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- PDB-6rzb: Cryo-EM structure of mouse cytoplasmic dynein-1 microtubule bindi... -

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Basic information

Entry
Database: PDB / ID: 6rzb
TitleCryo-EM structure of mouse cytoplasmic dynein-1 microtubule binding domain bound to microtubules
Components
  • MKIAA0325 protein
  • Tubulin alpha-1B chain
  • Tubulin beta chain
KeywordsMOTOR PROTEIN / filament / complex
Function / homology
Function and homology information


manchette / cilium movement / positive regulation of intracellular transport / positive regulation of spindle assembly / minus-end-directed vesicle transport along microtubule / establishment of spindle localization / ATP-dependent microtubule motor activity, minus-end-directed / cytoplasmic mRNA processing body assembly / dynein light intermediate chain binding / regulation of metaphase plate congression ...manchette / cilium movement / positive regulation of intracellular transport / positive regulation of spindle assembly / minus-end-directed vesicle transport along microtubule / establishment of spindle localization / ATP-dependent microtubule motor activity, minus-end-directed / cytoplasmic mRNA processing body assembly / dynein light intermediate chain binding / regulation of metaphase plate congression / cytoplasmic dynein complex / dynein complex / retrograde axonal transport / dynein intermediate chain binding / nuclear migration / regulation of mitotic spindle organization / stress granule assembly / cytoplasmic microtubule organization / microtubule motor activity / microtubule-based movement / cytoplasmic microtubule / axon cytoplasm / cellular response to interleukin-4 / microtubule-based process / filopodium / structural constituent of cytoskeleton / neuron migration / microtubule cytoskeleton organization / positive regulation of cold-induced thermogenesis / nuclear envelope / cell cortex / double-stranded RNA binding / mitotic cell cycle / microtubule / GTPase activity / cell division / centrosome / axon / GTP binding / neuronal cell body / ubiquitin protein ligase binding / go:0005623: / ATP binding / cytoplasm
Tubulin C-terminal domain / Dynein heavy chain, coiled coil stalk / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / AAA+ ATPase domain / Dynein heavy chain region D6 P-loop domain / Tubulin/FtsZ, C-terminal / Dynein heavy chain, domain-1 / Dynein heavy chain, domain-2 ...Tubulin C-terminal domain / Dynein heavy chain, coiled coil stalk / Alpha tubulin / Beta tubulin / Tubulin/FtsZ, GTPase domain / AAA+ ATPase domain / Dynein heavy chain region D6 P-loop domain / Tubulin/FtsZ, C-terminal / Dynein heavy chain, domain-1 / Dynein heavy chain, domain-2 / Beta tubulin, autoregulation binding site / Tubulin, conserved site / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin, C-terminal / Dynein heavy chain, AAA module D4 / P-loop containing nucleoside triphosphate hydrolase / Dynein heavy chain, hydrolytic ATP-binding dynein motor region / Dynein heavy chain AAA lid domain superfamily / Tubulin/FtsZ family, GTPase domain / Dynein heavy chain, C-terminal domain, barrel region / Dynein heavy chain, AAA1 domain, small subdomain / Dynein heavy chain, domain 2, C-terminal / Dynein heavy chain, domain 2, N-terminal / Dynein heavy chain AAA lid domain / Dynein heavy chain, AAA 5 extension domain / Dynein heavy chain, C-terminal domain / Tubulin/FtsZ, C-terminal domain superfamily / Tubulin/FtsZ, GTPase domain superfamily / Dynein heavy chain, ATP-binding dynein motor region / Tubulin
Tubulin beta chain / Tubulin alpha-1B chain / MKIAA0325 protein / Cytoplasmic dynein 1 heavy chain 1
Biological speciesMus musculus (house mouse)
Sus scrofa (pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsLacey, S.E. / He, S. / Scheres, S.H.W. / Carter, A.P.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_A025_1011 United Kingdom
Wellcome TrustWT210711 United Kingdom
CitationJournal: Elife / Year: 2019
Title: Cryo-EM of dynein microtubule-binding domains shows how an axonemal dynein distorts the microtubule.
Authors: Samuel E Lacey / Shaoda He / Sjors Hw Scheres / Andrew P Carter /
Abstract: Dyneins are motor proteins responsible for transport in the cytoplasm and the beating of axonemes in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain ...Dyneins are motor proteins responsible for transport in the cytoplasm and the beating of axonemes in cilia and flagella. They bind and release microtubules via a compact microtubule-binding domain (MTBD) at the end of a coiled-coil stalk. We address how cytoplasmic and axonemal dynein MTBDs bind microtubules at near atomic resolution. We decorated microtubules with MTBDs of cytoplasmic dynein-1 and axonemal dynein DNAH7 and determined their cryo-EM structures using helical Relion. The majority of the MTBD is rigid upon binding, with the transition to the high-affinity state controlled by the movement of a single helix at the MTBD interface. DNAH7 contains an 18-residue insertion, found in many axonemal dyneins, that contacts the adjacent protofilament. Unexpectedly, we observe that DNAH7, but not dynein-1, induces large distortions in the microtubule cross-sectional curvature. This raises the possibility that dynein coordination in axonemes is mediated via conformational changes in the microtubule.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 13, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 10, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Tubulin alpha-1B chain
B: Tubulin beta chain
C: MKIAA0325 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)115,2437
Polymers113,3983
Non-polymers1,8454
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8970 Å2
ΔGint-47 kcal/mol
Surface area34880 Å2
MethodPISA

