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UVA

UVA. Progress in WhoVille. Surface Entropy Reduction Alters surface features that inhibit crystallization. Large, flexible residues on the surface can inhibit crystallization. Lysines and Glutamates are primarily responsible for an “entropy shield”. Lysine Glutamate Rotamers Rotamers.

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UVA

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  1. UVA Progress in WhoVille

  2. Surface Entropy ReductionAlters surface features that inhibit crystallization. • Large, flexible residues on the surface can inhibit crystallization. • Lysines and Glutamates are primarily responsible for an “entropy shield” Lysine Glutamate Rotamers 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.

  3. The Standard process

  4. 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

  5. Streamlining our Pipeline Protein 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

  6. Streamlining our Pipeline Crystallization • 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.html Can be used as a calculator for manual pipetting, too. BioRobot3000 Mosquito

  7. Tm1865 Site 1) K49, E50, E51 Site 2) K173, E174 Site 3) K25, K26, K28

  8. Tm1865 Predicted as 5’ Endonuclease V FUNCTION: 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

  9. Tm1865

  10. Tm1865 P212121 a=69.6 b=72.0 c=120.3 3 copies / ASU Solvent Content 37.3% solution # 1 with overall quality = 83.31606

  11. Tm1024 Site 1 = K45,K46 Site 2 = E98,Q100,E101 Site 3 = K63, E64

  12. Tm1024

  13. Lots of poorly diffracting crystals

  14. We are getting better. SeMet Crystals of Tm1024 -1A – needs cryo conditions.

  15. Tm0439 Site 1) E188,K119,K122 Site 2) K2, K3 Site 3) E30, K31

  16. Tm0439 1A – Data collected

  17. TM0439-Looking for a Model

  18. Tm0439 N-terminal Domain

  19. Tm0439 – C terminal Domain ~90° The C-terminal domain has some internal symmetry that may complicate MR.

  20. 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.

  21. Tm1382 Site 1) K158,E159,K160 Site 2) K77,Q78,E80 Site 3) E47, E49

  22. Tm1382

  23. Tm1382

  24. Tm1382 -2A 2 minute NaBr soak 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

  25. Tm1679 Site 1) K159,E160 Site 2) K78,E79 Site 3) K100, K101

  26. Tm1679

  27. Tm0260 Site 1) K153,E154,K155 Site 2) E10,E11 Site 3) E78,K79

  28. Tm0260

  29. Tm0493 Site 1) E80, E81, K82 Site 2) K23, K24, K25 Site 3) K88, K89, E90

  30. Tm0152 Site 1) K25,K26,K27 Site 2) K11,K12,E13 Site 3) K84

  31. Tm0152

  32. Tm0493 Site 1) E80, E81, K82 Site 2) K23, K24, K25 Site 3) K88, K89, E90

  33. 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

  34. But Not Always! 1dmt, 696 AA Cluster 3 is in an Internal cavity 1cb8, 674 AA Cluster 3 buried 1cjc, 456 AA Cluster 1 buried

  35. DinB – Apc36150 Site 1) K70, E72, E73 Site 2) E87, K88 Site 3) K42, Q43

  36. DinB (Apc36150)

  37. How it was built

  38. But where are they?

  39. DinB Dimerization ~890 Å2 is burried

  40. DinB overlap

  41. APC1446 – hypothetical protein of unknown function 144 sequences, primarily from Bacteroidetes and Bacillales identified using BLAST Prediction by 3D Jury suggests similarity to thioredoxins and related protein, but the J-score is below the suggested threshold of significance

  42. APC 1446 has an unusual putative active site sequence V V V N S V C G _C A A

  43. SER server predicts only three suitable sites AA A YY Y AA YY AA YY

  44. APC1446 crystallizes with four molecules in the asymmetric unit, generating a non-crystallographic two-fold

  45. The four molecules in the AU are virtually identical A B C D

  46. Only one of the several crystal contacts appears to be mediated by the mutated patch

  47. DALI detects structural relationship of APC1446 to the thioredoxins 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 Human - reduced DUF1094 – B. subtilis APC1446 Trypanosoma brucei

  48. Cys 53 appears to have a pKa under tight regulation by a network of specific hydrogen bonds Glu125 Cys53 Arg121 Ser51 Cys55

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