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- EMDB-23829: Structure of yeast cytoplasmic dynein with AAA3 Walker B mutation... -

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Basic information

Entry
Database: EMDB / ID: EMD-23829
TitleStructure of yeast cytoplasmic dynein with AAA3 Walker B mutation bound to Lis1
Map datascDynein E2488Q bound to two Lis1s
Sample
  • Complex: Complex of yeast dynein bound by two Lis1s in the presence of ATP-Va
    • Protein or peptide: dynein AAA3-WalkerB mutant (E2488Q)
    • Protein or peptide: Nuclear distribution protein PAC1
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


positive regulation of microtubule plus-end binding / microtubule sliding / microtubule organizing center organization / nuclear migration along microtubule / vesicle transport along microtubule / Antigen processing: Ubiquitination & Proteasome degradation / dynein complex / microtubule plus-end binding / minus-end-directed microtubule motor activity / nuclear migration ...positive regulation of microtubule plus-end binding / microtubule sliding / microtubule organizing center organization / nuclear migration along microtubule / vesicle transport along microtubule / Antigen processing: Ubiquitination & Proteasome degradation / dynein complex / microtubule plus-end binding / minus-end-directed microtubule motor activity / nuclear migration / microtubule associated complex / dynein complex binding / microtubule-based movement / establishment of mitotic spindle orientation / cytoplasmic microtubule / kinetochore / spindle pole / nuclear envelope / microtubule / cell division / ATP binding / identical protein binding / nucleus / cytoplasm
Similarity search - Function
Dynein regulator LIS1 / LIS1, N-terminal / Dynein heavy chain 3, AAA+ lid domain / AAA+ lid domain / 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 regulator LIS1 / LIS1, N-terminal / Dynein heavy chain 3, AAA+ lid domain / AAA+ lid domain / 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. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / 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
Dynein heavy chain, cytoplasmic / Nuclear distribution protein PAC1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / baker's yeast (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLahiri I / Reimer JM / Leschziner AE
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM107214 United States
CitationJournal: Elife / Year: 2022
Title: Structural basis for cytoplasmic dynein-1 regulation by Lis1.
Authors: John P Gillies / Janice M Reimer / Eva P Karasmanis / Indrajit Lahiri / Zaw Min Htet / Andres E Leschziner / Samara L Reck-Peterson /
Abstract: The lissencephaly 1 gene, , is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein- ...The lissencephaly 1 gene, , is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator known to bind directly to dynein's motor domain, and by doing so alters dynein's mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein-Lis1 complex. Our 3.1 Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1's ß-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1's ability to form fully active human dynein complexes and to regulate yeast dynein's mechanochemistry and in vivo function.
History
DepositionApr 12, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateJan 19, 2022-
Current statusJan 19, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mgm
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23829.png

Downloads & links

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Map

FileDownload / File: emd_23829.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationscDynein E2488Q bound to two Lis1s
Voxel sizeX=Y=Z: 1.31 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.022621905 - 0.14397681
Average (Standard dev.)0.00015329351 (±0.0046286006)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 392.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.311.311.31
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z393.000393.000393.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0230.1440.000

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Supplemental data

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Half map: Half map 1

Fileemd_23829_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_23829_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of yeast dynein bound by two Lis1s in the presence of ATP-Va

EntireName: Complex of yeast dynein bound by two Lis1s in the presence of ATP-Va
Components
  • Complex: Complex of yeast dynein bound by two Lis1s in the presence of ATP-Va
    • Protein or peptide: dynein AAA3-WalkerB mutant (E2488Q)
    • Protein or peptide: Nuclear distribution protein PAC1
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of yeast dynein bound by two Lis1s in the presence of ATP-Va

SupramoleculeName: Complex of yeast dynein bound by two Lis1s in the presence of ATP-Va
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: dynein AAA3-WalkerB mutant (E2488Q)

