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- PDB-6cfz: Structure of the DASH/Dam1 complex shows its role at the yeast ki... -

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Basic information

Entry
Database: PDB / ID: 6cfz
TitleStructure of the DASH/Dam1 complex shows its role at the yeast kinetochore-microtubule interface
Components
  • Ask1
  • Dad1,Dad1
  • Dad2
  • Dad3
  • Dad4
  • Dam1
  • Duo1
  • Hsk3
  • Spc19
  • Spc34
KeywordsNUCLEAR PROTEIN / DASH / Dam1 complex / Ask1 / Dad1 / Dad2 / Dad3 / Dad4 / Dam1 / Duo1 / Hsk3 / Spc19 / Spc34 / kinetochore / kinetochore-microtubule interface / chromosome segregation / cell-division / point-centromere yeast
Function / homology
Function and homology information


DASH complex / protein transport along microtubule to mitotic spindle pole body / mitotic sister chromatid biorientation / attachment of spindle microtubules to kinetochore / attachment of mitotic spindle microtubules to kinetochore / mitotic spindle organization / chromosome segregation / spindle microtubule / mitotic spindle / kinetochore ...DASH complex / protein transport along microtubule to mitotic spindle pole body / mitotic sister chromatid biorientation / attachment of spindle microtubules to kinetochore / attachment of mitotic spindle microtubules to kinetochore / mitotic spindle organization / chromosome segregation / spindle microtubule / mitotic spindle / kinetochore / mitotic cell cycle / microtubule / cell division / cytoplasm
Similarity search - Function
DASH complex subunit Duo1 / DASH complex subunit Spc34 / DASH complex subunit Duo1 / DASH complex subunit Spc34 / DASH complex subunit Spc19 / DASH complex subunit Dad1 / DASH complex subunit Dad2 / DASH complex subunit Ask1 / DASH complex subunit Dad3 / Spc19 ...DASH complex subunit Duo1 / DASH complex subunit Spc34 / DASH complex subunit Duo1 / DASH complex subunit Spc34 / DASH complex subunit Spc19 / DASH complex subunit Dad1 / DASH complex subunit Dad2 / DASH complex subunit Ask1 / DASH complex subunit Dad3 / Spc19 / DASH complex subunit Dad1 / DASH complex subunit Dad2 / DASH complex subunit Ask1 / DASH complex subunit Dad3 / DASH complex subunit Dam1 / DASH complex subunit Dam1 / DASH complex subunit Dad4 / DASH complex subunit Dad4 / Actin / Actin family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Uncharacterized protein / DASH complex subunit DAD1 / DASH complex subunit DAD2 / DASH complex subunit SPC19 / Uncharacterized protein / DASH complex subunit DAM1 / Uncharacterized protein / Associated with spindles and kinetochores protein 1 / DASH complex subunit duo1-like protein
Similarity search - Component
Biological speciesChaetomium thermophilum (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsJenni, S. / Harrison, S.C.
Funding support United States, 1items
OrganizationGrant numberCountry
Damon Runyon Cancer Research FoundationDRG-2004-09 United States
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 19, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 2, 2018Provider: repository / Type: Initial release
Revision 1.1May 16, 2018Group: Data collection / Data processing / Category: em_3d_reconstruction / Item: _em_3d_reconstruction.resolution
Revision 1.2May 23, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Nov 27, 2019Group: Data collection / Category: em_software
Item: _em_software.details / _em_software.name / _em_software.version
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ask1
B: Dad3
C: Dad2
D: Duo1
E: Dad4
F: Dad1,Dad1
G: Hsk3
H: Dam1
I: Spc19
J: Spc34


Theoretical massNumber of molelcules
Total (without water)116,14910
Polymers116,14910
Non-polymers00
Water0
1
A: Ask1
B: Dad3
C: Dad2
D: Duo1
E: Dad4
F: Dad1,Dad1
G: Hsk3
H: Dam1
I: Spc19
J: Spc34
x 17


Theoretical massNumber of molelcules
Total (without water)1,974,525170
Polymers1,974,525170
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation16
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C17 (17 fold cyclic))

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Components

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Protein , 10 types, 10 molecules ABCDEFGHIJ

#1: Protein Ask1 / / DASH/Dam1 complex subunit Ask1


Mass: 8897.934 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Gene: CTHT_0029740 / Production host: Escherichia coli (E. coli) / References: UniProt: G0S8F9
#2: Protein Dad3 / DASH/Dam1 complex subunit Dad3


Mass: 7599.788 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Gene: CTHT_0005680 / Production host: Escherichia coli (E. coli) / References: UniProt: G0RY74
#3: Protein Dad2 / DASH/Dam1 complex subunit Dad2


Mass: 10229.733 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Gene: CTHT_0002350 / Production host: Escherichia coli (E. coli) / References: UniProt: G0RZB3
#4: Protein Duo1 / DASH/Dam1 complex subunit Duo1


Mass: 12907.559 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Gene: CTHT_0042930 / Production host: Escherichia coli (E. coli) / References: UniProt: G0SAN7
#5: Protein Dad4 / DASH/Dam1 complex subunit Dad4


Mass: 8400.615 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Gene: CTHT_0032660 / Production host: Escherichia coli (E. coli) / References: UniProt: G0S589
#6: Protein Dad1,Dad1 / DASH/Dam1 complex subunit Dad1


Mass: 10684.892 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: DASH/Dam1 complex subunit Dad1 / Source: (gene. exp.) Chaetomium thermophilum (fungus) / Gene: CTHT_0006450 / Production host: Escherichia coli (E. coli) / References: UniProt: G0RYE9
#7: Protein Hsk3 / DASH/Dam1 complex subunit Hsk3


Mass: 6641.681 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Production host: Escherichia coli (E. coli)
#8: Protein Dam1 / DASH/Dam1 complex subunit Dam1


Mass: 6314.139 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Gene: CTHT_0017540 / Production host: Escherichia coli (E. coli) / References: UniProt: G0S2K4
#9: Protein Spc19 / DASH/Dam1 complex subunit Spc19


Mass: 17028.398 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Gene: CTHT_0010600 / Production host: Escherichia coli (E. coli) / References: UniProt: G0S0N0
#10: Protein Spc34 / DASH/Dam1 complex subunit Spc34


Mass: 27443.814 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Gene: CTHT_0016520 / Production host: Escherichia coli (E. coli) / References: UniProt: G0S2A3

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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: DASH/Dam1 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.115 MDa / Experimental value: NO
Source (natural)Organism: Chaetomium thermophilum (fungus) / Cellular location: Nucleus
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 2.6 mg/ml / Embedding 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-1.2/1.3 4C
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Humidity: 89 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 30488 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 1.115 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2723
Details: Movies collected: 50 frames with 1.115 e/A2 per frame

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1EMAN22.1particle selectione2helixboxer.py
2SerialEMimage acquisition
4Gctf1.06CTF correction
7PHENIXmodel fitting
8Omodel fitting
10SPARXinitial Euler assignment
11EMAN2initial Euler assignment
12cisTEMfinal Euler assignmentFrealignX
14cisTEM3D reconstructionFrealignX
15Rosettamodel refinement
16PHENIX1.12model refinementRelease tag: 2829
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 34997
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34997 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 240 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: CC / Details: phenix.real_space_refine
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00712093
ELECTRON MICROSCOPYf_angle_d1.11821878
ELECTRON MICROSCOPYf_dihedral_angle_d7.8984849
ELECTRON MICROSCOPYf_chiral_restr0.053958
ELECTRON MICROSCOPYf_plane_restr0.0082534

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