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- EMDB-11559: mechanosensitive channel YnaI in a closed-like conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-11559
Titlemechanosensitive channel YnaI in a closed-like conformation
Map data
SampleYnaI in a closed-like conformation:
YnaI
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsFlegler VJ / Rasmussen A / Rao S / Wu N / Zenobi R / Sansom MSP / Hedrich R / Rasmussen T / Boettcher B
Funding support Germany, 2 items
OrganizationGrant numberCountry
German Research Foundation (DFG)Bo1150/15-1 Germany
German Research Foundation (DFG)INST 93/903-1 FUGG Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: The MscS-like channel YnaI has a gating mechanism based on flexible pore helices.
Authors: Vanessa Judith Flegler / Akiko Rasmussen / Shanlin Rao / Na Wu / Renato Zenobi / Mark S P Sansom / Rainer Hedrich / Tim Rasmussen / Bettina Böttcher /
Abstract: The mechanosensitive channel of small conductance (MscS) is the prototype of an evolutionarily diversified large family that fine-tunes osmoregulation but is likely to fulfill additional functions. ...The mechanosensitive channel of small conductance (MscS) is the prototype of an evolutionarily diversified large family that fine-tunes osmoregulation but is likely to fulfill additional functions. has six osmoprotective paralogs with different numbers of transmembrane helices. These helices are important for gating and sensing in MscS but the role of the additional helices in the paralogs is not understood. The medium-sized channel YnaI was extracted and delivered in native nanodiscs in closed-like and open-like conformations using the copolymer diisobutylene/maleic acid (DIBMA) for structural studies. Here we show by electron cryomicroscopy that YnaI has an extended sensor paddle that during gating relocates relative to the pore concomitant with bending of a GGxGG motif in the pore helices. YnaI is the only one of the six paralogs that has this GGxGG motif allowing the sensor paddle to move outward. Access to the pore is through a vestibule on the cytosolic side that is fenestrated by side portals. In YnaI, these portals are obstructed by aromatic side chains but are still fully hydrated and thus support conductance. For comparison with large-sized channels, we determined the structure of YbiO, which showed larger portals and a wider pore with no GGxGG motif. Further in silico comparison of MscS, YnaI, and YbiO highlighted differences in the hydrophobicity and wettability of their pores and vestibule interiors. Thus, MscS-like channels of different sizes have a common core architecture but show different gating mechanisms and fine-tuned conductive properties.
History
DepositionJul 31, 2020-
Header (metadata) releaseNov 18, 2020-
Map releaseNov 18, 2020-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_11559.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 200 pix.
= 212.7 Å
1.06 Å/pix.
x 200 pix.
= 212.7 Å
1.06 Å/pix.
x 200 pix.
= 212.7 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0635 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.06541595 - 0.15670171
Average (Standard dev.)0.0000322424 (±0.008643265)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 212.70001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.06351.06351.0635
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z212.700212.700212.700
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ172172172
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0650.1570.000

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Supplemental data

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Segmentation: #1

Fileemd_11559_msk_1.map
Projections & Slices
AxesZYX

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Additional map: rae map: from Relion Refine3D

Fileemd_11559_additional_1.map
Annotationrae map: from Relion Refine3D
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_11559_half_map_1.map
Projections & Slices
AxesZYX

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Half map: #1

Fileemd_11559_half_map_2.map
Projections & Slices
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Sample components

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Entire YnaI in a closed-like conformation

EntireName: YnaI in a closed-like conformation
Details: YnaI was purified with the detergent DDM and reconstituted into liposomes. After addition of LPC it was extracted using DIBMA co-polymer.
Number of components: 2

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Component #1: protein, YnaI in a closed-like conformation

ProteinName: YnaI in a closed-like conformation
Details: YnaI was purified with the detergent DDM and reconstituted into liposomes. After addition of LPC it was extracted using DIBMA co-polymer.
Recombinant expression: No
MassExperimental: 278 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, YnaI

ProteinName: YnaI / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.4 mg/mL / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 70 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 75000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 2200.0 - 2800.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: FEI FALCON III (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4161

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C7 (7 fold cyclic) / Number of projections: 20016
3D reconstructionSoftware: RELION / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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