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- EMDB-23841: Yeast dynein motor domain in the presence of a pyrazolo-pyrimidin... -

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Basic information

Entry
Database: EMDB / ID: EMD-23841
TitleYeast dynein motor domain in the presence of a pyrazolo-pyrimidinone-based compound, Map 1Motor protein
Map data
Sample
  • Complex: Dynein motor domain in the presence of a pyrazolo-pyrimidinone-based compoundMotor protein
    • Protein or peptide: Fusion protein of Dynein and Endolysin
  • Ligand: (8S)-6-(3-bromophenoxy)-2-[1-(4-chlorophenyl)cyclopropyl]-7-hydroxypyrazolo[1,5-a]pyrimidine-3-carbonitrile
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSantarossa CC / Urnavicius L / Coudray N / Ekeirt DC / Bhabha G / Kapoor TM
Funding support United States, 4 items
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, 2021-
Header (metadata) releaseMay 26, 2021-
Map releaseMay 26, 2021-
UpdateNov 3, 2021-
Current statusNov 3, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.004
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.004
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mi6
  • Surface level: 0.004
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23841.png

Downloads & links

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Map

FileDownload / File: emd_23841.map.gz / Format: CCP4 / Size: 190.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.518 Å
Density
Contour LevelBy AUTHOR: 0.004 / Movie #1: 0.004
Minimum - Maximum-0.008519489 - 0.017683027
Average (Standard dev.)0.00014691667 (±0.0010664521)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions368368368
Spacing368368368
CellA=B=C: 190.62401 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.5180.5180.518
M x/y/z368368368
origin x/y/z0.0000.0000.000
length x/y/z190.624190.624190.624
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS368368368
D min/max/mean-0.0090.0180.000

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Supplemental data

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Half map: #2

Fileemd_23841_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_23841_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dynein motor domain in the presence of a pyrazolo-pyrimidinone-ba...

EntireName: Dynein motor domain in the presence of a pyrazolo-pyrimidinone-based compoundMotor protein
Components
  • Complex: Dynein motor domain in the presence of a pyrazolo-pyrimidinone-based compoundMotor protein
    • Protein or peptide: Fusion protein of Dynein and Endolysin
  • Ligand: (8S)-6-(3-bromophenoxy)-2-[1-(4-chlorophenyl)cyclopropyl]-7-hydroxypyrazolo[1,5-a]pyrimidine-3-carbonitrile
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Dynein motor domain in the presence of a pyrazolo-pyrimidinone-ba...

SupramoleculeName: Dynein motor domain in the presence of a pyrazolo-pyrimidinone-based compound
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: VY972
Molecular weightTheoretical: 0.3 kDa/nm

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Macromolecule #1: Fusion protein of Dynein and Endolysin

