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- EMDB-9997: Complex of yeast cytoplasmic dynein MTBD-High and MT with DTT -

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Basic information

Entry
Database: EMDB / ID: EMD-9997
TitleComplex of yeast cytoplasmic dynein MTBD-High and MT with DTT
Map dataCryo-EM map of MTBD-High/MT complex, filtered to the local resolution.
Sample
  • Cell: MTBD-High/MT complex with DTT
    • Protein or peptide: Alpha tubulin
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Dynein heavy chain, cytoplasmic
KeywordsDynein / Microtubule / MOTOR PROTEIN-STRUCTURAL PROTEIN complex
Function / homology
Function and homology information


karyogamy / establishment of mitotic spindle localization / astral microtubule / nuclear migration along microtubule / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / dynein light intermediate chain binding / spindle pole body / nuclear migration / dynein intermediate chain binding ...karyogamy / establishment of mitotic spindle localization / astral microtubule / nuclear migration along microtubule / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / dynein light intermediate chain binding / spindle pole body / nuclear migration / dynein intermediate chain binding / mitotic sister chromatid segregation / establishment of mitotic spindle orientation / cytoplasmic microtubule / cytoplasmic microtubule organization / Neutrophil degranulation / mitotic spindle organization / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / mitotic cell cycle / cell cortex / microtubule / hydrolase activity / GTPase activity / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / cytoplasm
Similarity search - Function
: / DYN1, AAA+ ATPase lid domain / Dynein heavy chain 3, AAA+ lid domain / AAA+ lid domain / P-loop containing dynein motor region / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / Dynein heavy chain / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, linker ...: / DYN1, AAA+ ATPase lid domain / Dynein heavy chain 3, AAA+ lid domain / AAA+ lid domain / P-loop containing dynein motor region / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / Dynein heavy chain / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, linker / Dynein heavy chain, AAA module D4 / Dynein heavy chain, coiled coil stalk / Dynein heavy chain, hydrolytic ATP-binding dynein motor region / Dynein heavy chain, ATP-binding dynein motor region / Dynein heavy chain AAA lid domain / Dynein heavy chain AAA lid domain superfamily / Dynein heavy chain, domain 2, N-terminal / Dynein heavy chain, linker, subdomain 3 / Dynein heavy chain, AAA1 domain, small subdomain / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, N-terminal region 2 / Hydrolytic ATP binding site of dynein motor region / Microtubule-binding stalk of dynein motor / P-loop containing dynein motor region D4 / ATP-binding dynein motor region / Dynein heavy chain AAA lid domain / 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Tubulin alpha-1A chain / Tubulin beta chain / Dynein heavy chain, cytoplasmic
Similarity search - Component
Biological speciesSus scrofa (pig) / Saccharomyces cerevisiae S288c (yeast)
Methodhelical reconstruction / cryo EM / Resolution: 3.62 Å
AuthorsKomori Y / Nishida N
Funding support Japan, 2 items
OrganizationGrant numberCountry
Japan Science and TechnologyJPMJCR14M1 Japan
Japan Agency for Medical Research and Development (AMED)JP19am0101115j003 Japan
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis for two-way communication between dynein and microtubules.
Authors: Noritaka Nishida / Yuta Komori / Osamu Takarada / Atsushi Watanabe / Satoko Tamura / Satoshi Kubo / Ichio Shimada / Masahide Kikkawa /
Abstract: The movements of cytoplasmic dynein on microtubule (MT) tracks is achieved by two-way communication between the microtubule-binding domain (MTBD) and the ATPase domain via a coiled-coil stalk, but ...The movements of cytoplasmic dynein on microtubule (MT) tracks is achieved by two-way communication between the microtubule-binding domain (MTBD) and the ATPase domain via a coiled-coil stalk, but the structural basis of this communication remains elusive. Here, we regulate MTBD either in high-affinity or low-affinity states by introducing a disulfide bond to the stalk and analyze the resulting structures by NMR and cryo-EM. In the MT-unbound state, the affinity changes of MTBD are achieved by sliding of the stalk α-helix by a half-turn, which suggests that structural changes propagate from the ATPase-domain to MTBD. In addition, MT binding induces further sliding of the stalk α-helix even without the disulfide bond, suggesting how the MT-induced conformational changes propagate toward the ATPase domain. Based on differences in the MT-binding surface between the high- and low-affinity states, we propose a potential mechanism for the directional bias of dynein movement on MT tracks.
History
DepositionJul 19, 2019-
Header (metadata) releaseMar 4, 2020-
Map releaseMar 4, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.125
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.125
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6kiq
  • Surface level: 0.125
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6kiq
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9997.png

Downloads & links

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Map

FileDownload / File: emd_9997.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of MTBD-High/MT complex, filtered to the local resolution.
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.125 / Movie #1: 0.125
Minimum - Maximum-0.36650035 - 0.8984432
Average (Standard dev.)0.0096481275 (±0.054478526)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 237.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z237.600237.600237.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ512512512
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.3670.8980.010

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Supplemental data

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Sample components

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Entire : MTBD-High/MT complex with DTT

EntireName: MTBD-High/MT complex with DTT
Components
  • Cell: MTBD-High/MT complex with DTT
    • Protein or peptide: Alpha tubulin
    • Protein or peptide: Tubulin beta chain
    • Protein or peptide: Dynein heavy chain, cytoplasmic

