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- EMDB-9953: Structural insights into stressosome assembly -

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Basic information

Entry
Database: EMDB / ID: EMD-9953
TitleStructural insights into stressosome assembly
Map dataRsbRA/S complex
Sample
  • Complex: RsbRA/RsbS complex from Bacillus subtilis
Biological speciesBacillus subtilis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.3 Å
AuthorsKim H / Kwon E / Pathak D / Kim DY / Jung HS
Funding support Korea, Republic Of, 2 items
OrganizationGrant numberCountry
National Research Foundation (Korea)2018R1D1AB07045580 Korea, Republic Of
Rural Development AdministrationSSAC-PJ013273042019 Korea, Republic Of
CitationJournal: J Mol Biol / Year: 2011
Title: Role of the tail in the regulated state of myosin 2.
Authors: Hyun Suk Jung / Neil Billington / Kavitha Thirumurugan / Bridget Salzameda / Christine R Cremo / Joseph M Chalovich / Peter D Chantler / Peter J Knight /
Abstract: Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two ...Myosin 2 from vertebrate smooth muscle or non-muscle sources is in equilibrium between compact, inactive monomers and thick filaments under physiological conditions. In the inactive monomer, the two heads pack compactly together, and the long tail is folded into three closely packed segments that are associated chiefly with one of the heads. The molecular basis of the folding of the tail remains unexplained. By using electron microscopy, we show that compact monomers of smooth muscle myosin 2 have the same structure in both the native state and following specific, intramolecular photo-cross-linking between Cys109 of the regulatory light chain (RLC) and segment 3 of the tail. Nonspecific cross-linking between lysine residues of the folded monomer by glutaraldehyde also does not perturb the compact conformation and stabilizes it against unfolding at high ionic strength. Sequence comparisons across phyla and myosin 2 isoforms suggest that the folding of the tail is stabilized by ionic interactions between the positively charged N-terminal sequence of the RLC and a negatively charged region near the start of tail segment 3 and that phosphorylation of the RLC could perturb these interactions. Our results support the view that interactions between the heads and the distal tail perform a critical role in regulating activity of myosin 2 molecules through stabilizing the compact monomer conformation.
History
DepositionJun 18, 2019-
Header (metadata) releaseNov 6, 2019-
Map releaseNov 6, 2019-
UpdateMar 23, 2022-
Current statusMar 23, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9953.png

Downloads & links

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Map

FileDownload / File: emd_9953.map.gz / Format: CCP4 / Size: 137.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRsbRA/S complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 330 pix.
= 462. Å		1.4 Å/pix.
x 330 pix.
= 462. Å		1.4 Å/pix.
x 330 pix.
= 462. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.030700725 - 0.18443754
Average (Standard dev.)0.0013268844 (±0.009094623)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions330330330
Spacing330330330
CellA=B=C: 462.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.41.41.4
M x/y/z330330330
origin x/y/z0.0000.0000.000
length x/y/z462.000462.000462.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS330330330
D min/max/mean-0.0310.1840.001

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Supplemental data

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Mask #1

Fileemd_9953_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Half map: half map 1

Fileemd_9953_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_9953_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

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Sample components

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Entire : RsbRA/RsbS complex from Bacillus subtilis

EntireName: RsbRA/RsbS complex from Bacillus subtilis
Components
  • Complex: RsbRA/RsbS complex from Bacillus subtilis

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Supramolecule #1: RsbRA/RsbS complex from Bacillus subtilis

SupramoleculeName: RsbRA/RsbS complex from Bacillus subtilis / type: complex / ID: 1 / Parent: 0
Details: Stressosome (RsbRA/RsbS) complex from Bacillus subtilis
Source (natural)Organism: Bacillus subtilis (bacteria) / Strain: 168
Recombinant expressionOrganism: Enterobacteria phage L1 (virus) / Recombinant strain: BL-21star (DE3)
Molecular weightExperimental: 1.8 MDa

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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

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMNaClsodium chloride
20.0 mMHEPESHEPES
0.2 mMTCEPTCEP
5.0 %GlycerolGlycerol

Details: 20 mM HEPES, 50 mM NaCl, 0.2 mM TCEP, 5 % glycerol, pH 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 39.2 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 7 seconds before plunging.
DetailsRsbRA/RsbS complex buffer: 20 mM HEPES, 50 mM NaCl, 0.2 mM TCEP, 5 % glycerol, pH 7.5

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: OTHER / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 1788 / Average exposure time: 1.0 sec. / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 3.0 µm / Calibrated defocus min: 1.5 µm / Calibrated magnification: 47000 / Illumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 47000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 54798
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: from RELION 2.1
Final reconstructionNumber classes used: 37 / Applied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 42623
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 100 / Avg.num./class: 540 / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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