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- PDB-3j1t: High affinity dynein microtubule binding domain - tubulin complex -

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Basic information

Entry
Database: PDB / ID: 3j1t
TitleHigh affinity dynein microtubule binding domain - tubulin complex
Components
  • Cytoplasmic dynein 1 heavy chain 1, seryl t-RNA synthetase chimera
  • Tubulin alpha-1B chain
  • Tubulin beta-2B chain
KeywordsMOTOR PROTEIN/STRUCTURAL PROTEIN / MOTOR PROTEIN-STRUCTURAL PROTEIN complex
Function / homology
Function and homology information


COPI-independent Golgi-to-ER retrograde traffic / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Aggrephagy / COPI-mediated anterograde transport / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / cilium movement / RHO GTPases Activate Formins ...COPI-independent Golgi-to-ER retrograde traffic / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / Aggrephagy / COPI-mediated anterograde transport / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / cilium movement / RHO GTPases Activate Formins / Separation of Sister Chromatids / Loss of Nlp from mitotic centrosomes / Recruitment of mitotic centrosome proteins and complexes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of NuMA to mitotic centrosomes / Anchoring of the basal body to the plasma membrane / AURKA Activation by TPX2 / Regulation of PLK1 Activity at G2/M Transition / positive regulation of intracellular transport / regulation of metaphase plate congression / positive regulation of spindle assembly / MHC class II antigen presentation / establishment of spindle localization / positive regulation of axon guidance / dynein complex / P-body assembly / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / dynein light intermediate chain binding / dynein intermediate chain binding / microtubule-based process / cytoplasmic microtubule / Neutrophil degranulation / stress granule assembly / cellular response to interleukin-4 / regulation of mitotic spindle organization / filopodium / structural constituent of cytoskeleton / microtubule cytoskeleton organization / neuron migration / mitotic cell cycle / double-stranded RNA binding / positive regulation of cold-induced thermogenesis / microtubule cytoskeleton / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / cilium / protein heterodimerization activity / cell division / GTPase activity / ubiquitin protein ligase binding / centrosome / GTP binding / ATP hydrolysis activity / ATP binding / metal ion binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Dynein heavy chain, AAA 5 extension domain / Dynein heavy chain AAA lid domain / Dynein heavy chain, C-terminal domain / Dynein heavy chain, C-terminal domain, barrel region / Dynein heavy chain C-terminal domain / : / Dynein heavy chain, ATPase lid domain / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / P-loop containing dynein motor region ...Dynein heavy chain, AAA 5 extension domain / Dynein heavy chain AAA lid domain / Dynein heavy chain, C-terminal domain / Dynein heavy chain, C-terminal domain, barrel region / Dynein heavy chain C-terminal domain / : / Dynein heavy chain, ATPase lid domain / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / P-loop containing dynein motor region / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, linker / Dynein heavy chain, AAA module D4 / Dynein heavy chain, coiled coil stalk / Dynein heavy chain / Dynein heavy chain, hydrolytic ATP-binding dynein motor region / Dynein heavy chain, ATP-binding dynein motor region / Dynein heavy chain AAA lid domain / Dynein heavy chain AAA lid domain superfamily / Dynein heavy chain, domain 2, N-terminal / Dynein heavy chain, linker, subdomain 3 / Dynein heavy chain, AAA1 domain, small subdomain / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, N-terminal region 2 / Hydrolytic ATP binding site of dynein motor region / Microtubule-binding stalk of dynein motor / P-loop containing dynein motor region D4 / ATP-binding dynein motor region / Dynein heavy chain AAA lid domain / 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Tubulin alpha-1B chain / Tubulin beta-2B chain / Cytoplasmic dynein 1 heavy chain 1
Similarity search - Component
Biological speciesMus musculus (house mouse)
Bos taurus (domestic cattle)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 9.7 Å
AuthorsRedwine, W.B. / Hernandez-Lopez, R. / Zou, S. / Huang, J. / Reck-Peterson, S.L. / Leschziner, A.E.
CitationJournal: Science / Year: 2012
Title: Structural basis for microtubule binding and release by dynein.
Authors: W B Redwine / R Hernandez-Lopez / S Zou / J Huang / S L Reck-Peterson / A E Leschziner /
Abstract: Cytoplasmic dynein is a microtubule-based motor required for intracellular transport and cell division. Its movement involves coupling cycles of track binding and release with cycles of force- ...Cytoplasmic dynein is a microtubule-based motor required for intracellular transport and cell division. Its movement involves coupling cycles of track binding and release with cycles of force-generating nucleotide hydrolysis. How this is accomplished given the ~25 nanometers separating dynein's track- and nucleotide-binding sites is not understood. Here, we present a subnanometer-resolution structure of dynein's microtubule-binding domain bound to microtubules by cryo-electron microscopy that was used to generate a pseudo-atomic model of the complex with molecular dynamics. We identified large rearrangements triggered by track binding and specific interactions, confirmed by mutagenesis and single-molecule motility assays, which tune dynein's affinity for microtubules. Our results provide a molecular model for how dynein's binding to microtubules is communicated to the rest of the motor.
History
DepositionJun 25, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 26, 2012Provider: repository / Type: Initial release
Revision 1.1Oct 3, 2012Group: Database references
Revision 1.2Aug 27, 2014Group: Other
Revision 1.3Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.4Feb 21, 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: Cytoplasmic dynein 1 heavy chain 1, seryl t-RNA synthetase chimera
B: Tubulin alpha-1B chain
C: Tubulin beta-2B chain


