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- EMDB-26080: CaKip3[2-482] - AMP-PNP in complex with a dolastatin-10-stabilize... -

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Entry
Database: EMDB / ID: EMD-26080
TitleCaKip3[2-482] - AMP-PNP in complex with a dolastatin-10-stabilized tubulin ring
Map dataPrimary map, locally refined on the central asymmetric unit
Sample
  • Complex: caKip3[2-482] - AMP-PNP in complex with a dolastatin-10-stabilized tubulin ring
    • Complex: caKip3[2-482] - AMP-PNP
      • Protein or peptide: Kinesin-like protein
    • Organelle or cellular component: dolastatin-10 stabilized tubulin ring
      • Protein or peptide: Tubulin alpha-1B chain
      • Protein or peptide: Tubulin beta-2B chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: dolastatin-10
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
KeywordsKip3 / kinesin / tubulin / dolastatin-10 / MOTOR PROTEIN
Function / homology
Function and homology information


tubulin-dependent ATPase activity / plus-end specific microtubule depolymerization / regulation of mitotic spindle elongation / meiotic sister chromatid segregation / mitotic spindle astral microtubule / nuclear microtubule / mitotic spindle midzone / nuclear migration along microtubule / microtubule plus-end / mitotic spindle disassembly ...tubulin-dependent ATPase activity / plus-end specific microtubule depolymerization / regulation of mitotic spindle elongation / meiotic sister chromatid segregation / mitotic spindle astral microtubule / nuclear microtubule / mitotic spindle midzone / nuclear migration along microtubule / microtubule plus-end / mitotic spindle disassembly / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / Hedgehog 'off' state / Cilium Assembly / Intraflagellar transport / COPI-dependent Golgi-to-ER retrograde traffic / Carboxyterminal post-translational modifications of tubulin / RHOH GTPase cycle / Sealing of the nuclear envelope (NE) by ESCRT-III / Kinesins / PKR-mediated signaling / Resolution of Sister Chromatid Cohesion / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Separation of Sister Chromatids / The role of GTSE1 in G2/M progression after G2 checkpoint / Aggrephagy / plus-end-directed microtubule motor activity / Recruitment of NuMA to mitotic centrosomes / RHO GTPases activate IQGAPs / RHO GTPases Activate Formins / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / COPI-independent Golgi-to-ER retrograde traffic / MHC class II antigen presentation / COPI-mediated anterograde transport / microtubule depolymerization / negative regulation of microtubule polymerization / kinesin complex / microtubule-based movement / mitotic sister chromatid segregation / establishment of mitotic spindle orientation / mitotic spindle assembly / microtubule-based process / mitotic spindle organization / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / microtubule binding / microtubule / GTPase activity / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Kinesin-like protein / 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 ...Kinesin-like protein / 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 / Tubulin beta chain / Tubulin alpha-1B chain
Similarity search - Component
Biological speciesCandida albicans (yeast) / Sus scrofa (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsBenoit MPMH / Asenjo AB / Hunter B / Allingham JS / Sosa H
Funding support United States, Canada, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM113164 United States
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN/356025-2013 Canada
Canadian Institutes of Health Research (CIHR)MOP-97832 Canada
CitationJournal: Nat Commun / Year: 2022
Title: Kinesin-8-specific loop-2 controls the dual activities of the motor domain according to tubulin protofilament shape.
Authors: Byron Hunter / Matthieu P M H Benoit / Ana B Asenjo / Caitlin Doubleday / Daria Trofimova / Corey Frazer / Irsa Shoukat / Hernando Sosa / John S Allingham /
Abstract: Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains ...Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 Å) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic. We also determined a crystal structure of microtubule-unbound CaKip3-ADP (2.0 Å) and analyzed the biochemical activity of CaKip3 and kinesin-1 mutants. These data reveal that the microtubule depolymerization activity of kinesin-8 originates from conformational changes of its motor core that are amplified by dynamic contacts between its extended loop-2 and tubulin. On curved microtubule ends, loop-1 inserts into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin and assist depolymerization. On straight tubulin protofilaments in the microtubule lattice, loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. We propose that these tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.
History
DepositionJan 27, 2022-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateFeb 21, 2024-
Current statusFeb 21, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26080.png

Downloads & links

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Map

FileDownload / File: emd_26080.map.gz / Format: CCP4 / Size: 274.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary map, locally refined on the central asymmetric unit
Voxel sizeX=Y=Z: 1.083 Å
Density
Contour LevelBy AUTHOR: 0.00653
Minimum - Maximum0.0 - 0.041720927
Average (Standard dev.)0.000047241225 (±0.0004720857)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions416416416
Spacing416416416
CellA=B=C: 450.52798 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26080_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Mask #2

Fileemd_26080_msk_2.map
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Mask #3

Fileemd_26080_msk_3.map
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Mask #4

Fileemd_26080_msk_4.map
Projections & Slices
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Additional map: Map locally refined on the 3 central asymmetric units

Fileemd_26080_additional_1.map
AnnotationMap locally refined on the 3 central asymmetric units
Projections & Slices
AxesZYX

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Half map: Half map from the gold Standard refinement

Fileemd_26080_half_map_1.map
AnnotationHalf map from the gold Standard refinement
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map from the gold Standard refinement

Fileemd_26080_half_map_2.map
AnnotationHalf map from the gold Standard refinement
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Sample components

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Entire : caKip3[2-482] - AMP-PNP in complex with a dolastatin-10-stabilize...

