UVA

Progress in WhoVille

• 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.

The Standard process

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

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

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

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

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

Tm1865

WT Preliminary

Structure

1A Crystals

1Y Crystals

2A Clones Verified

2Y Crystals

3A Clones Verified

3Y Clones Verified

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%

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

Tm1024

WT Small crystals

1A Diffraction

1Y Diffraction

2A Concentration problems

2Y Concentration problems

3A Concentration problems

3Y Concentration problems

Lots of poorly diffracting crystals

We are getting better.

SeMet Crystals of Tm1024 -1A – needs cryo conditions.

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

Tm0439

WT Crystals

1A Data Collected

Diffracted to ~2.5 Å

1Y Expressed

2A Crystals

2Y Expressed

3A Crystals

3Y Expressed 1A – Data collected

TM0439-Looking for a Model

Tm0439 N-terminal Domain

Tm0439 – C terminal Domain

~90°

The C-terminal domain has some internal symmetry that may complicate MR.

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.

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

Tm1382WT Crystals

1A Crystals

1Y Screened – no hits

2A Crystals

2Y Screened

3A Crystals

3Y Crystals

Tm1382

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

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

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

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

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

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

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

Tm0152

WT Screened- no hits

1A Screened- no hits

1Y Screened- no hits

2A Expressed

2Y Expressed

3A Expressed

3Y Expressed

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

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

But Not Always!

1dmt, 696 AACluster 3 is in anInternal cavity

1cb8, 674 AACluster 3 buried

1cjc, 456 AACluster 1 buried

DinB – Apc36150

Site 1) K70, E72, E73Site 2) E87, K88Site 3) K42, Q43

DinB (Apc36150)

How it was built

But where are they?

DinB Dimerization

~890 Å2 is burried

DinB overlap

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

V V V N S V C G _C A A

APC 1446 has an unusual putative active site sequence

SER server predicts only three suitable sites

AA A

YY Y

AA

YY

AA

YY

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

The four molecules in the AU are virtually identical

A B C D

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

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

Cys53

Cys55

Arg121

Glu125

Ser51

Cys 53 appears to have a pKa under tight regulation by a network of specific hydrogen bonds

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