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- EMDB-3445: cryo-electron microscopy reconstruction of microtubule-bound S.po... -

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Entry
Database: EMDB / ID: 3445
Titlecryo-electron microscopy reconstruction of microtubule-bound S.pombe kinesin-5 motor domain in the AMPPNP state
Map dataCryo-electron microscopy of microtubule-bound S. pombe kinesin-5 motor domain in the AMPPNP state
Samplemicrotubule-bound S.pombe kinesin-5 motor domain in the AMPPNP state:
Tubulin alpha-1D chain / Tubulin beta-2B chain / Kinesin-like protein cut7 / (ligand) x 5
Function / homologyTubulin, conserved site / Kinesin motor domain / Tubulin-beta mRNA autoregulation signal. / Tubulin subunits alpha, beta, and gamma signature. / Tubulin C-terminal domain / Kinesin motor domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Kinesin motor domain superfamily / Tubulin ...Tubulin, conserved site / Kinesin motor domain / Tubulin-beta mRNA autoregulation signal. / Tubulin subunits alpha, beta, and gamma signature. / Tubulin C-terminal domain / Kinesin motor domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Kinesin motor domain superfamily / Tubulin / Alpha tubulin / Kinesin motor domain profile. / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding site / Tubulin/FtsZ, GTPase domain superfamily / Tubulin/FtsZ, 2-layer sandwich domain / Kinesin motor domain, conserved site / Tubulin, C-terminal / Kinesin motor domain signature. / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Kinesin-like protein / RHO GTPases Activate Formins / Kinesins / Kinesins / Carboxyterminal post-translational modifications of tubulin / The role of GTSE1 in G2/M progression after G2 checkpoint / Mitotic Prometaphase / COPI-independent Golgi-to-ER retrograde traffic / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / RHO GTPases activate IQGAPs / MHC class II antigen presentation / Intraflagellar transport / Cilium Assembly / Hedgehog 'off' state / Recycling pathway of L1 / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / P-loop containing nucleoside triphosphate hydrolase / mitotic spindle pole body separation / microtubule plus-end directed mitotic chromosome migration / mitotic spindle midzone assembly / mitotic spindle midzone / mitotic spindle pole body / condensed nuclear chromosome kinetochore / ATP-dependent microtubule motor activity, plus-end-directed / positive regulation of axon guidance / microtubule associated complex / meiotic spindle / kinesin complex / microtubule motor activity / mitotic spindle assembly / microtubule-based movement / microtubule-based process / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / microtubule cytoskeleton / spindle microtubule / mitotic cell cycle / microtubule / microtubule binding / ATPase activity / GTPase activity / cell division / GTP binding / protein heterodimerization activity / ATP binding / nucleus / cytoplasm / Kinesin-like protein cut7 / Tubulin alpha-1D chain / Tubulin beta-2B chain
Function and homology information
SourceBovine (cattle) / Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Methodsingle particle reconstruction / cryo EM / 9.3 Å resolution
AuthorsGoulet A / Moores CA / Cross RA
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2016
Title: Schizosaccharomyces pombe kinesin-5 switches direction using a steric blocking mechanism.
Authors: Mishan Britto / Adeline Goulet / Syeda Rizvi / Ottilie von Loeffelholz / Carolyn A Moores / Robert A Cross
Validation ReportPDB-ID: 5m5i

SummaryFull report
PDB-ID: 5m5l

SummaryFull report
PDB-ID: 5m5m

SummaryFull report
PDB-ID: 5m5n

SummaryFull report
PDB-ID: 5m5o

SummaryFull report
About validation report
DateDeposition: Oct 21, 2016 / Header (metadata) release: Nov 30, 2016 / Map release: Nov 30, 2016 / Last update: Jun 20, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1
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  • Surface view with fitted model
  • Atomic models: : PDB-5m5i
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5m5l
  • Surface level: 1
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  • Surface view with fitted model
  • Atomic models: : PDB-5m5m
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5m5n
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-5m5o
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3445.map.gz (map file in CCP4 format, 501 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
50 pix
2.2 Å/pix.
= 110. Å
50 pix
2.2 Å/pix.
= 110. Å
50 pix
2.2 Å/pix.
= 110. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2 Å
Density
Contour Level:1.0 (by author), 1 (movie #1):
Minimum - Maximum-5.693818 - 8.505580999999999
Average (Standard dev.)-0.026976306 (1.3084207)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions505050
Origin94.055.086.0
Limit143.0104.0135.0
Spacing505050
CellA=B=C: 110.0 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z505050
origin x/y/z0.0000.0000.000
length x/y/z110.000110.000110.000
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS559486
NC/NR/NS505050
D min/max/mean-5.6948.506-0.027

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Supplemental data

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Sample components

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Entire microtubule-bound S.pombe kinesin-5 motor domain in the AMPPNP state

EntireName: microtubule-bound S.pombe kinesin-5 motor domain in the AMPPNP state
Number of components: 9

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Component #1: protein, microtubule-bound S.pombe kinesin-5 motor domain in the ...

ProteinName: microtubule-bound S.pombe kinesin-5 motor domain in the AMPPNP state
Recombinant expression: No
MassTheoretical: 140 kDa

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Component #2: protein, Tubulin alpha-1D chain

ProteinName: Tubulin alpha-1D chain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 50.107238 kDa
SourceSpecies: Bovine (cattle)

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Component #3: protein, Tubulin beta-2B chain

ProteinName: Tubulin beta-2B chain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 49.90777 kDa
SourceSpecies: Bovine (cattle)

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Component #4: protein, Kinesin-like protein cut7

ProteinName: Kinesin-like protein cut7 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 40.737527 kDa
SourceSpecies: Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #6: ligand, GUANOSINE-5'-TRIPHOSPHATE

LigandName: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.52318 kDa

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Component #7: ligand, GUANOSINE-5'-DIPHOSPHATE

LigandName: GUANOSINE-5'-DIPHOSPHATE / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.443201 kDa

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Component #8: ligand, TAXOL

LigandName: TAXOLPaclitaxel / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.853906 kDa

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Component #9: ligand, PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

LigandName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.506196 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: helical array / Method: cryo EM
Sample solutionpH: 6.8
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temperature: 297 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 2 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 68000.0 X (calibrated) / Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 700.0 - nm
Specimen HolderModel: GATAN LIQUID NITROGEN
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 144300
3D reconstructionSoftware: FREALIGN / Resolution: 9.3 Å / Resolution method: FSC 0.5 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: Cross-correlation coefficient / Refinement space: REAL
Details: An initial homology model of S. pombe cut7 kinesin-5 motor domain based on human kinesin-5 structure (PDB 3HQD) was prepared using Modeller. The coordinates of motor bound to an alpha-beta tubulin dimer (PDB 1JFF) were rigidly fitted into the cryo-EM map using Chimera and refined by flexible fitting using Flex-EM. Structural models of loop5 and loop10 were generated using Modeller. The conformation of the neck-linker and the N-terminus were calculated using a conjugate-gradient energy minimization approach.
Output model

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