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- PDB-6rfd: Cryo-EM structure of the N-terminal DC repeat (NDC) of NDC-NDC ch... -

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Basic information

Entry
Database: PDB / ID: 6rfd
TitleCryo-EM structure of the N-terminal DC repeat (NDC) of NDC-NDC chimera (human sequence) bound to 14-protofilament GDP-microtubule
Components
  • Neuronal migration protein doublecortinDevelopment of the nervous system
  • Tubulin alpha-1B chain
  • Tubulin beta-2B chain
KeywordsCYTOSOLIC PROTEIN / Microtubule-associated protein chimera / ubiquitin-like fold / microtubule stabilisation
Function / homology
Function and homology information


axoneme assembly / Neurofascin interactions / positive regulation of axon guidance / microtubule associated complex / cytoplasmic microtubule / microtubule-based process / cellular response to interleukin-4 / central nervous system development / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / neuron migration ...axoneme assembly / Neurofascin interactions / positive regulation of axon guidance / microtubule associated complex / cytoplasmic microtubule / microtubule-based process / cellular response to interleukin-4 / central nervous system development / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / neuron migration / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / double-stranded RNA binding / retina development in camera-type eye / mitotic cell cycle / nervous system development / microtubule binding / microtubule / cytoskeleton / neuron projection / intracellular signal transduction / protein heterodimerization activity / GTPase activity / ubiquitin protein ligase binding / GTP binding / protein kinase binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Doublecortin domain / Neuronal migration protein doublecortin, chordata / : / Doublecortin / Doublecortin domain profile. / Domain in the Doublecortin (DCX) gene product / Doublecortin domain / Doublecortin domain superfamily / Helix hairpin bin / Tubulin/FtsZ, C-terminal domain ...Doublecortin domain / Neuronal migration protein doublecortin, chordata / : / Doublecortin / Doublecortin domain profile. / Domain in the Doublecortin (DCX) gene product / Doublecortin domain / Doublecortin domain superfamily / Helix hairpin bin / Tubulin/FtsZ, C-terminal domain / Tubulin/FtsZ, GTPase domain / 60s Ribosomal Protein L30; Chain: A; / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / 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 / Ubiquitin-like (UB roll) / Helix Hairpins / Roll / Rossmann fold / 2-Layer Sandwich / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / GUANOSINE-5'-TRIPHOSPHATE / Neuronal migration protein doublecortin / Tubulin alpha-1B chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Bos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 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
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
Revision 1.2Mar 3, 2021Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)206,17711
Polymers204,1955
Non-polymers1,9816
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy, The assembly is a fragment (repeating unit) of a 14-protofilament GDP-microtubule decorated with NDC-NDC chimera via either of the two N-terminal DC repeats of human ...Evidence: microscopy, The assembly is a fragment (repeating unit) of a 14-protofilament GDP-microtubule decorated with NDC-NDC chimera via either of the two N-terminal DC repeats of human doublecortin (DCX), as determined by Cryo-EM 3D reconstruction
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16040 Å2
ΔGint-77 kcal/mol
Surface area66920 Å2
MethodPISA

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Components

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Protein , 3 types, 5 molecules AaBbN

#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 (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 (cattle) / References: UniProt: Q6B856
#3: Protein Neuronal migration protein doublecortin / Development of the nervous system / Doublin / Lissencephalin-X / Lis-X


Mass: 11441.834 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 , 3 types, 6 molecules

#4: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine 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-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine 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 N-terminal DC domain (NDC) of NDC-NDC chimera (human doublecortin sequence) bound to 14-protofilament GDP-microtubule lattice site composed of 4 tubulin subunits (two alpha-subunits and two beta-subunits)COMPLEX#1-#30MULTIPLE SOURCES
2human doublecortinCOMPLEX#31RECOMBINANT
3tubulinCOMPLEX#2, #11NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Bos taurus (cattle)9913
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 FIELDBright-field microscopy
Image recordingElectron dose: 25 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model fitting
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 9984 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model buildingPDB-ID: 1MJD

1mjd

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