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- EMDB-9386: Cryo-EM reconstruction of delta T3046-T3055 mutant of yeast cytop... -

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Basic information

Entry
Database: EMDB / ID: EMD-9386
TitleCryo-EM reconstruction of delta T3046-T3055 mutant of yeast cytoplasmic dynein in the presence of ADP-Vi
Map dataMutant 5 of dynein in the ADP-vanadate (ADP-vi) state
Sample
  • Complex: delta T3046-T3055 mutant of yeast cytoplasmic dynein
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.2 Å
AuthorsNiekamp S / Coudray N / Zhang N / Vale RD / Bhabha G
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM112892 United States
Other privateDFS-20-16 (Damon Runyon Cancer Research Fundation) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM097312 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: EMBO J / Year: 2019
Title: Coupling of ATPase activity, microtubule binding, and mechanics in the dynein motor domain.
Authors: Stefan Niekamp / Nicolas Coudray / Nan Zhang / Ronald D Vale / Gira Bhabha /
Abstract: The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and ...The movement of a molecular motor protein along a cytoskeletal track requires communication between enzymatic, polymer-binding, and mechanical elements. Such communication is particularly complex and not well understood in the dynein motor, an ATPase that is comprised of a ring of six AAA domains, a large mechanical element (linker) spanning over the ring, and a microtubule-binding domain (MTBD) that is separated from the AAA ring by a ~ 135 Å coiled-coil stalk. We identified mutations in the stalk that disrupt directional motion, have microtubule-independent hyperactive ATPase activity, and nucleotide-independent low affinity for microtubules. Cryo-electron microscopy structures of a mutant that uncouples ATPase activity from directional movement reveal that nucleotide-dependent conformational changes occur normally in one-half of the AAA ring, but are disrupted in the other half. The large-scale linker conformational change observed in the wild-type protein is also inhibited, revealing that this conformational change is not required for ATP hydrolysis. These results demonstrate an essential role of the stalk in regulating motor activity and coupling conformational changes across the two halves of the AAA ring.
History
DepositionJan 3, 2019-
Header (metadata) releaseMar 6, 2019-
Map releaseMar 6, 2019-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.27
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.27
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9386.png

Downloads & links

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Map

FileDownload / File: emd_9386.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMutant 5 of dynein in the ADP-vanadate (ADP-vi) state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.16 Å/pix.
x 320 pix.
= 369.92 Å		1.16 Å/pix.
x 320 pix.
= 369.92 Å		1.16 Å/pix.
x 320 pix.
= 369.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.156 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.27 / Movie #1: 0.27
Minimum - Maximum-0.21422063 - 0.7299793
Average (Standard dev.)0.0024972884 (±0.035057202)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 369.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1561.1561.156
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z369.920369.920369.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-51-35-11
NX/NY/NZ11110799
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.2140.7300.002

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Supplemental data

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Sample components

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Entire : delta T3046-T3055 mutant of yeast cytoplasmic dynein

EntireName: delta T3046-T3055 mutant of yeast cytoplasmic dynein
Components
  • Complex: delta T3046-T3055 mutant of yeast cytoplasmic dynein

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Supramolecule #1: delta T3046-T3055 mutant of yeast cytoplasmic dynein

SupramoleculeName: delta T3046-T3055 mutant of yeast cytoplasmic dynein / type: complex / ID: 1 / Parent: 0
Details: Cryo-EM reconstruction of delta T3046-T3055 mutant of yeast cytoplasmic dynein in the presence of ADP-Vi
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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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: 8
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 664 / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 36000
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1.10)
Startup modelType of model: OTHER / Details: ab-initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 0.6) / Number images used: 8653
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 0.6)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 0.6)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 0.6)

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