Crystallization of Kinesin Family Motor Proteins
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Motor proteins of several kinesin family groups have now been crystallized: monomeric Kinesin-1 motor domains from human,rat and Neurospora (Kull et al.,1996; Sack et al.,1997; Song et al.,2001)and dimeric rat Kinesin-1 (two motor domains connected by a short coiled-coil; Kozielski et al.,1997); the Kinesin-14 (formerly C-terminal motor)proteins Drosophila Ncd in its monomeric and dimeric forms (Sablin & Fletterick,1995,Sablin et al.,1996,1998; Kozielski et al.,1997; Yun et al.,2003),yeast KAR3 (monomer)(Gulick et al.,1998; Yun et al.,2001)and three KAR3 mutants (Yun et al.,2001),and KCBP (Vinogradova et al.,2004); the Kinesin-3 (formerly Unc104/KIF1)motor,KIF1A bound to different nucleotides (KIF1A-ADP,KIF1A-AMPPCP,KIF1A-ADP-Vi,KIF1A-AlFx)(Kikkawa et al.,2001; Nitta et al.,2004); the Kinesin-5 (formerly BimC)motor,monomeric Eg5 (Turner et al.,2001); and the Kinesin-13 (formerly MCAK)motor,PfKinI (Shipley et al.,2004).The following tables summarize the crystallization conditions and some of the crystal parameters.
Crystals of monomeric rat Kinesin-1
Table 1: Crystallization conditions for kinesin motor protein constructs
| Construct | Method | Conditions | References |
| Human Kinesin-1 hK349 | Sitting drop 4°C | 5 mg/ml protein in 50 mM Na-acetate, pH 4.6, 75 mM KCl, 3.5% (w/v) PEG 4000, 2.5 mM ATP, 10 mM MgCl2 Reservoir: 100 mM Na-acetate pH 4.6, 150 mM KCl, 7% PEG 4000, 5 mM ATP, 20 mM MgCl2 | Kull et al., 1996 |
| Ncd 335-700 | Sitting drop Room temperature | 7 mg/ml protein in 10 mM Pipes, pH 7.2, 100 mM NaCl, 1 mM EGTA, 1 mM DTT, 7% (w/v) PEG 4000, 0.3% octyl-ß-D-glucoside, 2 mM ATP, 10 mM MgCl2 Reservoir: 15% (w/v) PEG 4000, 60 mM NaCl equally buffered | Sablin & Fletterick, 1995 Sablin et al., 1996 |
| Ncd 293-700 | Hanging drop 18°C | 17 mg/ml protein in 20 mM HEPES pH 7.4, 200 mM NaCl, 10 mM MgCl2, 2 mM DTT was pre-incubated with 4 mM AMundefinedPNP or ATP for 2 hours Crystals grew in 11% PEG 8000, 0.8 M NaCl, 50 mM Na2H2PO4, pH 6.8, 7 mM DTT | Yun et al., 2003 |
| Rat Kinesin-1 rK354 | Hanging drop Room temperature | 9-14 mg/ml protein 20 mM PIPES pH 7.5, 50 mM KCl, 1 mM EGTA, 1 mM DTT, 0.9 M Li2SO4 Reservoir: 20 mM PIPES pH 7.5, 1.8 M Li2SO4 or 15 mg/ml protein in 10 mM PIPES pH 7.5, 50 mM NaCl, 1 mM EGTA, 1 mM DTT, 0.5 mM NaN3, 0.9M (NH4)2SO4 Reservoir: 1.8M (NH4)2SO4,50 mM NaCl | Kozielski et al., 1997a Sack et al., 1997 |
| Rat Kinesin-1 rK379 | Hanging drop Room temperature | 15 mg/ml protein 10 mM PIPES pH 7.5, 200 mM NaCl, 1 mM EGTA, 1 mM DTT, 0.5 mM NaN3, 0.8M (NH4)2SO4 Reservoir: 1.6M (NH4)2SO4, 200mM NaCl | Kozielski et al. 1997a,b |
| Kar3 383-729 | Microbatch Room temperature | 11 mg/ml protein 10 mM HEPES pH 7.5, 150 mM NaCl, 1 mM DTT, 1 mM MgCl2, 0.2 mM NaN3, 2 mM ADP combined 1:1 with 22% methyl ether PEG 2000, 100 mM NaCl, 2% ethylene glycol, 50 mM HEPES pH 7.0 | Gulick et al., 1998 |
| Ncd 281-700 | Sitting drop 4°C | 20 mg/ml protein in 20 mM HEPES pH 7.5, 100 mM NaCl, 1 mM EGTA, 0.7 M Li2S04, 2 mM ADP, 10 mM MgCl2 Reservoir: 20 mM HEPES pH 7.5, 1.4 M Li2SO4, 1 mM EGTA, 1 mM DTT, 10 mM MgCl2 | Sablin et al., 1998 |
