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- EMDB-8706: Cryo-EM structure of yeast cytoplasmic dynein with Walker B mutat... -

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Basic information

Entry
Database: EMDB / ID: EMD-8706
TitleCryo-EM structure of yeast cytoplasmic dynein with Walker B mutation at AAA3 in presence of ATP-VO4
Map dataYeast cytoplasmic dynein with Walker B mutation at AAA3 in presence of ATP-VO4
Sample
  • Complex: Complex between yeast dynein (AAA3 Walker B) with Lis1 in the presence of ATP.VO4
    • Protein or peptide: Dynein heavy chain, cytoplasmic
    • Protein or peptide: Nuclear distribution protein PAC1
Function / homology
Function and homology information


microtubule sliding / karyogamy / establishment of mitotic spindle localization / nuclear migration along microtubule / astral microtubule / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / spindle pole body / dynein light intermediate chain binding / nuclear migration ...microtubule sliding / karyogamy / establishment of mitotic spindle localization / nuclear migration along microtubule / astral microtubule / minus-end-directed microtubule motor activity / cytoplasmic dynein complex / spindle pole body / dynein light intermediate chain binding / nuclear migration / microtubule associated complex / dynein intermediate chain binding / dynein complex binding / mitotic sister chromatid segregation / establishment of mitotic spindle orientation / cytoplasmic microtubule / cytoplasmic microtubule organization / Neutrophil degranulation / mitotic spindle organization / spindle pole / cell cortex / microtubule / cell division / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
Dynein regulator LIS1 / LIS1, N-terminal / : / 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 regulator LIS1 / LIS1, N-terminal / : / 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 / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Nuclear distribution protein PAC1 / Dynein heavy chain, cytoplasmic
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.5 Å
AuthorsCianfrocco MA / DeSantis ME / Htet ZM / Tran PT / Reck-Peterson SL / Leschziner AE
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM107214 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM100947 United States
CitationJournal: Cell / Year: 2017
Title: Lis1 Has Two Opposing Modes of Regulating Cytoplasmic Dynein.
Authors: Morgan E DeSantis / Michael A Cianfrocco / Zaw Min Htet / Phuoc Tien Tran / Samara L Reck-Peterson / Andres E Leschziner /
Abstract: Regulation is central to the functional versatility of cytoplasmic dynein, a motor involved in intracellular transport, cell division, and neurodevelopment. Previous work established that Lis1, a ...Regulation is central to the functional versatility of cytoplasmic dynein, a motor involved in intracellular transport, cell division, and neurodevelopment. Previous work established that Lis1, a conserved regulator of dynein, binds to its motor domain and induces a tight microtubule-binding state in dynein. The work we present here-a combination of biochemistry, single-molecule assays, and cryoelectron microscopy-led to the surprising discovery that Lis1 has two opposing modes of regulating dynein, being capable of inducing both low and high affinity for the microtubule. We show that these opposing modes depend on the stoichiometry of Lis1 binding to dynein and that this stoichiometry is regulated by the nucleotide state of dynein's AAA3 domain. The low-affinity state requires Lis1 to also bind to dynein at a novel conserved site, mutation of which disrupts Lis1's function in vivo. We propose a new model for the regulation of dynein by Lis1.
History
DepositionApr 25, 2017-
Header (metadata) releaseAug 23, 2017-
Map releaseSep 6, 2017-
UpdateJan 1, 2020-
Current statusJan 1, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.071
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.071
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5vlj
  • Surface level: 0.071
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8706.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationYeast cytoplasmic dynein with Walker B mutation at AAA3 in presence of ATP-VO4
Voxel sizeX=Y=Z: 2.4 Å
Density
Contour LevelBy AUTHOR: 0.071 / Movie #1: 0.071
Minimum - Maximum-0.04799215 - 0.18136224
Average (Standard dev.)0.0010077149 (±0.013684663)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 307.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.42.42.4
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z307.200307.200307.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0480.1810.001

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Supplemental data

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Sample components

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Entire : Complex between yeast dynein (AAA3 Walker B) with Lis1 in the pre...

