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- EMDB-5038: 11-Angstrom cryo-EM reconstruction of nucleotide-free Nod complex... -

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Basic information

Entry
Database: EMDB / ID: EMD-5038
Title11-Angstrom cryo-EM reconstruction of nucleotide-free Nod complexed to the 13-protofilament microtubule.
Map data11-Angstrom cryo-EM reconstruction of nucleotide-free Nod complexed to the 13-protofilament microtubule.
Sample
  • Sample: Nucleotide-free Nod complexed to the 13-protofilament microtubule
  • Protein or peptide: Nod
  • Protein or peptide: microtubule
Keywordskinesin / tubulin / nod / motor proteins / cytoskeletal motor
Function / homology
Function and homology information


establishment of meiotic spindle orientation / distributive segregation / COPI-dependent Golgi-to-ER retrograde traffic / Kinesins / meiotic chromosome segregation / female meiotic nuclear division / microtubule plus-end binding / spindle assembly involved in female meiosis I / positive regulation of axon guidance / tubulin complex ...establishment of meiotic spindle orientation / distributive segregation / COPI-dependent Golgi-to-ER retrograde traffic / Kinesins / meiotic chromosome segregation / female meiotic nuclear division / microtubule plus-end binding / spindle assembly involved in female meiosis I / positive regulation of axon guidance / tubulin complex / kinesin complex / microtubule motor activity / microtubule-based movement / cytoplasmic microtubule / microtubule-based process / positive regulation of microtubule polymerization / cellular response to interleukin-4 / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / double-stranded RNA binding / mitotic cell cycle / nervous system development / microtubule binding / microtubule / protein heterodimerization activity / GTPase activity / ubiquitin protein ligase binding / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Kinesin-like protein / RuvA domain 2-like / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Tubulin-beta mRNA autoregulation signal. ...Kinesin-like protein / RuvA domain 2-like / Kinesin motor domain signature. / Kinesin motor domain, conserved site / Kinesin motor domain / Kinesin motor domain profile. / Kinesin motor, catalytic domain. ATPase. / Kinesin motor domain / Kinesin motor domain superfamily / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Kinesin-like protein Nod / Tubulin alpha-1B chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesunidentified (others)
Methodhelical reconstruction / cryo EM / Resolution: 11.0 Å
AuthorsCochran JC / Sindelar CV / Mulko NK / Collins KA / Kong SE / Hawley RS / Kull FJ
CitationJournal: Cell / Year: 2009
Title: ATPase cycle of the nonmotile kinesin NOD allows microtubule end tracking and drives chromosome movement.
Authors: Jared C Cochran / Charles V Sindelar / Natasha K Mulko / Kimberly A Collins / Stephanie E Kong / R Scott Hawley / F Jon Kull /
Abstract: Segregation of nonexchange chromosomes during Drosophila melanogaster meiosis requires the proper function of NOD, a nonmotile kinesin-10. We have determined the X-ray crystal structure of the NOD ...Segregation of nonexchange chromosomes during Drosophila melanogaster meiosis requires the proper function of NOD, a nonmotile kinesin-10. We have determined the X-ray crystal structure of the NOD catalytic domain in the ADP- and AMPPNP-bound states. These structures reveal an alternate conformation of the microtubule binding region as well as a nucleotide-sensitive relay of hydrogen bonds at the active site. Additionally, a cryo-electron microscopy reconstruction of the nucleotide-free microtubule-NOD complex shows an atypical binding orientation. Thermodynamic studies show that NOD binds tightly to microtubules in the nucleotide-free state, yet other nucleotide states, including AMPPNP, are weakened. Our pre-steady-state kinetic analysis demonstrates that NOD interaction with microtubules occurs slowly with weak activation of ADP product release. Upon rapid substrate binding, NOD detaches from the microtubule prior to the rate-limiting step of ATP hydrolysis, which is also atypical for a kinesin. We propose a model for NOD's microtubule plus-end tracking that drives chromosome movement.
History
DepositionDec 16, 2008-
Header (metadata) releaseDec 18, 2008-
Map releaseMay 5, 2009-
UpdateDec 22, 2009-
Current statusDec 22, 2009Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3dco
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3dco
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5038.jpg

