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- EMDB-13151: Negative stain EM 3D Reconstruction of the Dam1cBim1p complex. -

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Basic information

Entry
Database: EMDB / ID: EMD-13151
TitleNegative stain EM 3D Reconstruction of the Dam1cBim1p complex.
Map dataA Dam1 complex dimer with an attached "arch" of one Bim1p. This sample was crosslinked with glutaraldehyde.
Sample
  • Complex: Dam1pBim1p Complex
    • Protein or peptide: x 11 types
Function / homology
Function and homology information


protein localization to microtubule plus-end / mitotic spindle orientation checkpoint signaling / mitotic spindle polar microtubule / 2-micrometer plasmid partitioning / centromere clustering / DASH complex / protein transport along microtubule to mitotic spindle pole body / mitotic sister chromatid biorientation / positive regulation of attachment of spindle microtubules to kinetochore / mitotic spindle midzone ...protein localization to microtubule plus-end / mitotic spindle orientation checkpoint signaling / mitotic spindle polar microtubule / 2-micrometer plasmid partitioning / centromere clustering / DASH complex / protein transport along microtubule to mitotic spindle pole body / mitotic sister chromatid biorientation / positive regulation of attachment of spindle microtubules to kinetochore / mitotic spindle midzone / mitotic spindle pole body / protein localization to microtubule / attachment of spindle microtubules to kinetochore / nuclear migration along microtubule / microtubule plus-end / protein localization to chromosome, centromeric region / negative regulation of microtubule depolymerization / microtubule nucleation / microtubule plus-end binding / microtubule organizing center / microtubule depolymerization / mitotic sister chromatid cohesion / cytoplasmic microtubule / spindle midzone / spindle assembly / regulation of microtubule polymerization or depolymerization / positive regulation of microtubule polymerization / spindle microtubule / mitotic spindle / spindle pole / microtubule binding / microtubule / cell division / identical protein binding / cytoplasm
Similarity search - Function
DASH complex subunit Hsk3 / 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 Hsk3 / 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 / DASH complex subunit Dad4 / DASH complex subunit Dad4 / EB1, C-terminal / Microtubule-associated protein RP/EB / EB1, C-terminal domain superfamily / EB1-C terminal (EB1-C) domain profile. / EB1-like C-terminal motif / Calponin homology domain / CH domain superfamily / Calponin homology (CH) domain profile.
Similarity search - Domain/homology
DASH complex subunit ASK1 / DASH complex subunit DAD2 / Protein BIM1 / 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)
Methodsingle particle reconstruction / negative staining / Resolution: 35.0 Å
AuthorsEngelhard L / Bourque CB / Klink BU / Gatsogiannis C
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG) Germany
CitationJournal: EMBO J / Year: 2021
Title: Phospho-regulated Bim1/EB1 interactions trigger Dam1c ring assembly at the budding yeast outer kinetochore.
Authors: Alexander Dudziak / Lena Engelhard / Cole Bourque / Björn Udo Klink / Pascaline Rombaut / Nikolay Kornakov / Karolin Jänen / Franz Herzog / Christos Gatsogiannis / Stefan Westermann /
Abstract: Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer ...Kinetochores form the link between chromosomes and microtubules of the mitotic spindle. The heterodecameric Dam1 complex (Dam1c) is a major component of the Saccharomyces cerevisiae outer kinetochore, assembling into 3 MDa-sized microtubule-embracing rings, but how ring assembly is specifically initiated in vivo remains to be understood. Here, we describe a molecular pathway that provides local control of ring assembly during the establishment of sister kinetochore bi-orientation. We show that Dam1c and the general microtubule plus end-associated protein (+TIP) Bim1/EB1 form a stable complex depending on a conserved motif in the Duo1 subunit of Dam1c. EM analyses reveal that Bim1 crosslinks protrusion domains of adjacent Dam1c heterodecamers and promotes the formation of oligomers with defined curvature. Disruption of the Dam1c-Bim1 interaction impairs kinetochore localization of Dam1c in metaphase and delays mitosis. Phosphorylation promotes Dam1c-Bim1 binding by relieving an intramolecular inhibition of the Dam1 C-terminus. In addition, Bim1 recruits Bik1/CLIP-170 to Dam1c and induces formation of full rings even in the absence of microtubules. Our data help to explain how new kinetochore end-on attachments are formed during the process of attachment error correction.
History
DepositionJun 30, 2021-
Header (metadata) releaseJul 28, 2021-
Map releaseJul 28, 2021-
UpdateSep 29, 2021-
Current statusSep 29, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 20
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 20
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13151.png

