+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2549 | |||||||||
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Title | 3D structure of the KMN network | |||||||||
Map data | Reconstruction of KMN network | |||||||||
Sample |
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Keywords | Mitosis / outer kinetochore / KMN network | |||||||||
Function / homology | Function and homology information G2/MI transition of meiotic cell cycle / kinetochore adaptor activity / Knl1/Spc105 complex / positive regulation of meiosis I spindle assembly checkpoint / homologous chromosome orientation in meiotic metaphase I / MIS12/MIND type complex / skeletal muscle satellite cell proliferation / Ndc80 complex / leucine zipper domain binding / kinetochore organization ...G2/MI transition of meiotic cell cycle / kinetochore adaptor activity / Knl1/Spc105 complex / positive regulation of meiosis I spindle assembly checkpoint / homologous chromosome orientation in meiotic metaphase I / MIS12/MIND type complex / skeletal muscle satellite cell proliferation / Ndc80 complex / leucine zipper domain binding / kinetochore organization / acrosome assembly / metaphase chromosome alignment / positive regulation of mitotic cell cycle spindle assembly checkpoint / meiotic chromosome segregation / regulation of mitotic cell cycle spindle assembly checkpoint / attachment of spindle microtubules to kinetochore / centrosome duplication / outer kinetochore / kinetochore assembly / attachment of mitotic spindle microtubules to kinetochore / spindle assembly involved in female meiosis I / protein localization to kinetochore / mitotic spindle assembly checkpoint signaling / establishment of mitotic spindle orientation / mitotic sister chromatid segregation / chromosome, centromeric region / cyclin binding / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Deposition of new CENPA-containing nucleosomes at the centromere / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / mitotic spindle organization / acrosomal vesicle / chromosome segregation / RHO GTPases Activate Formins / regulation of protein stability / kinetochore / fibrillar center / spindle pole / Separation of Sister Chromatids / azurophil granule lumen / mitotic cell cycle / microtubule binding / transcription regulator complex / transcription coactivator activity / transcription by RNA polymerase II / nuclear body / nuclear speck / cell division / intracellular membrane-bounded organelle / centrosome / Neutrophil degranulation / protein-containing complex binding / nucleolus / Golgi apparatus / extracellular region / nucleoplasm / identical protein binding / membrane / nucleus / cytosol Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 26.7 Å | |||||||||
Authors | Petrovic A / Mosalaganti S / Keller J / Mattiuzzo M / Overlack K / Krenn V / De Antoni A / Wohlgemuth S / Cecatiello V / Pasqualato S ...Petrovic A / Mosalaganti S / Keller J / Mattiuzzo M / Overlack K / Krenn V / De Antoni A / Wohlgemuth S / Cecatiello V / Pasqualato S / Raunser S / Musacchio A | |||||||||
Citation | Journal: Mol Cell / Year: 2014 Title: Modular assembly of RWD domains on the Mis12 complex underlies outer kinetochore organization. Authors: Arsen Petrovic / Shyamal Mosalaganti / Jenny Keller / Marta Mattiuzzo / Katharina Overlack / Veronica Krenn / Anna De Antoni / Sabine Wohlgemuth / Valentina Cecatiello / Sebastiano ...Authors: Arsen Petrovic / Shyamal Mosalaganti / Jenny Keller / Marta Mattiuzzo / Katharina Overlack / Veronica Krenn / Anna De Antoni / Sabine Wohlgemuth / Valentina Cecatiello / Sebastiano Pasqualato / Stefan Raunser / Andrea Musacchio / Abstract: Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between ...Faithful chromosome segregation is mandatory for cell and organismal viability. Kinetochores, large protein assemblies embedded in centromeric chromatin, establish a mechanical link between chromosomes and spindle microtubules. The KMN network, a conserved 10-subunit kinetochore complex, harbors the microtubule-binding interface. RWD domains in the KMN subunits Spc24 and Spc25 mediate kinetochore targeting of the microtubule-binding subunits by interacting with the Mis12 complex, a KMN subcomplex that tethers directly onto the underlying chromatin layer. Here, we show that Knl1, a KMN subunit involved in mitotic checkpoint signaling, also contains RWD domains that bind the Mis12 complex and that mediate kinetochore targeting of Knl1. By reporting the first 3D electron microscopy structure of the KMN network, we provide a comprehensive framework to interpret how interactions of RWD-containing proteins with the Mis12 complex shape KMN network topology. Our observations unveil a regular pattern in the construction of the outer kinetochore. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2549.map.gz | 7.2 MB | EMDB map data format | |
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Header (meta data) | emd-2549-v30.xml emd-2549.xml | 25.6 KB 25.6 KB | Display Display | EMDB header |
Images | emd_2549.tif | 222.5 KB | ||
Masks | emd_2549_msk_1.map | 8 MB | Mask map | |
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2549 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2549 | HTTPS FTP |
-Validation report
Summary document | emd_2549_validation.pdf.gz | 200.7 KB | Display | EMDB validaton report |
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Full document | emd_2549_full_validation.pdf.gz | 199.9 KB | Display | |
Data in XML | emd_2549_validation.xml.gz | 5.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2549 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2549 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_2549.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of KMN network | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.6 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Segmentation: Binary mask representing the whole structure
Annotation | Binary mask representing the whole structure | ||||||||||||
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File | emd_2549_msk_1.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : KMN network reconstituted as follows: human Mis12complex bound to...
+Supramolecule #1000: KMN network reconstituted as follows: human Mis12complex bound to...
+Macromolecule #1: Mis12
+Macromolecule #2: Polyamine-modulated factor 1
+Macromolecule #3: Dsn1
+Macromolecule #4: Nsl1
+Macromolecule #5: Bub-linking kinetochore protein
+Macromolecule #6: Highly expressed in cancer-1
+Macromolecule #7: Spc25
+Macromolecule #8: Spc24
+Macromolecule #9: Cell division cycle-assoiciated protein1
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.01 mg/mL |
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Buffer | pH: 8 / Details: 20mM Tris-HCl, 150mM NaCl, 1mM TCEP |
Staining | Type: NEGATIVE Details: Grids with adsorbed protein floated on 0.07% Uranyl formate |
Grid | Details: 200 mesh copper grids with thin carbon support, glow discharged |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | JEOL 1400 |
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Details | Minimal Dose system |
Date | Oct 14, 2013 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 248 / Average electron dose: 19 e/Å2 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 67200 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: JEOL / Tilt angle min: -50 |
-Image processing
Details | Tilt pairs were collected at 50 and 0 degrees. Particle pairs were manually selected using e2RCTboxer program. RCT reconstruction of tilted particles was calculated from best class average by back projection and was followed by back projection refinement. |
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Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 26.7 Å / Resolution method: OTHER / Software - Name: EMAN2/sparx / Number images used: 3764 |
Final two d classification | Number classes: 10 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: C |
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Software | Name: Chimera |
Details | The crystal structures were fit into the overall density manually. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
-Atomic model buiding 2
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: Chimera |
Details | The crystal structures were fit into the overall density manually. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |