Shotgun crystallization of the Shotgun crystallization of the Thermotoga maritimaThermotoga maritima proteome proteome

Protein properties and crystallization Protein properties and crystallization conditions that correlate with conditions that correlate with

crystallization successcrystallization success

Rebecca PageRebecca PageThe Scripps Research InstituteThe Scripps Research Institute3.30.2004 – PSI, NIH3.30.2004 – PSI, NIH

Data mining for faster structure Data mining for faster structure determinationdetermination

Crystallization Conditions

Protein Properties

Data mining for faster structure Data mining for faster structure determinationdetermination

Crystallization Conditions

Protein Properties

Data mining for faster structure Data mining for faster structure determinationdetermination

Crystallization Conditions

Protein Properties

Data mining for faster structure Data mining for faster structure determinationdetermination

Crystallization Conditions

Protein Properties

• Minimize Minimize initial initial crystallization crystallization screensscreens

Data mining for faster structure Data mining for faster structure determinationdetermination

Crystallization Conditions

Protein Properties

• Minimize Minimize initial initial crystallization crystallization screensscreens

• Improve Improve target target selectionselection

Thermotoga maritimaThermotoga maritima

1877 ORFs1877 ORFs

Experimental designExperimental design

• Process all T. maritima proteins through the JCSG structure determination pipeline

• Targets are not prefiltered

• Targets are processed using identical experimental methods

Lesley, et al. (PNAS, 2002)

Thermotoga maritimaThermotoga maritima

1877 ORFs1877 ORFs

Experimental designExperimental design

A more complete, less A more complete, less biased crystallization biased crystallization

dataset for data miningdataset for data mining

Lesley, et al. (PNAS, 2002)

• 258720 crystallization experiments

• 465 of 539 (86%) proteins crystallized

• 472 of 480 (98%) conditions produced crystals

• 5546 total crystal hits

The NumbersThe Numbers

1791

539

1376

539

Targets

Data mining crystallization Data mining crystallization conditionsconditions

Minimize initial crystallization screensMinimize initial crystallization screens

Data mining crystallization Data mining crystallization conditionsconditions

Minimize initial crystallization screensMinimize initial crystallization screens

Many proteins crystallized in 5 or more Many proteins crystallized in 5 or more of the original 480 conditionsof the original 480 conditions

0 1 to 56 to 1011 to 1516 to 2021 to 2526 to 5051 or more

0

1-5

6-10

11-15

16-20

21-25

26-50

51 or more

73; 13.5%

249; 46.2%

24; 4.5%

47; 8.7%

73; 13.5%

19; 3.5%

32; 5.9%

21; 3.9%

MINCOVIterative selection algorithm that identifies minimal screens, subsets of the original 480 conditions that would have crystallized all 465 proteins

Repeat 472 times (each condition)

Slawomir Grzechnik

• 472 minimal 472 minimal screensscreens

• Each Each contained 108-contained 108-116 conditions116 conditions

• Intersection = Intersection = Core ScreenCore Screen

Identify minimalIdentify minimal crystallization screenscrystallization screens

0

20

40

60

80

100

120

140

160

180

High MWPEG

Low MWPEG

Salts Polyalcohols Organics

Core Screen

All Conditions

Core Screen• 67 conditions (14%)

• All precipitants

• 392 proteins crystallized (84%)

Expanded Core Screen• 96 conditions (20%)

• 448 proteins crystallized (96%)

Core ScreenCore ScreenBest 96 conditions crystallize 448 proteinsBest 96 conditions crystallize 448 proteins

Page, et al. (Acta Cryst D, 2003)

180

140

100

60

20

High MWPEG

Low MWPEG

Salts Poly-alcohols

Organics

Original ScreenCore Screen

Data mining protein propertiesData mining protein properties

Improve target selectionImprove target selection

Data mining protein propertiesData mining protein properties

Improve target selectionImprove target selection

20

10

15

5

Fre

qu

ency

-1.0 0.0 1.0

Gravy Index

Gravy Index- hydrophilic+ hydrophobic

Identify upper and lower bounds of crystallized proteins and use these limits in future target selection

Better target selection for JCSG pipelineBetter target selection for JCSG pipeline

Proteins with 40 or more SEG Proteins with 40 or more SEG residues rarely crystallizeresidues rarely crystallize

• SEG: Filtering to identify low complexity segments

• Long SEG segments can be unstructured

Low-complexity segments

TPPTMPPPPTT

GGGSSSSHS

PNGLPHPTPPPP

QQQGRQQQQQLK

Proteins with 40 or more SEG Proteins with 40 or more SEG residues rarely crystallizeresidues rarely crystallize

• SEG: Filtering to identify low complexity segments

• Long SEG segments can be unstructured

0 20 40 60 80 100

30

20

10

0

Number of SEG residues

% c

ryst

alli

zed

New target selectionNew target selection Characteristic Proteins

EliminatedCrystals

Eliminated

Protein Total

(1877)

Crystal Total

(465)

Length120 1 1757 464

Charged AA188 0 1602 464

Gravy204 0 1562 464

pI 57 0 1538 464

TMHMM; SignalP

538 15 1245 448

Coiled-Coil 72 4 1213 445

SEG 144 6 1187 63% 439 94%

Goal: more structures!Goal: more structures!

Crystallization Conditions

Protein Properties

Goal: more structures!Goal: more structures!

Crystallization Conditions

Protein Properties

Crystallization Conditions

Protein Properties

Goal: more structures!Goal: more structures!

Crystallization Conditions

Protein Properties

Goal: more structures!Goal: more structures!

UCSD - BICJohn WooleyAdam GodzikSusan TaylorSlawomir GrzechnikSlawomir Grzechnik Bill WestAndrew MorseJie QuyangXianhong WangJaume CanavesJaume CanavesLukasz JaroszewskiRobert SchwarzenbacherRay Bean, Josie Alaoen

SSRL - SDCKeith HodgsonAshley DeaconMitchell MillerHenry van den BedemGuenter WolfS. Michael SoltisR. Paul PhizackerleyIrimpan MathewsQingping XuAmanda PradoJohn KovarikHsiu-Ju ChiuRoss FloydInna LevinRonald ReyesFred Rezazadeh

GNF / TSRI - CCRay Stevens Ray Stevens Scott LesleyScott LesleyRebecca PageCarina GrittiniJeff VelasquezKin MoyEric SimsBernard CollinsTom Clayton Angela Walker Heath KlockHeath KlockEric KoesemaEric Hampton Jamison CampbellMike HornsbyTanya BioracDan McMullanDan McMullanKevin RodriguesMike DiDonatoMike DiDonatoAndreas KreuschAndreas KreuschGlen SpraggonGlen SpraggonMarianne PatchXiaoping DaiTerry CrossKevin RodriguesPolat AbdubekPolat AbdubekEileen AmbingEileen Ambing

TSRI - ACIan WilsonPeter KuhnMarc ElsligerFrank von DelftVandana SridharDan Taillac

Exploratory ProjectsKurt Wüthrich, TSRILinda ColumbusTouraj EtezadyMargaret JohnsonWolfgang PetiVirgil Wood, UCSD Phillip BourneBarbara CottrellRaymond DeemsJack KimDennis PantazatosGeoffrey Chang, TSRI