RAMC 2001 – Pharmacia Corporation Company Confidential! Exploring two variables in vapor diffusion...
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RAMC 2001 – RAMC 2001 – Pharmacia Corporation Company Confidential! Pharmacia Corporation Company Confidential! Exploring two variables in vapor diffusion crystallization: plate types and crystallization scans Jennifer Pawlitz, Stephanie Shieh, Jennifer Sharamitaro, Eric Sturman, Mairi Lough, Anna Stevens Discovery Research Pharmacia Corporation Chesterfield Village, St. Louis, Missouri W hen searching for novel crystallization conditions for a protein, there is a wide array ofvariables from w hich to begin screening. O ften the protein reagentsupply is lim ited and the field ofvariables m ustbe narrow ed in order to begin crystallization trials. In the experim ents presented w e chose to explore tw o such variables: vapor diffusion plate-types and random crystallization screens. The goal of our experim ents w as to find the plate-type and the random screen w hich w ould give the m ost “hits” (crystals) when screening for initial crystallization conditions. Several types ofplates forvapor diffusion (both hanging drop and sitting drop m ethods) are com m ercially available. In these experim ents w e com pared the VD X, Q -plate, Q - plate II, and C ryschem plates (available from Hampton Research) and the CombiCloverplate (available from Emerald Biostructures). Likewise, several crystallization screens thatincorporate the use ofsparse m atrix sam pling techniques (C arterand C arter,JBC ,1979)are com m ercially available and m any labs,including ours, have designed their own random screens. R andom screens com pared in these experim ents w ere C rystalScreen I,C rystalScreen II(available from Ham pton R esearch),W izard I,W izard II(available from Em erald Biostructures) and 2Poly72 and Peg72 (Pharm acia C rystallization Laboratory). For the analysis, four proteins that have crystallized successfully in prior experiments were compared: two com m ercially available proteins (lysozym e and trypsin) and two proteins that are drug-design targets in our laboratory (a kinase, and a m atrix m etalloproteinase). R esults ofthese experim ents w ill be presented. A bstract VDX •Hampton Research •Hanging Drop •750uL Reservoir Volume Q-Plate •Hampton Research •Sitting Drop •1mL Reservoir Volume Q-Plate II •Hampton Research •Sitting Drop •750uL Reservoir Volume Cryschem •Hampton Research •Sitting Drop •500uL Reservoir Volume CombiClover •Emerald BioStructures •Sitting Drop •750uL Reservoir Volume Description of Plate Types Precipitants Salts Buffers Hampton Research Crystal Screen I 48 Conditions (used solutions 1-48) Pegs(P400, 1500, 4K, 8K), isopropanol, MPD, salts (acetates, citrates, formates, phosphates, sulfates, tartrate) acetates, chlorides, citrates, formates, phosphates, sulfates, tartrate, none Imidazole, NaAcetate, NaCacodylate, NaCitrate, NaHEPES, Tris, none pH 4.6-8.5 Hampton Research Crystal Screen II 48 Conditions (used solutions 1-48) Pegs, Monomethyl Ether Pegs, Jeffamine, organics, non-volatile organics, salts chlorides, phosphates, sulfates, tartrate, none Bicine, HEPES, MES, NaAcetate, NaCitrate, Tris, none pH 4.6-9.0 Emerald Biostructures Wizard I 48 Conditions Pegs, organics, non- volatile organics, salts NaCl, MgCl 2 , ZnAcetate, CaAcetate, Li 2 SO 4 (all at 0.2M), none CHES, HEPES, imidazole, CAPS, citrate, MES, acetate, cacodylate, Na/K phosphate, phosphate-citrate pH 4.2-10.5 Emerald Biostructures Wizard II 48 Conditions Pegs, organics, non- volatile organics, salts NaCl, MgCl 2 , ZnAcetate, CaAcetate, Li 2 SO 4 (all at 0.2M), none CHES, HEPES, imidazole, CAPS, citrate, MES, acetate, cacodylate, Na/K phosphate, phosphate-citrate pH 4.2-10.5 Pharmacia 2Poly72 72 Conditions Pegs (200, 400, 1450, 2K, 3K, 3350, 4K, 6K, 8K, 10K, 20K) Monomethyl Ether Pegs(550, 750, 2K, 5K) NH 4 Phosphate, NaCitrate, Li 2 SO 4 , NaBr , CaCl 2 , MgCl 2 , NaCl, KCl, LiCl, none NaAcetate, MES ADA, PIPES, HEPES, EPPS, TAPS, CHES pH 4.5-9.0 Pharmacia Peg72 72 Conditions Pegs (P4K, P8K), Monomethyl Ether Peg 550 CaCl 2 , NH 4 Phosphate, NH 4 Sulfate, NaCitrate, Li 2 