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- EMDB-5011: Crosslinked kinesin head-tail complex bound to the microtubule -

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Basic information

Entry
Database: EMDB / ID: EMD-5011
TitleCrosslinked kinesin head-tail complex bound to the microtubule
Map datanull
Sample
  • Sample: Monomeric kinesin-1 head domain crosslinked in trans to dimerized kinesin-1 tail domain, bound to the microtubule
  • Protein or peptide: microtubule
  • Protein or peptide: kinesin tail domain
  • Protein or peptide: human kinesin monomeric construct cys-lite K349
Keywordskinesin / microtubule / tail domain / cargo / switch I / regulation
Function / homologykinesin complex / Kinesin motor domain / tubulin complex / Alpha tubulin / Beta tubulin
Function and homology information
Methodhelical reconstruction / cryo EM / Resolution: 8.0 Å
AuthorsDietrich KA / Sindelar CV / Brewer PD / Downing KH / Cremo CR / Rice SE
CitationJournal: Proc Natl Acad Sci U S A / Year: 2008
Title: The kinesin-1 motor protein is regulated by a direct interaction of its head and tail.
Authors: Kristen A Dietrich / Charles V Sindelar / Paul D Brewer / Kenneth H Downing / Christine R Cremo / Sarah E Rice /
Abstract: Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular ...Kinesin-1 is a molecular motor protein that transports cargo along microtubules. Inside cells, the vast majority of kinesin-1 is regulated to conserve ATP and to ensure its proper intracellular distribution and coordination with other molecular motors. Regulated kinesin-1 folds in half at a hinge in its coiled-coil stalk. Interactions between coiled-coil regions near the enzymatically active heads at the N terminus and the regulatory tails at the C terminus bring these globular elements in proximity and stabilize the folded conformation. However, it has remained a mystery how kinesin-1's microtubule-stimulated ATPase activity is regulated in this folded conformation. Here, we present evidence for a direct interaction between the kinesin-1 head and tail. We photochemically cross-linked heads and tails and produced an 8-A cryoEM reconstruction of the cross-linked head-tail complex on microtubules. These data demonstrate that a conserved essential regulatory element in the kinesin-1 tail interacts directly and specifically with the enzymatically critical Switch I region of the head. This interaction suggests a mechanism for tail-mediated regulation of the ATPase activity of kinesin-1. In our structure, the tail makes simultaneous contacts with the kinesin-1 head and the microtubule, suggesting the tail may both regulate kinesin-1 in solution and hold it in a paused state with high ADP affinity on microtubules. The interaction of the Switch I region of the kinesin-1 head with the tail is strikingly similar to the interactions of small GTPases with their regulators, indicating that other kinesin motors may share similar regulatory mechanisms.
History
DepositionApr 14, 2008-
Header (metadata) releaseApr 15, 2008-
Map releaseApr 22, 2009-
UpdateApr 22, 2009-
Current statusApr 22, 2009Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5011_1.tif

Downloads & links

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Map

FileDownload / File: emd_5011.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationnull
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 180 pix.
= 360. Å		2 Å/pix.
x 180 pix.
= 360. Å		2 Å/pix.
x 180 pix.
= 360. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.0475 / Movie #1: 0.03
Minimum - Maximum-0.0550477 - 0.123624
Average (Standard dev.)0.00687519 (±0.0216116)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 360 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z360.000360.000360.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-127-127-127
NX/NY/NZ255255255
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.0550.1240.007

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Supplemental data

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Sample components

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Entire : Monomeric kinesin-1 head domain crosslinked in trans to dimerized...

EntireName: Monomeric kinesin-1 head domain crosslinked in trans to dimerized kinesin-1 tail domain, bound to the microtubule
Components
  • Sample: Monomeric kinesin-1 head domain crosslinked in trans to dimerized kinesin-1 tail domain, bound to the microtubule
  • Protein or peptide: microtubule
  • Protein or peptide: kinesin tail domain
  • Protein or peptide: human kinesin monomeric construct cys-lite K349

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Supramolecule #1000: Monomeric kinesin-1 head domain crosslinked in trans to dimerized...

SupramoleculeName: Monomeric kinesin-1 head domain crosslinked in trans to dimerized kinesin-1 tail domain, bound to the microtubule
type: sample / ID: 1000
Oligomeric state: One head-tail complex binds to one tubulin alpha-beta dimer
Number unique components: 3
Molecular weightTheoretical: 148 KDa

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Macromolecule #1: microtubule

MacromoleculeName: microtubule / type: protein_or_peptide / ID: 1 / Name.synonym: microtubule
Details: 13-protofilament microtubules with an asymmetric seam were selected for image processing
Oligomeric state: quasi-helical assembly / Recombinant expression: No / Database: NCBI
Source (natural)Tissue: brain / Cell: cow / Location in cell: cytoplasm
Molecular weightTheoretical: 800 KDa
SequenceGO: tubulin complex / InterPro: Alpha tubulin, Beta tubulin

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Macromolecule #2: kinesin tail domain

MacromoleculeName: kinesin tail domain / type: protein_or_peptide / ID: 2 / Name.synonym: kinesin / Details: residues 823-944 of kinesin-1 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Cell: BL21 / Location in cell: cytoplasm
Molecular weightTheoretical: 13 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceGO: kinesin complex / InterPro: Kinesin motor domain

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Macromolecule #3: human kinesin monomeric construct cys-lite K349

MacromoleculeName: human kinesin monomeric construct cys-lite K349 / type: protein_or_peptide / ID: 3 / Name.synonym: kinesin / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Cell: BL21 / Location in cell: cytoplasm
Molecular weightTheoretical: 50 MDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceGO: kinesin complex / InterPro: Kinesin motor domain

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 6.8
Details: 2.5 mM PIPES, 5 mM NaCl, 2 mM MgCl2, 1 mM EGTA, 5 mM Imidazole, 5 mM BME, and 40 uM ADP
GridDetails: 300 mesh copper grid with homemade holey carbon
VitrificationCryogen name: ETHANE / Chamber temperature: 103 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: homemade. Vitrification carried out under ambient conditions
Method: excess solution wicked away from grid before blotting and plunging immediately. Less than 0.5 seconds elapsed between blotting and plunging.

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Electron microscopy

MicroscopeJEOL 4000EX
TemperatureMin: 98 K / Max: 108 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was approximately corrected at 400,000 times magnification
DateSep 1, 2007
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.3 µm / Number real images: 347 / Average electron dose: 16 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 400 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 4.1 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: OTHER / Software - Name: SPIDER
Details: image defocus and astigmatism were determined by ctffind3
CTF correctionDetails: Integrated with fourier inversion reconstruction in the manner of FREALIGN by a customized c program
Final angle assignmentDetails: SPIDER

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Atomic model buiding 1

Initial modelPDB ID:

1bg2

SoftwareName: UCSF Chimera
DetailsProtocol: Rigid Body. the menu option Fit Model in Map from UCSF Chimera was used to generate the fit
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: real-space density

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Atomic model buiding 2

Initial modelPDB ID:

1jff

SoftwareName: UCSF Chimera
DetailsProtocol: Rigid Body. the menu option Fit Model in Map from UCSF Chimera was used to generate the fit
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: real-space density

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