PLAPA

Phosphate Localization And Prediction Algorithm

How does it work?

• Generates a surface using Matt’s surface generation tool in PLOP

• Places a phosphate “ball” of -2 charge at each point

• Minimizes the position of the ball• Calculates energy• Ranks all positions and chooses the

lowest energy point – no averages among points

What Have I Done?

• Used five test cases to evaluate different strategies– 1gxc – FHA domain– 1g6g – FHA domain– 1fpr – SH2 domain– 1bm2 – SH2 domain– 1een – SH2 domain

FHA Domain

What Have I Done?

• Started by using a phosphate ball– This did not work at either of the trial radii of

1.4, 2.0, and 3.0 A, or the OPLSAA phosphate radius of 4.83 A

– Why?? (All following examples with 1BM2 only, Native is always BLUE, Decoy RED)

Radius vs Distance from Native Post Minimization

Radius of Sphere in A

Dis

tanc

e fr

om N

ativ

e

Energy of Native vs Decoy at Different Radii

Radius of Sphere in A

Ene

rgy

in K

cals

Energy of Native vs Decoy After Subtraction of LJ

Radius of Sphere in A

Ene

rgy

in K

cals

Energy of Native vs Decoy Using only Electrostatics

Radius of Sphere in A

Ene

rgy

in K

cals

Another Issue, 1G6G

1G6G Fix

• Added capping group (ACE)

• Lowest energy moved to within 5 A of native phosphate position in native vs decoy trials

Final Output for 1BM2

Distance from Native in A

Ene

rgy

in K

cals

Distance from Native with Minimization in GB and in vacuum

Protein With GB Vacuum

1BM2 0.278 0.361

1GXC 0.585 0.675

1EEN 0.805 1.966

1G6G 7.971 11.485

1FPR 11.453 6.116

Future Directions

• Investigate the two examples that are not working, haven’t had time due to CASP…

CASP Target T0196

Possible Elongation Factor from Pyrococcus furiosus

Initial AlignmentP.abyssi MLG-FFRRKKKEE-EEKI---TGKPVGKVKVENILIVGFK-TVIICEVLEGMV P.horikoshi MFK-FFKRKGEDE--KDV---TGKPVGKVKVESILKVGFR-DVIICEVLEGIV 1 AHHHHHHGSGLFDFLKRKEVKE-EEKIEILSKKPAGKVVVEEVVNIMGK-DVIIGTVESGMI TARGET 189 ----------AKILELAGFLDSYIPEPERAIDKP-FLLPIEDVFSISGRGTVVTGRVERGII 1dg1 210 ----------YNGPTLEEYLDQ-LELPPKPVDKP-LRIPIQDVYSISGVGTVPVGRVESGVL 1jny DKIWELLDAIDEYIPTPVRDVDKP-FLMPVEDVFTITGRGTVATGRIERGKV 1aipA DKIWELLDAIDEYIPTPVRDVDKP-FLMPVEDVFTITGRGTVATGRIERGKV 1b23 DKIWELLDAIDEYIRTRVRDVDKR-FLMRVEDVFTITGRGTVATGRIERGKV 1mj1 VKGKTLLEAIDA-IEQPSRPTDKP-LRLPLQDVYKIGGIGTVPVGRVETGVI 1f60 KSVQKLLDAVDTYIPVPTRDLEKP-FLLPVESVYSIPGRGTVVTGTLERGIL 1d2e ANIDVLVKAIEDFIPTPKRDPNKPMLVLRSFDVNKPKLVGGVLDGSIVQGKL 1kjz P.ab KVGYKVRK-GKK---VAGIVSMEREHK-KVEFAIPGDKIGIMLEKN-IGAEK-GDILEVFIV P.ho KVGYKVKK-GKK---VAGIVSMEREHK-KIEFAIPGDRVGMMLEKN-INAEK-DDILEVYLV 61 GVGFKVKG-PSG---IGGIVRIERNRE-KVEFAIAGDRIGISIEGK-IGKVKKGDVLEIYQT TARGET 238 KVGEEVEIVGIKET-QKSTCTGVEMFRKLLDEGRAGENVGVLLRGIKREEIERGQVLAKPGT 1dg1 260 KVGDKIVFMPAG---KVGEVRSIETHHTKMDKAEPGDNIGFNVRGVEKKDIKRGDVVGHPNN 1jny KVGDEVEIVGLAPETRRTVVTGVEMHRKTLQEGIAGDNVGVLLRGVSREEVERGQVLAKPGS 1iapA KVGDEVEIVGLAPETRKTVVTGVEMHRKTLQEGIAGDNVGLLLRGVSREEVERGQVLAKPGS 1b23 KVGDEVEIVGLARETRKTVVTGVEMHRKTLQEGIAGDNVGLLLRGVSREEVERGQVLAKRGS 1mj1 KPGMVVTFAPAG---VTTEVKSVEMHHEQLEQGVPGDNVGFNVKNVSVKEIRRGNVCGDAKN 1f60 KKGDECEFLGHSKN-IRTVVTGIEMFHKSLDRAEAGDNLGALVRGLKREDLRRGLVMAKPGS 1d2e KVGDEIEIRPGVPEPITTEIVSLQAGGQFVEEAYPGGLVGVGTKLD--PYLTKGDLMAGNVV 1kjz

* Poly His at the beginning of a sequence is usually a His tag…

Secondary Structure Prediction With and Without His Tag

Superposition on 1JNY

Ok, so it doesn’t quite match

• Genomic search – is it only a piece?