Theoretical massNumber of molelcules
Total (without water)116,7783
Polymers116,7783
Non-polymers00
Water00
1


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

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Components

#1: Protein Cytoplasmic dynein 1 heavy chain 1, seryl t-RNA synthetase chimera / Cytoplasmic dynein heavy chain 1 / Dynein heavy chain / cytosolic


Mass: 18729.609 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Dync1h1, Dhc1, Dnch1, Dnchc1, Dyhc / Production host: Escherichia coli (E. coli) / References: UniProt: Q9JHU4
#2: Protein Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 50107.238 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: P81947*PLUS
#3: Protein Tubulin beta-2B chain / beta tubulin


Mass: 47940.945 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (domestic cattle) / References: UniProt: Q6B856*PLUS
Sequence detailsCHAINS B AND C ARE DERIVED FROM PDB ENTRY 1JFF. AS A RESULT, THE MODELED SEQUENCES ARE FROM SUS ...CHAINS B AND C ARE DERIVED FROM PDB ENTRY 1JFF. AS A RESULT, THE MODELED SEQUENCES ARE FROM SUS SCROFA (UNP P02550 AND P02554, RESPECTIVELY). CHAIN A IS A CHIMERA COMPRISING THE 34 N-TERMINAL RESIDUES OF THERMUS THERMOPHILUS SERYL TRNA SYNTHETASE, THE MICROTUBULE BINDING DOMAIN (UNP RESIDUES 3264-3427) OF MUS MUSCULUS DYNEIN, THE 326 C-TERMINAL RESIDUES OF THERMUS THERMOPHILUS SERYL TRNA SYNTHETASE, AND A C-TERMINAL EXPRESSION TAG (GAAEQKLISEEDLNGLEHHHHHHHH). IN THIS ENTRY, ONLY THE DYNEIN RESIDUES WERE MODELED. THE FULL EXPERIMENTAL SEQUENCE WAS AS FOLLOWS: MVDLKRLRQEPEVFHRAIREKGVALDLEALLAVDKQQEVIADKQMSVKEDLDKVEPAVIEA QNAVKSIKKQHLVEVRSMANPPAAVKLALESICLLLGESTTDWKQIRSIIMRENFIPTIVN FSAEEISDAIREKMKKNYMSNPSYNYEIVNRASLACGPMVKWAIAQLNYADMLKRVEPLRN ELQKLEDDAKDNQQKLEALLLQVPLPPWPGAPVGGEEANREIKRVGGPPEFSFPPLDHVAL MEKNGWWEPRASQDSGSRSYALKGDLASYEEALLRFAADFMARRGFTPADTPSRAREKAFL GTGHFPAYRDQVRAESETDTYTTGTAEVVLNALHSGEILPYEALPLRYAGYAPAFRSEAGS FGKDVRGLMRVHQFHKVEQYVLTEASLEASDRAFQELLENAEEILRLLELPYRLVEVATGD MGPGKWRQVDIEVYLPSEGRYRETHSCSALLDWQARRANLRYRDPEGRVRYAYTLNNTALA TPRILAMLLENHQLQDGRVRVPQALIPYMGKEVLEPGAAEQKLISEEDLNGLEHHHHHHHH

