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- PDB-5m5l: Pseudo-atomic model of microtubule-bound S. pombe kinesin-5 motor... -
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Open data
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Basic information
Entry | Database: PDB / ID: 5m5l | ||||||
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Title | Pseudo-atomic model of microtubule-bound S. pombe kinesin-5 motor domain in the AMPPNP state (based on cryo-electron microscopy experiment): the N-terminus adopts multiple conformations | ||||||
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![]() | MOTOR PROTEIN / microtubule-bound S.pombe kinesin-5 / motor domain / AMPPNP bound state | ||||||
Function / homology | ![]() mitotic spindle formation (spindle phase one) / mitotic spindle elongation (spindle phase three) / Kinesins / initial mitotic spindle pole body separation / microtubule plus-end directed mitotic chromosome migration / meiotic spindle pole / meiotic spindle assembly / mitotic spindle midzone assembly / mitotic spindle midzone / mitotic spindle pole body ...mitotic spindle formation (spindle phase one) / mitotic spindle elongation (spindle phase three) / Kinesins / initial mitotic spindle pole body separation / microtubule plus-end directed mitotic chromosome migration / meiotic spindle pole / meiotic spindle assembly / mitotic spindle midzone assembly / mitotic spindle midzone / mitotic spindle pole body / spindle elongation / polar microtubule / minus-end-directed microtubule motor activity / plus-end-directed microtubule motor activity / meiotic spindle / positive regulation of axon guidance / microtubule associated complex / microtubule motor activity / microtubule-based process / mitotic spindle assembly / spindle microtubule / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / mitotic spindle / kinetochore / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / nervous system development / microtubule binding / microtubule / hydrolase activity / protein heterodimerization activity / cell division / GTPase activity / GTP binding / ATP hydrolysis activity / ATP binding / nucleus / metal ion binding / cytoplasm Similarity search - Function | ||||||
Biological species | ![]() ![]() ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.3 Å | ||||||
![]() | Goulet, A. / Moores, C.A. / Cross, R.A. | ||||||
![]() | ![]() 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 / ![]() Abstract: Cut7, the sole kinesin-5 in Schizosaccharomyces pombe, is essential for mitosis. Like other yeast kinesin-5 motors, Cut7 can reverse its stepping direction, by mechanisms that are currently unclear. ...Cut7, the sole kinesin-5 in Schizosaccharomyces pombe, is essential for mitosis. Like other yeast kinesin-5 motors, Cut7 can reverse its stepping direction, by mechanisms that are currently unclear. Here we show that for full-length Cut7, the key determinant of stepping direction is the degree of motor crowding on the microtubule lattice, with greater crowding converting the motor from minus end-directed to plus end-directed stepping. To explain how high Cut7 occupancy causes this reversal, we postulate a simple proximity sensing mechanism that operates via steric blocking. We propose that the minus end-directed stepping action of Cut7 is selectively inhibited by collisions with neighbors under crowded conditions, whereas its plus end-directed action, being less space-hungry, is not. In support of this idea, we show that the direction of Cut7-driven microtubule sliding can be reversed by crowding it with non-Cut7 proteins. Thus, crowding by either dynein microtubule binding domain or Klp2, a kinesin-14, converts Cut7 from net minus end-directed to net plus end-directed stepping. Biochemical assays confirm that the Cut7 N terminus increases Cut7 occupancy by binding directly to microtubules. Direct observation by cryoEM reveals that this occupancy-enhancing N-terminal domain is partially ordered. Overall, our data point to a steric blocking mechanism for directional reversal through which collisions of Cut7 motor domains with their neighbors inhibit their minus end-directed stepping action, but not their plus end-directed stepping action. Our model can potentially reconcile a number of previous, apparently conflicting, observations and proposals for the reversal mechanism of yeast kinesins-5. | ||||||
History |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 66.6 KB | Display | ![]() |
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PDB format | ![]() | 35.9 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 3445MC ![]() 5m5iC ![]() 5m5mC ![]() 5m5nC ![]() 5m5oC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-Protein , 3 types, 3 molecules ABC
#1: Protein | Mass: 50236.352 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
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#2: Protein | Mass: 49907.770 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() ![]() |
#3: Protein | Mass: 40737.527 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: cut7, SPAC25G10.07c / Production host: ![]() ![]() |
-Non-polymers , 5 types, 6 molecules ![](data/chem/img/MG.gif)
![](data/chem/img/GTP.gif)
![](data/chem/img/GDP.gif)
![](data/chem/img/TA1.gif)
![](data/chem/img/ANP.gif)
![](data/chem/img/GTP.gif)
![](data/chem/img/GDP.gif)
![](data/chem/img/TA1.gif)
![](data/chem/img/ANP.gif)
#4: Chemical | #5: Chemical | ChemComp-GTP / | #6: Chemical | ChemComp-GDP / | #7: Chemical | ChemComp-TA1 / | #8: Chemical | ChemComp-ANP / | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: HELICAL ARRAY / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: microtubule-bound S. pombe kinesin-5 motor domain in the AMPPNP state Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES | ||||||||||||||||||||||||
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Molecular weight | Value: 0.14 MDa / Experimental value: NO | ||||||||||||||||||||||||
Buffer solution | pH: 6.8 | ||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2 | ||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 294 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Microscopy | Model: FEI TECNAI F20 |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Calibrated magnification: 68000 X / Nominal defocus min: 700 nm / Cs: 2 mm / C2 aperture diameter: 70 µm |
Specimen holder | Cryogen: NITROGEN Specimen holder model: GATAN CT3500 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER |
Image recording | Electron dose: 20 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 9.3 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 144300 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient 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 ...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. |