MacromoleculeName: dynein AAA3-WalkerB mutant (E2488Q) / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: baker's yeast (brewer's yeast)
Molecular weightTheoretical: 331.524 KDa
SequenceString: GDQLTHVVEE VKTYDLVWRS IKNLWEDVQR TFETPWCRVD VLLLQSDLAN FLRRADELPR AVKQFEMYKS LFSQVNMLTS VNKILVELK DGALKPRHWN MIFRDIGKRQ IQKNLLDKLE FSLKDVMVLN LTLNEILLTK IIERAQKEFV IEKSLNRIKK F WKEAQYEV ...String:
GDQLTHVVEE VKTYDLVWRS IKNLWEDVQR TFETPWCRVD VLLLQSDLAN FLRRADELPR AVKQFEMYKS LFSQVNMLTS VNKILVELK DGALKPRHWN MIFRDIGKRQ IQKNLLDKLE FSLKDVMVLN LTLNEILLTK IIERAQKEFV IEKSLNRIKK F WKEAQYEV IEHSSGLKLV REWDVLEQAC KEDLEELVSM KASNYYKIFE QDCLDLESKL TKLSEIQVNW VEVQFYWLDL YG ILGENLD IQNFLPLETS KFKSLTSEYK MITTRAFQLD TTIEVIHIPN FDTTLKLTID SLKMIKSSLS TFLERQRRQF PRF YFLGND DLLKIIGSGK HHDQVSKFMK KMFGSIESII FFEDSITGVR SVEGEVLNLN EKIELKDSIQ AQEWLNILDT EIKL SVFTQ FRDCLGQLKD GTDIEVVVSK YIFQAILLSA QVMWTELVEK CLQTNEFSKY WKEVDMKIKG LLDKLNKSSD NVKKK IEAL LVEYLHFNNV IGQLKNCSTK EEARLLWAKV QKFYQKNDTL DDLNSVFISQ SGYLLQYKFE YIGIPERLIY TPLLLV GFA TLTDSLHQKY GGCFFGPAGT GKTETVKAFG QNLGRVVVVF NCDDSFDYQV LSRLLVGITQ IGAWGCFDEF NRLDEKV LS AVSANIQQIQ NGLQVGKSHI TLLEEETPLS PHTAVFITLN PGYNGRSELP ENLKKSFREF SMKSPQSGTI AEMILQIM G FEDSKSLASK IVHFLELLSS KCSSMNHYHF GLRTLKGVLR NCSPLVSEFG EGEKTVVESL KRVILPSLGD TDELVFKDE LSKIFDSAGT PLNSKAIVQC LKDAGQRSGF SMSEEFLKKC MQFYYMQKTQ QALILVGKAG CGKTATWKTV IDAMAIFDGH ANVVYVIDT KVLTKESLYG SMLKATLEWR DGLFTSILRR VNDDITGTFK NSRIWVVFDS DLDPEYVEAM NSVLDDNKIL T LPNGERLP IPPNFRILFE TDNLDHTTPA TITRCGLLWF STDVCSISSK IDHLLNKSYE ALDNKLSMFE LDKLKDLISD SF DMASLTN IFTCSNDLVH ILGVRTFNKL ETAVQLAVHL ISSYRQWFQN LDDKSLKDVI TLLIKRSLLY ALAGDSTGES QRA FIQTIN TYFGHDSQEL SDYSTIVIAN DKLSFSSFCS EIPSVSLEAH EVMRPDIVIP TIDTIKHEKI FYDLLNSKRG IILC GPPGS GKTMIMNNAL RNSSLYDVVG INFSKDTTTE HILSALHRHT NYVTTSKGLT LLPKSDIKNL VLFCDQINLP KLDKY GSQN VVLFLRQLME KQGFWKTPEN KWVTIERIHI VGACNPPTDP GRIPMSERFT RHAAILYLGY PSGKSLSQIY EIYYKA IFK LVPEFRSYTE PFARASVHLY NECKARYSTG LQSHYLFSPR ELTRLVRGVY TAINTGPRQT LRSLIRLWAY EAWRIFA DR LVGVKEKNSF EQLLYETVDK YLPNQDLGNI SSTSLLFSGL LSLDFKEVNK TDLVNFIEER FKTFCDEELE VPMVIHES M VDHILRIDRA LKQVQGHMML IGASRTGKTI LTRFVAWLNG LKIVQPKIHR HSNLSDFDMI LKKAISDCSL KESRTCLII DESNILETAF LERMNTLLAN ADIPDLFQGE EYDKLLNNLR NKTRSLGLLL DTEQELYDWF VGEIAKNLHV VFTICDPTNN KSSAMISSP ALFNRCIINW MGDWDTKTMS QVANNMVDVV PMEFTDFIVP EVNKELVFTE PIQTIRDAVV NILIHFDRNF Y QKMKVGVN PRSPGYFIDG LRALVKLVTA KYQDLQENQR FVNVGLEKLN ESVLKVNELN KTLSKKSTEL TEKEKEARST LD KMLMEQN ESERKQEATE EIKKILKVQE EDIRKRKEVV MKSIQDIEPT ILEAQRGVKN IKKQQLTEIR SMVNPPSGVK IVM EAVCAI LGYQFSNWRD IQQFIRKDDF IHNIVHYDTT LHMKPQIRKY MEEEFLSDPN FTYETINRAS KACGPLYQWV NAQI NFSKV LENVDPLRQE MKRIEFESLK TKANLLAAEE MTQDLEASIE VSKQKYSLLI RDVEAIKTEM SNVQANLDRS ISLVK SLTF EKERWLNTTK QFSKTSQELI GNCIISSIYE TYFGHLNERE RGDMLVILKR LLGKFAVKYD VNYRFIDYLV TLDEKM KWL ECGLDKNDYF LENMSIVMNS QDAVPFLLDP SSHMITVISN YYGNKTVLLS FLEEGFVKRL ENAVRFGSVV IIQDGEF FD PIISRLISRE FNHAGNRVTV EIGDHEVDVS GDFKLFIHSC DPSGDIPIFL RSRVRLVHFV TNKESIETRI FDITLTEE N AEMQRKREDL IKLNTEYRLK LKNLEKRLLE ELNNSQGNML ENDELMVTLN NLKKEAMNIE KKLSESEEFF PQFDNLVEE YSIIGKHSVK IFSMLEKFGQ FHWFYGISIG QFLSCFKRVF IKKSRETRAA RTRVDEILWL LYQEVYCQFS TALDKKFKMI MAMTMFCLY KFDIESEQYK EAVLTMIGVL SESSDGVPKL TVDTNDDLRY LWDYVTTKSY ISALNWFKNE FFVDEWNIAD V VANSENNY FTMASERDVD GTFKLIELAK ASKESLKIIP LGSIENLNYA QEEISKSKIE GGWILLQNIQ MSLSWVKTYL HK HVEETKA AEEHEKFKMF MTCHLTGDKL PAPLLQRTDR VVYEDIPGIL DTVKDLWGSQ FFTGKISGVW SVYCTFLLSW FHA LITART RLVPHGFSKK YYFNDCDFQF ASVYLENVLA TNSTNNIPWA QVRDHIATIV YGGKIDEEKD LEVVAKLCAH VFCG SDNLQ IVPGVRIPQP LLQQSEEEER ARLTAILSNT IEPADSLSSW LQLPRESILD YERLQAKEVA SSTEQLLQEM

