MD1-34 is presented as 96 1 ml conditions / MD1-34-FX is presented as 96 x 100 µl conditions

Transcription

1 NR-LBD + NR-LBD Extension HT-96 / FX-96 MD-34 / MD-34-FX A rational approach to screening initial crystallization conditions of nuclear receptor - ligand binding domains (NR-LBDs). With all 96 reagents for nuclear receptor ligand binding domains available in one 96- well block. MD-34 is presented as 96 ml conditions / MD-34-FX is presented as 96 x 00 µl conditions Features of NR-LBD : Suitable for all types of proteins- but particularly suited to nuclear receptors. Covers a wide range of PEGS. Includes sodium formate, sodium citrate and sodium acetate carboxylic acids. Ionic strength controlled with sodium chloride and ammonium acetate ph range is between 6.5 and 8.5. Introduction Nuclear receptors form the largest family of eukaryotic transcription factors, controlling numerous processes involved in development, in the cell cycle, and in the control of important metabolic pathways. In general, they activate the transcription of sets of genes in response to the binding of cognate ligands, usually small lipophilic molecules such as steroids, vitamins, and fatty acid derivatives. Their ligand-dependent activity makes nuclear receptors obvious targets for drug design in many therapeutic areas. Intensive efforts are devoted to the discovery of ligands for orphan nuclear receptors to unravel new signalling pathways, to identify new therapeutic targets and to the optimisation of ligands for classical nuclear receptors, most of which are validated drug targets. Hence the need for a specific crystallization kit for complexes between nuclear receptor ligand-binding domains, ligands and cofactors. This screen is very effective for NR-LBDs, and not only allows the user to reproduce previously obtained results but also to crystallize new LBDs. This screen is also very effective with all other types of soluble proteins. Validation of the Screen The validity of this screen has been checked using different proteins produced in the laboratory. See the table below for these results. In designing NR- LBD, crystallographers at the Department of Biology and Structural Genomics at CNRS, Illkirch, France, have collected and analysed crystallization conditions from research on various NR-LBD proteins. Their analyses show that NRs usually crystallize in the presence of two reagent families: polyethylene glycol (PEG) and carboxylic acids in the ph range Protein Crystallization hits Human retinoid all-trans 2 receptor gamma (RAR ) Human estrogen related receptor 3 (ERR3) Ultraspiracle protein (USP) 5 Human peroxisome proliferator activating receptor gamma (PPAR ) Human vitamin D receptor (VDR) mutant Human estrogen receptor alpha (ER ) triple mutant Rat retinoid acid-related orphan receptor beta (RORβ) Human retinoid X receptor alpha (RXR- ) This screen is suitable for vapour diffusion experiments using hanging drop, sitting drop or modified microbatch techniques

