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- PDB-7mgm: Structure of yeast cytoplasmic dynein with AAA3 Walker B mutation... -

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Basic information

Entry
Database: PDB / ID: 7mgm
TitleStructure of yeast cytoplasmic dynein with AAA3 Walker B mutation bound to Lis1
Components
  • Nuclear distribution protein PAC1
  • dynein AAA3-WalkerB mutant (E2488Q)
KeywordsMOTOR PROTEIN / motor / AAA
Function / homology
Function and homology information


microtubule sliding / microtubule organizing center organization / positive regulation of microtubule plus-end binding / nuclear migration along microtubule / vesicle transport along microtubule / minus-end-directed microtubule motor activity / microtubule plus-end binding / dynein complex / nuclear migration / dynein complex binding ...microtubule sliding / microtubule organizing center organization / positive regulation of microtubule plus-end binding / nuclear migration along microtubule / vesicle transport along microtubule / minus-end-directed microtubule motor activity / microtubule plus-end binding / dynein complex / nuclear migration / dynein complex binding / microtubule associated complex / establishment of mitotic spindle orientation / microtubule-based movement / 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 / Dynein heavy chain 3, AAA+ lid domain / AAA+ lid domain / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / Dynein heavy chain, linker / Dynein heavy chain AAA lid domain / Dynein heavy chain, N-terminal region 2 ...Dynein regulator LIS1 / LIS1, N-terminal / Dynein heavy chain / Dynein heavy chain 3, AAA+ lid domain / AAA+ lid domain / Dynein heavy chain, tail / Dynein heavy chain, N-terminal region 1 / Dynein heavy chain, linker / Dynein heavy chain AAA lid domain / Dynein heavy chain, N-terminal region 2 / Dynein heavy chain region D6 P-loop domain / Dynein heavy chain, AAA1 domain, small subdomain / Dynein heavy chain, linker, subdomain 3 / Hydrolytic ATP binding site of dynein motor region / Dynein heavy chain region D6 P-loop domain / Microtubule-binding stalk of dynein motor / Dynein heavy chain, AAA module D4 / Dynein heavy chain, domain 2, N-terminal / Dynein heavy chain, hydrolytic ATP-binding dynein motor region / Dynein heavy chain, ATP-binding dynein motor region / P-loop containing dynein motor region D4 / ATP-binding dynein motor region / Dynein heavy chain AAA lid domain superfamily / Dynein heavy chain AAA lid domain / Dynein heavy chain, coiled coil stalk / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats circular profile. / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / 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
Dynein heavy chain, cytoplasmic / ADENOSINE-5'-DIPHOSPHATE / ADENOSINE-5'-TRIPHOSPHATE / Nuclear distribution protein PAC1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLahiri, I. / Reimer, J.M. / Leschziner, A.E.
Funding support United States, 1items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 19, 2022Provider: repository / Type: Initial release

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Structure visualization

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  • EMDB-23829
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Assembly

Deposited unit
A: dynein AAA3-WalkerB mutant (E2488Q)
C: Nuclear distribution protein PAC1
B: Nuclear distribution protein PAC1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)447,5839
Polymers445,5853
Non-polymers1,9976
Water0
1


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

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Components

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


Mass: 331524.000 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: A0A7I9C1Z7
#2: Protein Nuclear distribution protein PAC1 / Lissencephaly-1 homolog / LIS-1 / nudF homolog


Mass: 57030.617 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P39946
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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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: Complex of yeast dynein bound by two Lis1s in the presence of ATP-Va
Type: COMPLEX / Entity ID: #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The yeast dynein was biotinylated prior to complex formation.
Specimen supportDetails: 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.
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 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: 2700 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingAverage exposure time: 10 sec. / Electron dose: 58.3 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: 2229
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83975 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00424600
ELECTRON MICROSCOPYf_angle_d0.4733304
ELECTRON MICROSCOPYf_dihedral_angle_d5.9483177
ELECTRON MICROSCOPYf_chiral_restr0.0423767
ELECTRON MICROSCOPYf_plane_restr0.0034169

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