Crystallization of Kinesin Family Motor Proteins相关交流-蚂蚁淘在线

Motorproteinsofseveralkinesinfamilygroupshavenowbeencrystallized:monomericKinesin-1motordomainsfromhuman,ratandNeurospora(Kulletal.,1996;Sacketal.,1997;Songetal.,2001)anddimericratKinesin-1(twomotordomainsconnectedbyashortcoiled-coil;Kozielskietal.,1997);theKinesin-14(formerlyC-terminalmotor)proteinsDrosophilaNcdinitsmonomericanddimericforms(Sablin&Fletterick,1995,Sablinetal.,1996,1998;Kozielskietal.,1997;Yunetal.,2003),yeastKAR3(monomer)(Gulicketal.,1998;Yunetal.,2001)andthreeKAR3mutants(Yunetal.,2001),andKCBP(Vinogradovaetal.,2004);theKinesin-3(formerlyUnc104/KIF1)motor,KIF1Aboundtodifferentnucleotides(KIF1A-ADP,KIF1A-AMPPCP,KIF1A-ADP-Vi,KIF1A-AlFx)(Kikkawaetal.,2001;Nittaetal.,2004);theKinesin-5(formerlyBimC)motor,monomericEg5(Turneretal.,2001);andtheKinesin-13(formerlyMCAK)motor,PfKinI(Shipleyetal.,2004).Thefollowingtablessummarizethecrystallizationconditionsandsomeofthecrystalparameters.