EntireName: caKip3[2-482] - AMP-PNP in complex with a dolastatin-10-stabilized tubulin ring
Components
  • Complex: caKip3[2-482] - AMP-PNP in complex with a dolastatin-10-stabilized tubulin ring
    • Complex: caKip3[2-482] - AMP-PNP
      • Protein or peptide: Kinesin-like protein
    • Organelle or cellular component: dolastatin-10 stabilized tubulin ring
      • Protein or peptide: Tubulin alpha-1B chain
      • Protein or peptide: Tubulin beta-2B chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: dolastatin-10
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: caKip3[2-482] - AMP-PNP in complex with a dolastatin-10-stabilize...

SupramoleculeName: caKip3[2-482] - AMP-PNP in complex with a dolastatin-10-stabilized tubulin ring
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: caKip3[2-482] - AMP-PNP

SupramoleculeName: caKip3[2-482] - AMP-PNP / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Candida albicans (yeast)

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Supramolecule #3: dolastatin-10 stabilized tubulin ring

SupramoleculeName: dolastatin-10 stabilized tubulin ring / type: organelle_or_cellular_component / ID: 3 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Sus scrofa (pig) / Organ: Brain

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Macromolecule #1: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig) / Organ: Brain
Molecular weightTheoretical: 50.204445 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPSDK TIGGGDDSFN TFFSETGAGK HVPRAVFVDL EPTVIDEVRT GTYRQLFHP EQLITGKEDA ANNYARGHYT IGKEIIDLVL DRIRKLADQC TGLQGFLVFH SFGGGTGSGF TSLLMERLSV D YGKKSKLE FSIYPAPQVS TAVVEPYNSI LTTHTTLEHS DCAFMVDNEA IYDICRRNLD IERPTYTNLN RLISQIVSSI TA SLRFDGA LNVDLTEFQT NLVPYPRIHF PLATYAPVIS AEKAYHEQLS VAEITNACFE PANQMVKCDP RHGKYMACCL LYR GDVVPK DVNAAIATIK TKRSIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVQRAVC MLSNTTAIAE AWARLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDM AALEKDYEEV GVDSVEGEGE EEGEEY

UniProtKB: Tubulin alpha-1B chain

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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: Sus scrofa (pig)
Molecular weightTheoretical: 49.999887 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEATGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEATGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VMPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDSK NMMAACDPRH GRYLTVAAIF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATADEQGE FEEEEGEDEA

UniProtKB: Tubulin beta chain

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Macromolecule #3: Kinesin-like protein

MacromoleculeName: Kinesin-like protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Candida albicans (yeast)
Molecular weightTheoretical: 54.869469 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASYPNSLGS PATVTSTSVP TAKQSSISVA VRVRPFTEAE SNRLVKIDND DVFLGDGCLT SDNNNNNNNS NSNGNGNGNG SSAANSSGA STSRRAIFNT LGGLRKIINV VDDRMLIFDP PETNPLTKMQ RNAFPNSFKG SRIREHRFVF DRLFDEDCTQ D QVYRNTTQ ...String:
MASYPNSLGS PATVTSTSVP TAKQSSISVA VRVRPFTEAE SNRLVKIDND DVFLGDGCLT SDNNNNNNNS NSNGNGNGNG SSAANSSGA STSRRAIFNT LGGLRKIINV VDDRMLIFDP PETNPLTKMQ RNAFPNSFKG SRIREHRFVF DRLFDEDCTQ D QVYRNTTQ PLLDSVLDGY NATVFAYGAT GCGKTHTISG TPEDPGVIFL TMKELYNRIE ELKDTKIIDI SLSYLEIYNE TI RDLLNPM TQCKNLVIRE DANNKISVSN LSRHRPNSVE EVMQLILEGN KNRTCSPTEA NATSSRSHAV LQINVIQKDR TGD ITEEHT FATLSIIDLA GSERAAATKN RGARLNEGAN INKSLLALGN CINALCDPRR RNHVPYRDSK LTRLLKFSLG GNCK TVMIV CVSPSSQHYD ETLNTLKYAD RAKEIKTKLI RNQHNLDRHV GSYLKMITEQ KQEIEELRAR ESKMVESTIN KRKDL ESKL EHHHHHH

UniProtKB: Kinesin-like protein

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / 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 #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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: dolastatin-10

MacromoleculeName: dolastatin-10 / type: ligand / ID: 7 / Number of copies: 1 / Formula: SR6
Molecular weightTheoretical: 785.091 Da
Chemical component information

ChemComp-SR6:
dolastatin-10

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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 statefilament

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Sample preparation

BufferpH: 6.8
Component:
ConcentrationName
80.0 mMK-PIPES
20.0 uMDolastatin-10
5.0 mMMagnesium chloride
1.0 mMEGTA
4.0 mMAMP-PNP
GridModel: UltrAuFoil R2/2 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.67 µm / Nominal defocus min: 0.81 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-50 / Average electron dose: 64.01 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C14 (14 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: The "Number of particles used" reported here is the number of asymmetric units used.
Number images used: 530936
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7tr3:
CaKip3[2-482] - AMP-PNP in complex with a dolastatin-10-stabilized tubulin ring

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