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Crystallization of Kinesin Family Motor Proteins

2019.4.23
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zhaochenxu

致力于为分析测试行业奉献终身
Motor proteins of several kinesin family groups have now been crystallized: monomeric Kinesin-1 motor domains from human, rat andNeurospora (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

ConstructMethodConditionsReferences
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 335-700Sitting 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 293-700Hanging 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 AMP*PNP 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 383-729Microbatch 
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 7.0Gulick et al., 1998
Ncd 281-700Sitting 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 295-700Hanging 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-ADPVapor diffusion2 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 dKikkawa et al., 2001
KIF1A-AMPPCP
(soaked)		Vapor diffusion15 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 diffusion15 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 acetateKikkawa et al., 2001
Nkin 1-355Sitting 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 7.0-8.0Yun et al., 2001
Kar3N650KHanging 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 7.0-8.0Yun et al., 2001
Kar3 R598AHanging 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 7.0-8.0Yun et al., 2001
Kar3 E631AHanging 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 7.0-8.0Yun et al., 2001
Ncd 293 - 70018°C17 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
KIF1AVapor 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
KinISitting 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
ConstructSpace groupUnit cellResolutionStructural determinationSpecial Features
hK349

PDB: 1BG2		P212121a=48.54 Å
b=67.94 Å
c=112.95 Å		1.8 ÅMultiple isomorphous replacement
rK354

PDB: 2KIN		P212121a=71.56 Å
b=73.67 Å
c=74.13 Å		1.9 ÅMolecular replacement
rK379

PDB: 3KIN		P212121a=72.2 Å
b=91.9 Å
c=141.7 Å		3.0 ÅMultiple isomorphous replacement
Kar3 382-729


PDB: 3KAR		P21a=44.1 Å
b=81.2 Å
c=48.3 Å
ß=105.8°		2.3 ÅMolecular replacement and phases of three heavy atom derivatives
Ncd 335-700

Coordinates		I222a=127.1 Å
b=122.3 Å
c=68.0 Å		2.5 ÅMultiple isomorphous replacement
Ncd 281-700

PDB: 2NCD		P6122a= 123.0 Å
b=123.0 Å
c=121.1 Å		2.5 ÅMolecular replacement
Ncd 295-700


PDB: 1CZ7		C2221a=116.19 Å
b=148.83 Å
c=261.52 Å
2.9 ÅMolecular replacement and phases of three heavy atom derivatives
Eg5

PDB: 1II6		P21a=53.08 Å
b=78.59 Å
c=94.15 Å
ß=93.84°		2.1 ÅMolecular replacement
KIF1A-ADP


PDB: 1I5S		P212121a=41.67 Å
b=51.92 Å
c=157.06 Å		2.2 ÅMolecular replacement
KIF1A-AMPPCP
(soaked)

PDB: 1I6I		P212121a=41.99 Å
b=56.40 Å
c=156.12 Å
2.0 ÅMolecular replacementMotor bound to AMPPCP
KIF1A-AMPPCP
(co-crystalized)

PDB: 1IA0		P212121a=42.42 Å
b=55.43 Å
c=157.27 Å		1.9 ÅMolecular replacementMotor bound to AMPPCP
Nkin 1-355

PDB: 1GOJ		P212121a=51.97 Å
b=72.73 Å
c=84.93 Å
2.3 ÅMolecular replacement
Kar3+N11 (WT)


PDB: 1F9T		P21a=43.6 Å
b=78.8 Å
c=47.2 Å
ß=105.0°		1.5 ÅMolecular replacement
Kar3 N650K


PDB: 1F9U		P21a=43.6 Å
b=78.0 Å
c=47.3 Å
ß=105.1°		1.7 ÅMolecular replacement
Kar3 R598A


PDB: 1F9V		P21a=43.9 Å
b=77.4 Å
c=47.7 Å
ß=105.9°		1.3 ÅMolecular replacement
Kar3 E631A


PDB: 1F9W		P43a=62.9 Å
c=153.6 Å		2.5 ÅMolecular replacement
Ncd 293 - 700


PDB: 1N6M		C2a= 162.6Å
b= 66.6Å 
c= 94.8Å		2.5 ÅMolecular replacementRotation 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 replacementMotor bound to AMPPNP, ADP-AlFx or ADP-Vi
KCBP


PDB: 1SDM		P21212a= 95.7 Å
b= 85.3 Å
c= 44.5 Å		2.3 ÅMolecular replacement
KinI


PDB: 1RY6		P3221a= 105.6 Å
c= 84.8 Å		1.6 ÅMolecular replacementNo nucleotide bound to motor

Contributed by J. Muller, J. Kull and E. Mandelkow


If you use methods from the Kinesin site, we ask that you cite the Kinesin Home Page and authors, or the appropriate source publication in your work.


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