salvage methods applied to failed pfam families anna grzechnik 1, dennis carlton 1, heath klock 2...
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SALVAGE METHODS APPLIED TO FAILED PFAM FAMILIES
Anna Grzechnik1, Dennis Carlton1, Heath Klock2 Mark W. Knuth2 and Scott A. Lesley1,2*
1 The Joint Center for Structural Genomics (JCSG), The Scripps Research Institute
2 JCSG, The Genomics Institute of the Novartis Research Institute *[email protected]
NIH Bottlenecks Meeting4/15/08
• Protein families come in all shapes and sizes
• Common traits which allow us to recognize them but individual characteristics can vary greatly
• Protein families are collections of related sequences.
• Draft 1 and 2 Pfam families are large with many potential targets.
With 100 or more potential family members, we have the pick of the litter
We want these targets.
Not these targets.
Target Selection Within Pfam Assignments
PG1132I - 2.00 A resolution
Practical Filters: genomic DNA, #met, #cys
8 targets with crystals to beamline
87 targets pursued from PF07726
http://ffas.burnham.org/XtalPred-cgi/xtal.pl
Just doing more targets is not enough.
Select 82 families from 400 Draft 1 and 2 targets which failed using multiple targets.
Find the best possible targets from all genomes and process through a full panel of salvage strategies.
Low Temperature Expression
Regular Temperature Expression Solubility (37C)1 2 3 4 5 6 7 8 9 10 11 12
A -0.023 -0.024 -0.021 0.113 -0.020 -0.021 0.367 0.586 -0.015 0.012 0.233 -0.032
B -0.012 0.322 -0.023 0.834 0.027 0.706 0.057 -0.024 0.043 0.474 -0.001 -0.018
C -0.006 0.335 -0.009 0.065 0.004 0.008 0.010 0.006 -0.032 0.003 0.244 -0.009
D -0.002 -0.004 0.013 -0.009 0.509 -0.025 -0.039 -0.012 -0.009 0.145 -0.009 -0.037
E 0.019 -0.002 0.041 0.128 0.622 0.335 0.029 0.322 0.163 0.241 -0.013 -0.005
F 0.154 0.357 -0.004 0.029 0.000 0.076 -0.014 0.002 -0.021 -0.012 0.014 0.023
G 1.023 0.103 0.006 -0.003 0.033 -0.036 0.049 0.002 0.033 0.037 0.053 0.436
H 0.280 0.021 0.030 0.002 0.056 0.019 0.006 0.000
Total: 24
Low Temperature Expression Solubility (25C)1 2 3 4 5 6 7 8 9 10 11 12
A 0.038 0.196 0.047 0.617 0.083 0.386 0.423 0.428 0.186 0.179 0.580 0.106
B 0.455 0.517 0.133 0.511 0.309 0.522 0.562 0.204 0.334 0.455 0.102 0.141
C 0.129 0.393 0.246 0.296 0.156 0.187 0.240 0.174 0.137 0.182 0.131 0.055
D 0.223 0.101 0.196 0.196 0.676 0.162 0.592 0.181 0.375 0.052 0.101 0.090
E 0.044 0.137 0.111 0.340 0.523 0.761 0.305 0.556 0.295 0.095 0.076 0.081
F 0.346 0.343 0.459 0.117 0.105 0.118 0.168 0.252 0.083 0.250 0.163 0.148
G 0.782 0.620 0.140 0.134 0.195 0.061 0.490 0.115 0.168 0.077 0.213 0.569
H 0.392 0.166 0.098 0.131 0.262 0.219 0.248 0.274
Total: 78
37C
25C
188 Targets from failed 82 families
Microscreen expression and purification with total yield solubility cutoff
Low Temperature Expression
Micro-ANSEC analysis. Highly parallel, quick (12 min) run times, minimal resolution for aggregation testing
Low Temperature Expression
Making Truncations
Klock HE, Koesema EJ, Knuth MW, Lesley SA (2008) Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins 7: 982-94.
PIPE cloning facilitates making truncations and point mutationsWhat truncations to make?
•nested N- and C-terminal•bioinformatic predictions•experimentally determined
1 MRGMMLGMLAETHIHSGAGRSEGFVDLPVA 30 31 REAVTSYPVIAGSSLKGALRDAARERGMDE 60 61 SIFGDQDRAGDVLVSDARLLLLPVRSLTGS 90 91 YRWVTCPHILERLSRDMRLCGISDGFEGAS 120 121 VERGKACCTDDLNQIFLEEREFQRSNGIDG 150 151 ALIDALKKMVPHKQTASRLERQLVIISDDD 180 181 FGWFASYGLPVIARNKLDDNKKSKNLWYEE 210 211 ALAPDTLMYAMVFERKDGALGKVQSMFETK 240 241 PYLQLGGNETVGMGWFAVKILEQGEGR 267
Un
cleaved
Clea
ved
1 MRGMMLGMLAETHIHSGAGRSEGFVDLPVA 30 31 REAVTSYPVIAGSSLKGALRDAARERGMDE 60 61 SIFGDQDRAGDVLVSDARLLLLPVRSLTGS 90 91 YRWVTCPHILERLSRDMRLCGISDGFEGAS 120 121 VERGKACCTDDLNQIFLEEREFQRSNGIDG 150 151 ALIDALKKMVPHKQTASRLERQLVIISDDD 180 181 FGWFASYGLPVIARNKLDDNKKSKNLWYEE 210 211 ALAPDTLMYAMVFERKDGALGKVQSMFETK 240 241 PYLQLGGNETVGMGWFAVKILEQGEGR 267
Overlapping fragment not pursued further.
