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- PDB-8q84: Outer kinetochore Dam1 protomer dimer Ndc80-Nuf2 coiled-coil complex -

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Basic information

Entry
Database: PDB / ID: 8q84
TitleOuter kinetochore Dam1 protomer dimer Ndc80-Nuf2 coiled-coil complex
Components
  • (DASH complex subunit ...) x 10
  • (Kinetochore protein ...) x 2
KeywordsCELL CYCLE / Kinetochore / microtubule / error correction / chromosome segregation
Function / homology
Function and homology information


mitotic spindle polar microtubule / Ndc80 complex / DASH complex / protein transport along microtubule to mitotic spindle pole body / mitotic sister chromatid biorientation / kinetochore organization / positive regulation of attachment of spindle microtubules to kinetochore / mitotic spindle pole body / meiotic chromosome segregation / mitotic spindle midzone ...mitotic spindle polar microtubule / Ndc80 complex / DASH complex / protein transport along microtubule to mitotic spindle pole body / mitotic sister chromatid biorientation / kinetochore organization / positive regulation of attachment of spindle microtubules to kinetochore / mitotic spindle pole body / meiotic chromosome segregation / mitotic spindle midzone / attachment of spindle microtubules to kinetochore / attachment of mitotic spindle microtubules to kinetochore / condensed chromosome, centromeric region / spindle pole body / protein localization to kinetochore / positive regulation of microtubule polymerization / mitotic spindle organization / chromosome segregation / spindle microtubule / mitotic spindle / kinetochore / spindle pole / microtubule / cell division / protein-containing complex binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
DASH complex subunit Duo1 / DASH complex subunit Spc34 / DASH complex subunit Hsk3 / DASH complex subunit Duo1 / DASH complex subunit Spc34 / DASH complex subunit Hsk3-like / DASH complex subunit Hsk3 like / DASH complex subunit Spc19 / DASH complex subunit Dad1 / DASH complex subunit Dad2 ...DASH complex subunit Duo1 / DASH complex subunit Spc34 / DASH complex subunit Hsk3 / DASH complex subunit Duo1 / DASH complex subunit Spc34 / DASH complex subunit Hsk3-like / DASH complex subunit Hsk3 like / 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 / Kinetochore protein Nuf2, N-terminal / Nuf2, N-terminal domain superfamily / Nuf2 family / Kinetochore protein Ndc80 / Ndc80 domain superfamily / Domain of unknown function DUF5595 / HEC/Ndc80p family / Domain of unknown function (DUF5595) / DASH complex subunit Dad4 / DASH complex subunit Dad4
Similarity search - Domain/homology
Kinetochore protein NUF2 / DASH complex subunit ASK1 / DASH complex subunit SPC34 / DASH complex subunit DAD2 / Kinetochore protein NDC80 / DASH complex subunit DUO1 / DASH complex subunit DAM1 / DASH complex subunit DAD3 / DASH complex subunit DAD4 / DASH complex subunit HSK3 ...Kinetochore protein NUF2 / DASH complex subunit ASK1 / DASH complex subunit SPC34 / DASH complex subunit DAD2 / Kinetochore protein NDC80 / DASH complex subunit DUO1 / DASH complex subunit DAM1 / DASH complex subunit DAD3 / DASH complex subunit DAD4 / DASH complex subunit HSK3 / DASH complex subunit SPC19 / DASH complex subunit DAD1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsMuir, K.W. / Barford, D.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)MC_UP_1201/6 United Kingdom
Cancer Research UKC576/A14109 United Kingdom
CitationJournal: Science / Year: 2023
Title: Structural mechanism of outer kinetochore Dam1-Ndc80 complex assembly on microtubules.
Authors: Kyle W Muir / Christopher Batters / Tom Dendooven / Jing Yang / Ziguo Zhang / Alister Burt / David Barford /
Abstract: Kinetochores couple chromosomes to the mitotic spindle to segregate the genome during cell division. An error correction mechanism drives the turnover of kinetochore-microtubule attachments until ...Kinetochores couple chromosomes to the mitotic spindle to segregate the genome during cell division. An error correction mechanism drives the turnover of kinetochore-microtubule attachments until biorientation is achieved. The structural basis for how kinetochore-mediated chromosome segregation is accomplished and regulated remains an outstanding question. In this work, we describe the cryo-electron microscopy structure of the budding yeast outer kinetochore Ndc80 and Dam1 ring complexes assembled onto microtubules. Complex assembly occurs through multiple interfaces, and a staple within Dam1 aids ring assembly. Perturbation of key interfaces suppresses yeast viability. Force-rupture assays indicated that this is a consequence of impaired kinetochore-microtubule attachment. The presence of error correction phosphorylation sites at Ndc80-Dam1 ring complex interfaces and the Dam1 staple explains how kinetochore-microtubule attachments are destabilized and reset.
History
DepositionAug 17, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 6, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 20, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Kinetochore protein NDC80
B: Kinetochore protein NUF2
F: Kinetochore protein NDC80
G: Kinetochore protein NUF2
I: DASH complex subunit DAM1
J: DASH complex subunit DUO1
K: DASH complex subunit DAD2
L: DASH complex subunit DAD1
M: DASH complex subunit DAD4
N: DASH complex subunit DAD3
O: DASH complex subunit SPC34
P: DASH complex subunit ASK1
Q: DASH complex subunit HSK3
R: DASH complex subunit SPC19
U: DASH complex subunit DAM1
V: DASH complex subunit DUO1
W: DASH complex subunit DAD2
X: DASH complex subunit DAD1
Y: DASH complex subunit DAD4
Z: DASH complex subunit DAD3
a: DASH complex subunit SPC34
b: DASH complex subunit ASK1
c: DASH complex subunit HSK3
d: DASH complex subunit SPC19
e: DASH complex subunit DAM1


