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- EMDB-2005: Subnanometer reconstruction of GTPgammaS microtubules decorated w... -

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Basic information

Entry
Database: EMDB / ID: EMD-2005
TitleSubnanometer reconstruction of GTPgammaS microtubules decorated with monomeric Mal3
Map dataSubnanometer reconstruction of GTPgammaS microtubules decorated with monomeric Mal3
Sample
  • Sample: GTPgammaS microtubules decorated with monomeric Mal3
  • Protein or peptide: Alpha tubulin
  • Protein or peptide: beta tubulin
  • Protein or peptide: Mal3
  • Ligand: guanosine 5'-O-(gamma-thio)triphosphate
Keywordscytoskeleton / GTPase / end binding / calponin homology
Function / homology
Function and homology information


dynein-driven meiotic oscillatory nuclear movement / post-anaphase array microtubule end / nuclear migration involved in conjugation with cellular fusion / cortical microtubule / cell cortex of cell tip / mitotic spindle astral microtubule / karyogamy involved in conjugation with cellular fusion / nuclear microtubule / mitotic spindle midzone / mitotic spindle pole body ...dynein-driven meiotic oscillatory nuclear movement / post-anaphase array microtubule end / nuclear migration involved in conjugation with cellular fusion / cortical microtubule / cell cortex of cell tip / mitotic spindle astral microtubule / karyogamy involved in conjugation with cellular fusion / nuclear microtubule / mitotic spindle midzone / mitotic spindle pole body / protein localization to microtubule / astral microtubule / microtubule plus-end / cytoskeletal anchor activity / attachment of mitotic spindle microtubules to kinetochore / microtubule plus-end binding / microtubule organizing center / microtubule lateral binding / cytoplasmic microtubule / ATPase activator activity / spindle midzone / spindle assembly / regulation of microtubule polymerization or depolymerization / molecular condensate scaffold activity / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule binding / microtubule / hydrolase activity / cell division / GTPase activity / GTP binding / nucleus / metal ion binding / cytoplasm
Similarity search - Function
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. / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. ...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. / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / 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
Similarity search - Domain/homology
Tubulin alpha-1A chain / Tubulin beta chain / Microtubule integrity protein mal3
Similarity search - Component
Biological speciesSus scrofa (pig) / Schizosaccharomyces pombe (fission yeast) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.6 Å
AuthorsMaurer SP / Fourniol FJ / Bohner G / Moores CA / Surrey T
CitationJournal: Cell / Year: 2012
Title: EBs recognize a nucleotide-dependent structural cap at growing microtubule ends.
Authors: Sebastian P Maurer / Franck J Fourniol / Gergő Bohner / Carolyn A Moores / Thomas Surrey /
Abstract: Growing microtubule ends serve as transient binding platforms for essential proteins that regulate microtubule dynamics and their interactions with cellular substructures. End-binding proteins (EBs) ...Growing microtubule ends serve as transient binding platforms for essential proteins that regulate microtubule dynamics and their interactions with cellular substructures. End-binding proteins (EBs) autonomously recognize an extended region at growing microtubule ends with unknown structural characteristics and then recruit other factors to the dynamic end structure. Using cryo-electron microscopy, subnanometer single-particle reconstruction, and fluorescence imaging, we present a pseudoatomic model of how the calponin homology (CH) domain of the fission yeast EB Mal3 binds to the end regions of growing microtubules. The Mal3 CH domain bridges protofilaments except at the microtubule seam. By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state. The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization. This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.
History
DepositionDec 2, 2011-
Header (metadata) releaseJan 20, 2012-
Map releaseMay 31, 2012-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 2.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4abo
  • Surface level: 2.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

2005-mal3-GT...

