Bootstrap and jackknife calculation resampling

1
Bootstrap and jack-knife calculation (resampling)

• General principal is to stress the data
  repeatedly and recompute the tree each time,
  looking for robust features.
• Jack-knife drop each of N sequences from the
  alignment and recompute the resulting N trees,
  testing whether they are compatible with
  original.
• Bootstrap recompute pairwise distances from a
  random sample of alignment columns (with
  replacement). Recompute the tree for each new
  distance set and see how often a particular tree
  branch is positioned the same.

(given in the distance method context, the same
methods works for any tree construction method)
2
Bootstrap (cont.)
real alignment
Similar resampling and distance derivation done
for each sequence pair, then a new tree
calculated. Repeat ad nauseum. Compare bootstrap
trees with each other.
3
The molecular clock concept

• divergence distance vs. time NOT the same!
• molecular clock is common default hypothesis
  for translating distance into time (assume that
  divergence time).
• known to be violated whenever sufficiently broad
  groups are considered.
• however, frequently approximately valid for a
  particular sequence family over relatively short
  times (e.g. most proteins in primates).

4
Things that tend to invalidate the molecular clock

• differential changes in generation time on some
  branches.
• differential changes in selective constraints on
  some branches (extreme example would be positive
  selection).
• depth of divergence - though correctable unless
  distances are too long.

5
Protein structure and alignment

• When you see a protein sequence alignment,
  notice the blocks with higher and lower
  similarity (they are almost always there).
• (most of the time) These are not simply
  stochastic variation they represent regions
  under more or less strong purifying selection.
• These blocks can vary from rather small segments
  to rather long domains (or both depending on your
  window).
• Longer blocks usually correspond to different
  protein domains (which can vary as a unit in
  selective pressure).
• Shorter blocks usually correspond to
  intra-domain structural features.

6
Kinases form a complex, diverse family
Example from a particular enzyme
(many subtypes)
(many subtypes)
7
CaM Kinase I and CaM Kinase II (CaMKII) (CaM
stands for calcium-calmodulin)

• Very similar in the kinase and calmodulin
  regulatory domains.
• CaMKI is monomeric, whereas CaMKII is a 10-12
  subunit multimer.
• CaMKII most likely arose after the CaM Kinase
  domain by fusing a multimer formation domain to
  the C-terminus.