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeParent-ID
1High affinity construct of dynein microtubule binding domain bound to microtubulesCOMPLEX0
2dynein microtubule binding domain1
3alpha-tubulin1
4beta-tubulin1
Buffer solutionName: 50 mM Tris HCl, 1 mM MgCl2, 1mM EGTA, 1mM DTT / pH: 8 / Details: 50 mM Tris HCl, 1 mM MgCl2, 1mM EGTA, 1mM DTT
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: C-flat 2/2-2C holey carbon grids (Protochips) were glow-discharged for 20 seconds at 30 mA in an Edwards carbon evaporator.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K
Details: The solution was blotted manually, and the process of addition and blotting of SRS-MTBD was repeated a total of three times. The final blotting was done inside the humidity chamber of a ...Details: The solution was blotted manually, and the process of addition and blotting of SRS-MTBD was repeated a total of three times. The final blotting was done inside the humidity chamber of a Vitrobot Mark IV (FEI) at 22 Celsius.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Apr 10, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 62000 X / Calibrated magnification: 63377 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.2 mm
Specimen holderTemperature: 100 K
Image recordingElectron dose: 15 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 225

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Processing

EM software
IDNameCategory
1NAMDmodel fitting
2UCSF Chimeramodel fitting
3FREALIGN3D reconstruction
4SPIDER3D reconstruction
CTF correctionDetails: phase and amplitude correction using Frealign
Helical symmertyAngular rotation/subunit: -25.76 ° / Axial rise/subunit: 9.26 Å / Axial symmetry: C1
3D reconstructionMethod: Projection matching and back-projection in Fourier space
Resolution: 9.7 Å / Num. of particles: 10419 / Nominal pixel size: 1.988 Å / Actual pixel size: 1.988 Å / Magnification calibration: TMV images
Details: Projection matching and Helical symmetry operator during reconstruction
Symmetry type: HELICAL
Atomic model building
IDProtocolSpaceDetails
1FLEXIBLE FITREALMETHOD--Molecular dynamics flexible fitting REFINEMENT PROTOCOL--Rigid body DETAILS--Explicit solvent molecular dynamics, molecular dynamics flexible fitting, target molecular dynamics
2FLEXIBLE FITREALMETHOD--Molecular dynamics flexible fitting REFINEMENT PROTOCOL--Rigid body DETAILS--Explicit solvent molecular dynamics, molecular dynamics flexible fitting
3FLEXIBLE FITREALMETHOD--Molecular dynamics flexible fitting REFINEMENT PROTOCOL--Rigid body DETAILS--Explicit solvent molecular dynamics, molecular dynamics flexible fitting
Atomic model building

Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-ID 3D fitting-IDAccession codeInitial refinement model-ID
13ERR

3err

A13ERR1
21JFF

1jff

A21JFF2
31JFF

1jff

B31JFF2
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms8091 0 0 0 8091

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