SO 4 MgCl 2 , NaCl, NaBr, NaCl, KCl, LiCl NaAcetate, MES ADA, PIPES, HEPES, EPPS, TAPS, CHES pH 4.5-9.0 B estP late Type by W eek 0 10 20 30 40 W eek 1 W eek 3 W eek 12 Tim e Point Num ber ofC rystal Hits VDX Q -Plate Q -Plate II C ryschem Com biClover B estPlate Type by Protein 0 2 4 6 8 10 12 14 16 18 VD X Q -Plat e Q -Plate II C ryschem Com biClover Plate Type Num ber ofC rystalH its Lysozym e Trypsin Kinase MMP Description of Crystallization Scans Conclusion s •Plate Type Experiment –Combiclover plate gave the most crystal hits over all proteins –14 conditions gave hits with the Combiclover plate and no others –3 conditions gave hits with Q- plate or Q-plate II and no others –2 conditions gave hits with all trays except VDX and Combiclover –For only 5 conditions (across all proteins) did all 5 trays give hits •Crystallization Scan Experiment –Hampton Research Crystal Screen I had the best overall crystallization rate of success –Overall, there was a ~5-15% chance of crystallizing a protein from any one of the scans –Only 11 conditions (from a total of 336) yielded crystals of 2 or more of the 4 proteins tested B estC onditions from C rystallization Scan Experim ent M es,pH 6.0 0.2M C a(O Ac) 2 20% P8K W izard II#28 M es,pH 5.5 20m M (NH 4 ) 2 HPO 4 15% P1450 2P oly72 #33 M es,pH 5.5 800m M KCl 30% M M P550 Peg72 #2 Acetate,pH 4.5 N one 0.8M Na/K PO 4 W izard II#35 C acodylate,pH 6.5 0.2M Li 2 SO 4 30% P400 W izard II#12 C acodylate,pH 6.5 0.2M C a(O Ac) 2 18% P8K C rystalScreen I#46 C acodylate,pH 6.5 0.2M NaO Ac 30% P8K C rystalScreen I#28 C acodylate,pH 6.5 0.2M M g(O Ac) 2 20% P8K C rystalScreen I#18 C acodylate,pH 6.5 0.2M (NH 4 ) 2 SO 4 30% P8K C rystalScreen I#15 C itrate,pH 5.6 0.2M NH 4 OAc 30% P4K C rystalScreen I#9 Tris,pH 8.5 0.2M NaOAc 30% P4K C rystalScreen I#22 B uffer Salt Precipitant C ondition
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Transcript of RAMC 2001 – Pharmacia Corporation Company Confidential! Exploring two variables in vapor diffusion...
RAMC 2001 – RAMC 2001 – Pharmacia Corporation Company Confidential!Pharmacia Corporation Company Confidential!RAMC 2001 – RAMC 2001 – Pharmacia Corporation Company Confidential!Pharmacia Corporation Company Confidential!
Exploring two variables in vapor diffusion crystallization: plate types and crystallization scans
Jennifer Pawlitz, Stephanie Shieh, Jennifer Sharamitaro, Eric Sturman, Mairi Lough,
Anna Stevens
Discovery ResearchPharmacia Corporation
Chesterfield Village, St. Louis, Missouri
When searching for novel crystallization conditions for a protein, there is a wide array of variables from which to begin screening. Often the protein reagent supply is limited and the field of variables must be narrowed in order to begin crystallization trials. In the experiments presented we chose to explore two such variables: vapor diffusion plate-types and random crystallization screens. The goal of our experiments was to find the plate-type and the random screen which would give the most “hits” (crystals) when screening for initial crystallization conditions. Several types of plates for vapor diffusion (both hanging drop and sitting drop methods) are commercially available. In these experiments we compared the VDX, Q-plate, Q-plate II, and Cryschem plates (available from Hampton Research) and theCombiClover plate (available from Emerald Biostructures). Likewise, several crystallization screens that incorporate the use of sparse matrix sampling techniques (Carter and Carter, JBC, 1979) are commercially available and many labs, including ours, have designed their own random screens. Random screens compared in these experiments were Crystal Screen I, Crystal Screen II (available from Hampton Research), Wizard I, Wizard II (available from Emerald Biostructures) and 2Poly72 and Peg72 (Pharmacia Crystallization Laboratory). For the analysis, four proteins that have crystallized successfully in prior experiments were compared: two commercially available proteins (lysozyme and trypsin) and two proteins that are drug-design targets in our laboratory (a kinase, and a matrix metalloproteinase). Results of these experiments will be presented.
