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- PDB-7mi8: Signal subtracted reconstruction of AAA5 and AAA6 domains of dyne... -

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Basic information

Entry
Database: PDB / ID: 7mi8
TitleSignal subtracted reconstruction of AAA5 and AAA6 domains of dynein in the presence of a pyrazolo-pyrimidinone-based compound, Model 5
ComponentsFusion protein of Dynein and Endolysin
KeywordsMOTOR PROTEIN / AAA ATPase / ATPase inhibitor
Function / homology
Function and homology information


karyogamy / establishment of mitotic spindle localization / astral microtubule / nuclear migration along microtubule / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / dynein light intermediate chain binding / spindle pole body / nuclear migration / dynein intermediate chain binding ...karyogamy / establishment of mitotic spindle localization / astral microtubule / nuclear migration along microtubule / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / dynein light intermediate chain binding / spindle pole body / nuclear migration / dynein intermediate chain binding / mitotic sister chromatid segregation / establishment of mitotic spindle orientation / cytoplasmic microtubule / cytoplasmic microtubule organization / viral release from host cell by cytolysis / Neutrophil degranulation / peptidoglycan catabolic process / mitotic spindle organization / cell wall macromolecule catabolic process / lysozyme / lysozyme activity / cell cortex / host cell cytoplasm / defense response to bacterium / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
: / DYN1, AAA+ ATPase lid domain / Dynein heavy chain 3, AAA+ lid domain / AAA+ lid 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 ...: / DYN1, AAA+ ATPase lid domain / Dynein heavy chain 3, AAA+ lid domain / AAA+ lid 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 / Endolysin T4 type / T4-type lysozyme / Glycoside hydrolase, family 24 / Lysozyme domain superfamily / Phage lysozyme / Lysozyme-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Endolysin / Dynein heavy chain, cytoplasmic
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Enterobacteria phage T4 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsSantarossa, C.C. / Coudray, N. / Urnavicius, L. / Ekiert, D.C. / Bhabha, G. / Kapoor, T.M.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM98579 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM130234-01 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM112982 United States
Damon Runyon Cancer Research FoundationDFS-20-16 United States
CitationJournal: Cell Chem Biol / Year: 2021
Title: Targeting allostery in the Dynein motor domain with small molecule inhibitors.
Authors: Cristina C Santarossa / Keith J Mickolajczyk / Jonathan B Steinman / Linas Urnavicius / Nan Chen / Yasuhiro Hirata / Yoshiyuki Fukase / Nicolas Coudray / Damian C Ekiert / Gira Bhabha / Tarun M Kapoor /
Abstract: Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein's unusually large size, ...Cytoplasmic dyneins are AAA (ATPase associated with diverse cellular activities) motor proteins responsible for microtubule minus-end-directed intracellular transport. Dynein's unusually large size, four distinct nucleotide-binding sites, and conformational dynamics pose challenges for the design of potent and selective chemical inhibitors. Here we use structural approaches to develop a model for the inhibition of a well-characterized S. cerevisiae dynein construct by pyrazolo-pyrimidinone-based compounds. These data, along with functional assays of dynein motility and mutagenesis studies, suggest that the compounds inhibit dynein by engaging the regulatory ATPase sites in the AAA3 and AAA4 domains, and not by interacting with dynein's main catalytic site in the AAA1 domain. A double Walker B mutation of the AAA3 and AAA4 sites substantially reduces enzyme activity, suggesting that targeting these regulatory domains is sufficient to inhibit dynein. Our findings reveal how chemical inhibitors can be designed to disrupt allosteric communication across dynein's AAA domains.
History
DepositionApr 16, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 26, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 2, 2021Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Nov 3, 2021Group: Database references / Category: citation / database_2
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Fusion protein of Dynein and Endolysin


Theoretical massNumber of molelcules
Total (without water)304,8901
Polymers304,8901
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Fusion protein of Dynein and Endolysin /


Mass: 304889.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast), (gene. exp.) Enterobacteria phage T4 (virus)
Gene: DYN1, DHC1, YKR054C / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): VY972 / References: UniProt: P36022, UniProt: P00720

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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: Dynein motor domain in the presence of a pyrazolo-pyrimidinone-based compoundMotor protein
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.3 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Saccharomyces cerevisiae (brewer's yeast)4932
21Enterobacteria phage T4 (virus)10665
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: VY972
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClTris1
250 mMPotassium AcetateK-Ac1
32 mMMagnesium AcetateMg(Ac)21
41 mMEGTA1
510 %Glycerol1
680 uMCompound 201
70.2 %DMSODimethyl sulfoxide1
81 mMTCEP1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 defocus max: 1500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1300 nm / Calibrated defocus max: 3700 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 10 sec. / Electron dose: 44 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4893
Image scansWidth: 7420 / Height: 7676 / Movie frames/image: 50

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7Coot0.8.9.2model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.17.1-3660model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 128004 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 7MI1

7mi1


Pdb chain-ID: A
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0067187
ELECTRON MICROSCOPYf_angle_d0.9639712
ELECTRON MICROSCOPYf_dihedral_angle_d10.913923
ELECTRON MICROSCOPYf_chiral_restr0.0541082
ELECTRON MICROSCOPYf_plane_restr0.0071225

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