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- EMDB-7446: Structure of the DASH/Dam1 complex shows its role at the yeast ki... -

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Basic information

Entry
Database: EMDB / ID: EMD-7446
TitleStructure of the DASH/Dam1 complex shows its role at the yeast kinetochore-microtubule interface (symmetric reconstruction)
Map dataDASH/Dam1c symmetric reconstruction
Sample
  • Complex: DASH/Dam1 complex
    • Protein or peptide: Ask1
    • Protein or peptide: Dad1
    • Protein or peptide: Dad2
    • Protein or peptide: Dad3
    • Protein or peptide: Dad4
    • Protein or peptide: Dam1
    • Protein or peptide: Duo1
    • Protein or peptide: Hsk3
    • Protein or peptide: Spc19
    • Protein or peptide: Spc34
Biological speciesChaetomium thermophilum (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsJenni S / Harrison SC
CitationJournal: Science / Year: 2018
Title: Structure of the DASH/Dam1 complex shows its role at the yeast kinetochore-microtubule interface.
Authors: Simon Jenni / Stephen C Harrison /
Abstract: Kinetochores connect mitotic-spindle microtubules with chromosomes, allowing microtubule depolymerization to pull chromosomes apart during anaphase while resisting detachment as the microtubule ...Kinetochores connect mitotic-spindle microtubules with chromosomes, allowing microtubule depolymerization to pull chromosomes apart during anaphase while resisting detachment as the microtubule shortens. The heterodecameric DASH/Dam1 complex (DASH/Dam1c), an essential component of yeast kinetochores, assembles into a microtubule-encircling ring. The ring associates with rodlike Ndc80 complexes to organize the kinetochore-microtubule interface. We report the cryo-electron microscopy structure (at ~4.5-angstrom resolution) of a DASH/Dam1c ring and a molecular model of its ordered components, validated by evolutionary direct-coupling analysis. Integrating this structure with that of the Ndc80 complex and with published interaction data yields a molecular picture of kinetochore-microtubule attachment, including how flexible, C-terminal extensions of DASH/Dam1c subunits project and contact widely separated sites on the Ndc80 complex rod and how phosphorylation at previously identified sites might regulate kinetochore assembly.
History
DepositionFeb 1, 2018-
Header (metadata) releaseMar 21, 2018-
Map releaseMay 23, 2018-
UpdateNov 27, 2019-
Current statusNov 27, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.017
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.017
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6cfz
  • Surface level: 0.017
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7446.png

Downloads & links

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Map

FileDownload / File: emd_7446.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDASH/Dam1c symmetric reconstruction
Voxel sizeX=Y=Z: 1.64 Å
Density
Contour LevelBy AUTHOR: 0.017 / Movie #1: 0.017
Minimum - Maximum-0.05638907 - 0.14141443
Average (Standard dev.)0.0002588731 (±0.003867113)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 839.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.641.641.64
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z839.680839.680839.680
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-0.0560.1410.000

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Supplemental data

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Mask #1

Fileemd_7446_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1 (not masked, not filtered)

Fileemd_7446_half_map_1.map
AnnotationHalf-map 1 (not masked, not filtered)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2 (not masked, not filtered)

Fileemd_7446_half_map_2.map
AnnotationHalf-map 2 (not masked, not filtered)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DASH/Dam1 complex

EntireName: DASH/Dam1 complex
Components
  • Complex: DASH/Dam1 complex
    • Protein or peptide: Ask1
    • Protein or peptide: Dad1
    • Protein or peptide: Dad2
    • Protein or peptide: Dad3
    • Protein or peptide: Dad4
    • Protein or peptide: Dam1
    • Protein or peptide: Duo1
    • Protein or peptide: Hsk3
    • Protein or peptide: Spc19
    • Protein or peptide: Spc34

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Supramolecule #1: DASH/Dam1 complex

SupramoleculeName: DASH/Dam1 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 115 KDa

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Macromolecule #1: Ask1

MacromoleculeName: Ask1 / type: protein_or_peptide / ID: 1 / Details: DASH/Dam1 complex subunit Ask1 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MTLTEELEKL EQQITLTLQE IDSNFAKAHR IVTTSILPLV EQYGEHSRAV WEATKFWKQF F EASANVSL SGYEELVD

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Macromolecule #2: Dad1

MacromoleculeName: Dad1 / type: protein_or_peptide / ID: 2 / Details: DASH/Dam1 complex subunit Dad1 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSYFEQQRQA LIEEIAMNFE HVLANINKLN RSLEAVIAVG NEFSSVEALW SQFENVMSWS H PQFEKGAM TGWSHPQFEK RSAGSWSHPQ FEK

