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- EMDB-4863: Cryo-EM structure of the N-terminal DC repeat (NDC) of NDC-NDC ch... -

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Entry
Database: EMDB / ID: EMD-4863
TitleCryo-EM structure of the N-terminal DC repeat (NDC) of NDC-NDC chimera (human sequence) bound to 14-protofilament GDP-microtubule
Map dataCryo-EM structure of the N-terminal DC repeat (NDC) of NDC-NDC chimera (human sequence) bound to 14-protofilament GDP-microtubule
Sample
  • Complex: 5-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: human doublecortin
      • Protein or peptide: Neuronal migration protein doublecortin
    • Complex: tubulin
      • Protein or peptide: Tubulin beta-2B chain
      • Protein or peptide: Tubulin alpha-1B chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
KeywordsMicrotubule-associated protein chimera / ubiquitin-like fold / microtubule stabilisation / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


axoneme assembly / Neurofascin interactions / positive regulation of axon guidance / microtubule associated complex / microtubule-based process / central nervous system development / neuron migration / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton ...axoneme assembly / Neurofascin interactions / positive regulation of axon guidance / microtubule associated complex / microtubule-based process / central nervous system development / neuron migration / structural constituent of cytoskeleton / microtubule cytoskeleton organization / microtubule cytoskeleton / mitotic cell cycle / retina development in camera-type eye / nervous system development / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule binding / microtubule / cytoskeleton / intracellular signal transduction / neuron projection / protein heterodimerization activity / GTPase activity / GTP binding / protein kinase binding / metal ion binding / cytosol / cytoplasm
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 ...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
Neuronal migration protein doublecortin / Tubulin alpha-1B chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesHomo sapiens (human) / Bos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsManka SW
Funding support United Kingdom, 1 items
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, 2019-
Header (metadata) releaseApr 24, 2019-
Map releaseMay 13, 2020-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6rfd
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4863.png

Downloads & links

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Map

FileDownload / File: emd_4863.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the N-terminal DC repeat (NDC) of NDC-NDC chimera (human sequence) bound to 14-protofilament GDP-microtubule
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.39 Å/pix.
x 92 pix.
= 127.88 Å		1.39 Å/pix.
x 96 pix.
= 133.44 Å		1.39 Å/pix.
x 60 pix.
= 83.4 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: 1.39 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.08062031 - 0.17560998
Average (Standard dev.)0.005633883 (±0.018871019)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions966092
Spacing609692
CellA: 83.4 Å / B: 133.44 Å / C: 127.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.391.391.39
M x/y/z609692
origin x/y/z0.0000.0000.000
length x/y/z83.400133.440127.880
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS609692
D min/max/mean-0.0810.1760.006

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Supplemental data

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Sample components

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Entire : 5-component complex of N-terminal DC domain (NDC) of NDC-NDC chim...

EntireName: 5-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 ...Name: 5-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)
Components
  • Complex: 5-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: human doublecortin
      • Protein or peptide: Neuronal migration protein doublecortin
    • Complex: tubulin
      • Protein or peptide: Tubulin beta-2B chain
      • Protein or peptide: Tubulin alpha-1B chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: 5-component complex of N-terminal DC domain (NDC) of NDC-NDC chim...

SupramoleculeName: 5-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 ...Name: 5-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)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: human doublecortin

SupramoleculeName: human doublecortin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: tubulin

SupramoleculeName: tubulin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2, #1
Source (natural)Organism: Bos taurus (cattle)

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Macromolecule #1: Tubulin alpha-1B chain

MacromoleculeName: Tubulin alpha-1B chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 48.263594 KDa
SequenceString: MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPDSF NTFFSETGAG KHVPRAVFVD LEPTVIDEVR TGTYRQLFHP EQLITGKED AANNYARGHY TIGKEIIDLV LDRIRKLADQ CTGLQGFLVF HSFGGGTGSG FTSLLMERLS VDYGKKSKLE F SIYPAPQV ...String:
MRECISIHVG QAGVQIGNAC WELYCLEHGI QPDGQMPDSF NTFFSETGAG KHVPRAVFVD LEPTVIDEVR TGTYRQLFHP EQLITGKED AANNYARGHY TIGKEIIDLV LDRIRKLADQ CTGLQGFLVF HSFGGGTGSG FTSLLMERLS VDYGKKSKLE F SIYPAPQV STAVVEPYNS ILTTHTTLEH SDCAFMVDNE AIYDICRRNL DIERPTYTNL NRLISQIVSS ITASLRFDGA LN VDLTEFQ TNLVPYPRIH FPLATYAPVI SAEKAYHEQL SVAEITNACF EPANQMVKCD PRHGKYMACC LLYRGDVVPK DVN AAIATI KTKRSIQFVD WCPTGFKVGI NYQPPTVVPG GDLAKVQRAV CMLSNTTAIA EAWARLDHKF DLMYAKRAFV HWYV GEGME EGEFSEARED MAALEKDYEE VGVDSVE

UniProtKB: Tubulin alpha-1B chain

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Macromolecule #2: Tubulin beta-2B chain

MacromoleculeName: Tubulin beta-2B chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 48.113129 KDa
SequenceString: MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS ...String:
MREIVHIQAG QCGNQIGAKF WEVISDEHGI DPTGSYHGDS DLQLERINVY YNEAAGNKYV PRAILVDLEP GTMDSVRSGP FGQIFRPDN FVFGQSGAGN NWAKGHYTEG AELVDSVLDV VRKESESCDC LQGFQLTHSL GGGTGSGMGT LLISKIREEY P DRIMNTFS VVPSPKVSDT VVEPYNATLS VHQLVENTDE TYCIDNEALY DICFRTLKLT TPTYGDLNHL VSATMSGVTT CL RFPGQLN ADLRKLAVNM VPFPRLHFFM PGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMAACDPRH GRYLTVAAVF RGR MSMKEV DEQMLNVQNK NSSYFVEWIP NNVKTAVCDI PPRGLKMSAT FIGNSTAIQE LFKRISEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDAT

UniProtKB: Tubulin beta-2B chain

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Macromolecule #3: Neuronal migration protein doublecortin

MacromoleculeName: Neuronal migration protein doublecortin / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.441834 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QALSNEKKAK KVRFYRNGDR YFKGIVYAVS SDRFRSFDAL LADLTRSLSD NINLPQGVRY IYTIDGSRKI GSMDELEEGE SYVCSSDNF FKKVEYTKNV

UniProtKB: Neuronal migration protein doublecortin

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Macromolecule #4: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 2 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 2 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI POLARA 300
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 9984
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: ANGULAR RECONSTITUTION

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Atomic model buiding 1

Initial modelPDB ID:

1mjd


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-6rfd:
Cryo-EM structure of the N-terminal DC repeat (NDC) of NDC-NDC chimera (human sequence) bound to 14-protofilament GDP-microtubule

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