Downloads & links

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Map

FileDownload / File: emd_13151.map.gz / Format: CCP4 / Size: 1.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationA Dam1 complex dimer with an attached "arch" of one Bim1p. This sample was crosslinked with glutaraldehyde.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.92 Å/pix.
x 76 pix.
= 373.92 Å		4.92 Å/pix.
x 76 pix.
= 373.92 Å		4.92 Å/pix.
x 76 pix.
= 373.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 4.92 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 20.0 / Movie #1: 20
Minimum - Maximum-0.775786 - 162.91025
Average (Standard dev.)0.9978052 (±6.8564415)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions767676
Spacing767676
CellA=B=C: 373.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.924.924.92
M x/y/z767676
origin x/y/z0.0000.0000.000
length x/y/z373.920373.920373.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS767676
D min/max/mean-0.776162.9100.998

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Supplemental data

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Sample components

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Entire : Dam1pBim1p Complex

EntireName: Dam1pBim1p Complex
Components
  • Complex: Dam1pBim1p Complex
    • Protein or peptide: Ask1 protein of the Dam1c from S. cerevisiae
    • Protein or peptide: Dam1 protein of the Dam1c from S. cerevisiae
    • Protein or peptide: Dam1 protein of the Dam1c from S. cerevisiae
    • Protein or peptide: Duo1 protein of the Dam1c from S. cerevisiae
    • Protein or peptide: Spc19 protein of the Dam1c from S. cerevisiae
    • Protein or peptide: Dad2p of the Dam1c from S. cerevisiae
    • Protein or peptide: Dad1p of the Dam1c from S. cerevisiae
    • Protein or peptide: Dad3p of the Dam1c from S. cerevisiae
    • Protein or peptide: Dad4p of the Dam1c from S. cerevisiae
    • Protein or peptide: Hsk3p of the Dam1c from S. cerevisiae
    • Protein or peptide: Bim1p of S. cerevisiae

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Supramolecule #1: Dam1pBim1p Complex

SupramoleculeName: Dam1pBim1p Complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: An unknown number of copies of Bim1p (2-4) binds to a dimeric complex formed by two Dam1c heterodecamers
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Ask1 protein of the Dam1c from S. cerevisiae

MacromoleculeName: Ask1 protein of the Dam1c from S. cerevisiae / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MDSASKEETL EKLDQEITVN LQKIDSNLSF CFHKITQDII PHVATYSEIC ERIMDSTEWL GTMFQETGLV NLQANAAAPV GNAPVKSLVS NNVGIFPTSA EEASRQSQTD NGPNEADSAV HVNRDVHSMF NNDSIDDFHT ANITSTGQIL KLPDSSDEDT GSEAVPSREQ ...String:
MDSASKEETL EKLDQEITVN LQKIDSNLSF CFHKITQDII PHVATYSEIC ERIMDSTEWL GTMFQETGLV NLQANAAAPV GNAPVKSLVS NNVGIFPTSA EEASRQSQTD NGPNEADSAV HVNRDVHSMF NNDSIDDFHT ANITSTGQIL KLPDSSDEDT GSEAVPSREQ TDLTGEGHGG ADDEQDESTI QRQSRKRKIS LLLQQQYGSS SSMVPSPIVP NKMRKQLAHE EHINNDGDND DENSNNIESS PLKQGHHHPK GQADDNNEGP DEEESTKEVP KPGTIIHFST NR

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Macromolecule #2: Dam1 protein of the Dam1c from S. cerevisiae

