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- EMDB-6307: Electron cryo-microscopy of microtubule-bound TTLL7 -

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Basic information

Entry
Database: EMDB / ID: EMD-6307
TitleElectron cryo-microscopy of microtubule-bound TTLL7
Map dataReconstruction of a Tubulin Tyrosine Ligase-Like Glutamylase bound to the microtubule
Sample
  • Sample: Tubulin Tyrosine Ligase-Like (TTLL7) bound to the microtubule
  • Protein or peptide: Tubulin Tyrosine Ligase-Like (TTLL7) Family Glutamylase
  • Protein or peptide: tubulin
Keywordsmicrotubule-bound TTLL7
Biological speciesHomo sapiens (human) / Bos taurus (cattle)
Methodhelical reconstruction / cryo EM / Resolution: 7.95 Å
AuthorsWilson-Kubalek EM / Garnham CP / Vemu A / Yu I / Szyk A / Lander GC / Milligan RA / Roll-Mecak A
CitationJournal: Cell / Year: 2015
Title: Multivalent Microtubule Recognition by Tubulin Tyrosine Ligase-like Family Glutamylases.
Authors: Christopher P Garnham / Annapurna Vemu / Elizabeth M Wilson-Kubalek / Ian Yu / Agnieszka Szyk / Gabriel C Lander / Ronald A Milligan / Antonina Roll-Mecak /
Abstract: Glutamylation, the most prevalent tubulin posttranslational modification, marks stable microtubules and regulates recruitment and activity of microtubule- interacting proteins. Nine enzymes of the ...Glutamylation, the most prevalent tubulin posttranslational modification, marks stable microtubules and regulates recruitment and activity of microtubule- interacting proteins. Nine enzymes of the tubulin tyrosine ligase-like (TTLL) family catalyze glutamylation. TTLL7, the most abundant neuronal glutamylase, adds glutamates preferentially to the β-tubulin tail. Coupled with ensemble and single-molecule biochemistry, our hybrid X-ray and cryo-electron microscopy structure of TTLL7 bound to the microtubule delineates a tripartite microtubule recognition strategy. The enzyme uses its core to engage the disordered anionic tails of α- and β-tubulin, and a flexible cationic domain to bind the microtubule and position itself for β-tail modification. Furthermore, we demonstrate that all single-chain TTLLs with known glutamylase activity utilize a cationic microtubule-binding domain analogous to that of TTLL7. Therefore, our work reveals the combined use of folded and intrinsically disordered substrate recognition elements as the molecular basis for specificity among the enzymes primarily responsible for chemically diversifying cellular microtubules.
History
DepositionMar 16, 2015-
Header (metadata) releaseApr 29, 2015-
Map releaseMay 27, 2015-
UpdateJun 10, 2015-
Current statusJun 10, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6307.gif

Downloads & links

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Map

FileDownload / File: emd_6307.map.gz / Format: CCP4 / Size: 1.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a Tubulin Tyrosine Ligase-Like Glutamylase bound to the microtubule
Voxel sizeX=Y=Z: 2.73 Å
Density
Contour LevelBy AUTHOR: 0.6 / Movie #1: 0.6
Minimum - Maximum-4.09821844 - 7.11796856
Average (Standard dev.)-0.04005896 (±1.32854939)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin14-31-36
Dimensions6267102
Spacing6267102
CellA: 182.91 Å / B: 169.26 Å / C: 278.46 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.732.732.73
M x/y/z6762102
origin x/y/z0.0000.0000.000
length x/y/z182.910169.260278.460
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-3114-36
NC/NR/NS6762102
D min/max/mean-4.0987.118-0.040

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Supplemental data

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Sample components

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Entire : Tubulin Tyrosine Ligase-Like (TTLL7) bound to the microtubule

EntireName: Tubulin Tyrosine Ligase-Like (TTLL7) bound to the microtubule
Components
  • Sample: Tubulin Tyrosine Ligase-Like (TTLL7) bound to the microtubule
  • Protein or peptide: Tubulin Tyrosine Ligase-Like (TTLL7) Family Glutamylase
  • Protein or peptide: tubulin

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Supramolecule #1000: Tubulin Tyrosine Ligase-Like (TTLL7) bound to the microtubule

SupramoleculeName: Tubulin Tyrosine Ligase-Like (TTLL7) bound to the microtubule
type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: one TTLL7 bound to tubulin dimer / Number unique components: 2
Molecular weightExperimental: 61 KDa / Theoretical: 61 KDa / Method: SDS PAGE

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Macromolecule #1: Tubulin Tyrosine Ligase-Like (TTLL7) Family Glutamylase

MacromoleculeName: Tubulin Tyrosine Ligase-Like (TTLL7) Family Glutamylase
type: protein_or_peptide / ID: 1 / Name.synonym: TTLL7 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 61 KDa / Theoretical: 61 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: tubulin

MacromoleculeName: tubulin / type: protein_or_peptide / ID: 2
Details: Two sources of tubulin were used: cytoskeletal tubulin from bovine brain and human tubulin from TCA 201 cells.
Recombinant expression: No / Database: NCBI
Source (natural)Organism: Bos taurus (cattle) / synonym: bovine / Tissue: brain / Location in cell: cytoskeleton

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation stateparticle

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Sample preparation

Concentration11 mg/mL
BufferpH: 7 / Details: 20 mM HEPES, 0.5 mM ATP, 1 mM TCEP, 50 mM NaCl
GridDetails: Protochips C-flat grid: holey carbon with 2 um holes and 400 mesh copper grid with 2 um spacing
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER
Method: Blot grid from behind for 3 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 62000x magnification.
DateAug 5, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 2.73 µm / Number real images: 614 / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFIND v3
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.95 Å / Resolution method: OTHER / Software - Name: EMAN2, FREALIGN
Details: Final maps were calculated from three averaged data sets.
DetailsWe used IHRSR adapted for microtubules with a dimer repeat.

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