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- PDB-5m5l: Pseudo-atomic model of microtubule-bound S. pombe kinesin-5 motor... -

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Basic information

Entry
Database: PDB / ID: 5m5l
TitlePseudo-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
Components
  • Kinesin-like protein cut7
  • Tubulin alpha-1D chain
  • Tubulin beta-2B chain
KeywordsMOTOR PROTEIN / microtubule-bound S.pombe kinesin-5 / motor domain / AMPPNP bound state
Function / homology
Function and homology information


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 assembly / meiotic spindle pole / 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 assembly / meiotic spindle pole / 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 / mitotic spindle / structural constituent of cytoskeleton / kinetochore / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / nervous system development / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / 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
: / : / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Alpha tubulin ...: / : / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / TAXOL / Kinesin-like protein cut7 / Tubulin alpha-1D chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesSchizosaccharomyces pombe (fission yeast)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.3 Å
AuthorsGoulet, A. / Moores, C.A. / Cross, R.A.
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 /
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
DepositionOct 21, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 30, 2016Provider: repository / Type: Initial release
Revision 1.1Dec 14, 2016Group: Database references
Revision 1.2Aug 2, 2017Group: Data collection / Derived calculations ...Data collection / Derived calculations / Experimental preparation / Refinement description
Category: em_3d_fitting / em_image_scans ...em_3d_fitting / em_image_scans / em_sample_support / em_software / pdbx_struct_conn_angle
Item: _em_3d_fitting.target_criteria / _em_sample_support.grid_type ..._em_3d_fitting.target_criteria / _em_sample_support.grid_type / _em_software.name / _em_software.version
Revision 1.3Dec 5, 2018Group: Data collection / Category: em_imaging / Item: _em_imaging.c2_aperture_diameter
Revision 1.4May 8, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • EMDB-3445
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: Tubulin alpha-1D chain
B: Tubulin beta-2B chain
C: Kinesin-like protein cut7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)143,2579
Polymers140,8823
Non-polymers2,3756
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Tubulin alpha-1D chain / Coordinate model: Cα atoms only


Mass: 50236.352 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: Q2HJ86
#2: Protein Tubulin beta-2B chain / Coordinate model: Cα atoms only


Mass: 49907.770 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / References: UniProt: Q6B856
#3: Protein Kinesin-like protein cut7 / Cell untimely torn protein 7 / Coordinate model: Cα atoms only


Mass: 40737.527 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Gene: cut7, SPAC25G10.07c / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P24339

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Non-polymers , 5 types, 6 molecules

#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#5: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#6: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#7: Chemical ChemComp-TA1 / TAXOL


Mass: 853.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM
#8: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: microtubule-bound S. pombe kinesin-5 motor domain in the AMPPNP state
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.14 MDa / Experimental value: NO
Buffer solutionpH: 6.8
Buffer component
IDConc.FormulaBuffer-ID
125 mMPIPES1
230 mMNaCl1
37 mMMgCl21
41 mMEGTA1
55 mMAMPPNP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: 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
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 68000 X / Nominal defocus min: 700 nm / Cs: 2 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN CT3500 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Image recordingElectron dose: 20 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1EMANparticle selection
4CTFFIND3CTF correction
5FREALIGNCTF correction
8Flex-EMmodel fitting
9UCSF Chimeramodel fitting
11SPIDERinitial Euler assignment
12FREALIGNfinal Euler assignment
14FREALIGN3D reconstruction
15Flex-EMmodel refinement
16MODELLERmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 9.3 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 144300 / Symmetry type: POINT
Atomic model buildingProtocol: 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.

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