UVA
description
Transcript of UVA
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UVA
Progress in WhoVille
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• Large, flexible residues on the surface can inhibit crystallization.
• Lysines and Glutamates are primarily responsible for an “entropy shield”
Surface Entropy ReductionAlters surface features that inhibit crystallization.
Lysine GlutamateRotamers Rotamers
Candidate Proteins:– Soluble and purify well.
– Difficult to crystallize or diffract poorly.
– Contain a cluster of highly-entropic residues.
– Don’t have other problems.
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The Standard process
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Streamlining our Pipeline• Using Google Calendar to schedule
equipment.• Using Google Docs and Spreadsheets to
track target progress.– Will be linked to ISFI website and TargetDB
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Streamlining our PipelineProtein Expression Highlights
– Using Pepsi Bottles doubles shaker space • Now 9 proteins a day capacity
• We get ours free from the local Pepsi Bottling Plant!
– Lining centrifuge bottles with zipper bags
(Dramatically reduces harvesting time)– Growth and harvesting are done by a 2 person team
(Reduces demand on 1 individual.)– Custom Web Interface for Akta Prime Systems
http://ginsberg.med.virginia.edu/akta.html
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Streamlining our PipelineCrystallization• Alternate reservoir and standard screening by default.• Mosquito Crystallization Robot for screening.• Custom BioRobot3000 application with web interface:
Crystallization Grid Screen Generator
http://ginsberg.med.virginia.edu/grid.htmlCan be used as a calculator for manual pipetting, too.
BioRobot3000 Mosquito
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Tm1865
Site 1) K49, E50, E51 Site 2) K173, E174 Site 3) K25, K26, K28
MW 25.5
# of Residues 225
pI 8.93
Gravy Index -.21
# of Mets 4
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Predicted as 5’ Endonuclease VFUNCTION: Selectively cleaves double-stranded DNA at the second phosphodiester bond 3' to a deoxyinosine leaving behind the intact lesion on the nicked DNA. Acts in DNA repair
Tm1865
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Tm1865
WT Preliminary
Structure
1A Crystals
1Y Crystals
2A Clones Verified
2Y Crystals
3A Clones Verified
3Y Clones Verified
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Tm1865
Peak Edge RemoteWavelength 0.97876 0.97876 0.97243
Resolution 40.0 -2.5
(2.59 – 2.5)
40.0 – 2.7
(2.8 – 2.7)
40.0 -2.5
(2.59 – 2.5)
Completeness 98.5(89.6) 99.8 (98.9) 99.8(98.6)
Rsym 13.6 (52) 13.1 (45.5) 13.4 (52)
I/ 12.5 (1.6) 14.7 (2.6) 14.4 (2.35)
solution # 1 with overall quality = 83.31606
P212121 a=69.6 b=72.0 c=120.3
3 copies / ASU Solvent Content 37.3%
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Tm1024
Site 1 = K45,K46 Site 2 = E98,Q100,E101 Site 3 = K63, E64
MW 17.6 kDa
# of Residues 153
pI 5.31
Gravy Index -.14
# of Mets 2
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Tm1024
WT Small crystals
1A Diffraction
1Y Diffraction
2A Concentration problems
2Y Concentration problems
3A Concentration problems
3Y Concentration problems
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Lots of poorly diffracting crystals
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We are getting better.
SeMet Crystals of Tm1024 -1A – needs cryo conditions.
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Tm0439
Site 1) E188,K119,K122Site 2) K2, K3Site 3) E30, K31
MW 25.0 kDa
# of Residues 214
pI 5.36
Gravy Index -.38
# of Mets
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Tm0439
WT Crystals
1A Data Collected
Diffracted to ~2.5 Å
1Y Expressed
2A Crystals
2Y Expressed
3A Crystals
3Y Expressed 1A – Data collected
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TM0439-Looking for a Model
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Tm0439 N-terminal Domain
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Tm0439 – C terminal Domain
~90°
The C-terminal domain has some internal symmetry that may complicate MR.
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Tm0439
There is a lot of variation of the relative orientations of the two domains within the family. I have tried all MR with complete models (unmodified and alanine-only models), with the N- and C- terminus independently and at the same time, and with multiple models for one “ensemble” in Phaser.
I still think a solution is possible, but lets just proceed with some SeMet.
