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 Na2 H2 PO4 , 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 Li2 SO4 Reservoir : 20 mM PIPES pH 7.5, 1.8 M Li2 SO4 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 )2 SO4 Reservoir : 1.8M (NH4 )2 SO4 ,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 )2 SO4 Reservoir : 1.6M (NH4 )2 SO4 , 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 Li2 S04 , 2 mM ADP, 10 mM MgCl2 Reservoir : 20 mM HEPES pH 7.5, 1.4 M Li2 SO4 , 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 Na2 PO4 , pH 6.8, 6.8% PEG 8000, 1 M NaCl, 2 mM ATP, 3.5 mM DTT, 10 mM MgCl2 Reservoir : 25 mM Na2 PO4 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 Na2 HPO4 /NaH2 PO4 (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 |
P21 21 21 |
a=48.54 Å b=67.94 Å c=112.95 Å |
1.8 Å | Multiple isomorphous replacement | |
|
rK354 PDB: 2KIN |
P21 21 21 |
a=71.56 Å b=73.67 Å c=74.13 Å |
1.9 Å | Molecular replacement | |
|
rK379 PDB: 3KIN |
P21 21 21 |
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 |
P61 22 |
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 |
P21 21 21 |
a=41.67 Å b=51.92 Å c=157.06 Å |
2.2 Å | Molecular replacement | |
|
KIF1A-AMPPCP (soaked) PDB: 1I6I |
P21 21 21 |
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 |
P21 21 21 |
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 |
P21 21 21 |
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: P21 21 21 AMPPNP2: P21 21 21 ADP-AlFx: P21 21 21 ADP-Vi: P21 21 21 |
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 |
P21 21 2 |
a= 95.7 Å b= 85.3 Å c= 44.5 Å |
2.3 Å | Molecular replacement | |
|
KinI PDB: 1RY6 |
P32 21 |
a= 105.6 Å c= 84.8 Å |
1.6 Å | Molecular replacement | No nucleotide bound to motor |
Contributed by J. Muller , J. Kull and E. Mandelkow
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