Pfam a resource for remote homology domain identification et al NAR 2014.
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Transcript of Pfam a resource for remote homology domain identification et al NAR 2014.
Build SEED MSA ofrepresentative members
Build Profile-HMM
Search UniProtKB
AnnotateEMBO Workshop, Cape Town, 2014
Building familiesIdentify target
QCs and fix Significance thresholds
Abandon
Abandon
Old Family
New Family
EMBO Workshop, Cape Town, 2014
SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN
QC: family overlaps
EMBO Workshop, Cape Town, 2014
Old Family
New Family
SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN
A – Old and New family are evolutionary related nature overlaps, profile-profile, functional residues, functional annotation, structure
QC: family overlaps
EMBO Workshop, Cape Town, 2014
A – Old and New family are evolutionary related
• Solution 1: Merge
Old Family
New Family
SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN
QC: family overlaps
EMBO Workshop, Cape Town, 2014
A – Old and New family are evolutionary related
• Solution 2: Create/Add to clan
Old Family
New Family
SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN
ClanQC: family overlaps
EMBO Workshop, Cape Town, 2014
A – Old and New family are NOT evolutionary related-> then overlaps might be false positives
Old Family
New Family
SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN
QC: family overlaps
A – Old and New family are NOT evolutionary related
Old Family
New Family
SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN
• Solution 1: Separate (expunge seqs from SEED, trim ends, raise threshold)
QC: family overlaps
A – Old and New family are NOT evolutionary related
Old Family
New Family
SNLVMYIVIIIHWNACVFYSISKAIGFGNDTWVYPDINDPEFGRLARKYVYSLYWSTLTLTTIGETPPPVRDSEYVFVVVDFLIGVLIFATIVGNIGSMISN
• Solution 2: Manually Edit (no change to family but sequence removed)
QC: family overlaps
• Overlaps
• Hits Score vs Taxonomic distribution
• Known annotation (e.g. functional/structural residues)
• Known structures
• …
EMBO Workshop, Cape Town, 2014
False positive detection
Build SEED MSA ofrepresentative members
Build Profile-HMM
Search UniProtKB
AnnotateEMBO Workshop, Cape Town, 2014
Building familiesIdentify target
QCs and fix Significance thresholds
Abandon
Abandon
• A - Domain• B - Metal
stabilised domain• C - 7 repeats form
domain• D - 9 repeats form
domain could be unlimited number
A B
C D
Domain and repeats
EMBO Workshop, Cape Town, 2014
Example: Lipoprotein attachment site, LPAM_1
Alignment coloured by Residue-type
Motifs
EMBO Workshop, Cape Town, 2014
TUM, January 2013
Some caveats
• Identifying repeats is challenging, especially with HMMER3 ->local
• Functional diversity within families and clans
• Domains of Unknown Function
• Family boundaries if no structure available
EMBO Workshop, Cape Town, 2014
TUM, January 2013
Comparison of Enolase clan/superfamily in Pfam and SFLD
SFLD: Akiva et al. NAR 2013Picture courtesy of Patsy Babbit (UCSF)
from the Pfam blog: at http://xfam.wordpress.com/tag/pfam/
How far from covering the sequence space: H. sapiens
EMBO Workshop, Cape Town, 2014
TUM, January 2013
2KX7
Pick a target region
OPEN Chimera1.
File -> Open “2KX7.pdb”2.
EMBO Workshop, Cape Town, 2014
TUM, January 2013
SELECT “2KX7.pdb (#0.1) chain A”
Actions-> Ribbon-> hide
2KX7 model 1
1.
Actions -> Ribbon -> show
2.
3.
EMBO Workshop, Cape Town, 2014
Pick a target region
TUM, January 2013Schmöe et al. Structure 2011
2KX7
EMBO Workshop, Cape Town, 2014
Rcs-signaling systembacterial two component system (sensor kinase +response regulator)
TUM, January 2013EMBO Workshop, Cape Town, 2014
Pick a target regionLook-up UniprotKB ID: P39838 on the Pfam website (http://pfam.xfam.org)
TUM, January 2013EMBO Workshop, Cape Town, 2014
Pick a target regionLook-up UniprotKB ID: P39838 on the Pfam website (http://pfam.xfam.org)
TUM, January 2013
2KX7
EMBO Workshop, Cape Town, 2014Schmöe et al. Structure 2011
HK
S
ABL
HPt
Pick a target region
TUM, January 2013
2KX7
EMBO Workshop, Cape Town, 2014Schmöe et al. Structure 2011
HK
S
ABL
HPt
Pick a target region
Look for homologs
EMBO Workshop, Cape Town, 2014
http://hmmer.janelia.org
Click Start
HMMER website: Finn et al. NAR 2011
Look for homologs
EMBO Workshop, Cape Town, 2014
http://hmmer.janelia.org
Choose “Marco-Data/Other/2KX7.fasta”
Download aligned hits
EMBO Workshop, Cape Town, 2014
CLICK on Download and then on Aligned FASTA1.
Save as “RcsD-ABL-hmmer-ali.fasta”2.