31790.801
18035.201
9017.601
PT03787B-mth-267-18-182
Partial Proteolysis
PT03787B-mth-267-46-127
Fragment was reconstructed, re-expressedand is currently in crystal trials.
Deuterium Exchange Mass Spectrometry (DXMS)
DXMS identifies regions of disorder and flexibility by mapping the location of rapid hydrogen/deuterium exchange to peptides derived from targets of interest. The information can be used to design expression constructs with improved crystallization properties (Spraggon et al., 2004).
PFAM 6249: Ethanolamine utilization protein eutQ from Salmonella typhimurium LT2
FL-protein: poor crystallization Truncation: 1.9Å structure
High-Exchange Location WT Targets Salvage ConstructsClass Number % N I C To Beam Solved Solved Rate Solved Salvage rate Total Solved Overall Success
I 30 10% n/a n/a n/a 16 13 43.3% 0 0.0% 13 43.3%II 106 35% 51 85 55 43 16 15.1% 7 7.8% 23 21.7%III 86 28% 58 78 59 32 7 8.1% 4 5.1% 11 12.8%IV 77 25% 44 27 48 32 5 6.5% 12 16.7% 17 22.1%V 8 3% 8 8 8 1 0 0.0% 0 0.0% 0 0.0%
307 161 198 170 124 41 23 64 20.8%
I
II
III
IV
V
Surface Mutagenesis
Ligand ScreeningTarget: PG1132F
Target PG1132F binds Blue resin suggesting nucleotide binding. Target is screened against nucleotide ligand library by thermofluor. Binding to ADP and GDP is observed.
185 Targets
74 Non binding targets
Targets that do not bind to the resin are screened against a 300 ligand library
111 Targets bindAffi-Gel Blue
Method courtesy of Chang Yub-Kim
Methods of ligand screening used:•Thermofluor: fluorescent detection of melting temperature•DSC: detection of differences in protein heat capacity•Stargazer: light scattering detection of aggregation
Nucleotide-LikeLigand Library:ADP GDPADP Ribose GTPAMP NADATP NADPCDP NADPH CMP TryptophanCTPcAMP
Thermofluor Ligand Screen on PG1132F
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20 30 40 50 60 70 80 90 100
Temperature (C)
Flu
ore
scen
ce None
ADP
GDP
Target PS04248
RM
Target PS03963
No mountable crystals were available from native protein. After reductive
methylation, two conditions produced harvestable crystals which are currently in finescreening.
Structure of PS04248 was solved with Reductive Methylation
RM
Reductive Methylation
UCSD & BurnhamBioinformatics CoreJohn WooleyAdam GodzikLukasz JaroszewskiSri Krishna SubramanianAndrew MorseTamara AstakhovaLian DuanPiotr KozbialDana WeekesNatasha SefcovicPrasad BurraJosie AlaoenCindy Cook
GNF & TSRICrystallomics CoreScott LesleyMark KnuthHeath KlockDennis CarltonThomas ClaytonChristina TroutMarc DellerDaniel McMullanPolat Abdubek Julie FeuerhelmJoanna HaleJessica PaulsenThamara JanaratneHope JohnsonEdward NigoghossianLinda OkachSebastian SudekGlen SpraggonSanjay AgarwallaAnna GrzechnikRegina GorskiConnie ChenDustin Ernst
TSRINMR CoreKurt Wüthrich Reto Horst Maggie JohnsonAmaranth ChatterjeeMichael GeraltWojtek AugustyniakPedro SerranoBill PedriniWilliam Placzek
TSRI Administrative CoreIan WilsonMarc ElsligerGye Won HanDavid MarcianoHenry TienLisa van Veen
Stanford /SSRLStructure Determination CoreKeith HodgsonAshley DeaconMitchell Miller Herbert AxelrodHsiu-Ju (Jessica) ChiuKevin JinChristopher RifeQingping XuSilvya OommachenHenry van den BedemScott TalafuseRonald ReyesAbhinav KumarChristine Trame
The JCSG is supported by the NIH Protein Structure Initiative (PSI) Grant U54 GM074898 from NIGMS (www.nigms.nih.gov).