Downloads & links

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Map

FileDownload / File: emd_5038.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation11-Angstrom cryo-EM reconstruction of nucleotide-free Nod complexed to the 13-protofilament microtubule.
Voxel sizeX=Y=Z: 2 Å
Density
Contour LevelBy EMDB: 0.05 / Movie #1: 0.02
Minimum - Maximum-0.0572441 - 0.0998985
Average (Standard dev.)0.00314966 (±0.02226)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-90-90-90
Dimensions180180180
Spacing180180180
CellA=B=C: 360 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z360.000360.000360.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-20-28-19
NX/NY/NZ415638
MAP C/R/S123
start NC/NR/NS-90-90-90
NC/NR/NS180180180
D min/max/mean-0.0570.1000.003

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Supplemental data

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Sample components

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Entire : Nucleotide-free Nod complexed to the 13-protofilament microtubule

EntireName: Nucleotide-free Nod complexed to the 13-protofilament microtubule
Components
  • Sample: Nucleotide-free Nod complexed to the 13-protofilament microtubule
  • Protein or peptide: Nod
  • Protein or peptide: microtubule

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Supramolecule #1000: Nucleotide-free Nod complexed to the 13-protofilament microtubule

SupramoleculeName: Nucleotide-free Nod complexed to the 13-protofilament microtubule
type: sample / ID: 1000
Oligomeric state: One monomer of Nod binds to one heterodimer of tubulin
Number unique components: 3
Molecular weightTheoretical: 164 KDa
Method: Theoretical MW of one Nod monomer complexed to one tubulin dimer, based on length of the protein chains.

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Macromolecule #1: Nod

MacromoleculeName: Nod / type: protein_or_peptide / ID: 1 / Name.synonym: Nod / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: unidentified (others) / Strain: B834(DE3) / Location in cell: cytosol
Molecular weightTheoretical: 43 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET21a

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Macromolecule #2: microtubule

MacromoleculeName: microtubule / type: protein_or_peptide / ID: 2 / Name.synonym: tubulin / Oligomeric state: quasi-helical assembly / Recombinant expression: No / Database: NCBI
Source (natural)Organism: unidentified (others) / Tissue: brain / Cell: cow / Location in cell: cytoplasm
Molecular weightTheoretical: 120 KDa
SequenceGO: tubulin complex / InterPro: Alpha tubulin, Beta tubulin

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8 / Details: see publication
GridDetails: 300 mesh copper grid with homemade holey carbon film
VitrificationCryogen name: ETHANE / Chamber temperature: 103 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: homemade. Carried out under ambient conditions. No glow discharge was applied to the grids prior to sample application.
Method: Excess solution wicked away from grid with filter paper before blotting and plunging immediately. Less than 0.5 seconds elapsed between blotting and plunging.

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Electron microscopy

MicroscopeJEOL 4000EX
Electron beamAcceleration voltage: 400 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 4.1 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 60000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 98 K / Max: 108 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: crude, software correction used
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.3 µm / Number real images: 130 / Average electron dose: 16 e/Å2
Details: Data from only 17 micrographs were selected for the final reconstruction.
Bits/pixel: 16

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Image processing

CTF correctionDetails: Integrated with fourier inversion (similar to FREALIGN)
Final angle assignmentDetails: SPIDER
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.0 Å / Resolution method: OTHER / Software - Name: SPIDER
Details: Image defocus and astigmatism were determined by ctffind3.

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Atomic model buiding 1

Initial modelPDB ID:

3dc4

SoftwareName: SITUS
DetailsProtocol: Rigid Body. Because Nod occupancy was low, most tubulin density was deleted using UCSF Chimera"s volume eraser prior to automatic fitting to avoid spurious fits.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation
Output model

PDB-3dco:
Drosophila NOD (3DC4) and Bovine Tubulin (1JFF) Docked into the 11-Angstrom Cryo-EM Map of Nucleotide-Free NOD Complexed to the Microtubule

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Atomic model buiding 2

Initial modelPDB ID:

1jff

SoftwareName: SITUS
DetailsProtocol: Rigid Body
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation
Output model

PDB-3dco:
Drosophila NOD (3DC4) and Bovine Tubulin (1JFF) Docked into the 11-Angstrom Cryo-EM Map of Nucleotide-Free NOD Complexed to the Microtubule

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