2 References: Billas, I. M. et al. (200) J. Biol. Chem. 276, Bourguet, W. et al. (995) Nature 375, Brzozowski, A. M. et al. (200) J. Appl. Cryst. 34, Carter, C. W. Jr et al. (979) J. Biol. Chem. 254, Cudney B. et al. Acta Cryst. D50, Darimont, B. D. et al. (998) Genes Dev. 2, Egea, P. F. et al. (2000) EMBO J. 9, Egea, P. F. et al. (200) Mol. endocrinology in press. Gangloff et al. (200) J. Biol. Chem. 276, Greschik, H. et al. (2002) Mol. Cell 9, Jancarik J. et al. (99) J. Appl. Cryst. 24, Klaholz, B. P. et al. (998) Nat. Struct. Biol. 5, Lamour, V. et al. (2002) J. Biol. Chem. in press. Nolte, R. T. et al. (998) Nature, 395, Pike, A. C. et al. (999) EMBO J. 8, Renaud, J. P. et al. (995) Nature 378, Renaud, J. P. et al (2000) Rochel, N. et al. (2000) Mol. Cell 5, Segelke, B. W. (200) J. Cryst. Growth 232, Shiau, A. K. et al. (998) Cell 95, Stehlin, C. et al. (200) EMBO J. 20, Uppenberg, J. et al. (998) J. Biol. Chem. 273, Xu, H. E. et al (999) Mol. Cell 3, Formulation Notes: NR-LBD and NR-LBD Extension reagents are formulated using ultrapure water (>8.0 M ) and are sterile-filtered using 0.22 µm filters. No preservatives are added. Final ph may vary from that specified on the datasheet. Molecular Dimensions will be happy to discuss the precise formulation of individual reagents. Individual reagents and stock solutions for optimization are available from Molecular Dimensions. Enquiries regarding formulation, interpretation of results or optimization strategies are welcome. Please , fax or phone your query to Molecular Dimensions. Contact and product details can be found at NR-LBD and NR-LBD Extension were developed by Denis Zeyer, Sylvie Duclaud, Dino Moras and Jean-Paul Renaud at The Institute of Genetics and Molecular and Cell Biology, CNRS, UMR 704, Illkirch, France and is manufactured under an exclusive licence from CNRS, France. 2

3 NR-LBD + NR-LBD Extension HT-96 / FX-96 Conditions A D2 MD-34 / MD-34-FX Well # Conc. Salt Conc. Salt 2 Conc. Buffer ph Conc. Precipitant A 0. M Sodium HEPES % w/v PEG 8000 A2 0.9 M Sodium chloride 0. M Tris % w/v PEG 8000 A3 0.5 M Sodium chloride 0. M Bis-Tris % w/v PEG 4000 A4 0.2 M Ammonium acetate 0. M Tris % w/v PEG 6000 A5 0. M Tris % w/v PEG 8000 A6 0.2 M Sodium chloride 0. M Sodium HEPES % w/v PEG 4000 A7 0. M Sodium HEPES % w/v PEG 8000 A8 0. M Sodium HEPES % w/v PEG 6000 A9 0. M PIPES % w/v PEG 8000 A0 0. M Ammonium acetate 0. M Sodium HEPES % w/v PEG 4000 A 0. M Ammonium acetate 0. M Sodium HEPES % w/v PEG 4000 A2 0.3 M Sodium chloride 0. M Bis-Tris % w/v PEG 4000 B 0.3 M Sodium chloride 0. M Bis-Tris % w/v PEG 4000 B2 0. M PIPES % w/v PEG 4000 B3.2 M Ammonium acetate 0. M Tris % w/v PEG 4000 B4 0.4 M Sodium chloride 0. M Tris % w/v PEG 4000 B5 0.6 M Sodium chloride 0. M PIPES % w/v PEG 4000 B6 0. M Tris % w/v PEG 6000 B7 0. M Tris % w/v PEG 8000 B8 