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- PDB-5nug: Motor domains from human cytoplasmic dynein-1 in the phi-particle... -

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Basic information

Entry
Database: PDB / ID: 5nug
TitleMotor domains from human cytoplasmic dynein-1 in the phi-particle conformation
ComponentsCytoplasmic dynein 1 heavy chain 1
KeywordsMOTOR PROTEIN / dynein / motor domain / AAA+
Function / homology
Function and homology information


positive regulation of intracellular transport / regulation of metaphase plate congression / establishment of spindle localization / positive regulation of spindle assembly / P-body assembly / dynein complex / COPI-independent Golgi-to-ER retrograde traffic / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / retrograde axonal transport ...positive regulation of intracellular transport / regulation of metaphase plate congression / establishment of spindle localization / positive regulation of spindle assembly / P-body assembly / dynein complex / COPI-independent Golgi-to-ER retrograde traffic / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / retrograde axonal transport / dynein light intermediate chain binding / nuclear migration / dynein intermediate chain binding / cytoplasmic microtubule / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / COPI-mediated anterograde transport / cytoplasmic microtubule organization / stress granule assembly / Mitotic Prometaphase / regulation of mitotic spindle organization / EML4 and NUDC in mitotic spindle formation / axon cytoplasm / Loss of Nlp from mitotic centrosomes / Loss of proteins required for interphase microtubule organization from the centrosome / Recruitment of mitotic centrosome proteins and complexes / Resolution of Sister Chromatid Cohesion / Recruitment of NuMA to mitotic centrosomes / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Anchoring of the basal body to the plasma membrane / MHC class II antigen presentation / AURKA Activation by TPX2 / mitotic spindle organization / filopodium / RHO GTPases Activate Formins / Aggrephagy / HCMV Early Events / Separation of Sister Chromatids / azurophil granule lumen / Regulation of PLK1 Activity at G2/M Transition / positive regulation of cold-induced thermogenesis / cell cortex / microtubule / cell division / centrosome / Neutrophil degranulation / ATP hydrolysis activity / RNA binding / extracellular exosome / extracellular region / ATP binding / membrane / cytosol
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 / P-loop containing dynein motor region / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / Dynein heavy chain / Dynein heavy chain region D6 P-loop domain ...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 / P-loop containing dynein motor region / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / Dynein heavy chain / 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, 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 / Cytoplasmic dynein 1 heavy chain 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsZhang, K. / Foster, H.E. / Carter, A.P.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome TrustWT100387 United Kingdom
Medical Research Council (United Kingdom)MC_UP_A025_1011 United Kingdom
CitationJournal: Cell / Year: 2017
Title: Cryo-EM Reveals How Human Cytoplasmic Dynein Is Auto-inhibited and Activated.
Authors: Kai Zhang / Helen E Foster / Arnaud Rondelet / Samuel E Lacey / Nadia Bahi-Buisson / Alexander W Bird / Andrew P Carter /
Abstract: Cytoplasmic dynein-1 binds dynactin and cargo adaptor proteins to form a transport machine capable of long-distance processive movement along microtubules. However, it is unclear why dynein-1 moves ...Cytoplasmic dynein-1 binds dynactin and cargo adaptor proteins to form a transport machine capable of long-distance processive movement along microtubules. However, it is unclear why dynein-1 moves poorly on its own or how it is activated by dynactin. Here, we present a cryoelectron microscopy structure of the complete 1.4-megadalton human dynein-1 complex in an inhibited state known as the phi-particle. We reveal the 3D structure of the cargo binding dynein tail and show how self-dimerization of the motor domains locks them in a conformation with low microtubule affinity. Disrupting motor dimerization with structure-based mutagenesis drives dynein-1 into an open form with higher affinity for both microtubules and dynactin. We find the open form is also inhibited for movement and that dynactin relieves this by reorienting the motor domains to interact correctly with microtubules. Our model explains how dynactin binding to the dynein-1 tail directly stimulates its motor activity.
History
DepositionApr 30, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 28, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 2, 2017Group: Data collection / Derived calculations / Category: em_software / pdbx_struct_conn_angle / Item: _em_software.name
Revision 1.2Oct 17, 2018Group: Data collection / Refinement description / Category: refine
Revision 1.3Oct 23, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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Assembly