| Ncd 295-700 | Hanging drop Room temperature | 5 mg/ml protein in 25 mM Na2PO4, pH 6.8, 6.8% PEG 8000, 1 M NaCl, 2 mM ATP, 3.5 mM DTT, 10 mM MgCl2 Reservoir: 25 mM Na2PO4 pH 6.8, 13.5% PEG 8000, 2 M NaCl, 7 mM DTT | Kozielski et al., 1999 |
| Eg5 1-368 HsKSP | Sitting Drop 4°C | 5 mg/ml protein in 9% PEG-3350, 50 mM PIPES pH 6.8, 100 mM NaNO3 Reservoir: 18% PEG-3350, 100 mM PIPES pH 6.8, 200 mM NaNO3 Imperfect crystals were crushed and used to seed 5 mg/ml Eg5 in 7.5% PEG-3350, 50 mM MES pH 5.6, 100 mM NaNO3 Reservoir: 15% PEG-3350, 100 mM MES pH 5.6, 200 mM NaNO3 | Turner et al., 2001 |
| KIF1A-ADP | Vapor diffusion | 2 microliters of protein (15 mg/ml) + 2 microliters of reservoir buffer (RB1) composed of 30% w/v PEG4000, 100 mM Tris-HCl pH 8.5, 200 mM sodium acetate, 8% w/v sucrose, 4 mM ADP, 10 mM MgCl2, equilibrated against RB1 for 5 d | Kikkawa et al., 2001 |
| KIF1A-AMPPCP (soaked) | Vapor diffusion | 15 mg/ml protein + reservoir buffer (RB2) composed of 27% w/v PEG4000, 100 mM MES-NaOH pH 6.5, 200 mM sodium acetate, then soaked in RB2 + 20 mM AMPPCP + 40 mM MgCl2 for 24 hrs. | Kikkawa et al., 2001 |
| KIF1A-AMPPCP (co-crystalized) | Vapor diffusion | 15 mg/ml protein co-crystalized with 5 mM AMPPCP + 20 mM MgCl2 in reservoir buffer (RB3) composed of 30% w/v PEG4000, 100 mM Tris-HCl pH 8.5, 200 mM sodium acetate | Kikkawa et al., 2001 |
| Nkin 1-355 | Sitting Drop 19°C | 7.5-15 mg/ml protein in 20 mM Tris pH 7.9, 5 mM MgCl2, 0.5 mM ADP Reservoir: 17.5% PEGMME 2000, 100 mM HEPES pH 6.5-7.5, 3% glycerol | Song et al., 2001 |
| Kar3 +N11 (WT) | Hanging Drop 18°C | 2 microliters of protein (15 mg/ml) + 2 microliters of well solution containing 20-26% PEG2000ME, 0.2 M NaCl, 50 mM HEPES buffer pH 7.0-8.0 | Yun et al., 2001 |
| Kar3 N650K | Hanging Drop 18°C | 2 microliters of protein (15 mg/ml) + 2 microliters of well solution containing 20-26% PEG2000ME, 0.2 M NaCl, 50 mM HEPES buffer pH 7.0-8.0 | Yun et al., 2001 |
| Kar3 R598A | Hanging Drop 4°C | 2 microliters of protein (15 mg/ml) + 2 microliters of well solution containing 20-26% PEG2000ME, 0.2 M NaCl, 50 mM HEPES buffer pH 7.0-8.0 | Yun et al., 2001 |
| Kar3 E631A | Hanging Drop 4°C | 2 microliters of protein (15 mg/ml) + 2 microliters of well solution containing 20-26% PEG2000ME, 0.2 M NaCl, 50 mM HEPES buffer pH 7.0-8.0 | Yun et al., 2001 |
| Ncd 293 - 700 | 18°C | 17 mg/ml protein in 20 mM HEPES pH 7.4, 200 mM NaCl, 10 mM MgCl2 and 2 mM DTT, pre-incubated with 4 mM AMP PNP or ATP for 2 h Crystals grew in 11.0% PEG 8000, 0.8 M NaCl, 50 mM Na2HPO4/NaH2PO4 (pH 6.8) and 7 mM DTT at 18°C | Yun et al., 2003 |
| KIF1A | Vapor diffusion at 20°C for 24 h | AMPPNP: 27% w/v PEG4000, 100 mM Tris-HCl pH 8.5, 200 mM sodium acetate and 3% w/v xylitol with a final concentration of 5 mM AMPPNP and 1 mM MgCl2 ADP-AlFx: 29% w/v PEG4000, 100 mM Tris-HCl pH 8.5, 200 mM sodium acetate and 3% w/v xylitol with a final concentration of 1 mM ADP, 1 mM MgCl2 and 1 mM AlF3 ADP-Vi; 28% w/v PEG4000, 100 mM Tris-HCl pH 8.5, 200 mM sodium acetate and 3% w/v xylitol with a final concentration of 1 mM ADP, 1 mM MgCl2 and 1 mM NaVO4 | Nitta et al., 2004 |