0. M PIPES % w/v PEG 4000 B9 0.2 M Sodium chloride 0. M Tris % w/v PEG 8000 B0 0.5 M Sodium chloride 0. M PIPES % w/v PEG 6000 B 0.4 M Sodium chloride 0. M PIPES % w/v PEG 4000 B2 0. M PIPES % w/v PEG 4000 C.0 M Sodium acetate trihydrate 0. M PIPES 7.0 C2.3 M Sodium citrate tribasic dihydrate 0. M Sodium HEPES 7.5 C3 3.0 M Sodium formate 0.2 M Ammonium acetate 0. M Tris 8.5 C4.0 M Sodium citrate tribasic dihydrate 0. M Ammonium acetate 0. M Tris 8.0 C5.6 M Sodium acetate trihydrate 0. M Ammonium acetate 0. M Sodium HEPES 7.5 C6.2 M Sodium citrate tribasic dihydrate 0.2 M Ammonium acetate 0. M Sodium HEPES 7.5 C7.3 M Sodium citrate tribasic dihydrate 0.4 M Sodium chloride 0. M Sodium HEPES 7.5 C8.6 M Sodium acetate trihydrate 0. M Sodium HEPES 7.5 C9 0.9 M Sodium acetate trihydrate 0.5 M Sodium chloride 0. M Tris 8.0 C0.0 M Sodium citrate tribasic dihydrate 0. M Ammonium acetate 0. M Bis-Tris 6.5 C.5 M Sodium acetate trihydrate 0.3 M Sodium chloride 0. M Sodium HEPES 7.5 C2 2.6 M Sodium formate 0.3 M Sodium chloride 0. M PIPES 7.0 D.4 M Sodium acetate trihydrate 0. M Bis-Tris 6.5 D2.5 M Sodium citrate tribasic dihydrate 0. M PIPES 7.0 D3.2 M Sodium citrate tribasic dihydrate 0.8 M Sodium chloride 0. M PIPES 7.0 D4 2.5 M Sodium formate 0. M Ammonium acetate 0. M PIPES 7.0 D5 2.0 M Sodium formate 0. M Sodium chloride 0. M Bis-Tris 6.5 D6.0 M Sodium citrate tribasic dihydrate 0.4 M Sodium chloride 0. M Bis-Tris 6.5 D7.0 M Sodium citrate tribasic dihydrate 0. M Ammonium acetate 0. M Sodium HEPES 7.5 D8.3 M Sodium acetate trihydrate 0. M Tris 8.5 D9 0.8 M Sodium acetate trihydrate 0. M Sodium HEPES 7.5 D0 3.5 M Sodium formate 0. M Tris 8.0 D 2.7 M Sodium formate 0.2 M Ammonium acetate 0. M PIPES 7.0 D2 2.2 M Sodium formate 0.5 M Sodium chloride 0. M Sodium HEPES 7.5 3

4 NR-LBD + NR-LBD Extension HT-96 / FX-96 Conditions E H2 MD-34 / MD-34-FX Well # Conc. Salt Conc. Salt 2 Conc. Buffer ph Conc. Precipitant E 0. M Ammonium acetate 0. M PIPES % v/v PEG 400 E2 0.3 M Sodium chloride 0. M Sodium HEPES % v/v PEG 400 E3 0.2 M Ammonium sulfate 0. M Bis-Tris % v/v PEG 400 E4 0.2 M Magnesium chloride hexahydrate 0. M Tris % w/v PEG 2000 MME E5 0. M Sodium chloride 0. M PIPES % w/v PEG 2000 MME E6 0.2 M Sodium chloride 0. M Bis-Tris % w/v PEG 2000 MME E7 0. M Sodium HEPES % w/v PEG 2000 MME E8 0.3 M Ammonium sulfate 0. M PIPES 7.0 % w/v PEG 4000 E9 0.5 M Ammonium sulfate 0. M PIPES % w/v PEG 4000 E0 0.4 M Sodium thiocyanate 0. M Tris % w/v PEG 4000 E 0.2 M Magnesium chloride hexahydrate 0. M Sodium HEPES % w/v PEG 4000 E2 0.2 M Magnesium chloride hexahydrate 0. M PIPES % w/v PEG 4000 F 0.2 M Sodium thiocyanate 0. M Bis-Tris % w/v PEG 4000 F2 0. M Sodium HEPES % w/v PEG 5000 MME F3 0.3 M Ammonium sulfate 0. M Tris % w/v PEG 5000 MME F4 0.2 M Ammonium acetate 0. M PIPES % w/v PEG 5000 MME F5 0.2 M Magnesium chloride hexahydrate 0. M Tris % w/v PEG 6000 F6 0.2 M Ammonium sulfate 0. M Sodium