Deposited unit
A: Cytoplasmic dynein 1 heavy chain 1
B: Cytoplasmic dynein 1 heavy chain 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,069,79312
Polymers1,066,1672
Non-polymers3,62610
Water0
1
A: Cytoplasmic dynein 1 heavy chain 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)534,8966
Polymers533,0831
Non-polymers1,8135
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Cytoplasmic dynein 1 heavy chain 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)534,8966
Polymers533,0831
Non-polymers1,8135
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Protein Cytoplasmic dynein 1 heavy chain 1 / Cytoplasmic dynein heavy chain 1 / Dynein heavy chain / cytosolic


Mass: 533083.250 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DYNC1H1, DHC1, DNCH1, DNCL, DNECL, DYHC, KIAA0325 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q14204
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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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: Motor domains from human cytoplasmic dynein-1 in the phi-particle conformation
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
Details: 20mM HEPES pH7.4, 150 mM KCl, 1mM MgCl2, 5 mM DTT, 0.1 mM ATP
SpecimenConc.: 0.15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: vitrification was performed immediately after gel filtration.
Specimen supportGrid material: GOLD / Grid mesh size: 300 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

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 106061 X / Nominal defocus max: 5000 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1500 nm / Calibrated defocus max: 5000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 90 K
Image recordingAverage exposure time: 2 sec. / Electron dose: 1.6 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of grids imaged: 6746 / Num. of real images: 312198
Details: Images were also collected using K2 summit detetor in counting mode
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096 / Movie frames/image: 34 / Used frames/image: 3-34

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Processing

SoftwareName: REFMAC / Version: 5.8.0166 / Classification: refinement
EM software
IDNameVersionCategoryDetails
1Gautomatch0.53particle selection
2EPUimage acquisitionManually & automatically collection
4Gctf1.06CTF correctionlocal CTF and equiphase averaging(EPA) was performed; phase flip was applied for picking but not for particle extraction
7Coot0.83model fitting
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
11RELION1.4classification
12RELION1.43D reconstruction
13REFMAC5.8.0166model refinement
Image processingDetails: Negative stain dataset was processed as
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1
Details: Negative stain dataset was processed to generate templates.
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 233227 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 145 / Protocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 3.8→232.32 Å / Cor.coef. Fo:Fc: 0.865 / SU B: 59.043 / SU ML: 0.777 / ESU R: 0.879
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.3694 --
obs0.3694 302957 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 145.21 Å2
Baniso -1Baniso -2Baniso -3
1-2.67 Å2-0.12 Å20 Å2
2--1.83 Å2-0 Å2
3----4.5 Å2
Refinement stepCycle: 1 / Total: 46232
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.01947188
ELECTRON MICROSCOPYr_bond_other_d0.0020.0243942
ELECTRON MICROSCOPYr_angle_refined_deg1.3011.96764066
ELECTRON MICROSCOPYr_angle_other_deg0.9153101546
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.67755808
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.41523.8342102
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.806158100
ELECTRON MICROSCOPYr_dihedral_angle_4_deg11.81115336
ELECTRON MICROSCOPYr_chiral_restr0.0830.27340
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.02151902
ELECTRON MICROSCOPYr_gen_planes_other0.0020.029642
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it7.43814.54823328
ELECTRON MICROSCOPYr_mcbond_other7.43814.54723327
ELECTRON MICROSCOPYr_mcangle_it13.02521.80329104
ELECTRON MICROSCOPYr_mcangle_other13.02521.80429105
ELECTRON MICROSCOPYr_scbond_it7.90615.11823860
ELECTRON MICROSCOPYr_scbond_other7.90615.11823861
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other14.52222.27434963
ELECTRON MICROSCOPYr_long_range_B_refined23.43787802
ELECTRON MICROSCOPYr_long_range_B_other23.43787803
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.8→3.899 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork1.259 22478 -
Rfree-0 -
obs--100 %

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