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- EMDB-11561: mechanosensitive channel YnaI in an intermediate conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-11561
Titlemechanosensitive channel YnaI in an intermediate conformation
Map datamap from Relion Post-process, sharpened with a B-factor of -134, without map
Sample
  • Complex: YnaI in an intermediate conformation
    • Protein or peptide: YnaI
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.32 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: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_11561.png

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Map

FileDownload / File: emd_11561.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap from Relion Post-process, sharpened with a B-factor of -134, without map
Voxel sizeX=Y=Z: 1.0635 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.12256649 - 0.21898198
Average (Standard dev.)0.00001492059 (±0.011440354)
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-0.1230.2190.000

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Supplemental data

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Mask #1

Fileemd_11561_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Additional map: raw map: from Relion Refine3D

Fileemd_11561_additional_1.map
Annotationraw map: from Relion Refine3D
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_11561_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_11561_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : YnaI in an intermediate conformation

EntireName: YnaI in an intermediate conformation
Components
  • Complex: YnaI in an intermediate conformation
    • Protein or peptide: YnaI

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Supramolecule #1: YnaI in an intermediate conformation

SupramoleculeName: YnaI in an intermediate conformation / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: YnaI was purified with the detergent DDM and reconstituted into liposomes. After addition of LPC it was extracted using DIBMA co-polymer.
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 278 KDa

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Macromolecule #1: YnaI

MacromoleculeName: YnaI / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIAELFTNNA LNLVIIFGSC AALILMSFWF RRGNRKRKGF LFHAVQFLIY TIIISAVGSI INYVIENYKL KFITPGVID FICTSLIAVI LTIKLFLLIN QFEKQQIKKG RDITSARIMS RIIKITIIVV LVLLYGEHFG M SLSGLLTF AGIAGLAVGM AGKDILSNFF ...String:
MIAELFTNNA LNLVIIFGSC AALILMSFWF RRGNRKRKGF LFHAVQFLIY TIIISAVGSI INYVIENYKL KFITPGVID FICTSLIAVI LTIKLFLLIN QFEKQQIKKG RDITSARIMS RIIKITIIVV LVLLYGEHFG M SLSGLLTF AGIAGLAVGM AGKDILSNFF SGIMLYFDRP FSIGDWIRSP DRNIEGTVAE IGWRITKIKT FD NRPLYVP NSLFSSISVE NPGRMTNRRI TTTIGLRYED AAKVGVIVEA VREMLKNHPA IDQRQTLLVY FNQ FADSSL NIMVYCFTKT TVWAEWLAAQ QDVYLKIIDI VQSHGADFAF PSQTLYMDNI TPPDQGRLEH HHHHH

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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

Concentration0.4 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 4161 / Average exposure time: 63.0 sec. / Average electron dose: 70.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.1)
Details: 3D classification revealed the presence of three different conformations in the data set.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 9309
FSC plot (resolution estimation)

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