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Supramolecule #1: MTBD-High/MT complex with DTT

SupramoleculeName: MTBD-High/MT complex with DTT / type: cell / ID: 1 / Parent: 0 / Macromolecule list: all
Details: DTT was added to cleave the disulfide bond between CC1 and CC2 of MTBD-High
Source (natural)Organism: Sus scrofa (pig)

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Macromolecule #1: Alpha tubulin

MacromoleculeName: Alpha tubulin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 45.95998 KDa
SequenceString: RECISIHVGQ AGVQIGNACW ELYCLEHGIQ PDGHVPRAVF VDLEPTVIDE VRTGTYRQLF HPEQLITGKE DAANNYARGH YTIGKEIID LVLDRIRKLA DQCTGLQGFS VFHSFGGGTG SGFTSLLMER LSVDYGKKSK LEFSIYPAPQ VSTAVVEPYN S ILTTHTTL ...String:
RECISIHVGQ AGVQIGNACW ELYCLEHGIQ PDGHVPRAVF VDLEPTVIDE VRTGTYRQLF HPEQLITGKE DAANNYARGH YTIGKEIID LVLDRIRKLA DQCTGLQGFS VFHSFGGGTG SGFTSLLMER LSVDYGKKSK LEFSIYPAPQ VSTAVVEPYN S ILTTHTTL EHSDCAFMVD NEAIYDICRR NLDIERPTYT NLNRLIGQIV SSITASLRFD GALNVDLTEF QTNLVPYPRG HF PLATYAP VISAEKAYHE QLSVAEITNA CFEPANQMVK CDPRHGKYMA CCLLYRGDVV PKDVNAAIAT IKTKRTIQFV DWC PTGFKV GINYEPPTVV PGGDLAKVQR AVCMLSNTTA IAEAWARLDH KFDLMYAKRA FVHWYVGEGM EEGEFSEARE DMAA LEKDY EEVGVDS

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Sus scrofa (pig)
Molecular weightTheoretical: 47.809746 KDa
SequenceString: REIVHIQAGQ CGNQIGAKFW EVISDEHGID PTGSYHGDSD LQLERINVYY NEAAGNKYVP RAILVDLEPG TMDSVRSGPF GQIFRPDNF VFGQSGAGNN WAKGHYTEGA ELVDSVLDVV RKESESCDCL QGFQLTHSLG GGTGSGMGTL LISKIREEYP D RIMNTFSV ...String:
REIVHIQAGQ CGNQIGAKFW EVISDEHGID PTGSYHGDSD LQLERINVYY NEAAGNKYVP RAILVDLEPG TMDSVRSGPF GQIFRPDNF VFGQSGAGNN WAKGHYTEGA ELVDSVLDVV RKESESCDCL QGFQLTHSLG GGTGSGMGTL LISKIREEYP D RIMNTFSV VPSPKVSDTV VEPYNATLSV HQLVENTDET YCIDNEALYD ICFRTLKLTT PTYGDLNHLV SATMSGVTTC LR FPGQLNA DLRKLAVNMV PFPRLHFFMP GFAPLTSRGS QQYRALTVPE LTQQMFDAKN MMAACDPRHG RYLTVAAVFR GRM SMKEVD EQMLNVQNKN SSYFVEWIPN NVKTAVCDIP PRGLKMSATF IGNSTAIQEL FKRISEQFTA MFRRKAFLHW YTGE GMDEM EFTEAESNMN DLVSEYQQYQ D

UniProtKB: Tubulin beta chain

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Macromolecule #3: Dynein heavy chain, cytoplasmic

MacromoleculeName: Dynein heavy chain, cytoplasmic / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast) / Strain: S288c
Molecular weightTheoretical: 15.264564 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MKSIQDCEPT ILEAQRGVKN IKKQQLTEIR SMVNPPSGVK IVMEAVCAIL GYQFSNWRDI QQFIRKDDFI HNIVHYDTTL HMKPQIRKY MEEEFLSDPN FTYETINRAS KACGPLYQWV NAQINFSKCL E

UniProtKB: Dynein heavy chain, cytoplasmic

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
Component:
ConcentrationFormulaName
80.0 mMC8H18N2O6S2PIPES pH 6.8
1.0 mMC14H24N2O10EGTA
1.0 mMMgCl2magnesium chloride
0.01 %C14H22O(C2H4O)nNP40

Details: DTT was added to the final concentration of 1 mM.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER/RHODIUM
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 279.0 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: OTHER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 621 / Average exposure time: 10.0 sec. / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 35636 / Details: particles were collected using PyFilamentPicker
Startup modelType of model: OTHER / Details: Cryo-EM map of microtubule filtered to 8 Angstroms
Final angle assignmentType: NOT APPLICABLE / Software - Name: FREALIGN (ver. 9.11)
Details: FREALIGN v9.11 and "super-particle" based approach were used to refine the angular assignment
Final reconstructionApplied symmetry - Helical parameters - Δz: 9.2219 Å
Applied symmetry - Helical parameters - Δ&Phi: -25.7519 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Details: FSCtrue was calculated to validate the resolution. / Number images used: 32666

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-6kiq:
Complex of yeast cytoplasmic dynein MTBD-High and MT with DTT

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