MacromoleculeName: Fusion protein of Dynein and Endolysin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 304.889875 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: GEFVIEKSLN RIKKFWKEAQ YEVIEHSSGL KLVREWDVLE QACKEDLEEL VSMKASNYYK IFEQDCLDLE SKLTKLSEIQ VNWVEVQFY WLDLYGILGE NLDIQNFLPL ETSKFKSLTS EYKMITTRAF QLDTTIEVIH IPNFDTTLKL TIDSLKMIKS S LSTFLERQ ...String:
GEFVIEKSLN RIKKFWKEAQ YEVIEHSSGL KLVREWDVLE QACKEDLEEL VSMKASNYYK IFEQDCLDLE SKLTKLSEIQ VNWVEVQFY WLDLYGILGE NLDIQNFLPL ETSKFKSLTS EYKMITTRAF QLDTTIEVIH IPNFDTTLKL TIDSLKMIKS S LSTFLERQ RRQFPRFYFL GNDDLLKIIG SGKHHDQVSK FMKKMFGSIE SIIFLEDFIT GVRSVEGEVL NLNEKIELKD SI QAQEWLN ILDTEIKLSV FTQFRDCLGQ LKDGTDIEVV VSKYIFQAIL LSAQVMWTEL VEKCLQTNQF SKYWKEVDMK IKG LLDKLN KSSDNVKKKI EALLVEYLHF NNVIGQLKNC STKEEARLLW AKVQKFYQKN DTLDDLNSVF ISQSGYLLQY KFEY IGIPE RLIYTPLLLI GFATLTDSLH QKYGGCFFGP AGTGKTETVK AFGQNLGRVV VVFNCDDSFD YQVLSRLLVG ITQIG AWGC FDQFNRLDEK VLSAVSANIQ QIQNGLQVGK SHITLLEEET PLSPHTAVFI TLNPGYNGRS ELPENLKKSF REFSMK SPQ SGTIAEMILQ IMGFEDSKSL ASKIVHFLEL LSSKCSSMNH YHFGLRTLKG VLRNCSPLIS EFGEGEKTVV ESLKRVI LP SLGDTDELVF KDELSKIFDS AGTPLNSKAI VQCLKDAGQR SGFSMSEEFL KKCMQFYYMQ KTQQALILVG KAGCGKTA T WKTVIDAMAI FDGHANVVYV IDTKVLTKES LYGSMLKATL EWRDGLFTSI LRRVNDDITG TFKNSRIWVV FDSDLDPEY VEAMNSVLDD NKILTLPNGE RLPIPPNFRI LFETDNLDHT TPATITRCGL LWFSTDVCSI SSKIDHLLNK SYEALDNKLS MFELDKLKD LISDSFDMAS LTNIFTCSND LVHILGVRTF NKLETAVQLA VHLISSYRQW FQNLDDKSLK DVITLLIKRS L LYALAGDS TGESQRAFIQ TINTYFGHDS QELSDYSTIV IANDKLSFSS FCSEIPSVSL EAHEVMRPDI VIPTIDTIKH EK IFYDLLN SKRGIILCGP PGSGKTMIMN NALRNSSLYD VVGINFSKDT TTEHILSALH RHTNYVTTSK GLTLLPKSDI KNL VLFCDE INLPKLDKYG SQNVVLFLRQ LMEKQGFWKT PENKWVTIER IHIVGACNPP TDPGRIPMSE RFTRHAAILY LGYP SGKSL SQIYEIYYKA IFKLVPEFRS YTEPFARASV HLYNECKARY STGLQSHYLF SPRELTRLVR GVYTAINTGP RQTLR SLIR LWAYEAWRIF ADRLVGVKEK NSFEQLLYET VDKYLPNQDL GNISSTSLLF SGLLSLDFKE VNKTDLVNFI EERFKT FCD EELEVPMVIH ESMVDHILRI DRALKQVQGH MMLIGASRTG KTILTRFVAW LNGLKIVQPK IHRHSNLSDF DMILKKA IS DCSLKESRTC LIIDESNILE TAFLERMNTL LANADIPDLF QGEEYDKLLN NLRNKTRSLG LLLDTEQELY DWFVGEIA K NLHVVFTICD PTNNKSSAMI SSPALFNRCI INWMGDWDTK TMSQVANNMV DVIPMEFTDF IVPEVNKELV FTEPIQTIR DAVVNILIHF DRNFYQKMKV GVNPRSPGYF IDGLRALVKL VTAKYQDLQE NQRFVNVGLE KLNESVLKVN ELNKTLGSGS GSNIFEMLR IDEGLRLKIY KDTEGYYTIG IGHLLTKSPS LNAAKSELDK AIGRNTNGVI TKDEAEKLFN QDVDAAVRGI L RNAKLKPV YDSLDAVRRA ALINMVFQMG ETGVAGFTNS LRMLQQKRWD EAAVNLAKSR WYNQTPNRAK RVITTFRTGT WD AYGSGSG SSISLVKSLT FEKERWLNTT KQFSKTSQEL IGNCIISSIY ETYFGHLNER ERADMLVILK RLLGKFAVKY DVN YRFIDY LVTLDEKMKW LECGLDKNDY FLENMSIVMN SQDAVPFLLD PSSHMITVIS NYYGNKTVLL SFLEEGFVKR LENA IRFGS VVIIQDGEFF DPIISRLISR EFNHAGNRVT VEIGDHEVDV SGDFKLFIHS CDPSGDIPIF LRSRVRLVHF VTNKE SIET RIFDITLTEE NAEMQRKRED LIKLNTEYKL KLKNLEKRLL EELNNSQGNM LENDELMVTL NNLKKEAMNI EKKLSE SEE FFPQFDNLVE EYSIIGKHSV KIFSMLEKFG QFHWFYGISI GQFLSCFKRV FIKKSRETRA ARTRVDEILW LLYQEVY CQ FSTALDKKFK MIMAMTMFCL YKFDIESEQY KEAVLTMIGV LSESSDGVPK LTVDTNDDLR YLWDYVTTKS YISALNWF K NEFFVDEWNI ADVVANSENN YFTMASERDV DGTFKLIELA KASKESLKII PLGSIENLNY AQEEISKSKI EGGWILLQN IQMSLSWVKT YLHKHVEETK AAEEHEKFKM FMTCHLTGDK LPAPLLQRTD RVVYEDIPGI LDTVKDLWGS QFFTGKISGV WSVYCTFLL SWFHALITAR TRLVPHGFSK KYYFNDCDFQ FASVYLENVL ATNSTNNIPW AQVRDHIATI VYGGKIDEEK D LEVVAKLC AHVFCGSDNL QIVPGVRIPQ PLLQQSEEEE RARLTAILSN TIEPADSLSS WLQLPRESIL DYERLQAKEV AS STEQLLQ EMGSGSGSHH HHHH

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Macromolecule #2: (8S)-6-(3-bromophenoxy)-2-[1-(4-chlorophenyl)cyclopropyl]-7-hydro...

MacromoleculeName: (8S)-6-(3-bromophenoxy)-2-[1-(4-chlorophenyl)cyclopropyl]-7-hydroxypyrazolo[1,5-a]pyrimidine-3-carbonitrile
type: ligand / ID: 2 / Number of copies: 2 / Formula: ZG7
Molecular weightTheoretical: 481.729 Da
Chemical component information

ChemComp-ZG7:
(8S)-6-(3-bromophenoxy)-2-[1-(4-chlorophenyl)cyclopropyl]-7-hydroxypyrazolo[1,5-a]pyrimidine-3-carbonitrile

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris-HClTris
50.0 mMPotassium AcetateK-Ac
2.0 mMMagnesium AcetateMg(Ac)2
1.0 mMEGTA
10.0 %Glycerol
80.0 uMCompound 20
0.2 %DMSODimethyl sulfoxide
1.0 mMTCEP
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.7 µm / Calibrated defocus min: 1.3 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7420 pixel / Digitization - Dimensions - Height: 7676 pixel / Number grids imaged: 1 / Number real images: 4893 / Average exposure time: 10.0 sec. / Average electron dose: 44.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7mi1

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 136180

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Atomic model buiding 1

Initial modelPDB ID:

7mi1


Chain - Chain ID: A
RefinementSpace: REAL
Output model

PDB-7mi6:
Yeast dynein motor domain in the presence of a pyrazolo-pyrimidinone-based compound, Model 1

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