HEPES 7.5 % w/v PEG 6000 F7 0.4 M Sodium thiocyanate 0. M Bis-Tris % w/v PEG 6000 F8 0.2 M Sodium thiocyanate 0. M Bis-Tris % w/v PEG 8000 F9 0.2 M Magnesium chloride hexahydrate 0. M PIPES % w/v PEG 8000 F0 0.2 M Ammonium sulfate 0. M Bis-Tris % w/v PEG 8000 F 0. M Sodium thiocyanate 0. M PIPES % w/v PEG 8000 F2 0. M Magnesium chloride hexahydrate 0. M Sodium HEPES % w/v PEG 8000 G 0. M Sodium thiocyanate 0. M Tris % w/v PEG 0,000 G2 0. M Ammonium sulfate 0. M Sodium HEPES % w/v PEG 0,000 G3 0.2 M Ammonium acetate 0. M PIPES % w/v PEG 0,000 G4.0 M Sodium acetate trihydrate 0.2 M Sodium thiocyanate 0. M Tris 8.5 G5. M Sodium citrate tribasic dihydrate 0. M Ammonium sulfate 0. M Tris 8.0 G6 2.7 M Sodium formate 0.2 M Magnesium chloride hexahydrate 0. M Sodium HEPES 7.5 G7 0.3 M Ammonium acetate 0.8 M Sodium tartrate dibasic dihydrate 0. M Sodium HEPES 7.5 G8 0.2 M Sodium chloride 0.8 M Sodium tartrate dibasic dihydrate 0. M Bis-Tris 6.5 G9 0. M Sodium thiocyanate 0.9 M Sodium tartrate dibasic dihydrate 0. M Tris 8.0 G0 0.5 M Ammonium sulfate 0.9 M Sodium tartrate dibasic dihydrate 0. M PIPES 7.0 G 0.2 M Ammonium sulfate. M Sodium tartrate dibasic dihydrate 0. M Tris 8.0 G2 0. M Magnesium chloride hexahydrate. M Sodium tartrate dibasic dihydrate 0. M Sodium HEPES 7.5 H 0.7 M Lithium sulfate 0.2 M Sodium thiocyanate 0. M Bis-Tris 6.5 H2 0.8 M Lithium sulfate 0. M Ammonium acetate 0. M Sodium HEPES 7.5 H3.0 M Lithium sulfate 0.2 M Sodium chloride 0. M Tris 8.5 H4. M Lithium sulfate 0. M Magnesium chloride hexahydrate 0. M Sodium HEPES 7.5 H5.3 M Lithium sulfate 0. M Sodium HEPES 7.5 H6.4 M Lithium sulfate 0. M Bis-Tris 6.5 H7.3 M Ammonium sulfate 0.2 M Sodium chloride 0. M PIPES 7.0 H8.4 M Ammonium sulfate 0.2 M Ammonium acetate 0. M Sodium HEPES 7.5 H9.5 M Ammonium sulfate 0. M Magnesium chloride hexahydrate 0. M Tris 8.5 H0.6 M Ammonium sulfate 0.2 M Sodium thiocyanate 0. M PIPES 7.0 H.7 M Ammonium sulfate 0.3 M Sodium chloride 0. M Tris 8.0 H2 2.0 M Ammonium sulfate 0.2 M Sodium chloride 0. M Tris 8.0 Abbreviations: Sodium HEPES; 2-(4-(2-Hydroxyethyl)--piperazinyl)ethanesulfonic Acid Sodium Salt, MME; monomethyl ether, PEG; Polyethylene glycol, PIPES; Piperazine-,4-bis(2-ethanesulfonic acid, Tris; 2-Amino-2-(hydroxymethyl)propane-,3-diol. 4

5 Manufacturer s safety data sheets are available from our website or by scanning the QR code here: Re-Ordering details: Catalogue Description Catalogue Code NR-LBD 48 x 0 ml MD-24 NR-LBD Extension 48 x 0 ml MD-26 The Nuclear Receptor Combination 96 x 0 ml MD-27 (NR-LBD + NR-LBD Extension) NR-LBD + NR-LBD Extension HT x ml MD-34 NR-LBD + NR-LBD Extension FX x 00 µl MD-34-FX Single Reagents NR-LBD single reagents 00 ml MDSR-24-tube number NR-LBD Extension single reagents 00 ml MDSR-26-tube number NR-LBD + NR-LDB Extension HT-96 single 00 ml MDSR-34-well number reagents For NR-LBD stock reagents visit our Optimization page on our website. 5