8
CaM Kinase II structure
N
C
multimer
serine-threonine
calmodulin
formation
protein kinase
regulation
12 subunits
with the catalytic
domains facing out
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unc-43 --------------------MQLQQINSGAFSVV
RRCVHKTTGLEFAAKIINTKKLSARD rCaMKII
-------MATITCTRFTEEYQLFEELGKGAFSVVRRCVKVLAGQEYPAKI
INTKKLSARD hCaMKI MLGAVEGPRWKQAEDIRDIYDFR
DVLGTGAFSEVILAEDKRTQKLVAIKCIAKEALEGKE rCaMKI
MPGAVEGPRWKQAEDIRDIYDFRDVLGTGAFSEVILAEDKRTQKLV
AIKCIAKKALEGKE
.. . .
..   unc-43 FQKLEREARICRKLQHPNIVRLHDSIQEE
SFHYLVFDLVTGGELFEDIVAREFYSEADAS rCaMKII
HQKLEREARICRLLKHPNIVRLHDSISEEGHHYLIFDLVTGGELFEDIVA
REYYSEADAS hCaMKI GS-MENEIAVLHKIKHPNIVALD
DIYESGGHLYLIMQLVSGGELFDRIVEKGFYTERDAS rCaMKI
GS-MENEIAVLHKIKHPNIVALDDIYESGGHLYLIMQLVSGGELFD
RIVEKGFYTERDAS . . .
.. . .. . ..
  unc-43 HCIQQILESIAYCHSNGIVHRDLKPENL
LLASKAKGAAVKLADFGLAIEVN-DSEAWHGF rCaMKII
HCIQQILEAVLHCHQMGVVHRDLKPENLLLASKLKGAAVKLADFGLAIEV
EGEQQRWFGF hCaMKI RLIFQVLDAVKYLHDLGIVHRDL
KPENLLYYSLDEDSKIMISDFGLSKMED-PGSVLSTA rCaMKI
RLIFQVLDAVKYLHDLGIVHRDLKPENLLYYSLDEDSKIMISDFGL
SKMED-PGSVLSTA . ....
. . . ...
  unc-43 AGTPGYLSPEVLKKDPYSKPVDIWACGVILY
ILLVGYPPFWDEDQHRLYAQIKAGAYDYP rCaMKII
AGTPGYLSPEVLRKDPYGKPVDLWACGVILYILLVGYPPFWDEDQHRLYQ
QIKARAYDFP hCaMKI CGTPGYVAPEVLAQKPYSKAVDC
WSIGVIAYILLCGYPPFYDENDAKLFEQILKAEYEFD rCaMKI
CGTPGYVAPEVLAQKPYSKAVDCWSIGVIAYILLCGYPPFYDENDA
KLFEQILKAEYEFD ... .
. .. ..
  unc-43 SPEWDTVTPEAKSLIDSMLTVNPKKRITADQ
ALKVPWICNRERVASAIHRQDTVDCLKKF rCaMKII
SPEWDTVTPEAKDLINKMLTINPSKRITAAEALKHPWISHRSTVASCMHR
QETVDCLKKF hCaMKI SPYWDDISDSAKDFIRHLMEKDP
EKRFTCEQALQHPWIAGDTALDKNIH-QSVSEQIKKN rCaMKI
SPYWDDISDSAKDFIRHLMEKDPEKRFTCEQALQHPWIAGDTALDK
NIH-QSVSEQIKKN ..
.. . .. . . . . .
  unc-43 NARRKLKGAILTTMIATRNLSSKRSYRLTLG
AEKLVISMKNIEYWQVLLNKIFATYKIKM rCaMKII
NARRKLKGAILTTMLATRNFSGG---------------------------
--------KS hCaMKI FAKSKWKQAFNATAVVRHMR---
------------------------------------- rCaMKI
FAKSKWKQAFNATAVVRHMR--------------------------
-------------- . . . .

continued
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continued (overlapped)
unc-43 SPEWDTVTPEAKSLIDSMLTVNPKKRITADQALK
VPWICNRERVASAIHRQDTVDCLKKF rCaMKII
SPEWDTVTPEAKDLINKMLTINPSKRITAAEALKHPWISHRSTVASCMHR
QETVDCLKKF hCaMKI SPYWDDISDSAKDFIRHLMEKDP
EKRFTCEQALQHPWIAGDTALDKNIH-QSVSEQIKKN rCaMKI
SPYWDDISDSAKDFIRHLMEKDPEKRFTCEQALQHPWIAGDTALDK
NIH-QSVSEQIKKN ..
.. . .. . . . . .
  unc-43 NARRKLKGAILTTMIATRNLSSKRSYRLTLG
AEKLVISMKNIEYWQVLLNKIFATYKIKM rCaMKII
NARRKLKGAILTTMLATRNFSGG---------------------------
--------KS hCaMKI FAKSKWKQAFNATAVVRHMR---
------------------------------------- rCaMKI
FAKSKWKQAFNATAVVRHMR--------------------------
-------------- . . . .

