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- PDB-27pz: Dictyostelium discoideum cytoplasmic dynein motor domain in the p... -

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Basic information

Entry
Database: PDB / ID: 27pz
TitleDictyostelium discoideum cytoplasmic dynein motor domain in the presence of ADP (ADP state 2)
ComponentsDynein heavy chain, cytoplasmic
KeywordsMOTOR PROTEIN / dynein / ATPase / AAA+ protein
Function / homology
Function and homology information


minus-end-directed vesicle transport along microtubule / early phagosome membrane / Aggrephagy / COPI-mediated anterograde transport / phagolysosome membrane / CTPase activity / Neutrophil degranulation / phagosome maturation / ATPase complex / minus-end-directed microtubule motor activity ...minus-end-directed vesicle transport along microtubule / early phagosome membrane / Aggrephagy / COPI-mediated anterograde transport / phagolysosome membrane / CTPase activity / Neutrophil degranulation / phagosome maturation / ATPase complex / minus-end-directed microtubule motor activity / dynein light intermediate chain binding / cytoplasmic dynein complex / nuclear migration / microtubule motor activity / dynein intermediate chain binding / mitotic spindle assembly / endocytic vesicle / cytoplasmic microtubule / cytoplasmic microtubule organization / mitotic spindle organization / tubulin binding / cell cortex / microtubule binding / microtubule / centrosome / ATP binding / identical protein binding / 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 / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Dynein heavy chain, cytoplasmic
Similarity search - Component
Biological speciesDictyostelium discoideum AX2 (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsShimo-Kon, R. / Tokita, H. / Imai, H. / Maeshima, T. / Kon, T.
Funding support Japan, 6items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP16K07327 Japan
Japan Society for the Promotion of Science (JSPS)JP21K06096 Japan
Japan Society for the Promotion of Science (JSPS)JP24K09395 Japan
Japan Society for the Promotion of Science (JSPS)JP17H03665 Japan
Japan Society for the Promotion of Science (JSPS)JP24H02259 Japan
Japan Society for the Promotion of Science (JSPS)JP23H02445 Japan
CitationJournal: J Mol Biol / Year: 2026
Title: ADP-Bound States of Cytoplasmic Dynein: Cryo-electron Microscopy Reveals a Two-Step Post-Power-Stroke Transition and Roles of Regulatory ATPase Sites.
Authors: Hiroshi Imai / Riko Kanazawa / Toshihisa Maeshima / Duy Phuoc Tran / Rieko Shimo-Kon / Hisaya Tokita / Tomoko Miyata / Fumiaki Makino / Junso Fujita / Takayuki Kato / Akio Kitao / Keiichi Namba / Takahide Kon /
Abstract: Cytoplasmic dynein is essential for intracellular transport and cell division, and its dysfunction is implicated in severe neurological disorders. Its motility is driven by the primary ATPase site in ...Cytoplasmic dynein is essential for intracellular transport and cell division, and its dysfunction is implicated in severe neurological disorders. Its motility is driven by the primary ATPase site in the AAA1 module and allosterically modulated by regulatory ATPase sites in the AAA3 and AAA4 modules. Despite recent structural advances, post-force-generating (post-power-stroke) transitions and the structural basis of AAA3/AAA4-mediated regulation remain elusive. Using cryo-electron microscopy, we captured intermediate structures of Dictyostelium discoideum cytoplasmic dynein. We identified two distinct ADP-bound states and two apo states, the latter comprising a partially nucleotide-free state with ADP retained at the AAA3 and AAA4 ATPase sites and a fully nucleotide-free state. For the AAA1 ATPase site, our structures and molecular dynamics simulations suggest a dynamic equilibrium between two ADP-bound states. In this model, linker swing and docking onto the AAA5 module can be driven by thermal fluctuations while ADP remains bound at the AAA1 ATPase site, shifting the motor from a canonical ADP-bound state to a metastable apo-like conformation with reduced ADP affinity. This transition could provide a framework for tension-sensing coordination. For the AAA3 and AAA4 ATPase sites, comparison of the partially and fully nucleotide-free apo structures indicates that ADP release induces localized outward tilting of the corresponding large submodules. We propose that this rearrangement displaces the AAA4 Pre-Sensor-I insert from the linker, thereby disfavoring the recovery stroke and stabilizing an inactive state. Together, our findings provide a mechanistic model in which coordinated conformational dynamics across multiple ATPase sites govern dynein's chemomechanical cycle.
History
DepositionJun 9, 2026Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 26, 2026Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 26, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Dynein heavy chain, cytoplasmic
hetero molecules