Theoretical massNumber of molelcules
Total (without water)713,54425
Polymers713,54425
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Kinetochore protein ... , 2 types, 4 molecules AFBG

#1: Protein Kinetochore protein NDC80 /


Mass: 80609.375 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: NDC80 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P40460
#2: Protein Kinetochore protein NUF2 /


Mass: 53025.949 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: NUF2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P33895

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DASH complex subunit ... , 10 types, 21 molecules IUeJVKWLXMYNZOaPbQcRd

#3: Protein DASH complex subunit DAM1


Mass: 38477.871 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: DAM1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P53267
#4: Protein DASH complex subunit DUO1


Mass: 27515.008 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: DUO1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P53168
#5: Protein DASH complex subunit DAD2


Mass: 15084.501 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: DAD2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P36162
#6: Protein DASH complex subunit DAD1


Mass: 10522.616 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: DAD1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q12248
#7: Protein DASH complex subunit DAD4


Mass: 8164.255 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: DAD4 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P69851
#8: Protein DASH complex subunit DAD3


Mass: 10862.234 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: DAD3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P69850
#9: Protein DASH complex subunit SPC34


Mass: 34131.812 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SPC34 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P36131
#10: Protein DASH complex subunit ASK1


Mass: 32106.631 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ASK1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P35734
#11: Protein DASH complex subunit HSK3


Mass: 8097.205 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: HSK3 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P69852
#12: Protein DASH complex subunit SPC19


Mass: 18935.559 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SPC19 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q03954

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Outer kinetochore Dam1 protomer dimer with staple and Ndc80-Nuf2 coiled-coils
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 673772.36 / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 6.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
7UCSF Chimera1.12model fitting
8Coot0.9.3model fitting
10PHENIXdev-4620-000model refinement
11cryoSPARC3.3.2initial Euler assignment
12RELION4.0-devinitial Euler assignment
13cryoSPARC4.2final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 248732 / Symmetry type: POINT
Atomic model buildingB value: 540.98 / Protocol: OTHER / Space: REAL
Details: Initial rigid body fitting was performed in chimera, with manual correction in coot and real-space refinement in PHENIX
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 540.98 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003226920
ELECTRON MICROSCOPYf_angle_d0.600836178
ELECTRON MICROSCOPYf_chiral_restr0.0364189
ELECTRON MICROSCOPYf_plane_restr0.00414688
ELECTRON MICROSCOPYf_dihedral_angle_d4.40183489

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