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- EMDB-7976: Preformed GMPCPP microtubule washed with EB3, class 1 -

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Basic information

Entry
Database: EMDB / ID: 7976
TitlePreformed GMPCPP microtubule washed with EB3, class 1
Map dataPreformed GMPCPP microtubule washed with EB3, class 1
SamplePreformed GMPCPP microtubule washed with EB3, class 1:
microtubule / (EB3) x 2 / alpha tubulin / beta tubulin
SourceSus scrofa (pig) / Homo sapiens (human)
Methodhelical reconstruction / cryo EM / 4.3 Å resolution
AuthorsZhang R / Nogales E
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2018
Title: Separating the effects of nucleotide and EB binding on microtubule structure.
Authors: Rui Zhang / Benjamin LaFrance / Eva Nogales
Abstract: Microtubules (MTs) are polymers assembled from αβ-tubulin heterodimers that display the hallmark behavior of dynamic instability. MT dynamics are driven by GTP hydrolysis within the MT lattice, and ...Microtubules (MTs) are polymers assembled from αβ-tubulin heterodimers that display the hallmark behavior of dynamic instability. MT dynamics are driven by GTP hydrolysis within the MT lattice, and are highly regulated by a number of MT-associated proteins (MAPs). How MAPs affect MTs is still not fully understood, partly due to a lack of high-resolution structural data on undecorated MTs, which need to serve as a baseline for further comparisons. Here we report three structures of MTs in different nucleotide states (GMPCPP, GDP, and GTPγS) at near-atomic resolution and in the absence of any binding proteins. These structures allowed us to differentiate the effects of nucleotide state versus MAP binding on MT structure. Kinesin binding has a small effect on the extended, GMPCPP-bound lattice, but hardly affects the compacted GDP-MT lattice, while binding of end-binding (EB) proteins can induce lattice compaction (together with lattice twist) in MTs that were initially in an extended and more stable state. We propose a MT lattice-centric model in which the MT lattice serves as a platform that integrates internal tubulin signals, such as nucleotide state, with outside signals, such as binding of MAPs or mechanical forces, resulting in global lattice rearrangements that in turn affect the affinity of other MT partners and result in the exquisite regulation of MT dynamics.
DateDeposition: Jun 9, 2018 / Header (metadata) release: Jul 4, 2018 / Map release: Jul 4, 2018 / Last update: Jul 18, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 3.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_7976.map.gz (map file in CCP4 format, 536871 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
512 pix
1.33 Å/pix.
= 680.96 Å
512 pix
1.33 Å/pix.
= 680.96 Å
512 pix
1.33 Å/pix.
= 680.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.33 Å
Density
Contour Level:3.8 (by author), 3.8 (movie #1):
Minimum - Maximum-7.958745 - 17.437935
Average (Standard dev.)-0.036095407 (0.901363)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions512512512
Origin0.0.0.
Limit511.511.511.
Spacing512512512
CellA=B=C: 680.96 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.331.331.33
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z680.960680.960680.960
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS512512512
D min/max/mean-7.95917.438-0.036

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Supplemental data

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Sample components

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Entire Preformed GMPCPP microtubule washed with EB3, class 1

EntireName: Preformed GMPCPP microtubule washed with EB3, class 1 / Number of components: 6

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Component #1: cellular-component, Preformed GMPCPP microtubule washed with EB3,...

Cellular-componentName: Preformed GMPCPP microtubule washed with EB3, class 1 / Recombinant expression: No

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Component #2: cellular-component, microtubule

Cellular-componentName: microtubule / Recombinant expression: No
SourceSpecies: Sus scrofa (pig)

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Component #3: protein, EB3

ProteinName: EB3 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli K-12 (bacteria) / Strain: K-12

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Component #4: protein, alpha tubulin

ProteinName: alpha tubulin / Details: GTP / Recombinant expression: No
SourceSpecies: Sus scrofa (pig)

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Component #5: protein, beta tubulin

ProteinName: beta tubulin / Details: GMPCPP / Recombinant expression: No
SourceSpecies: Sus scrofa (pig)

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Component #6: protein, EB3

ProteinName: EB3 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Escherichia coli K-12 (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: helical array / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Hand: RIGHT HANDED / Delta z: 8.957 Å / Delta phi: -25.769 deg.
Sample solutionSpecimen conc.: 3 mg/ml
Buffer solution: BRB80 buffer (80 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM MgCl2) supplemented with 1 mM GMPCPP, 1 mM DTT, and 0.05% Nonident P-40
pH: 6.8
Support filmThe grid was glow discharged with Solarus (Gatan Inc).
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 95 % / Details: Blot for 4 seconds before plunging..

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Electron microscopy imaging

ImagingMicroscope: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.44 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 27500. X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 2500.0 nm
Specimen HolderModel: GATAN LIQUID NITROGEN
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 182

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionAlgorithm: FOURIER SPACE / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF

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