Downloads & links

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Map

FileDownload / File: emd_2005.map.gz / Format: CCP4 / Size: 859.4 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubnanometer reconstruction of GTPgammaS microtubules decorated with monomeric Mal3
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.2 Å/pix.
x 90 pix.
= 198. Å		2.2 Å/pix.
x 50 pix.
= 110. Å		2.2 Å/pix.
x 50 pix.
= 110. 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: 2.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.9 / Movie #1: 2.2
Minimum - Maximum-3.88709569 - 8.56416321
Average (Standard dev.)0.98212427 (±1.79068077)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin5611191
Dimensions505090
Spacing505090
CellA: 110.0 Å / B: 110.0 Å / C: 198.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z505090
origin x/y/z0.0000.0000.000
length x/y/z110.000110.000198.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-56-56-55
NX/NY/NZ112112112
MAP C/R/S123
start NC/NR/NS1115691
NC/NR/NS505090
D min/max/mean-3.8878.5640.982

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Supplemental data

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Sample components

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Entire : GTPgammaS microtubules decorated with monomeric Mal3

EntireName: GTPgammaS microtubules decorated with monomeric Mal3
Components
  • Sample: GTPgammaS microtubules decorated with monomeric Mal3
  • Protein or peptide: Alpha tubulin
  • Protein or peptide: beta tubulin
  • Protein or peptide: Mal3
  • Ligand: guanosine 5'-O-(gamma-thio)triphosphate

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Supramolecule #1000: GTPgammaS microtubules decorated with monomeric Mal3

SupramoleculeName: GTPgammaS microtubules decorated with monomeric Mal3 / type: sample / ID: 1000
Details: tubulin was mixed with GMPCPP microtubule seeds, GTPgammaS and monomeric Mal3, and incubated 3-10min at 37degC
Oligomeric state: 13-protofilament microtubule / Number unique components: 4

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Macromolecule #1: Alpha tubulin

MacromoleculeName: Alpha tubulin / type: protein_or_peptide / ID: 1 / Name.synonym: Alpha tubulin dimer / Oligomeric state: Dimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / synonym: Pig / Tissue: Brain / Location in cell: cytoplasmic
Molecular weightExperimental: 50 KDa / Theoretical: 50 KDa

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Macromolecule #2: beta tubulin

MacromoleculeName: beta tubulin / type: protein_or_peptide / ID: 2 / Name.synonym: beta tubulin / Oligomeric state: Dimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Sus scrofa (pig) / synonym: Pig / Tissue: Brain / Location in cell: cytoplasmic
Molecular weightExperimental: 50 KDa / Theoretical: 50 KDa

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Macromolecule #4: Mal3

MacromoleculeName: Mal3 / type: protein_or_peptide / ID: 4 / Name.synonym: Mal3 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Schizosaccharomyces pombe (fission yeast) / synonym: fission yeast / Location in cell: cytoplasmic
Molecular weightExperimental: 16 KDa / Theoretical: 16 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #3: guanosine 5'-O-(gamma-thio)triphosphate

MacromoleculeName: guanosine 5'-O-(gamma-thio)triphosphate / type: ligand / ID: 3 / Name.synonym: GTPgammaS / Recombinant expression: No / Database: NCBI
Source (natural)Organism: synthetic construct (others)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6.8 / Details: 40mM Pipes, 1mM MgCl2, 1mM EGTA
GridDetails: 300 mesh lacey carbon grid
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot (FEI) / Method: Chamber at 37 degrees C, blot 2s

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 88 K / Max: 98 K / Average: 93 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 162 / Average electron dose: 17 e/Å2 / Details: sampling size 2.2 A per pixel
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 0.7 µm / Nominal magnification: 68000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: FREALIGN
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, FREALIGN
Details: approximately 129000 tubulin dimers were averaged together in the final map
Number images used: 129000

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Atomic model buiding 1

Initial modelPDB ID:

2xrp

SoftwareName: UCSF Chimera
DetailsProtocol: Rigid body. Each tubulin monomer and a homology model for Mal3 CH domain were separately fitted using automatic fitting, and the multi-subunit fitting was refined in Flex-EM
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-4abo:
Mal3 CH domain homology model and mammalian tubulin (2XRP) docked into the 8.6-Angstrom cryo-EM map of Mal3-GTPgammaS-microtubules

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