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- PDB-2xrp: Human Doublecortin N-DC Repeat (1MJD) and Mammalian Tubulin (1JFF... -

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Basic information

Entry
Database: PDB / ID: 2xrp
TitleHuman Doublecortin N-DC Repeat (1MJD) and Mammalian Tubulin (1JFF and 3HKE) Docked into the 8-Angstrom Cryo-EM Map of Doublecortin- Stabilised Microtubules
Components
  • NEURONAL MIGRATION PROTEIN DOUBLECORTIN
  • TUBULIN ALPHA-1D CHAIN
  • TUBULIN BETA-2B CHAIN
KeywordsSTRUCTURAL 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 / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / nervous system development ...axoneme assembly / Neurofascin interactions / positive regulation of axon guidance / microtubule associated complex / microtubule-based process / central nervous system development / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / nervous system development / mitotic cell cycle / 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 / hydrolase activity / neuron projection / intracellular signal transduction / protein heterodimerization activity / GTPase activity / 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 / Neuronal migration protein doublecortin / Tubulin alpha-1D chain / Tubulin beta-2B chain
Similarity search - Component
Biological speciesHOMO SAPIENS (human)
BOS TAURUS (domestic cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsFourniol, F.J. / Sindelar, C.V. / Amigues, B. / Clare, D.K. / Thomas, G. / Perderiset, M. / Francis, F. / Houdusse, A. / Moores, C.A.
CitationJournal: J Cell Biol / Year: 2010
Title: Template-free 13-protofilament microtubule-MAP assembly visualized at 8 A resolution.
Authors: Franck J Fourniol / Charles V Sindelar / Béatrice Amigues / Daniel K Clare / Geraint Thomas / Mylène Perderiset / Fiona Francis / Anne Houdusse / Carolyn A Moores /
Abstract: Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed ...Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX's unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin-tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX's exquisite binding selectivity uncovers important insights into regulation of cellular MTs.
History
DepositionSep 18, 2010Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 24, 2010Provider: repository / Type: Initial release
Revision 1.1Mar 21, 2012Group: Other / Version format compliance
Revision 2.0Aug 23, 2017Group: Atomic model / Data collection / Refinement description
Category: atom_site / em_3d_fitting / em_software
Item: _atom_site.Cartn_x / _atom_site.Cartn_y ..._atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _em_software.name
Revision 2.1May 8, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / 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 / struct_site
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 / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-1788
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: TUBULIN BETA-2B CHAIN
B: TUBULIN ALPHA-1D CHAIN
C: TUBULIN BETA-2B CHAIN
D: TUBULIN ALPHA-1D CHAIN
E: TUBULIN BETA-2B CHAIN
F: TUBULIN ALPHA-1D CHAIN
G: TUBULIN BETA-2B CHAIN
H: TUBULIN ALPHA-1D CHAIN
I: NEURONAL MIGRATION PROTEIN DOUBLECORTIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)415,47117
Polymers411,6069
Non-polymers3,8668
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Protein
TUBULIN BETA-2B CHAIN / TUBULIN BETA CHAIN


Mass: 49907.770 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) BOS TAURUS (domestic cattle) / Organ: BRAIN / References: UniProt: Q6B856, EC: 3.6.5.6
#2: Protein
TUBULIN ALPHA-1D CHAIN / TUBULIN ALPHA CHAIN


Mass: 50236.352 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) BOS TAURUS (domestic cattle) / Organ: BRAIN / References: UniProt: Q2HJ86, EC: 3.6.5.6
#3: Protein NEURONAL MIGRATION PROTEIN DOUBLECORTIN / DOUBLECORTIN / LISSENCEPHALIN-X / LIS-X / DOUBLIN


Mass: 11029.393 Da / Num. of mol.: 1 / Fragment: RESIDUES 46-140
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) HOMO SAPIENS (human) / Organ: BRAIN / Plasmid: PFASTBAC / Production host: SPODOPTERA FRUGIPERDA (fall armyworm) / Strain (production host): SF9 / References: UniProt: O43602
#4: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#5: Chemical
ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
Sequence detailsCHIMERIC SEQUENCE OF SHEEP AND CATTLE MAIN ALPHA-TUBULIN ISOFORMS (1JFF AND 3HKE)

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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

ComponentName: MICROTUBULES NUCLEATED AND STABILISED BY DOUBLECORTIN / Type: COMPLEX
Buffer solutionName: 80MM PIPES, 1MM EGTA, 3MM MGCL2, 1MM TCEP, 0.5MM GTP / pH: 6.8
Details: 80MM PIPES, 1MM EGTA, 3MM MGCL2, 1MM TCEP, 0.5MM GTP
SpecimenConc.: 1.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Details: LIQUID ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 50000 X / Nominal defocus max: 2900 nm / Nominal defocus min: 760 nm / Cs: 2 mm
Specimen holderTemperature: 93 K
Image recordingElectron dose: 15 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 63
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1Flex-EMmodel fitting
2UCSF Chimeramodel fitting
3FREALIGN3D reconstruction
4SPIDER3D reconstruction
CTF correctionDetails: DONE IN FREALIGN
3D reconstructionMethod: CUSTOM SCRIPTS, PROJECTION MATCHING / Resolution: 8.2 Å / Num. of particles: 168000 / Nominal pixel size: 2.8 Å
Details: THE STRUCTURE WAS DETERMINED IN THE ABSENCE OF A STABILISING DRUG. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-1788. (DEPOSITION ID: 7535).
Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER / Target criteria: Cross-correlation coefficient
Details: METHOD--LOCAL CORRELATION REFINEMENT PROTOCOL--ELECTRON CRYSTALLOGRAPHY
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
11JFF

1jff

11JFF1PDBexperimental model
23HKE

3hke

13HKE2PDBexperimental model
31MJD

1mjd

11MJD3PDBexperimental model
RefinementHighest resolution: 8.2 Å
Refinement stepCycle: LAST / Highest resolution: 8.2 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms27516 0 240 0 27756

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