| KCBP 884 - 1252 | Sitting drop vapor diffusion at 4°C | 10 mg/mL protein in 50 mM Tris, pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 1 mM ATP, 1 mM Tris(2-carboxyethyl)-phosphine Reservoir: 20% polyethylene glycol 3350 in 0.2 M di-sodium hydrogen phosphate, pH 9.1 | Vinogradova et al., 2004 |
| KinI | Sitting drop 4°C | 10 - 20 mg/ml protein Reservoir: 1.4-1.8 M ammonium sulfate, 100 mM sodium acetate (pH 5.0), 200 mM sodium nitrate | Shipley et al., 2004 |
Table 2: Crystallographic parameters
| Construct | Space group | Unit cell | Resolution | Structural determination | Special Features |
| hK349 PDB: 1BG2 | P212121 | a=48.54 Å b=67.94 Å c=112.95 Å | 1.8 Å | Multiple isomorphous replacement | |
| rK354 PDB: 2KIN | P212121 | a=71.56 Å b=73.67 Å c=74.13 Å | 1.9 Å | Molecular replacement | |
| rK379 PDB: 3KIN | P212121 | a=72.2 Å b=91.9 Å c=141.7 Å | 3.0 Å | Multiple isomorphous replacement | |
| Kar3 382-729 PDB: 3KAR | P21 | a=44.1 Å b=81.2 Å c=48.3 Å ß=105.8° | 2.3 Å | Molecular replacement and phases of three heavy atom derivatives | |
| Ncd 335-700 Coordinates | I222 | a=127.1 Å b=122.3 Å c=68.0 Å | 2.5 Å | Multiple isomorphous replacement | |
| Ncd 281-700 PDB: 2NCD | P6122 | a= 123.0 Å b=123.0 Å c=121.1 Å | 2.5 Å | Molecular replacement | |
| Ncd 295-700 PDB: 1CZ7 | C2221 | a=116.19 Å b=148.83 Å c=261.52 Å | 2.9 Å | Molecular replacement and phases of three heavy atom derivatives | |
| Eg5 PDB: 1II6 | P21 | a=53.08 Å b=78.59 Å c=94.15 Å ß=93.84° | 2.1 Å | Molecular replacement | |
| KIF1A-ADP PDB: 1I5S | P212121 | a=41.67 Å b=51.92 Å c=157.06 Å | 2.2 Å | Molecular replacement | |
| KIF1A-AMPPCP (soaked) PDB: 1I6I | P212121 | a=41.99 Å b=56.40 Å c=156.12 Å | 2.0 Å | Molecular replacement | Motor bound to AMPPCP |
| KIF1A-AMPPCP (co-crystalized) PDB: 1IA0 | P212121 | a=42.42 Å b=55.43 Å c=157.27 Å | 1.9 Å | Molecular replacement | Motor bound to AMPPCP |
| Nkin 1-355 PDB: 1GOJ | P212121 | a=51.97 Å b=72.73 Å c=84.93 Å | 2.3 Å | Molecular replacement | |
| Kar3+N11 (WT) PDB: 1F9T | P21 | a=43.6 Å b=78.8 Å c=47.2 Å ß=105.0° | 1.5 Å | Molecular replacement | |
| Kar3 N650K PDB: 1F9U | P21 | a=43.6 Å b=78.0 Å c=47.3 Å ß=105.1° | 1.7 Å | Molecular replacement | |
| Kar3 R598A PDB: 1F9V | P21 | a=43.9 Å b=77.4 Å c=47.7 Å ß=105.9° | 1.3 Å | Molecular replacement | |
| Kar3 E631A PDB: 1F9W | P43 | a=62.9 Å c=153.6 Å | 2.5 Å | Molecular replacement | |
| Ncd 293 - 700 PDB: 1N6M | C2 | a= 162.6Å b= 66.6Å c= 94.8Å | 2.5 Å | Molecular replacement | Rotation of stalk by 75° |
| KIF1A AMPPNP: PDB: 1VFV PDB: 1VFW ADP-AlFx: PDB: 1VFX ADP-Vi: PDB: 1VFZ | AMPPNP1: P212121 AMPPNP2: P212121 ADP-AlFx: P212121 ADP-Vi: P212121 | AMPPNP1: a= 42.6 Å b= 55.2 Å c= 157.0 Å AMPPNP2: a= 42.6 Å b= 55.5 Å c= 156.7 Å ADP-AlFx: a= 41.9 Å b= 54.6 Å c= 156.8 Å ADP-Vi: a= 41.5 Å b= 51.8 Å c= 157.0 Å | AMPPNP1: 1.85 Å AMPPNP2: 2.2 Å ADP-AlFx: 2.6 Å ADP-Vi: 2.2 Å | Molecular replacement | Motor bound to AMPPNP, ADP-AlFx or ADP-Vi |
| KCBP PDB: 1SDM | P21212 | a= 95.7 Å b= 85.3 Å c= 44.5 Å | 2.3 Å | Molecular replacement | |
| KinI PDB: 1RY6 | P3221 | a= 105.6 Å c= 84.8 Å | 1.6 Å | Molecular replacement | No nucleotide bound to motor |
