+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6307 | |||||||||
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Title | Electron cryo-microscopy of microtubule-bound TTLL7 | |||||||||
Map data | Reconstruction of a Tubulin Tyrosine Ligase-Like Glutamylase bound to the microtubule | |||||||||
Sample |
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Keywords | microtubule-bound TTLL7 | |||||||||
Biological species | Homo sapiens (human) / Bos taurus (cattle) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 7.95 Å | |||||||||
Authors | Wilson-Kubalek EM / Garnham CP / Vemu A / Yu I / Szyk A / Lander GC / Milligan RA / Roll-Mecak A | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6307.map.gz | 1.5 MB | EMDB map data format | |
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Header (meta data) | emd-6307-v30.xml emd-6307.xml | 10.7 KB 10.7 KB | Display Display | EMDB header |
Images | 400_6307.gif 80_6307.gif | 123.6 KB 7.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6307 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6307 | HTTPS FTP |
-Validation report
Summary document | emd_6307_validation.pdf.gz | 78.5 KB | Display | EMDB validaton report |
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Full document | emd_6307_full_validation.pdf.gz | 77.6 KB | Display | |
Data in XML | emd_6307_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6307 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6307 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6307.map.gz / Format: CCP4 / Size: 1.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of a Tubulin Tyrosine Ligase-Like Glutamylase bound to the microtubule | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.73 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Tubulin Tyrosine Ligase-Like (TTLL7) bound to the microtubule
Entire | Name: Tubulin Tyrosine Ligase-Like (TTLL7) bound to the microtubule |
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Components |
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-Supramolecule #1000: Tubulin Tyrosine Ligase-Like (TTLL7) bound to the microtubule
Supramolecule | Name: 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 |
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Molecular weight | Experimental: 61 KDa / Theoretical: 61 KDa / Method: SDS PAGE |
-Macromolecule #1: Tubulin Tyrosine Ligase-Like (TTLL7) Family Glutamylase
Macromolecule | Name: 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 |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Molecular weight | Experimental: 61 KDa / Theoretical: 61 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Macromolecule #2: tubulin
Macromolecule | Name: 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 |
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Source (natural) | Organism: Bos taurus (cattle) / synonym: bovine / Tissue: brain / Location in cell: cytoskeleton |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 11 mg/mL |
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Buffer | pH: 7 / Details: 20 mM HEPES, 0.5 mM ATP, 1 mM TCEP, 50 mM NaCl |
Grid | Details: Protochips C-flat grid: holey carbon with 2 um holes and 400 mesh copper grid with 2 um spacing |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER Method: Blot grid from behind for 3 seconds before plunging. |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 62000x magnification. |
Date | Aug 5, 2014 |
Image recording | Category: 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 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 62000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Details | We used IHRSR adapted for microtubules with a dimer repeat. |
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Final reconstruction | Algorithm: 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. |
CTF correction | Details: CTFIND v3 |