MacromoleculeName: Dam1 protein of the Dam1c from S. cerevisiae / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSEDKAKLGT TRSATEYRLS IGSAPTSRRS SMGESSSLMK FADQEGLTSS VGEYNENTIQ QLLLPKIREL SDSIITLDSN FTRLNFIHES LADLNESLGS LLYGIMSNSW CVEFSQAPHD IQDDLIAIKQ LKSLEDEKNN LVMELSNMER GIKRKKDEQG ENDLAKASQN ...String:
MSEDKAKLGT TRSATEYRLS IGSAPTSRRS SMGESSSLMK FADQEGLTSS VGEYNENTIQ QLLLPKIREL SDSIITLDSN FTRLNFIHES LADLNESLGS LLYGIMSNSW CVEFSQAPHD IQDDLIAIKQ LKSLEDEKNN LVMELSNMER GIKRKKDEQG ENDLAKASQN KQFNQPLFPS SQVRKYRSYD NRDKRKPSKI GNNLQVENEE DYEDDTSSEA SFVLNPTNIG MSKSSQGHVT KTTRLNNNTN SKLRRKSILH TIRNSIASGA DLPIENDNVV NLGDLHPNNR ISLGSGAARV VNGPVTKNRN SMFSGRAERK PTESRHSVAK KTEKKINTRP PFR

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Macromolecule #3: Dam1 protein of the Dam1c from S. cerevisiae

MacromoleculeName: Dam1 protein of the Dam1c from S. cerevisiae / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MGESLDRCID DINRAVDSMS TLYFKPPGIF HNAILQGASN KASIRKDITR LIKDCNHDEA YLLFKVNPEK QSVSRRDGKE GVFDYVIKRD TDMKRNRRLG RPGEKPIIHV PKEVYLNKDR LDLNNKRRRT ATTSGGGLNG FIFDTDLIGS SVISNSSSGT FKALSAVFKD ...String:
MGESLDRCID DINRAVDSMS TLYFKPPGIF HNAILQGASN KASIRKDITR LIKDCNHDEA YLLFKVNPEK QSVSRRDGKE GVFDYVIKRD TDMKRNRRLG RPGEKPIIHV PKEVYLNKDR LDLNNKRRRT ATTSGGGLNG FIFDTDLIGS SVISNSSSGT FKALSAVFKD DPQIQRLLYA LENGSVLMEE ESNNQRRKTI FVEDFPTDLI LKVMAEVTDL WPLTEFKQDY DQLYHNYEQL SSKLRFIKKE VLLQDDRLKT MSQYHPSSSH DVAKIIRKEK DEIRRLEMEI ANLQE

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Macromolecule #4: Duo1 protein of the Dam1c from S. cerevisiae

MacromoleculeName: Duo1 protein of the Dam1c from S. cerevisiae / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSEQSQLDDS TIDKLIPQIF NEMRSNLNNT TNKFPKSTGG GASDNISANS NSIRSFNSIT TQSLLKESES LDKITAMIKN VTAALKNNLP VYVNQVHEVC KSTNSILDSW INIHSQAGYI HKLMSDQTYL KLINDRLHNE NVNTNDEDGS TLHNVIALKK KEILDLRQKL ...String:
MSEQSQLDDS TIDKLIPQIF NEMRSNLNNT TNKFPKSTGG GASDNISANS NSIRSFNSIT TQSLLKESES LDKITAMIKN VTAALKNNLP VYVNQVHEVC KSTNSILDSW INIHSQAGYI HKLMSDQTYL KLINDRLHNE NVNTNDEDGS TLHNVIALKK KEILDLRQKL ENRKGEKDAA PAKPPNQGLN PRYGVQSGRR PVPSAGISNN GRVRKTHVPA SKRPSGIPRV TNRWTKPTAS SSRKMFR

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Macromolecule #5: Spc19 protein of the Dam1c from S. cerevisiae

MacromoleculeName: Spc19 protein of the Dam1c from S. cerevisiae / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MTDALEQSVL ALEGTVSVLK DSVESLKCAN EPSTNLASTM LQTKRVFRLV PEYDVERSKL DLIEEVEPLV RTLGDKLRKS MGRMQRELDT LQQTYELNDL RLKKNISMDD DDALNSPDMG QEYEGRDADD VVMMASSTNE ELEELKKLKE KKKQLENKLE ILKQK

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Macromolecule #6: Dad2p of the Dam1c from S. cerevisiae

MacromoleculeName: Dad2p of the Dam1c from S. cerevisiae / type: protein_or_peptide / ID: 6 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MDSIDEQIAI KRKELQSLQK ITSLTDGLKI QLTELNEQIK EMGMNADSVA QLMNNWDSII NNISQASLGL LQYAEGDYEI GPWKDSKKKE SEQSNETGLE AQENDKNDED NDEDEDLVPL PETMVRIRVD GNE

