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Progress in Who-Ville

Progress in Who-Ville. David Cooper Zygmunt Derewenda Lab. 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”.

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Progress in Who-Ville

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  1. Progress in Who-Ville David Cooper Zygmunt Derewenda Lab

  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 Ni Affinity  (Gel Filtration)  Concentrate Screened with JCSG+ suite with standard and alternate reservoir screns Some clones express better than others. High purity can be achieved.

  4. Streamlining our Pipeline • Using Google Calendar to schedule equipment. • Using Google Docs and Spreadsheets to track target progress.

  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.)

  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 1A WT 1Y 2A

  13. Lots of poorly diffracting crystals

  14. They are getting better. Minmin’s new crystals! SeMet Crystals of Tm1024 -1A – needs better cryo conditions.

  15. Data Sets • March Tm1024-1A (Low resolution – maybe 5.5 Tm1024-1Y w/ Br (incomplete crystal died) (Potentially I212121 a=69.5 b=120.5 c=164.5) • April Tm1024_1Y w/ Br to ~ 3.5 ish P3121 or P3221 a=70.5 b=70.5 c=81.3 Weak anomalous signal • June Tm1024-1A p3121 a=72.2 b=72.2 c=82.1 ~3.1Å Tm1024-1A p3121 a=72.7 b=72.7 c=82.1 ~3.1Å • July Tm1024-1A P3121 a=73.9 b=73.9 c=82.7 3 Wavelength Br MAD to ~2.7

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

  17. Tm0439 Tm0439-1A – Data collected

  18. TM0439-Looking for a Model

  19. Tm0439 N-terminal Domain

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

  21. 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- termini 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.

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

  23. Tm1382

  24. Tm1382

  25. 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

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

  27. Tm1679

  28. Tm1679 Tm1679 – 3Y Native SeMet Crystals

  29. 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

  30. Data collection was tricky Because of a long unit cell (307 – 314 Å)

  31. Data collection was tricky And died quickly giving low completeness. Notice the burning of this crystal. Then Minmin worked her magic.

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

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

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

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

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

  37. 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

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

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

  40. DinB (Apc36150)

  41. DinB Dimerization ~890 Å2 is burried

  42. But where are the predicted sites? In crystal contacts.

  43. DinB Metal Binding Site

  44. DinB overlap w/ 1RXQ

  45. DinB / 1RXQ Packing

  46. UVA Zygmunt Derewenda WonChan Choi Ulla Derewenda Darkhan Utepbergenov Meiying Zheng Natalya Olekhnovich Eliza Zylkiewicz Tomek Boczek Kasia Grelewska Gosia Pinkowska Michal Zawadzki Los Alamos Nat’l Lab Tom Terwilliger Chang Yub Kim UCLA David Eisenberg Luki Goldschmidt Tom Holton Lawrence Berkeley Nat’l Lab Li-Wei Hung Minmin Yu And ALL ISFI members! The ISFI is funded by NIH U54 GM074946.

  47. End of show

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

  49. Tm0493

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