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Tubulin alpha-1B chain / Alpha-tubulin ubiquitous / Tubulin K-alpha-1 / Tubulin alpha-ubiquitous chain


Mass: 48679.051 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / Organ: Brain / References: UniProt: Q2XVP4
#2: Protein Tubulin beta chain / Beta-tubulin


Mass: 47825.859 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa (pig) / References: UniProt: P02554
#3: Protein MKIAA0325 protein


Mass: 16893.574 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Dync1h1, Dnchc1, mKIAA0325 / Production host: Escherichia coli (E. coli) / References: UniProt: Q80U36, UniProt: Q9JHU4*PLUS

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Non-polymers , 4 types, 4 molecules

#4: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#7: Chemical ChemComp-TA1 / TAXOL / Paclitaxel


Mass: 853.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C47H51NO14 / Comment: medication, chemotherapy*YM

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Details

Has ligand of interestN

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Mouse cytoplasmic dynein-1 microtubule binding domain decorating microtubulesCOMPLEX1, 2, 30MULTIPLE SOURCES
2TubulinCOMPLEX1, 21NATURAL
3dynein-1 microtubule bindingCOMPLEX31NATURAL
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Sus scrofa (pig)9823
23Mus musculus (house mouse)10090
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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
Image recordingElectron dose: 60 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0238 / Classification: refinement
EM software
IDNameVersionCategory
10RELION3final Euler assignment
12RELION33D reconstruction
13REFMAC5model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59100 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
RefinementResolution: 5→187.2 Å / Cor.coef. Fo:Fc: 0.532 / SU B: 171.433 / SU ML: 1.972 / ESU R: 1.061
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.66163 --
Obs0.66163 109888 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 162.659 Å2
Baniso -1Baniso -2Baniso -3
1-5.51 Å21.22 Å2-0.51 Å2
2---1.29 Å2-1.62 Å2
3----4.23 Å2
Refinement stepCycle: 1 / Resolution: 5→187.2 Å / Total: 8001
Refine LS restraints
Refinement-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.0128178
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.5121.64511109
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.1215997
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.02122.627434
ELECTRON MICROSCOPYr_dihedral_angle_3_deg18.471151370
ELECTRON MICROSCOPYr_dihedral_angle_4_deg16.6031550
ELECTRON MICROSCOPYr_chiral_restr0.1070.21073
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.026291
ELECTRON MICROSCOPYr_gen_planes_other
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_it15.7314.4084000
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it26.62221.564993
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it24.48117.3994178
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined58.60635231
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 5.001→5.131 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.828 7966 -
Obs--100 %

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