut Frequency
Isoschizomers
AciI
1
Bsc4I
1
BsiSI
1
BssKI
1
Bsu6I
1
HhaI
2
Hin6I
2
HinP1I,HspAI
TaqI
1
# Enzymes that do not cut
AclI
BamHI
BceAI
Bse1I
BshI
ClaI
EcoRI
EcoRII
Hin4I
HindII
HindIII
HpyCH4IV KpnI
NotI
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Example: cirdna
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File: ../../data/data.cirp
Start
1001
End
4270
group
label
Block
1011
ex1
endlabel
label
Tick
1610
EcoR1
endlabel
label
Block
1647
endlabel
label
Tick
2459
BamH1
endlabel
label
Block
4139
ex2
endlabel
endgroup
group
label
Range
2541
Alu
endlabel
label
Range
3322
MER13
endlabel
endgroup
1362
3
8
1815
1
8
4258
3
2812
[
]
5
3497
>
<
5
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Example: plotorf
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
EMBOSS format input/output
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UFO Universal Feature Object
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Alignments
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Multiple and pairwise, many flavors (FASTA, MSF, SRS…)
Reports
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gff, swissprot, embl, pir, nbrf (with or without sequence)
Feature (UFO), SRS, motif, seqtable, excel, diffseq, listfile (USA),
etc…
Sequences (compatible with USA)
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Many!!! E.g., fasta, clustal, gcg, paup, gff, embl, swissprot, acedb,
abi, etc…
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Web interfaces
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PISE (Pasteur Institute Software Environment)
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http://www-alt.pasteur.fr/~letondal/Pise/
wEMBOSS (Belgium&Argentina) (not yet at
SIB)
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http://www.wemboss.org
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Pise
http://emboss.ch.embnet.org/Pise
a tool to generate Web interfaces for Molecular Biology programs
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
http://www.wemboss.org
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Launch Jemboss
http://emboss.ch.embnet.org/Jemboss
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Launch Jemboss
First time only…
Each time…
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Jemboss windows
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Jemboss windows other systems
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Summary
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Anonymous web access through Pise
Registered access through Jemboss
Registered access through command-line
(requires UNIX skills)
Please report problems!
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08
Exercises
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DEA Exercises web based sequence analysis
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The goal of this exercise is to use web based tools for protein sequence analysis
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List of useful links:
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a) Take this TrEMBL sequence (Q9X252) and try a BLAST against swissprot with the complete protein or
with the first 70 residues. Explain the difference. Use TMPred, SignalP, and COILS to help you.
b) Pass this sequence through PFSCAN and search all databases. Compare with this command on
ludwig-sun1/2: hits -b "prf pat pfam" tr:Q9X252
c) use the different profile, motifs, pattern databases to get more information about the domain(s) you
found.
d) How do you evaluate the PRINTS tropomyosin annotation in this TrEMBL entry (Q9WZH0)?
basic BLAST or advanced BLAST or PSI-BLAST
TMPred prediction tool for transmembrane regions (or TMHMM)
COILS prediction tool for coiled-coil regions
SignalP prediction tool for signal-peptide cleavage site
Profile, domain, motifs databases and search sites:
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PFSCAN
InterPro (Pfam, PRINTS, PROSITE, SMART)
HITS
Swiss Institute of Bioinformatics
Institut Suisse de Bioinformatique
LF-2004.08