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- EMDB-0612: CryoEM Density of Acetylated Microtubules -

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Basic information

Entry
Database: EMDB / ID: EMD-0612
TitleCryoEM Density of Acetylated Microtubules
Map data
SampleAcetylated Microtubule:
Tubulin alpha-1B chain / Tubulin beta chain / (ligand) x 3
Function / homology
Function and homology information


RHO GTPases activate IQGAPs / Intraflagellar transport / Cilium Assembly / Hedgehog 'off' state / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / COPI-dependent Golgi-to-ER retrograde traffic ...RHO GTPases activate IQGAPs / Intraflagellar transport / Cilium Assembly / Hedgehog 'off' state / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) / Resolution of Sister Chromatid Cohesion / Separation of Sister Chromatids / Microtubule-dependent trafficking of connexons from Golgi to the plasma membrane / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / Kinesins / Carboxyterminal post-translational modifications of tubulin / The role of GTSE1 in G2/M progression after G2 checkpoint / Mitotic Prometaphase / COPI-independent Golgi-to-ER retrograde traffic / RHO GTPases Activate Formins / microtubule-based process / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / microtubule / GTPase activity / GTP binding / cytoplasm
Tubulin C-terminal domain / Tubulin/FtsZ, GTPase domain superfamily / Tubulin-beta mRNA autoregulation signal. / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin / Alpha tubulin ...Tubulin C-terminal domain / Tubulin/FtsZ, GTPase domain superfamily / Tubulin-beta mRNA autoregulation signal. / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, C-terminal / Beta tubulin, autoregulation binding site / Tubulin, conserved site
Tubulin beta chain / Tubulin alpha-1B chain
Biological speciesPig (pig)
Methodhelical reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsEshun-Wilson L / Zhang R / Portran D / Nachury MV / Toso D / Lohr T / Vendruscolo M / Bonomi M / Fraser JS / Nogales E
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2019
Title: Effects of α-tubulin acetylation on microtubule structure and stability.
Authors: Lisa Eshun-Wilson / Rui Zhang / Didier Portran / Maxence V Nachury / Daniel B Toso / Thomas Löhr / Michele Vendruscolo / Massimiliano Bonomi / James S Fraser / Eva Nogales /
Abstract: Acetylation of K40 in α-tubulin is the sole posttranslational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule ...Acetylation of K40 in α-tubulin is the sole posttranslational modification to mark the luminal surface of microtubules. It is still controversial whether its relationship with microtubule stabilization is correlative or causative. We have obtained high-resolution cryo-electron microscopy (cryo-EM) reconstructions of pure samples of αTAT1-acetylated and SIRT2-deacetylated microtubules to visualize the structural consequences of this modification and reveal its potential for influencing the larger assembly properties of microtubules. We modeled the conformational ensembles of the unmodified and acetylated states by using the experimental cryo-EM density as a structural restraint in molecular dynamics simulations. We found that acetylation alters the conformational landscape of the flexible loop that contains αK40. Modification of αK40 reduces the disorder of the loop and restricts the states that it samples. We propose that the change in conformational sampling that we describe, at a location very close to the lateral contacts site, is likely to affect microtubule stability and function.
Validation ReportPDB-ID: 6o2q

SummaryFull reportAbout validation report
History
DepositionFeb 24, 2019-
Header (metadata) releaseMar 20, 2019-
Map releaseMay 22, 2019-
UpdateJun 5, 2019-
Current statusJun 5, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 2.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6o2q
  • Surface level: 2.1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6o2q
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0612.map.gz / Format: CCP4 / Size: 10.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.07 Å/pix.
x 157 pix.
= 167.99 Å
1.07 Å/pix.
x 93 pix.
= 99.51 Å
1.07 Å/pix.
x 194 pix.
= 207.58 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: 1.07 Å
Density
Contour LevelBy AUTHOR: 1.1 / Movie #1: 2.1
Minimum - Maximum-5.2240195 - 9.120468000000001
Average (Standard dev.)-0.000000000002012 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions93194157
Spacing15793194
CellA: 167.99 Å / B: 99.51 Å / C: 207.58002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z15793194
origin x/y/z0.0000.0000.000
length x/y/z167.99099.510207.580
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ15793194
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS19493157
D min/max/mean-5.2249.120-0.000

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Supplemental data

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Sample components

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Entire Acetylated Microtubule

EntireName: Acetylated Microtubule / Number of components: 6

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Component #1: cellular-component, Acetylated Microtubule

Cellular-componentName: Acetylated Microtubule / Recombinant expression: No

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Component #2: protein, Tubulin alpha-1B chain

ProteinName: Tubulin alpha-1B chain / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 50.204445 kDa
SourceSpecies: Pig (pig)
Source (natural)Organ or tissue: Brain

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Component #3: protein, Tubulin beta chain

ProteinName: Tubulin beta chain / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 49.90777 kDa
SourceSpecies: Pig (pig)
Source (natural)Organ or tissue: Brain

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Component #4: ligand, GUANOSINE-5'-TRIPHOSPHATE

LigandName: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 0.52318 kDa

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Component #5: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #6: ligand, GUANOSINE-5'-DIPHOSPHATE

LigandName: GUANOSINE-5'-DIPHOSPHATE / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 0.443201 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: helical array / Method: cryo EM
Helical parametersAxial symmetry: C1 (asymmetric) / Delta z: 9.3 Å / Delta phi: -27.7 %deg;
Sample solutionSpecimen conc.: 10 mg/mL
Buffer solution: Contains 80 mM PIPES, 1 mM MgCl2, 1 mM EGTA, pH 6.8 with KOH (stored at 4 degrees Celsius).
pH: 6.8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 310.15 K / Humidity: 100 % / Details: Blotted for 4 seconds at blot force 10..

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Details: Preliminary grid screening was performed manually and all of the alignments were initially done using a gold calibration grid.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 22500.0 X (nominal), 23364.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500.0 - 2500.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: (77.0 - 77.0 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 287

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Image processing

ProcessingMethod: helical reconstruction
3D reconstructionAlgorithm: FOURIER SPACE / Software: FREALIGN / CTF correction: CTFFIND4 / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Target criteria: 0.5 / Refinement space: REAL
Details: We use PHENIX to perform real space refinement and sharpen our cryoEM maps.
Input PDB model: 3JAR
Overall bvalue: 126
Output model

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