• Bacterial gene prediction

• Operon search (functional proteins are grouped onto operons in prokaryotes)

• Similar operons = more sequences for the alignment

• It’s a real protein…

Structural Alignment

• Schrodinger Structural Alignment Tool– ~jacobson/bin/dinger/utilities/structalign pdb1 pdb2 pdbN– Must use PDB files with only one chain– ~jacobson/bin/dinger/utilities/getpdb pdbID:chain (CAPS)– BUGS

• Rotation matrix a bit screwy for some pdbs• Some of your pdbs will appear in alignment but will not

appear as files at the end of the run• This can be fixed by creating a .fasta file with the

structural alignment, loading into chimera, and aligning the structures

Structural Alignment

• Whole protein structural alignment performed with CE and Schrodinger– CE provided a good alignment, but output pdb

file useless since the other domains influenced position

– Schrodinger script could not handle this

• Domain only alignment based on sequence alignment– CE could not do this– Schrodinger could…with bugs

Domain Alignment

Domain Alignment

Domain Alignment without 1KJZ

Domain Alignment without 1KJZ

Interlude

• Here is where I bitch about how chimera crashed my system about 5 times so I downloaded the linux version, in which I could not figure out how to change the background to white…so its black.

No Gaps: 1B23, 1MJ1, 1AIP

One Gap: 1DG1, 1D2E, 1EFC

Two Gaps: 1JNY, 1F60

Different Full Templates

Template After Min

1b23 -5800.713823

1mj1 -5779.877660

1jny -5764.675874

1aip -5759.879378

1d2e -5753.214127

1efc -5747.859993

1f60 -5693.832217

1dg1 -5601.769850

1kjz -3393.077255

Template After Min

1b23 -4390.297563

1jny -4388.786342

1d2e -4351.241514

1aip -4345.402888

1dg1 -4327.787431

1f60 -4212.589465

1efc -4187.567498

1mj1 -4151.266300

1kjz -1905.964341

Different Partial Templates

AlignmentP.abyssi MLG-FFRRKKKEE-EEKI---TGKPVGKVKVENILIVGFK-TVIICEVLEGMV P.horikoshi MFK-FFKRKGEDE--KDV---TGKPVGKVKVESILKVGFR-DVIICEVLEGIV 1 AHHHHHHGSGLFDFLKRKEVKE-EEKIEILSKKPAGKVVVEEVVNIMGK-DVIIGTVESGMI TARGET 189 ----------AKILELAGFLDSYIPEPERAIDKP-FLLPIEDVFSISGRGTVVTGRVERGII 1dg1 210 ----------YNGPTLEEYLDQ-LELPPKPVDKP-LRIPIQDVYSISGVGTVPVGRVESGVL 1jny DKIWELLDAIDEYIPTPVRDVDKP-FLMPVEDVFTITGRGTVATGRIERGKV 1aipA DKIWELLDAIDEYIPTPVRDVDKP-FLMPVEDVFTITGRGTVATGRIERGKV 1b23 DKIWELLDAIDEYIRTRVRDVDKR-FLMRVEDVFTITGRGTVATGRIERGKV 1mj1 VKGKTLLEAIDA-IEQPSRPTDKP-LRLPLQDVYKIGGIGTVPVGRVETGVI 1f60 KSVQKLLDAVDTYIPVPTRDLEKP-FLLPVESVYSIPGRGTVVTGTLERGIL 1d2e ANIDVLVKAIEDFIPTPKRDPNKPMLVLRSFDVNKPKLVGGVLDGSIVQGKL 1kjz P.ab KVGYKVRK-GKK---VAGIVSMEREHK-KVEFAIPGDKIGIMLEKN-IGAEK-GDILEVFIV P.ho KVGYKVKK-GKK---VAGIVSMEREHK-KIEFAIPGDRVGMMLEKN-INAEK-DDILEVYLV 61 GVGFKVKG-PSG---IGGIVRIERNRE-KVEFAIAGDRIGISIEGK-IGKVKKGDVLEIYQT TARGET 238 KVGEEVEIVGIKET-QKSTCTGVEMFRKLLDEGRAGENVGVLLRGIKREEIERGQVLAKPGT 1dg1 260 KVGDKIVFMPAG---KVGEVRSIETHHTKMDKAEPGDNIGFNVRGVEKKDIKRGDVVGHPNN 1jny KVGDEVEIVGLAPETRRTVVTGVEMHRKTLQEGIAGDNVGVLLRGVSREEVERGQVLAKPGS 1iapA KVGDEVEIVGLAPETRKTVVTGVEMHRKTLQEGIAGDNVGLLLRGVSREEVERGQVLAKPGS 1b23 KVGDEVEIVGLARETRKTVVTGVEMHRKTLQEGIAGDNVGLLLRGVSREEVERGQVLAKRGS 1mj1 KPGMVVTFAPAG---VTTEVKSVEMHHEQLEQGVPGDNVGFNVKNVSVKEIRRGNVCGDAKN 1f60 KKGDECEFLGHSKN-IRTVVTGIEMFHKSLDRAEAGDNLGALVRGLKREDLRRGLVMAKPGS 1d2e KVGDEIEIRPGVPEPITTEIVSLQAGGQFVEEAYPGGLVGVGTKLD--PYLTKGDLMAGNVV 1kjz

All Models

No Gaps: 1B23, 1MJ1, 1AIP

One Gap: 1DG1, 1D2E, 1EFC

Two Gaps: 1JNY, 1F60

Two Lowest Energy Models

1JNY and 1B23 structures

Further Directions

• Build longer models

• Look over surface of final model to see whether there is a hydrophobic patch for helix packing

• Get your suggestions…

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