Theoretical massNumber of molelcules
Total (without water)386,1446
Polymers384,3311
Non-polymers1,8135
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Dynein heavy chain, cytoplasmic / Dynein heavy chain / cytosolic / DYHC


Mass: 384331.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: N-terminal His6-FLAG-tagged HF380 motor-domain construct of Dictyostelium discoideum cytoplasmic dynein heavy chain.
Source: (gene. exp.) Dictyostelium discoideum AX2 (eukaryote)
Gene: dhcA, DDB_G0276355 / Plasmid: pMB38-HF380 / Production host: Dictyostelium discoideum AX2 (eukaryote) / References: UniProt: P34036
#2: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Dictyostelium discoideum HF380 cytoplasmic dynein motor domain in the presence of ADP
Type: COMPLEX
Details: Recombinantly expressed and purified HF380 was prepared with ADP before vitrification.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Dictyostelium discoideum AX2 (eukaryote)
Source (recombinant)Organism: Dictyostelium discoideum AX2 (eukaryote) / Plasmid: pMB38-HF380
Buffer solutionpH: 7.1
Details: 10 mM K-PIPES, 4 mM MgSO4, 1 mM EGTA, 0.05 mM ADP, 0.1% (v/v) Tween-20, pH 7.1
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMK-PIPESK-PIPES1
24 mMmagnesium sulfateMgSO41
31 mMEGTAEGTA1
40.05 mMADPADP1
50.1 % v/vTween-201
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportDetails: Grids were glow-discharged using a JEC-3000FC sputter coater (JEOL, Japan) at 20 mA for 20 s.
Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 60000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 32531
Details: Images were collected in movie-mode. Dose rate was 2.0 electrons/angstrom squared/frame.
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC4.4.1particle selectionBlob and tempalte-based picker
2SerialEM3.8 or 4.050image acquisitionAutomated data collection using together with yoneo-Locr (version 1.051).
4cryoSPARC4.4.1CTF correctionPatch CTFestimation; grobal CTF refinemnt; local CTF refinement
7UCSF ChimeraX1.7model fittingRigid-body fitting and model inspection.
8Coot0.9.8.92model fittingManual model building and local adjustment.
10PHENIX1.21.1_5286model refinementReal-space refinement.
11cryoSPARC4.4.1initial Euler assignmentInitial orientation assignment during ab initio reconstruction.
12cryoSPARC4.4.1final Euler assignmentFinal orientation refinement during non-uniform refinement.
13cryoSPARC4.4.1classification3D valiability
14cryoSPARC4.4.13D reconstructionNon-uniform refinement was used.
Image processingDetails: Movies were motion-corrected using Patch Motion Correction, and CTF parameters were estimated using Patch CTF estimation in cryoSPARC 4.4.1. Micrographs with CTF-estimated maximum ...Details: Movies were motion-corrected using Patch Motion Correction, and CTF parameters were estimated using Patch CTF estimation in cryoSPARC 4.4.1. Micrographs with CTF-estimated maximum resolutions better than 6 angstrom were retained for further processing.
CTF correctionDetails: CTF parameters were estimated using Patch CTF estimation in cryoSPARC and further refined by global and local CTF refinement.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4072620
Details: Particles were selected by blob picking and template-based picking in cryoSPARC, followed by 2D classification and 3D refinement to retain 4,072,620 particles for 3D variability analysis.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 596990 / Algorithm: FOURIER SPACE
Details: The final ADP state 2 map was reconstructed from the particle subset selected after 3D variability analysis and 3D Variability Display in cluster mode. The selected particles were refined by ...Details: The final ADP state 2 map was reconstructed from the particle subset selected after 3D variability analysis and 3D Variability Display in cluster mode. The selected particles were refined by non-uniform refinement in cryoSPARC with global and local CTF refinement.
Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingB value: 118.4 / Protocol: FLEXIBLE FIT / Space: REAL
Target criteria: Real-space target; weighted map sum at atom centers
Details: A monomeric HF380 motor-domain model was fitted into cryo-EM map and refined by phenix.real_space_refine.
Atomic model buildingPDB-ID: 3VKG

3vkg


Pdb chain-ID: A / Accession code: 3VKG / Chain residue range: 1410-4729 / Details: Initial model for the HF380 dynein motor domain. / Pdb chain residue range: 1410-4729 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.2 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00325173
ELECTRON MICROSCOPYf_angle_d0.52934126
ELECTRON MICROSCOPYf_dihedral_angle_d5.5773336
ELECTRON MICROSCOPYf_chiral_restr0.043833
ELECTRON MICROSCOPYf_plane_restr0.0044352

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