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Macromolecule #3: Dad2

MacromoleculeName: Dad2 / type: protein_or_peptide / ID: 3 / Details: DASH/Dam1 complex subunit Dad2 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSPALLARVN EKKAELENLK ELRDLSAAVA AQMEALEQKL STLSSGTEAI ATVLANWHNV L RAISMASA KIPEPKEETE ENTVPLPQTL VRIPT

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Macromolecule #4: Dad3

MacromoleculeName: Dad3 / type: protein_or_peptide / ID: 4 / Details: DASH/Dam1 complex subunit Dad3 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MLSPLEQEVL DEYERLSENM KKLAVLLDEL ASAPATEILD GLRELERKTS LVFTLLKASV Y SIVLQQ

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Macromolecule #5: Dad4

MacromoleculeName: Dad4 / type: protein_or_peptide / ID: 5 / Details: DASH/Dam1 complex subunit Dad4 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MESPHEHQQN LLLSRIITNV EKLNEAIMVM NKTLQEINIQ NMNIELVAQM FKNYQSNVLF H LEATDNLK DPA

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Macromolecule #6: Dam1

MacromoleculeName: Dam1 / type: protein_or_peptide / ID: 6 / Details: DASH/Dam1 complex subunit Dam1 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MNAFEPAFAE LADAVADLEA NMMHFQLMHE SLARFSESFA SFLYGLNMNA FCVDFP

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Macromolecule #7: Duo1

MacromoleculeName: Duo1 / type: protein_or_peptide / ID: 7 / Details: DASH/Dam1 complex subunit Duo1 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MEAREAALRK ELEGVRKINE VIEGMIGTLE RAKGNMGTVS QTVTNATTLL NTWTRMLSQT E HNQRLILN PEWKGATQDL LELEAEERRR QEEVERRAAE AERRREEARR KA

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Macromolecule #8: Hsk3

MacromoleculeName: Hsk3 / type: protein_or_peptide / ID: 8 / Details: DASH/Dam1 complex subunit Hsk3 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MAVKARQLAH LHSQLTQLSH NLATTENLMR MTAVQAEAMR GLGSWHAGLF MAASKVLGEE S

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Macromolecule #9: Spc19

MacromoleculeName: Spc19 / type: protein_or_peptide / ID: 9 / Details: DASH/Dam1 complex subunit Spc19 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSYADCVCSL RTSLAFLESS VATLDNGVQD FPRLCHVLRT VRHYELIPQT TLAAAEASLR D EIGPFIQL LLDRAEKHLD RQARRIETLK ARAELNAGRL SQYSGDGHNN GKFSGQGMDF RK SRPLNGE AALRAKVVRQ RKEALKYSVE RLE

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Macromolecule #10: Spc34

MacromoleculeName: Spc34 / type: protein_or_peptide / ID: 10 / Details: DASH/Dam1 complex subunit Spc34 / Enantiomer: LEVO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSLLSAHLEQ ISISCQGIDS LPFPPPKIFT NALLSNPDIT SLIRDTEAHE RALFSVPPPP P RQTTLTAE QQQQQKPSNR RQTVFNVTGG EIRTGGVGSA STARRNTAVA AVLGGDLHAQ IM RGTRARP GQQPGSGDID MEVLLRGVEK LCAVYPLPGA LERVPVIRQK ...String:
MSLLSAHLEQ ISISCQGIDS LPFPPPKIFT NALLSNPDIT SLIRDTEAHE RALFSVPPPP P RQTTLTAE QQQQQKPSNR RQTVFNVTGG EIRTGGVGSA STARRNTAVA AVLGGDLHAQ IM RGTRARP GQQPGSGDID MEVLLRGVEK LCAVYPLPGA LERVPVIRQK WQAQSNTLAY YEA KIAEQQ EMLDRIAQER MMNDGDGDVE MEDVEEVGMT EEDLRREEEE VRELDKRKRD LQHH HHHH

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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

Concentration2.6 mg/mL
BufferpH: 7.5
GridModel: C-flat-1.2/1.3 4C / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 89 % / Chamber temperature: 293 K / Instrument: GATAN CRYOPLUNGE 3

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 30488 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.2 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: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 2723 / Average electron dose: 1.115 e/Å2
Details: Movies collected: 50 frames with 1.115 e/A2/per frame
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 34997
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: OTHER / Details: Hollow cylinder with random noise
Initial angle assignmentType: PROJECTION MATCHING / Software: (Name: SPARX, EMAN2)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 9.11)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D17 (2x17 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 9.11) / Number images used: 34997
FSC plot (resolution estimation)

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