EntireName: Complex between yeast dynein (AAA3 Walker B) with Lis1 in the presence of ATP.VO4
Components
  • Complex: Complex between yeast dynein (AAA3 Walker B) with Lis1 in the presence of ATP.VO4
    • Protein or peptide: Dynein heavy chain, cytoplasmic
    • Protein or peptide: Nuclear distribution protein PAC1

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Supramolecule #1: Complex between yeast dynein (AAA3 Walker B) with Lis1 in the pre...

SupramoleculeName: Complex between yeast dynein (AAA3 Walker B) with Lis1 in the presence of ATP.VO4
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightExperimental: 444 KDa

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Macromolecule #1: Dynein heavy chain, cytoplasmic

MacromoleculeName: Dynein heavy chain, cytoplasmic / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 272.66625 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: VQFYWLDLYG ILGENLDIQN FLPLETSKFK SLTSEYKMIT TRAFQLDTTI EVIHIPNFDT TLKLTIDSLK MIKSSLSTFL ERQRRQFPR FYFLGNDDLL KIIGSGKHHD QVSKFMKKMF GSIESIIFFE DSITGVRSVE GEVLNLNEKI ELKDSIQAQE W LNILDTEI ...String:
VQFYWLDLYG ILGENLDIQN FLPLETSKFK SLTSEYKMIT TRAFQLDTTI EVIHIPNFDT TLKLTIDSLK MIKSSLSTFL ERQRRQFPR FYFLGNDDLL KIIGSGKHHD QVSKFMKKMF GSIESIIFFE DSITGVRSVE GEVLNLNEKI ELKDSIQAQE W LNILDTEI KLSVFTQFRD CLGQLKDGTD IEVVVSKYIF QAILLSAQVM WTELVEKCLQ TNEFSKYWKE VDMKIKGLLD KL NKSSDNV KKKIEALLVE YLHFNNVIGQ LKNCSTKEEA RLLWAKVQKF YQKNDTLDDL NSVFISQSGY LLQYKFEYIG IPE RLIYTP LLLVGFATLT DSLHQKYGGC FFGPAGTGKT ETVKAFGQNL GRVVVVFNCD DSFDYQVLSR LLVGITQIGA WGCF DEFNR LDEKVLSAVS ANIQQIQNGL QVGKSHITLL EEETPLSPHT AVFITLNPGY NGRSELPENL KKSFREFSMK SPQSG TIAE MILQIMGFED SKSLASKIVH FLELLSSKCS SMNHYHFGLR TLKGVLRNCS PLVSEFGEGE KTVVESLKRV ILPSLG DTD ELVFKDELSK IFDSAGTPLN SKAIVQCLKD AGQRSGFSMS EEFLKKCMQF YYMQKTQQAL ILVGKAGCGK TATWKTV ID AMAIFDGHAN VVYVIDTKVL TKESLYGSML KATLEWRDGL FTSILRRVND DITGTFKNSR IWVVFDSDLD PEYVEAMN S VLDDNKILTL PNGERLPIPP NFRILFETDN LDHTTPATIT RCGLLWFSTD VCSISSKIDH LLNKSYEALD NKLSMFELD KLKDLISDSF DMASLTNIFT CSNDLVHILG VRTFNKLETA VQLAVHLISS YRQWFQNLDD KSLKDVITLL IKRSLLYALA GDSTGESQR AFIQTINTYF GHDSQELSDY STIVIANDKL SFSSFCSEIP SVSLEAHEVM RPDIVIPTID TIKHEKIFYD L LNSKRGII LCGPPGSGKT MIMNNALRNS SLYDVVGINF SKDTTTEHIL SALHRHTNYV TTSKGLTLLP KSDIKNLVLF CD EINLPKL DKYGSQNVVL FLRQLMEKQG FWKTPENKWV TIERIHIVGA CNPPTDPGRI PMSERFTRHA AILYLGYPSG KSL SQIYEI YYKAIFKLVP EFRSYTEPFA RASVHLYNEC KARYSTGLQS HYLFSPRELT RLVRGVYTAI NTGPRQTLRS LIRL WAYEA WRIFADRLVG VKEKNSFEQL LYETVDKYLP NQDLGNISST SLLFSGLLSL DFKEVNKTDL VNFIEERFKT FCDEE LEVP MVIHESMVDH ILRIDRALKQ VQGHMMLIGA SRTGKTILTR FVAWLNGLKI VQPKIHRHSN LSDFDMILKK AISDCS LKE SRTCLIIDES NILETAFLER MNTLLANADI PDLFQGEEYD KLLNNLRNKT RSLGLLLDTE QELYDWFVGE IAKNLHV VF TICDPTNNKS SAMISSPALF NRCIINWMGD WDTKTMSQVA NNMVDVVPME FTDFIVPEVN KELVFTEPIQ TIRDAVVN I LIHFDRNFYQ KMKVGVNPRS PGYFIDGLRA LVKLVTAKYQ DLQENQRFVN VGLEKLNESV SLTFEKERWL NTTKQFSKT SQELIGNCII SSIYETYFGH LNERERGDML VILKRLLGKF AVKYDVNYRF IDYLVTLDEK MKWLECGLDK NDYFLENMSI VMNSQDAVP FLLDPSSHMI TVISNYYGNK TVLLSFLEEG FVKRLENAVR FGSVVIIQDG EFFDPIISRL ISREFNHAGN R VTVEIGDH EVDVSGDFKL FIHSCDPSGD IPIFLRSRVR LVHFVTNKES IETRIFDITL TEENAEMQRK REDLIKLNTE YR LKLKNLE KRLLEELNNS QGNMLENDEL MVTLNNLKKE AMNIEKKLSE SEEFFPQFDN LVEEYSIIGK HSVKIFSMLE KFG QFHWFY GISIGQFLSC FKRVFIKKSR ETRAARTRVD EILWLLYQEV YCQFSTALDK KFKMIMAMTM FCLYKFDIES EQYK EAVLT MIGVLSESSD GVPKLTVDTN DDLRYLWDYV TTKSYISALN WFKNEFFVDE WNIADVVANS ENNYFTMASE RDVDG TFKL IELAKASKES LKIIPLGSIE NLNYAQEEIS KSKIEGGWIL LQNIQMSLSW VKTYLHKHVE ETKAAEEHEK FKMFMT CHL TGDKLPAPLL QRTDRVVYED IPGILDTVKD LWGSQFFTGK ISGVWSVYCT FLLSWFHALI TARTRLVPHG FSKKYYF ND CDFQFASVYL ENVLATNSTN NIPWAQVRDH IATIVYGGKI DEEKDLEVVA KLCAHVFCGS DNLQIVPGVR IPQPLLQQ S EEEERARLTA ILSNTIEPAD SLSSWLQLPR ESILDYERLQ AKEVASSTEQ LLQEM