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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: baker's yeast (brewer's yeast)
Molecular weightTheoretical: 57.030617 KDa
SequenceString: GMTNWQQQLP LTDTQKNELD KSVLRYLNWN YKQTVRHEHA QDYESVRHAI VTLSGFLLQE SVDRQEFISN NDTSNESMVD IDELLLPKK WNSIVRLQKK IIELEQNTET LVSQIKDLNT QVSELAQFKP TTSNGTSAHN VLKWIPRNLP SCLINVESSV T SVKLHPNL ...String:
GMTNWQQQLP LTDTQKNELD KSVLRYLNWN YKQTVRHEHA QDYESVRHAI VTLSGFLLQE SVDRQEFISN NDTSNESMVD IDELLLPKK WNSIVRLQKK IIELEQNTET LVSQIKDLNT QVSELAQFKP TTSNGTSAHN VLKWIPRNLP SCLINVESSV T SVKLHPNL PIVFVATDHG KLYAFDLFNY TIPLASLQSH TKAITSMDVL FTNYTNSSKK NYLVIVTASK DLQIHVFKWV SE ECKFQQI RSLLGHEHIV SAVKIWQKNN DVHIASCSRD QTVKIWDFHN GWSLKTFQPH SQWVRSIDVL GDYIISGSHD TTL RLTHWP SGNGLSVGTG HEFPIEKVKF IHFIEDSPEI RFRTPSTDRY KNWGMQYCVS ASRDRTIKIW EIPLPTLMAH RAPI PNPTD SNFRCVLTLK GHLSWVRDIS IRGQYLFSCA DDKSVRCWDL NTGQCLHVWE KLHTGFVNCL DLDVDFDSNV TPRQM MVTG GLDCKSNVFM R

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 3 / 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: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / 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

BufferpH: 7.4
GridDetails: Quantifoil R2/2 grids with gold foil were used. A monolayer of streptavidin crystals was deposited prior to applying the sample. The streptavidin monolayer acted as an affinity surface for ...Details: Quantifoil R2/2 grids with gold foil were used. A monolayer of streptavidin crystals was deposited prior to applying the sample. The streptavidin monolayer acted as an affinity surface for the biotinylated sample.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK II
DetailsThe yeast dynein was biotinylated prior to complex formation.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 2.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 70.0 K / Max: 70.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2229 / Average exposure time: 10.0 sec. / Average electron dose: 58.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 83975
FSC plot (resolution estimation)

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