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- EMDB-6594: Cryo-EM of human Ndc80-broccoli bound to microtubules -

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Basic information

Entry
Database: EMDB / ID: EMD-6594
TitleCryo-EM of human Ndc80-broccoli bound to microtubules
Map data3D reconstruction of the human Ndc80 broccoli complex bound to microtubules
Sample
  • Sample: human Ndc80 Broccoli complex bound to microtubules
  • Organelle or cellular component: microtubule
  • Protein or peptide: Ndc80
KeywordsKinetochore protein complex
Biological speciesunidentified (others) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsWilson-Kubalek EM / Cheeseman IM / Milligan RA
CitationJournal: Mol Biol Cell / Year: 2016
Title: Structural comparison of the Caenorhabditis elegans and human Ndc80 complexes bound to microtubules reveals distinct binding behavior.
Authors: Elizabeth M Wilson-Kubalek / Iain M Cheeseman / Ronald A Milligan /
Abstract: During cell division, kinetochores must remain tethered to the plus ends of dynamic microtubule polymers. However, the molecular basis for robust kinetochore-microtubule interactions remains poorly ...During cell division, kinetochores must remain tethered to the plus ends of dynamic microtubule polymers. However, the molecular basis for robust kinetochore-microtubule interactions remains poorly understood. The conserved four-subunit Ndc80 complex plays an essential and direct role in generating dynamic kinetochore-microtubule attachments. Here we compare the binding of theCaenorhabditis elegansand human Ndc80 complexes to microtubules at high resolution using cryo-electron microscopy reconstructions. Despite the conserved roles of the Ndc80 complex in diverse organisms, we find that the attachment mode of these complexes for microtubules is distinct. The human Ndc80 complex binds every tubulin monomer along the microtubule protofilament, whereas theC. elegansNdc80 complex binds more tightly to β-tubulin. In addition, theC. elegansNdc80 complex tilts more toward the adjacent protofilament. These structural differences in the Ndc80 complex between different species may play significant roles in the nature of kinetochore-microtubule interactions.
History
DepositionFeb 23, 2016-
Header (metadata) releaseMar 23, 2016-
Map releaseMar 23, 2016-
UpdateApr 27, 2016-
Current statusApr 27, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6594.gif

Downloads & links

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Map

FileDownload / File: emd_6594.map.gz / Format: CCP4 / Size: 27.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of the human Ndc80 broccoli complex bound to microtubules
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 111 pix.
= 144.3 Å		1.3 Å/pix.
x 257 pix.
= 334.1 Å		1.3 Å/pix.
x 257 pix.
= 334.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.3 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.8 / Movie #1: 2.8
Minimum - Maximum-7.39598846 - 13.738596920000001
Average (Standard dev.)0.01343017 (±1.17511463)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00110
Dimensions257257111
Spacing257257111
CellA: 334.09998 Å / B: 334.09998 Å / C: 144.29999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.31.31.3
M x/y/z257257111
origin x/y/z0.0000.0000.000
length x/y/z334.100334.100144.300
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00110
NC/NR/NS257257111
D min/max/mean-7.39613.7390.013

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Supplemental data

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Sample components

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Entire : human Ndc80 Broccoli complex bound to microtubules

EntireName: human Ndc80 Broccoli complex bound to microtubules
Components
  • Sample: human Ndc80 Broccoli complex bound to microtubules
  • Organelle or cellular component: microtubule
  • Protein or peptide: Ndc80

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Supramolecule #1000: human Ndc80 Broccoli complex bound to microtubules

SupramoleculeName: human Ndc80 Broccoli complex bound to microtubules / type: sample / ID: 1000 / Oligomeric state: helical / Number unique components: 2

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Supramolecule #1: microtubule

SupramoleculeName: microtubule / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others)

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Macromolecule #1: Ndc80

MacromoleculeName: Ndc80 / type: protein_or_peptide / ID: 1 / Oligomeric state: helical / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant plasmid: pST39

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

BufferpH: 6.8 / Details: 80 mM PIPES, pH 6.8, 1 mM MgCl2, 1 mM EGTA
GridDetails: 400-mesh C-flat grids (Protochips, Inc) containing 2.0 micron holes separated by 2.0 micron spacing
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 120 K / Instrument: HOMEMADE PLUNGER / Method: Blotted back of grid for 3 seconds.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateOct 7, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 1.3 µm / Number real images: 838 / Average electron dose: 40 e/Å2 / Details: Each image is an average of 30 frames.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsEMAN2 and FREALIGN IHRSR adapted for microtubules with a dimer repeat were used.
Final reconstructionApplied symmetry - Helical parameters - Δz: 11.2 Å
Applied symmetry - Helical parameters - Δ&Phi: 23.817 °
Applied symmetry - Helical parameters - Axial symmetry: C15 (15 fold cyclic)
Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: OTHER / Software - Name: EMAN2, FREALIGN, IHRSR
Details: The final map was calculated from two averaged data sets.
CTF correctionDetails: CTFIND v3

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