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Macromolecule #7: Dad1p of the Dam1c from S. cerevisiae

MacromoleculeName: Dad1p of the Dam1c from S. cerevisiae / type: protein_or_peptide / ID: 7 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MMASTSNDEE KLISTTDKYF IEQRNIVLQE INETMNSILN GLNGLNISLE SSIAVGREFQ SVSDLWKTLY DGLESLSDEA PIDEQPTLSQ SKTK

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Macromolecule #8: Dad3p of the Dam1c from S. cerevisiae

MacromoleculeName: Dad3p of the Dam1c from S. cerevisiae / type: protein_or_peptide / ID: 8 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MEHNLSPLQQ EVLDKYKQLS LDLKALDETI KELNYSQHRQ QHSQQETVSP DEILQEMRDI EVKIGLVGTL LKGSVYSLIL QRKQEQESLG SNSK

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Macromolecule #9: Dad4p of the Dam1c from S. cerevisiae

MacromoleculeName: Dad4p of the Dam1c from S. cerevisiae / type: protein_or_peptide / ID: 9 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MENPHEQVQA NILSRIIGNV KRLNESVAIL NQELVTINNR NKNLEIMGAI CDNYHSSVQF NLEATNNKKP PL

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Macromolecule #10: Hsk3p of the Dam1c from S. cerevisiae

MacromoleculeName: Hsk3p of the Dam1c from S. cerevisiae / type: protein_or_peptide / ID: 10 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MNANKQRQYN QLAHELRELQ TNLQETTKQL DIMSKQCNEN LVGQLGKVHG SWLIGSYIYY MEQMLGKTQ

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Macromolecule #11: Bim1p of S. cerevisiae

MacromoleculeName: Bim1p of S. cerevisiae / type: protein_or_peptide / ID: 11 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSAGIGESRT ELLTWLNGLL NLNYKKIEEC GTGAAYCQIM DSIYGDLPMN RVKFNATAEY EFQTNYKILQ SCFSRHGIEK TVYVDKLIRC KFQDNLEFLQ WLKKHWIRHK DESVYDPDAR RKYRPIITNN SATKPRTVSN PTTAKRSSST GTGSAMSGGL ATRHSSLGIN ...String:
MSAGIGESRT ELLTWLNGLL NLNYKKIEEC GTGAAYCQIM DSIYGDLPMN RVKFNATAEY EFQTNYKILQ SCFSRHGIEK TVYVDKLIRC KFQDNLEFLQ WLKKHWIRHK DESVYDPDAR RKYRPIITNN SATKPRTVSN PTTAKRSSST GTGSAMSGGL ATRHSSLGIN GSRKTSVTQG QLVAIQAELT KSQETIGSLN EEIEQYKGTV STLEIEREFY FNKLRDIEIL VHTTQDLINE GVYKFNDETI TGHGNGNGGA LLRFVKKVES ILYATAEGFE MNDGEDELND KNLGEHGTVP NQGGYANSNG EVNGNEGSNH DVIMQNDEGE VGVSNNLIID EETF

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.4 / Component: (Formula: HEPES, NaCl, TCEP, Glycerol)
Details: The sample was crosslinked using 0.5 % glutaraldehyde. The crosslinking reaction was stopped after 60 seconds by the addition of TRIS
StainingType: NEGATIVE / Material: Uranyl Formate
Details: 4 microl of complex were applied on freshly glow-discharged carbon-coated copper grids (Agar Scientific, G400C). After an incubation of 2 minutes, the sample was blotted with Whatman no. 4 ...Details: 4 microl of complex were applied on freshly glow-discharged carbon-coated copper grids (Agar Scientific, G400C). After an incubation of 2 minutes, the sample was blotted with Whatman no. 4 filter paper, washed 2 times with ddH2O and stained with 0.75 % uranyl formate.
GridModel: Homemade / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE
Details25 mM HEPES, pH 7.4, 200 mM NaCl, 1 mM MgCl2, 0.5 mM TCEP and 2.5 (v/v) % glycerol

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Electron microscopy

MicroscopeJEOL 1400
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 5.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 3.4 mm
Sample stageSpecimen holder model: JEOL

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: Initial Model was computed using SPHIRE (VIPER module)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 35.0 Å / Resolution method: OTHER / Software - Name: SPHIRE / Number images used: 36980
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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