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- EMDB-13328: Time-resolved cryo-EM structures of ATP-induced actomyosin dissoc... -

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Basic information

Entry
Database: EMDB / ID: EMD-13328
TitleTime-resolved cryo-EM structures of ATP-induced actomyosin dissociation
Map datasharpened map of actomyosin from all timepoints combined
Sample
  • Complex: Skeletal myosin subfragment 1 (A1 fraction) from rabbit in complex with filamentous actin from rabbit
Biological speciesOryctolagus cuniculus (rabbit)
Methodhelical reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsKlebl DP / White HD / Sobott F / Muench SP
Funding support3 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/P0263971/1
American Heart AssociationAMR21-236078
Wellcome Trust08466/Z/15/Z
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2021
Title: On-grid and in-flow mixing for time-resolved cryo-EM.
Authors: David P Klebl / Howard D White / Frank Sobott / Stephen P Muench /
Abstract: Time-resolved cryo-electron microscopy (TrEM) allows the study of proteins under non-equilibrium conditions on the millisecond timescale, permitting the analysis of large-scale conformational changes ...Time-resolved cryo-electron microscopy (TrEM) allows the study of proteins under non-equilibrium conditions on the millisecond timescale, permitting the analysis of large-scale conformational changes or assembly and disassembly processes. However, the technique is developing and there have been few comparisons with other biochemical kinetic studies. Using current methods, the shortest time delay is on the millisecond timescale (∼5-10 ms), given by the delay between sample application and vitrification, and generating longer time points requires additional approaches such as using a longer delay line between the mixing element and nozzle, or an incubation step on the grid. To compare approaches, the reaction of ATP with the skeletal actomyosin S1 complex was followed on grids prepared with a 7-700 ms delay between mixing and vitrification. Classification of the cryo-EM data allows kinetic information to be derived which agrees with previous biochemical measurements, showing fast dissociation, low occupancy during steady-state hydrolysis and rebinding once ATP has been hydrolysed. However, this rebinding effect is much less pronounced when on-grid mixing is used and may be influenced by interactions with the air-water interface. Moreover, in-flow mixing results in a broader distribution of reaction times due to the range of velocities in a laminar flow profile (temporal spread), especially for longer time delays. This work shows the potential of TrEM, but also highlights challenges and opportunities for further development.
History
DepositionAug 4, 2021-
Header (metadata) releaseOct 13, 2021-
Map releaseOct 13, 2021-
UpdateOct 13, 2021-
Current statusOct 13, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13328.png

Downloads & links

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Map

FileDownload / File: emd_13328.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map of actomyosin from all timepoints combined
Voxel sizeX=Y=Z: 2.13 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.043096405 - 0.14011763
Average (Standard dev.)0.00021942142 (±0.0042872727)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 426.00003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.132.132.13
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z426.000426.000426.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0430.1400.000

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Supplemental data

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Mask #1

Fileemd_13328_msk_1.map
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Additional map: unsharpened reconstruction from all 13 ms particles

Fileemd_13328_additional_1.map
Annotationunsharpened reconstruction from all 13 ms particles
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Additional map: unsharpened reconstruction from all 340 ms particles

Fileemd_13328_additional_10.map
Annotationunsharpened reconstruction from all 340 ms particles
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Additional map: unsharpened reconstruction from all 400 ms particles

Fileemd_13328_additional_11.map
Annotationunsharpened reconstruction from all 400 ms particles
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Additional map: unsharpened reconstruction from all 640 ms particles

Fileemd_13328_additional_2.map
Annotationunsharpened reconstruction from all 640 ms particles
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Additional map: halfmap 2 of actin from all timepoints combined

Fileemd_13328_additional_3.map
Annotationhalfmap 2 of actin from all timepoints combined
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Additional map: unsharpened map of actomyosin from all timepoints combined

Fileemd_13328_additional_4.map
Annotationunsharpened map of actomyosin from all timepoints combined
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Additional map: sharpened map of actin from all timepoints combined

Fileemd_13328_additional_5.map
Annotationsharpened map of actin from all timepoints combined
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Additional map: unsharpened map of actin from all timepoints combined

Fileemd_13328_additional_6.map
Annotationunsharpened map of actin from all timepoints combined
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Additional map: halfmap 1 of actin from all timepoints combined

Fileemd_13328_additional_7.map
Annotationhalfmap 1 of actin from all timepoints combined
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Additional map: unsharpened reconstruction from all 700 ms particles

Fileemd_13328_additional_8.map
Annotationunsharpened reconstruction from all 700 ms particles
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Additional map: unsharpened reconstruction from all 7 ms particles

Fileemd_13328_additional_9.map
Annotationunsharpened reconstruction from all 7 ms particles
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Half map: halfmap 1 of actomyosin from all timepoints combined

Fileemd_13328_half_map_1.map
Annotationhalfmap 1 of actomyosin from all timepoints combined
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Half map: halfmap 2 of actomyosin from all timepoints combined

Fileemd_13328_half_map_2.map
Annotationhalfmap 2 of actomyosin from all timepoints combined
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Sample components

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Entire : Skeletal myosin subfragment 1 (A1 fraction) from rabbit in comple...

EntireName: Skeletal myosin subfragment 1 (A1 fraction) from rabbit in complex with filamentous actin from rabbit
Components
  • Complex: Skeletal myosin subfragment 1 (A1 fraction) from rabbit in complex with filamentous actin from rabbit

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Supramolecule #1: Skeletal myosin subfragment 1 (A1 fraction) from rabbit in comple...

SupramoleculeName: Skeletal myosin subfragment 1 (A1 fraction) from rabbit in complex with filamentous actin from rabbit
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber temperature: 293 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 6 / Average electron dose: 62.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 27.56 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.25 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 74556
Startup modelType of model: OTHER / Details: featureless cylinder
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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