AbstractVDX
•Hampton Research
•Hanging Drop
•750uL Reservoir Volume
Q-Plate
•Hampton Research
•Sitting Drop
•1mL Reservoir Volume
Q-Plate II
•Hampton Research
•Sitting Drop
•750uL Reservoir Volume
Cryschem
•Hampton Research
•Sitting Drop
•500uL Reservoir Volume
CombiClover
•Emerald BioStructures
•Sitting Drop
•750uL Reservoir Volume
Description of Plate Types
Precipitants Salts Buffers
Hampton Research Crystal Screen I
48 Conditions (used solutions 1-48)
Pegs(P400, 1500, 4K, 8K), isopropanol, MPD, salts (acetates, citrates, formates, phosphates, sulfates, tartrate)
acetates, chlorides, citrates, formates, phosphates, sulfates, tartrate, none
Imidazole, NaAcetate, NaCacodylate, NaCitrate, NaHEPES, Tris, none
pH 4.6-8.5
Hampton Research Crystal Screen II
48 Conditions (used solutions 1-48)
Pegs, Monomethyl Ether Pegs, Jeffamine, organics, non-volatile organics, salts
chlorides, phosphates, sulfates, tartrate, none
Bicine, HEPES, MES, NaAcetate, NaCitrate, Tris, none
pH 4.6-9.0
Emerald Biostructures Wizard I
48 Conditions
Pegs, organics, non-volatile organics, salts
NaCl, MgCl2, ZnAcetate, CaAcetate, Li2SO4 (all at 0.2M), none
CHES, HEPES, imidazole, CAPS, citrate, MES, acetate, cacodylate, Na/K phosphate, phosphate-citrate
pH 4.2-10.5
Emerald Biostructures Wizard II
48 Conditions
Pegs, organics, non-volatile organics, salts
NaCl, MgCl2, ZnAcetate, CaAcetate, Li2SO4 (all at 0.2M), none
CHES, HEPES, imidazole, CAPS, citrate, MES, acetate, cacodylate, Na/K phosphate, phosphate-citrate
pH 4.2-10.5
Pharmacia 2Poly72
72 Conditions
Pegs (200, 400, 1450, 2K, 3K, 3350, 4K, 6K, 8K, 10K, 20K)
Monomethyl Ether Pegs(550, 750, 2K, 5K)
NH4Phosphate, NaCitrate, Li2SO4, NaBr , CaCl2 , MgCl2, NaCl, KCl, LiCl, none
NaAcetate, MES ADA, PIPES, HEPES, EPPS, TAPS, CHES
pH 4.5-9.0
Pharmacia Peg72
72 Conditions
Pegs (P4K, P8K), Monomethyl Ether Peg 550
CaCl2, NH4Phosphate, NH4Sulfate, NaCitrate, Li2SO4
MgCl2, NaCl, NaBr, NaCl, KCl, LiCl
NaAcetate, MES ADA, PIPES, HEPES, EPPS, TAPS, CHES
pH 4.5-9.0
Best Plate Type by Week
0
10
20
30
40
Week 1 Week 3 Week 12
Time Point
Nu
mb
er o
f C
ryst
al
Hit
s
VDX
Q-Plate
Q-Plate II
Cryschem
CombiClover
Best Plate Type by Protein
0
2
4
6
8
10
12
14
16
18
VDX
Q-P
late
Q-P
late
II
Crysc
hem
CombiC
lover
Plate Type
Nu
mb
er o
f C
ryst
al H
its
Lysozyme
Trypsin
Kinase
MMP
Description of Crystallization Scans
Conclusions
•Plate Type Experiment–Combiclover plate gave the most crystal hits over all proteins–14 conditions gave hits with the Combiclover plate and no others–3 conditions gave hits with Q-plate or Q-plate II and no others–2 conditions gave hits with all trays except VDX and Combiclover–For only 5 conditions (across all proteins) did all 5 trays give hits
•Crystallization Scan Experiment–Hampton Research Crystal Screen I had the best overall crystallization rate of success–Overall, there was a ~5-15% chance of crystallizing a protein from any one of the scans–Only 11 conditions (from a total of 336) yielded crystals of 2 or more of the 4 proteins tested
Best Conditions from Crystallization Scan Experiment
Mes, pH 6.00.2M Ca(OAc) 220%P8KWizard II #28
Mes, pH 5.520mM (NH4)2HPO4
15%P14502Poly72 #33
Mes, pH 5.5800mM KCl30%MMP550Peg72 #2
Acetate, pH 4.5None0.8M Na/K PO4
Wizard II #35
Cacodylate, pH 6.50.2M Li 2SO430%P400Wizard II #12
Cacodylate, pH 6.50.2M Ca(OAc) 218%P8KCrystal Screen I #46
Cacodylate, pH 6.50.2M NaOAc30%P8KCrystal Screen I #28
Cacodylate, pH 6.50.2M Mg(OAc) 220%P8KCrystal Screen I #18
Cacodylate, pH 6.50.2M (NH4)2SO430%P8KCrystal Screen I #15
Citrate, pH 5.60.2M NH4OAc30%P4KCrystal Screen I #9
Tris, pH 8.50.2M NaOAc30%P4KCrystal Screen I #22
BufferSaltPrecipitantCondition