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Tm1382
Site 1) K158,E159,K160Site 2) K77,Q78,E80Site 3) E47, E49
MW 22.9 kDa
# of Residues 199
pI 4.98
Gravy Index -.31
# of Mets 4
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Tm1382WT Crystals
1A Crystals
1Y Screened – no hits
2A Crystals
2Y Screened
3A Crystals
3Y Crystals
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Tm1382
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Tm1382 -2A 2 minute NaBr soak
Spacegroup P21
Cell a=47.6 b=62.6 c=74.0 β=98.0
Resolution 50-2.6 (2.66 – 2.57)
Completeness 99.5 (96.3)
Rsym 13.2 (55.2)
Average I/ 17.5 (2.2)
There’s just not an anomalous signal.
Resl. Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.8 - 3.6 - 3.4 - 3.2 - 3.0 - 2.8 - 2.58 N(data) 461 645 818 1790 620 751 919 1208 1517 1981 2878 <I/sig> 51.9 32.5 31.1 32.4 24.7 22.2 18.1 13.3 8.9 6.0 3.2 %Complete 94.9 99.8 99.9 99.9 100.0 99.9 100.0 99.9 99.9 99.9 94.6 <d"/sig> 0.84 0.93 1.04 0.99 0.87 0.91 0.86 0.81 0.77 0.83 0.79
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Tm1679
Site 1) K159,E160Site 2) K78,E79Site 3) K100, K101
MW 28.5
# of Residues 255
pI 5.95
Gravey Index -.25
# of Mets 4
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Tm1679
WT Screened – no hits
1A Screened – no hits
1Y Screened – no hits
2A Crystals
2Y Screened – no hits
3A Screened – no hits
3Y Crystals-diffraction to 2.5
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Tm0260
Site 1) K153,E154,K155Site 2) E10,E11Site 3) E78,K79
MW 25.8 kDa
# of Residues 222
pI 5.05
Gravy Index -.36
# of Mets 8
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Tm0260
WT Screened- no hits
1A Crystals
1Y Screened – no hits
2A Crystals
2Y Screened – no hits
3A Screened – no hits
3Y Screened – no hits
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Tm0493
Site 1) E80, E81, K82 Site 2) K23, K24, K25 Site 3) K88, K89, E90
MW 27.3 kDa
# of Residues 229
pI 8.52
Gravy Index -.34
# of Mets 9
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Tm0152
Site 1) K25,K26,K27Site 2) K11,K12,E13Site 3) K84
MW 24.3 kDa
# of Residues 215
pI 8.28
Gravy Index -.3
# of Mets 2
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Tm0152
WT Screened- no hits
1A Screened- no hits
1Y Screened- no hits
2A Expressed
2Y Expressed
3A Expressed
3Y Expressed
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Tm0493
WT Purified – need repeating
1A Primers Ordered
1Y Purified
2A Clone Verified
2Y Expressed
3A Primers Ordered
3Y Clone Verified
Site 1) E80, E81, K82 Site 2) K23, K24, K25 Site 3) K88, K89, E90
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Predicted Sites are usually Surface Exposed
1dqg,134 AA 1b2v,173 AA 1c1k,217 AA 1dyp,266 AA
1fy7,273 AA 1bqc,302 AA 1ds1,323 AA 1dkq,410 AA
1cwy,500 AA 1dab,539 AA 1dab,539 AA 1cb6,689 AA
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But Not Always!
1dmt, 696 AACluster 3 is in anInternal cavity
1cb8, 674 AACluster 3 buried
1cjc, 456 AACluster 1 buried
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DinB – Apc36150
Site 1) K70, E72, E73Site 2) E87, K88Site 3) K42, Q43
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DinB (Apc36150)
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How it was built
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But where are they?