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Macromolecule #2: Nuclear distribution protein PAC1

MacromoleculeName: Nuclear distribution protein PAC1 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 40.645449 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: LKWIPRNLPS CLINVESSVT SVKLHPNLPI VFVATDHGKL YAFDLFNYTI PLASLQSHTK AITSMDVLFT NYTNSSKKNY LVIVTASKD LQIHVFKWVS EECKFQQIRS LLGHEHIVSA VKIWQKNNDV HIASCSRDQT VKIWDFHNGW SLKTFQPHSQ W VRSIDVLG ...String:
LKWIPRNLPS CLINVESSVT SVKLHPNLPI VFVATDHGKL YAFDLFNYTI PLASLQSHTK AITSMDVLFT NYTNSSKKNY LVIVTASKD LQIHVFKWVS EECKFQQIRS LLGHEHIVSA VKIWQKNNDV HIASCSRDQT VKIWDFHNGW SLKTFQPHSQ W VRSIDVLG DYIISGSHDT TLRLTHWPSG NGLSVGTGHE FPIEKVKFIH FIEDSPEIRF RTPSTDRYKN WGMQYCVSAS RD RTIKIWE IPLPTLMAHR APIPNPTDSN FRCVLTLKGH LSWVRDISIR GQYLFSCADD KSVRCWDLNT GQCLHVWEKL HTG FVNCLD LDVDFDSNVT PRQMMVTGGL DCKSNVFM

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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

Concentration0.23 mg/mL
BufferpH: 8
GridModel: Quantifoil UltrAuFoil 1.2/1.3 / Material: GOLD / Mesh: 300 / Details: No grid pretreatment
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 276 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 4826 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 10.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 27807

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 800
Output model

PDB-5vlj:
Cryo-EM structure of yeast cytoplasmic dynein with Walker B mutation at AAA3 in presence of ATP-VO4

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