CrystalsofmonomericratKinesin-1

Table1:Crystallizationconditionsforkinesinmotorproteinconstructs

Construct	Method	Conditions	References
HumanKinesin-1hK349	Sittingdrop4°C	5mg/mlproteinin50mMNa-acetate,pH4.6,75mMKCl,3.5%(w/v)PEG4000,2.5mMATP,10mMMgCl₂Reservoir:100mMNa-acetatepH4.6,150mMKCl,7%PEG4000,5mMATP,20mMMgCl₂	Kulletal.,1996
Ncd335-700	SittingdropRoomtemperature	7mg/mlproteinin10mMPipes,pH7.2,100mMNaCl,1mMEGTA,1mMDTT,7%(w/v)PEG4000,0.3%octyl-ß-D-glucoside,2mMATP,10mMMgCl₂Reservoir:15%(w/v)PEG4000,60mMNaClequallybuffered	Sablin&Fletterick,1995Sablinetal.,1996
Ncd293-700	Hangingdrop18°C	17mg/mlproteinin20mMHEPESpH7.4,200mMNaCl,10mMMgCl₂,2mMDTTwaspre-incubatedwith4mMAMP*PNPorATPfor2hoursCrystalsgrewin11%PEG8000,0.8MNaCl,50mMNa₂H₂PO₄,pH6.8,7mMDTT	Yunetal.,2003
RatKinesin-1rK354	HangingdropRoomtemperature	9-14mg/mlprotein20mMPIPESpH7.5,50mMKCl,1mMEGTA,1mMDTT,0.9MLi₂SO₄Reservoir:20mMPIPESpH7.5,1.8MLi₂SO₄or15mg/mlproteinin10mMPIPESpH7.5,50mMNaCl,1mMEGTA,1mMDTT,0.5mMNaN₃,0.9M(NH₄)₂SO₄Reservoir:1.8M(NH₄)₂SO₄,50mMNaCl	Kozielskietal.,1997aSacketal.,1997
RatKinesin-1rK379	HangingdropRoomtemperature	15mg/mlprotein10mMPIPESpH7.5,200mMNaCl,1mMEGTA,1mMDTT,0.5mMNaN₃,0.8M(NH₄)₂SO₄Reservoir:1.6M(NH₄)₂SO₄,200mMNaCl	Kozielskietal.1997a,b
Kar3383-729	MicrobatchRoomtemperature	11mg/mlprotein10mMHEPESpH7.5,150mMNaCl,1mMDTT,1mMMgCl₂,0.2mMNaN₃,2mMADPcombined1:1with22%methyletherPEG2000,100mMNaCl,2%ethyleneglycol,50mMHEPESpH7.0	Gulicketal.,1998
Ncd281-700	Sittingdrop4°C	20mg/mlproteinin20mMHEPESpH7.5,100mMNaCl,1mMEGTA,0.7MLi₂S0₄,2mMADP,10mMMgCl₂Reservoir:20mMHEPESpH7.5,1.4MLi₂SO₄,1mMEGTA,1mMDTT,10mMMgCl₂	Sablinetal.,1998
Ncd295-700	HangingdropRoomtemperature	5mg/mlproteinin25mMNa₂PO₄,pH6.8,6.8%PEG8000,1MNaCl,2mMATP,3.5mMDTT,10mMMgCl₂Reservoir:25mMNa₂PO₄pH6.8,13.5%PEG8000,2MNaCl,7mMDTT	Kozielskietal.,1999
Eg51-368HsKSP	SittingDrop4°C	5mg/mlproteinin9%PEG-3350,50mMPIPESpH6.8,100mMNaNO₃Reservoir:18%PEG-3350,100mMPIPESpH6.8,200mMNaNO₃Imperfectcrystalswerecrushedandusedtoseed5mg/mlEg5in7.5%PEG-3350,50mMMESpH5.6,100mMNaNO₃Reservoir:15%PEG-3350,100mMMESpH5.6,200mMNaNO₃	Turneretal.,2001
KIF1A-ADP	Vapordiffusion	2microlitersofprotein(15mg/ml)+2microlitersofreservoirbuffer(RB1)composedof30%w/vPEG4000,100mMTris-HClpH8.5,200mMsodiumacetate,8%w/vsucrose,4mMADP,10mMMgCl₂,equilibratedagainstRB1for5d	Kikkawaetal.,2001
KIF1A-AMPPCP(soaked)	Vapordiffusion	15mg/mlprotein+reservoirbuffer(RB2)composedof27%w/vPEG4000,100mMMES-NaOHpH6.5,200mMsodiumacetate,thensoakedinRB2+20mMAMPPCP+40mMMgCl₂for24hrs.	Kikkawaetal.,2001
KIF1A-AMPPCP(co-crystalized)	Vapordiffusion	15mg/mlproteinco-crystalizedwith5mMAMPPCP+20mMMgCl₂inreservoirbuffer(RB3)composedof30%w/vPEG4000,100mMTris-HClpH8.5,200mMsodiumacetate	Kikkawaetal.,2001
Nkin1-355	SittingDrop19°C	7.5-15mg/mlproteinin20mMTrispH7.9,5mMMgCl₂,0.5mMADPReservoir:17.5%PEGMME2000,100mMHEPESpH6.5-7.5,3%glycerol	Songetal.,2001
Kar3+N11(WT)	HangingDrop18°C	2microlitersofprotein(15mg/ml)+2microlitersofwellsolutioncontaining20-26%PEG2000ME,0.2MNaCl,50mMHEPESbufferpH7.0-8.0	Yunetal.,2001
Kar3N650K	HangingDrop18°C	2microlitersofprotein(15mg/ml)+2microlitersofwellsolutioncontaining20-26%PEG2000ME,0.2MNaCl,50mMHEPESbufferpH7.0-8.0	Yunetal.,2001
Kar3R598A	HangingDrop4°C	2microlitersofprotein(15mg/ml)+2microlitersofwellsolutioncontaining20-26%PEG2000ME,0.2MNaCl,50mMHEPESbufferpH7.0-8.0	Yunetal.,2001
Kar3E631A	HangingDrop4°C	2microlitersofprotein(15mg/ml)+2microlitersofwellsolutioncontaining20-26%PEG2000ME,0.2MNaCl,50mMHEPESbufferpH7.0-8.0	Yunetal.,2001
Ncd293-700	18°C	17mg/mlproteinin20mMHEPESpH7.4,200mMNaCl,10mMMgCl₂and2mMDTT,pre-incubatedwith4mMAMPPNPorATPfor2hCrystalsgrewin11.0%PEG8000,0.8MNaCl,50mMNa₂HPO₄/NaH₂PO₄(pH6.8)and7mMDTTat18°C	Yunetal.,2003
KIF1A	Vapordiffusionat20°Cfor24h	AMPPNP:27%w/vPEG4000,100mMTris-HClpH8.5,200mMsodiumacetateand3%w/vxylitolwithafinalconcentrationof5mMAMPPNPand1mMMgCl₂ADP-AlFx:29%w/vPEG4000,100mMTris-HClpH8.5,200mMsodiumacetateand3%w/vxylitolwithafinalconcentrationof1mMADP,1mMMgCl₂and1mMAlF₃ADP-Vi;28%w/vPEG4000,100mMTris-HClpH8.5,200mMsodiumacetateand3%w/vxylitolwithafinalconcentrationof1mMADP,1mMMgCl₂and1mMNaVO₄	Nittaetal.,2004
KCBP884-1252	Sittingdropvapordiffusionat4°C	10mg/mLproteinin50mMTris,pH7.5,50mMNaCl,2mMMgCl₂,1mMEGTA,1mMATP,1mMTris(2-carboxyethyl)-phosphineReservoir:20%polyethyleneglycol3350in0.2Mdi-sodiumhydrogenphosphate,pH9.1	Vinogradovaetal.,2004
KinI	Sittingdrop4°C	10-20mg/mlproteinReservoir:1.4-1.8Mammoniumsulfate,100mMsodiumacetate(pH5.0),200mMsodiumnitrate	Shipleyetal.,2004