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DinB Dimerization
~890 Å2 is burried
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DinB overlap
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APC1446 – hypothetical protein of unknown function
Prediction by 3D Jury suggests similarity to thioredoxins and related protein, but the J-score is below the suggested threshold of significance
144 sequences, primarily from Bacteroidetes and Bacillales identified using BLAST
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V V V N S V C G _C A A
APC 1446 has an unusual putative active site sequence
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SER server predicts only three suitable sites
AA A
YY Y
AA
YY
AA
YY
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APC1446 crystallizes with four molecules in the asymmetric unit, generating a non-crystallographic two-fold
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The four molecules in the AU are virtually identical
A B C D
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Only one of the several crystal contacts appears to be mediated by the mutated patch
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Trypanosoma bruceiHuman - reduced DUF1094 – B. subtilis APC1446
1: 6187-A 1erv 11.7 2.2 99 105 12 0 0 7 S OXIDOREDUCTASE thioredoxin Mutant (homo sapiens) huma 2: 6187-A 1thx 11.2 2.4 101 108 23 0 0 7 S ELECTRON TRANSPORT thioredoxin (thioredoxin 2) (anaba 3: 6187-A 2d08-A 10.0 2.4 102 135 12 0 0 7 S 4: 6187-A 2b5e-A 10.0 2.6 106 483 11 0 0 11 S ISOMERASE protein disulfide-isomerase (pdi, thioredoxi 5: 6187-A 2es7-A 9.2 3.0 100 101 8 0 0 10 S ISOMERASE putative thiol-disulfide isomerase and thior 6: 6187-A 1a8y 9.2 2.6 96 338 14 0 0 9 S CALCIUM-BINDING PROTEIN calsequestrin (oryctolagus cu 7: 6187-A 1uc7-A 8.9 2.8 105 124 11 0 0 12 S OXIDOREDUCTASE thiol:disulfide interchange protein dsb 8: 6187-A 2c0e-A 8.8 2.9 99 228 24 0 0 10 S CHAPERONE windbeutel protein (wind mutant, erp29 homol 9: 6187-A 1ovn-A 8.8 2.7 98 229 24 0 0 10 S CHAPERONE windbeutel (drosophila melanogaster) fruit 10: 6187-A 2dj0-A 8.6 2.3 102 137 13 0 0 10 S STRUCTURAL GENOMICS, UNKNOWN FUNCTION thioredoxin-rela 11: 6187-A 1qgv-A 8.6 2.9 105 130 7 0 0 10 S TRANSCRIPTION spliceosomal protein u5-15kd fragment ( 12: 6187-A 1b9y-C 8.6 3.1 101 167 14 0 0 10 S SIGNALING PROTEIN transducin fragment (gt beta) transd 13: 6187-A 1zma-A 8.5 3.2 107 116 9 0 0 11 S TRANSPORT PROTEIN bacterocin transport accessory prote 14: 6187-A 1y9n-A 8.4 2.6 96 107 16 0 0 9 S 15: 6187-A 2dbc-A 8.0 3.0 101 135 11 0 0 9 S SIGNALING PROTEIN unnamed protein product fragment (pd 16: 6187-A 1v9w-A 8.0 2.4 98 130 11 0 0 9 S STRUCTURAL GENOMICS, UNKNOWN FUNCTION putative 42-9-9
DALI detects structural relationship of APC1446 to the thioredoxins
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Cys53
Cys55
Arg121
Glu125
Ser51
Cys 53 appears to have a pKa under tight regulation by a network of specific hydrogen bonds
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Tm043922.5% identity in 222 residues overlap; Score: 87.02di3 - 8 VMDWVTEELRSGRLKIGDHLPSERALSETLGVSRSSLREALRVLEALGTISTATGSGPRSTm0439- 13 VYNLLKEMILNHELKLGEKL-NVRELSEKLGISFTPVRDALLQLATEGLVKVV----PRV - * * ** * * * *** ** * * ** * * **
2di3 - 68 GTIITAAPGQALSLSVTLQLVTNQVGHHDIYETRQLLEGWAALHSSAERGDWDVAEALLETm0439- 68 GFFVTDVDEKFIRETI---------------ETRIMMEVFCLENYFDKIAGSEELLEIKG - * * *** *
2di3 - 128 KMDD--PSLPLEDFLRFDAEFHVVISKGAENPLISTLMEAL--RLSVADHTVARARALPDTm0439- 113 EIDDVEKSAKREIFDDSDERLHKLFIRASGNELIISLYEKIWDRIDLVRHLNERYVVS-- - ** * * * * * * ** * * * * *
2di3 - 184 WRATSARLQKEHRAILAALRAGESTVAATLIKEHIEGYYEETTm0439- 171 --------NREHKELIERIISGDKEGAIEKLKEHLKNVEAET