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- PDB-6rf2: Cryo-EM structure of the C-terminal DC repeat (CDC) of human doub... -

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Basic information

Entry
Database: PDB / ID: 6rf2
TitleCryo-EM structure of the C-terminal DC repeat (CDC) of human doublecortin (DCX) bound to 13-protofilament GDP.Pi-microtubule
Components
  • Neuronal migration protein doublecortin
  • Tubulin alpha-1B chain
  • Tubulin beta-2B chain
KeywordsCYTOSOLIC PROTEIN / Microtubule-associated protein / neuronal migration protein / microtubule nucleation and stabilisation
Function / homology
Function and homology information


axoneme assembly / Neurofascin interactions / positive regulation of axon guidance / microtubule associated complex / microtubule-based process / cytoplasmic microtubule / cellular response to interleukin-4 / central nervous system development / structural constituent of cytoskeleton / microtubule cytoskeleton organization ...axoneme assembly / Neurofascin interactions / positive regulation of axon guidance / microtubule associated complex / microtubule-based process / cytoplasmic microtubule / cellular response to interleukin-4 / central nervous system development / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / nervous system development / mitotic cell cycle / double-stranded RNA binding / retina development in camera-type eye / microtubule cytoskeleton / microtubule binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / cytoskeleton / neuron projection / intracellular signal transduction / cilium / protein heterodimerization activity / GTPase activity / ubiquitin protein ligase binding / protein kinase binding / GTP binding / metal ion binding / cytoplasm / cytosol
Similarity search - Function
Neuronal migration protein doublecortin, chordata / Doublecortin / Doublecortin domain profile. / Domain in the Doublecortin (DCX) gene product / Doublecortin domain / Doublecortin domain superfamily / Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / Beta tubulin, autoregulation binding site / Beta tubulin ...Neuronal migration protein doublecortin, chordata / Doublecortin / Doublecortin domain profile. / Domain in the Doublecortin (DCX) gene product / Doublecortin domain / Doublecortin domain superfamily / 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
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / PHOSPHATE ION / Neuronal migration protein doublecortin / Tubulin alpha-1B chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Bos taurus (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsManka, S.W.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MR/R000352/1 United Kingdom
CitationJournal: EMBO Rep / Year: 2020
Title: Pseudo-repeats in doublecortin make distinct mechanistic contributions to microtubule regulation.
Authors: Szymon W Manka / Carolyn A Moores /
Abstract: Doublecortin (DCX) is a neuronal microtubule-associated protein (MAP) indispensable for brain development. Its flexibly linked doublecortin (DC) domains-NDC and CDC-mediate microtubule (MT) ...Doublecortin (DCX) is a neuronal microtubule-associated protein (MAP) indispensable for brain development. Its flexibly linked doublecortin (DC) domains-NDC and CDC-mediate microtubule (MT) nucleation and stabilization, but it is unclear how. Using high-resolution time-resolved cryo-EM, we mapped NDC and CDC interactions with tubulin at different MT polymerization stages and studied their functional effects on MT dynamics using TIRF microscopy. Although coupled, each DC repeat within DCX appears to have a distinct role in MT nucleation and stabilization: CDC is a conformationally plastic module that appears to facilitate MT nucleation and stabilize tubulin-tubulin contacts in the nascent MT lattice, while NDC appears to be favored along the mature lattice, providing MT stabilization. Our structures of MT-bound DC domains also explain in unprecedented detail the DCX mutation-related brain defects observed in the clinic. This modular composition of DCX reflects a common design principle among MAPs where pseudo-repeats of tubulin/MT binding elements chaperone or stabilize distinct conformational transitions to regulate distinct stages of MT dynamic instability.
History
DepositionApr 12, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 13, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Mar 3, 2021Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3May 22, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
a: Tubulin alpha-1B chain
B: Tubulin beta-2B chain
b: Tubulin beta-2B chain
A: Tubulin alpha-1B chain
C: Neuronal migration protein doublecortin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)204,82513
Polymers202,6545
Non-polymers2,1718
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, The assembly is a fragment (repeating unit) of a 13-protofilament GDP.Pi-microtubule decorated with doublecortin (DCX) via the C-terminal DC repeat, as determined by Cryo-EM 3D reconstruction
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15870 Å2
ΔGint-95 kcal/mol
Surface area68450 Å2
MethodPISA

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Components

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Protein , 3 types, 5 molecules aABbC

#1: Protein Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 48263.594 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: P81947
#2: Protein Tubulin beta-2B chain


Mass: 48113.129 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: Q6B856
#3: Protein Neuronal migration protein doublecortin / Doublin / Lissencephalin-X / Lis-X


Mass: 9900.500 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DCX, DBCN, LISX / Production host: Escherichia coli (E. coli) / References: UniProt: O43602

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Non-polymers , 4 types, 8 molecules

#4: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: PO4
#7: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
15-component complex of C-terminal DC domain of human doublecortin bound to 13-protofilament GDP.Pi-microtubule lattice site composed of 4 tubulin subunits (two alpha-subunits and two beta-subunits)COMPLEX#1-#30MULTIPLE SOURCES
2tubulinCOMPLEX#1-#21NATURAL
3human doublecortinCOMPLEX#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Bos taurus (domestic cattle)9913
23Homo sapiens (human)9606
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 6.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 25 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 6591 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5IP4

5ip4


Accession code: 5IP4 / Source name: PDB / Type: experimental model

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