Table2:Crystallographicparameters

Construct	Spacegroup	Unitcell	Resolution	Structuraldetermination	SpecialFeatures
hK349PDB:1BG2	P2₁2₁2₁	a=48.54Åb=67.94Åc=112.95Å	1.8Å	Multipleisomorphousreplacement
rK354PDB:2KIN	P2₁2₁2₁	a=71.56Åb=73.67Åc=74.13Å	1.9Å	Molecularreplacement
rK379PDB:3KIN	P2₁2₁2₁	a=72.2Åb=91.9Åc=141.7Å	3.0Å	Multipleisomorphousreplacement
Kar3382-729PDB:3KAR	P2₁	a=44.1Åb=81.2Åc=48.3Åß=105.8°	2.3Å	Molecularreplacementandphasesofthreeheavyatomderivatives
Ncd335-700Coordinates	I222	a=127.1Åb=122.3Åc=68.0Å	2.5Å	Multipleisomorphousreplacement
Ncd281-700PDB:2NCD	P6₁22	a=123.0Åb=123.0Åc=121.1Å	2.5Å	Molecularreplacement
Ncd295-700PDB:1CZ7	C222₁	a=116.19Åb=148.83Åc=261.52Å	2.9Å	Molecularreplacementandphasesofthreeheavyatomderivatives
Eg5PDB:1II6	P2₁	a=53.08Åb=78.59Åc=94.15Åß=93.84°	2.1Å	Molecularreplacement
KIF1A-ADPPDB:1I5S	P2₁2₁2₁	a=41.67Åb=51.92Åc=157.06Å	2.2Å	Molecularreplacement
KIF1A-AMPPCP(soaked)PDB:1I6I	P2₁2₁2₁	a=41.99Åb=56.40Åc=156.12Å	2.0Å	Molecularreplacement	MotorboundtoAMPPCP
KIF1A-AMPPCP(co-crystalized)PDB:1IA0	P2₁2₁2₁	a=42.42Åb=55.43Åc=157.27Å	1.9Å	Molecularreplacement	MotorboundtoAMPPCP
Nkin1-355PDB:1GOJ	P2₁2₁2₁	a=51.97Åb=72.73Åc=84.93Å	2.3Å	Molecularreplacement
Kar3+N11(WT)PDB:1F9T	P2₁	a=43.6Åb=78.8Åc=47.2Åß=105.0°	1.5Å	Molecularreplacement
Kar3N650KPDB:1F9U	P2₁	a=43.6Åb=78.0Åc=47.3Åß=105.1°	1.7Å	Molecularreplacement
Kar3R598APDB:1F9V	P2₁	a=43.9Åb=77.4Åc=47.7Åß=105.9°	1.3Å	Molecularreplacement
Kar3E631APDB:1F9W	P4₃	a=62.9Åc=153.6Å	2.5Å	Molecularreplacement
Ncd293-700PDB:1N6M	C2	a=162.6Åb=66.6Åc=94.8Å	2.5Å	Molecularreplacement	Rotationofstalkby75°
KIF1AAMPPNP:PDB:1VFVPDB:1VFWADP-AlFx:PDB:1VFXADP-Vi:PDB:1VFZ	AMPPNP1:P2₁2₁2₁AMPPNP2:P2₁2₁2₁ADP-AlFx:P2₁2₁2₁ADP-Vi:P2₁2₁2₁	AMPPNP1:a=42.6Åb=55.2Åc=157.0ÅAMPPNP2:a=42.6Åb=55.5Åc=156.7ÅADP-AlFx:a=41.9Åb=54.6Åc=156.8ÅADP-Vi:a=41.5Åb=51.8Åc=157.0Å	AMPPNP1:1.85ÅAMPPNP2:2.2ÅADP-AlFx:2.6ÅADP-Vi:2.2Å	Molecularreplacement	MotorboundtoAMPPNP,ADP-AlFxorADP-Vi
KCBPPDB:1SDM	P2₁2₁2	a=95.7Åb=85.3Åc=44.5Å	2.3Å	Molecularreplacement
KinIPDB:1RY6	P3₂21	a=105.6Åc=84.8Å	1.6Å	Molecularreplacement	Nonucleotideboundtomotor

ContributedbyJ.Muller,J.KullandE.Mandelkow

IfyouusemethodsfromtheKinesinsite,weaskthatyoucitetheKinesinHomePageandauthors,ortheappropriatesourcepublicationinyourwork.

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