  unc-43 KQCRNLLNKKEQGPPSTIKESSESS-QTIDD
NDSEKGGGQLKHENTVVRADGATGIVSSS rCaMKII
G--G---NKKNDG----VKESSESTNTTIEDED-----------------
---------- .
.. ..   unc-43
NSSTASKSSSTNLSAQKQDIVRVTQTLLDAISCKDFETYTRLCDTSMTCF
EPEALGNLIE rCaMKII ------------TKVRKQEIIKV
TEQLIEAISNGDFESYTKMCDPGMTAFEPEALGNLVE
.... .. ...
..   unc-43
GIEFHRFYFD--GNRKNQ-VHTTMLNPNVHIIGEDAACVAYVKLTQFLDR
NGEAHTRQSQ rCaMKII GLDFHRFYFENLWSRNSKPVHTT
ILNPHIHLMGDESACIAYIRITQYLDAGGIPRTAQSE
... . .....
..... .   unc-43
ESRVWSKKQGRWVCVHVHRSTQPSTNTTVSEF rCaMKII
ETRVWHRRDGKWQIVHFHRSGAPSVLPH----
. .. . .
(note both inter- and intra-domain differences in
conservation)
11
Protein structure basics

• proteins consist mostly of a-helices, b-sheets,
  and turns.
• the a-helices and b-sheets typically form the
  framework of the protein.
• the turns and other atypical structures often
  play important binding and catalytic roles.
• the core of the protein is hydrophobic, whereas
  the surface is usually polar or charged.
• most sharp turns (kinks) have glycine or proline.

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alpha helix
13
three-stranded antiparallel b-sheet
14
three-stranded antiparallel b-sheet, space filled
15
substrate binding cleft
rCaMKII SPEWDTVTPEAKDLINKMLTINPSKRITAAEALK
HPWISHRSTVASCMHRQETVDCLKKF rCaMKI
SPYWDDISDSAKDFIRHLMEKDPEKRFTCEQALQHPWIAGDTALDKNIH-
QSVSEQIKKN 297 ..
.. . . ... . . . . .
   rCaMKII NARRKLKGAILTTMLATRN rCaMKI
FAKSKWKQAFNATAVVRHM 316
. . . . .
 
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sliced half-way through the protein
red - charged blue - polar green - hydrophobic
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rCaMKII HQKLEREARICRLLKHPNIVRLHDSISEEGHHYL
IFDLVTGGELFEDIVAREYYSEADAS rCaMKI
GS-MENEIAVLHKIKHPNIVALDDIYESGGHLYLIMQLVSGGELFDRIVE
KGFYTERDAS 119 . . .
. ... . ..
  rCaMKII HCIQQILEAVLHCHQMGVVHRDLKPENL
LLASKLKGAAVKLADFGLAIEVEGEQQRWFGF rCaMKI
RLIFQVLDAVKYLHDLGIVHRDLKPENLLYYSLDEDSKIMISDFGLSKME
D-PGSVLSTA 178 . ..
. . . ... .
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rCaMKII HCIQQILEAVLHCHQMGVVHRDLKPENLLLASKL
KGAAVKLADFGLAIEVEGEQQRWFGF rCaMKI
RLIFQVLDAVKYLHDLGIVHRDLKPENLLYYSLDEDSKIMISDFGLSKME
D-PGSVLSTA 178 . ..
. . . ... .
    rCaMKII AGTPGYLSPEVLRKDPYGKPVDLWACGVI
LYILLVGYPPFWDEDQHRLYQQIKARAYDFP rCaMKI
CGTPGYVAPEVLAQKPYSKAVDCWSIGVIAYILLCGYPPFYDENDAKLFE
QILKAEYEFD 238 ... .
. .. ... .
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Measuring structural similarity

• Structural similarity can persist after sequence
  similarity has reached noise levels.
• More generally, how do you measure two
  structures for degree of similarity?
• Commonly used approach is root mean square
  deviation (RMSD) between the positions of matched
  backbone atoms.

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No statistically significant sequence similarity
RMSD for shared regions 3.5 Angstroms
23
Illustration of three points on a structure of
poorly known function

• gaps in alignments tend to be on surface loops
• areas of highest conservation tend to be at key
  sites (e.g. active sites of enzymes) and in core
  structural elements
• BUT when positive selection acts, binding faces
  may tend to be the parts that vary.

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MATH domain containing genes a mystery family
in C. elegans
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No assignment for Thursday. Final assignment
will be posted by this evening.