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- EMDB-13346: Single-particle cryo-EM reconstruction of the tetrahedral 24mer o... -

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Basic information

Entry
Database: EMDB / ID: EMD-13346
TitleSingle-particle cryo-EM reconstruction of the tetrahedral 24mer of Hsp17 from Caenorhabditis elegans
Map data
Sample
  • Complex: Tetrahedral oligomer (24mer) of Hsp17
    • Protein or peptide: SHSP domain-containing protein
Function / homologyAlpha crystallin/Small heat shock protein, animal type / Hsp20/alpha crystallin family / Small heat shock protein (sHSP) domain profile. / Alpha crystallin/Hsp20 domain / HSP20-like chaperone / unfolded protein binding / response to heat / protein refolding / SHSP domain-containing protein
Function and homology information
Biological speciesCaenorhabditis elegans (invertebrata)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.49 Å
AuthorsRossa B / Weinkauf S / Buchner J
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB1035 Germany
CitationJournal: J Biol Chem / Year: 2023
Title: The permanently chaperone-active small heat shock protein Hsp17 from Caenorhabditis elegans exhibits topological separation of its N-terminal regions.
Authors: Annika Strauch / Benjamin Rossa / Fabian Köhler / Simon Haeussler / Moritz Mühlhofer / Florian Rührnößl / Caroline Körösy / Yevheniia Bushman / Barbara Conradt / Martin Haslbeck / ...Authors: Annika Strauch / Benjamin Rossa / Fabian Köhler / Simon Haeussler / Moritz Mühlhofer / Florian Rührnößl / Caroline Körösy / Yevheniia Bushman / Barbara Conradt / Martin Haslbeck / Sevil Weinkauf / Johannes Buchner /
Abstract: Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind nonnative proteins in an ATP-independent manner. Caenorhabditis elegans encodes 16 different sHsps, among them Hsp17, ...Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind nonnative proteins in an ATP-independent manner. Caenorhabditis elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear. Here, we find that Hsp17 has a distinct expression pattern, structural organization, and chaperone function. Consistent with its presence under nonstress conditions, and in contrast to many other sHsps, we determined that Hsp17 is a mono-disperse, permanently active chaperone in vitro, which interacts with hundreds of different C. elegans proteins under physiological conditions. Additionally, our cryo-EM structure of Hsp17 reveals that in the 24-mer complex, 12 N-terminal regions are involved in its chaperone function. These flexible regions are located on the outside of the spherical oligomer, whereas the other 12 N-terminal regions are engaged in stabilizing interactions in its interior. This allows the same region in Hsp17 to perform different functions depending on the topological context. Taken together, our results reveal structural and functional features that further define the structural basis of permanently active sHsps.
History
DepositionAug 9, 2021-
Header (metadata) releaseDec 14, 2022-
Map releaseDec 14, 2022-
UpdateJan 4, 2023-
Current statusJan 4, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13346.png

Downloads & links

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Map

FileDownload / File: emd_13346.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.09 Å
Density
Contour LevelBy AUTHOR: 0.37
Minimum - Maximum-0.55706316 - 1.4202365
Average (Standard dev.)0.0012313685 (±0.084626175)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 327.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_13346_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_13346_half_map_1.map
Projections & Slices
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Histogram

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Half map: #1

Fileemd_13346_half_map_2.map
Projections & Slices
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Sample components

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Entire : Tetrahedral oligomer (24mer) of Hsp17

EntireName: Tetrahedral oligomer (24mer) of Hsp17
Components
  • Complex: Tetrahedral oligomer (24mer) of Hsp17
    • Protein or peptide: SHSP domain-containing protein

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Supramolecule #1: Tetrahedral oligomer (24mer) of Hsp17

SupramoleculeName: Tetrahedral oligomer (24mer) of Hsp17 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Molecular weightTheoretical: 422 KDa

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Macromolecule #1: SHSP domain-containing protein

MacromoleculeName: SHSP domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Molecular weightTheoretical: 17.44251 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MDRRFPPFSP FFNHGRFFDD VDFDRHMIRP YWADQTMLTG HRVGDAIDVV NNDQEYNVSV DVSQFEPEEL KVNIVDNQLI IEGKHNEKT DKYGQVERHF VRKYNLPTGV RPEQIKSELS NNGVLTVKYE KNQEQQPKSI PITIVPKRN

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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

Concentration2.5 mg/mL
BufferpH: 7.4 / Details: PBS
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 20.0 nm / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: waiting time 0, blotting force 5, blotting time 2.5.
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 2568 / Average electron dose: 55.0 e/Å2
Details: Images were recorded in CDS mode as tif-stacks with 30 frames.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 663141
Details: Gautomatch automatic picking yielded an initial dataset that was used to generate templates for cryoSPARC2 template picking.
Startup modelType of model: OTHER / Details: Ab-initio reconstruction
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Resolution.type: BY AUTHOR / Resolution: 6.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 187116
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7pe3:
Pseudo-atomic model of the tetrahedral 24mer of Hsp17 from Caenorhabditis elegans

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