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Entry
Database: EMDB / ID: EMD-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
Sample
  • Complex: microtubule-bound S.pombe kinesin-5 motor domain in the AMPPNP state
    • Protein or peptide: Tubulin alpha-1D chain
    • Protein or peptide: Tubulin beta-2B chain
    • Protein or peptide: Kinesin-like protein cut7
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOLPaclitaxel
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
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 pole / meiotic spindle assembly / mitotic spindle pole body / mitotic spindle midzone assembly / mitotic spindle midzone ...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 pole body / mitotic spindle midzone assembly / mitotic spindle midzone / 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 / mitotic spindle assembly / microtubule-based process / spindle microtubule / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / mitotic spindle / microtubule cytoskeleton organization / kinetochore / 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 / metal ion binding / nucleus / 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 / Tubulin-beta mRNA autoregulation signal. ...: / : / 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 / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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
Kinesin-like protein cut7 / Tubulin alpha-1D chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesBovine (cattle) / Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.3 Å
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 /
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, 2016-
Header (metadata) releaseNov 30, 2016-
Map releaseNov 30, 2016-
UpdateJun 20, 2018-
Current statusJun 20, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
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  • Atomic models: PDB-5m5i
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  • Atomic models: PDB-5m5l
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  • Atomic models: PDB-5m5m
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  • Atomic models: PDB-5m5n
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  • Atomic models: PDB-5m5o
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3445.png

Downloads & links

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Map

FileDownload / File: emd_3445.map.gz / Format: CCP4 / Size: 489.3 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-electron microscopy of microtubule-bound S. pombe kinesin-5 motor domain in the AMPPNP state
Voxel sizeX=Y=Z: 2.2 Å
Density
Contour LevelBy AUTHOR: 1. / Movie #1: 1
Minimum - Maximum-5.693818 - 8.505580999999999
Average (Standard dev.)-0.026976306 (±1.3084207)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin945586
Dimensions505050
Spacing505050
CellA=B=C: 110.0 Å
α=β=γ: 90.0 °

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
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
Components
  • Complex: microtubule-bound S.pombe kinesin-5 motor domain in the AMPPNP state
    • Protein or peptide: Tubulin alpha-1D chain
    • Protein or peptide: Tubulin beta-2B chain
    • Protein or peptide: Kinesin-like protein cut7
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: TAXOLPaclitaxel
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: microtubule-bound S.pombe kinesin-5 motor domain in the AMPPNP state

SupramoleculeName: microtubule-bound S.pombe kinesin-5 motor domain in the AMPPNP state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 140 KDa

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Macromolecule #1: Tubulin alpha-1D chain

MacromoleculeName: Tubulin alpha-1D chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bovine (cattle)
Molecular weightTheoretical: 50.107238 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFSVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFSVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLIGQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRGHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRTIQFVDW CPTGFKVGIN YEPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

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Macromolecule #2: Tubulin beta-2B chain

MacromoleculeName: Tubulin beta-2B chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bovine (cattle)
Molecular weightTheoretical: 49.90777 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATADEQGE FEEEEGEDEA

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Macromolecule #3: Kinesin-like protein cut7

MacromoleculeName: Kinesin-like protein cut7 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Schizosaccharomyces pombe (strain 972 / ATCC 24843) (yeast)
Molecular weightTheoretical: 40.737527 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: ALHDENETNI NVVVRVRGRT DQEVRDNSSL AVSTSGAMGA ELAIQSDPSS MLVTKTYAFD KVFGPEADQL MLFENSVAPM LEQVLNGYN CTIFAYGQTG TGKTYTMSGD LSDSDGILSE GAGLIPRALY QLFSSLDNSN QEYAVKCSYY ELYNEEIRDL L VSEELRKP ...String:
ALHDENETNI NVVVRVRGRT DQEVRDNSSL AVSTSGAMGA ELAIQSDPSS MLVTKTYAFD KVFGPEADQL MLFENSVAPM LEQVLNGYN CTIFAYGQTG TGKTYTMSGD LSDSDGILSE GAGLIPRALY QLFSSLDNSN QEYAVKCSYY ELYNEEIRDL L VSEELRKP ARVFEDTSRR GNVVITGIEE SYIKNAGDGL RLLREGSHRR QVAATKCNDL SSRSHSIFTI TLHRKVSSGM TD ETNSLTI NNNSDDLLRA SKLHMVDLAG SENIGRSGAE NKRARETGMI NQSLLTLGRV INALVEKAHH IPYRESKLTR LLQ DSLGGK TKTSMIVTVS STNTNLEETI STLEYAARAK SIRNKPQNNQ LVF

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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Macromolecule #6: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM / Guanosine diphosphate

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Macromolecule #7: TAXOL

MacromoleculeName: TAXOL / type: ligand / ID: 7 / Number of copies: 1 / Formula: TA1
Molecular weightTheoretical: 853.906 Da
Chemical component information

ChemComp-TA1:
TAXOL / medication, chemotherapy*YM / Paclitaxel

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Macromolecule #8: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 8 / Number of copies: 1 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 6.8
Component:
ConcentrationFormula
25.0 mMPIPES
30.0 mMNaClSodium chloride
7.0 mMMgCl2
1.0 mMEGTA
5.0 mMAMPPNP
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 297 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 68000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus min: 0.7 µm
Sample stageSpecimen holder model: GATAN CT3500 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 20.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware: (Name: CTFFIND (ver. 3), FREALIGN)
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: SPIDER
Final angle assignmentType: PROJECTION MATCHING / Software - Name: FREALIGN
Final reconstructionResolution.type: BY AUTHOR / Resolution: 9.3 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: FREALIGN / Number images used: 144300

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Atomic model buiding 1

DetailsAn 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-5m5i:
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 conformation allows formation of a cover neck bundle.

PDB-5m5l:
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

PDB-5m5m:
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.

PDB-5m5n:
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.

PDB-5m5o:
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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