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- EMDB-44477: alphaB-crystallin N-terminal IXI variant in a fibril state -

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Basic information

Entry
Database: EMDB / ID: EMD-44477
TitlealphaB-crystallin N-terminal IXI variant in a fibril state
Map data
Sample
  • Complex: Asymmetric dimer of alphaB-crystallin filament state formed by an N-terminal IXI variant
    • Protein or peptide: Alpha-crystallin B chain
  • Protein or peptide: Alpha-crystallin B chain
Keywordssmall heat-shock protein fibril proteostasis cataract / CHAPERONE
Function / homology
Function and homology information


microtubule polymerization or depolymerization / negative regulation of intracellular transport / apoptotic process involved in morphogenesis / regulation of programmed cell death / cardiac myofibril / tubulin complex assembly / structural constituent of eye lens / negative regulation of amyloid fibril formation / M band / lens development in camera-type eye ...microtubule polymerization or depolymerization / negative regulation of intracellular transport / apoptotic process involved in morphogenesis / regulation of programmed cell death / cardiac myofibril / tubulin complex assembly / structural constituent of eye lens / negative regulation of amyloid fibril formation / M band / lens development in camera-type eye / muscle organ development / actin filament bundle / negative regulation of reactive oxygen species metabolic process / HSF1-dependent transactivation / negative regulation of protein-containing complex assembly / stress-activated MAPK cascade / muscle contraction / synaptic membrane / response to hydrogen peroxide / cellular response to gamma radiation / negative regulation of cell growth / Z disc / unfolded protein binding / protein folding / response to estradiol / amyloid-beta binding / response to heat / protein refolding / perikaryon / microtubule binding / dendritic spine / response to hypoxia / lysosome / protein stabilization / axon / negative regulation of gene expression / negative regulation of DNA-templated transcription / negative regulation of apoptotic process / protein-containing complex binding / structural molecule activity / cell surface / protein homodimerization activity / protein-containing complex / mitochondrion / extracellular exosome / nucleoplasm / metal ion binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Alpha-crystallin B chain, ACD domain / : / Alpha-crystallin, N-terminal / Alpha crystallin A chain, N terminal / Alpha 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
Similarity search - Domain/homology
Alpha-crystallin B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsMcFarland R / Reichow SL
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)R01EY030987 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM124779 United States
National Institutes of Health/National Eye Institute (NIH/NEI)F31EY033230 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129547 United States
CitationJournal: Nat Commun / Year: 2024
Title: Dynamic fibrillar assembly of αB-crystallin induced by perturbation of the conserved NT-IXI motif resolved by cryo-EM.
Authors: Russell McFarland / Rozhan Noroozi / Adam P Miller / Steve L Reichow /
Abstract: αB-crystallin is an archetypical member of the small heat shock proteins (sHSPs) vital for cellular proteostasis and mitigating protein misfolding diseases. Gaining insights into the principles ...αB-crystallin is an archetypical member of the small heat shock proteins (sHSPs) vital for cellular proteostasis and mitigating protein misfolding diseases. Gaining insights into the principles defining their molecular organization and chaperone function have been hindered by intrinsic dynamic properties and limited high-resolution structural analysis. To disentangle the mechanistic underpinnings of these dynamical properties, we ablate a conserved IXI-motif located within the N-terminal (NT) domain of human αB-crystallin implicated in subunit exchange dynamics and client sequestration. This results in a profound structural transformation, from highly polydispersed caged-like native assemblies into an elongated fibril state amenable to high-resolution cryo-EM analysis. The reversible nature of this variant facilitates interrogation of functional effects due to perturbation of the NT-IXI motif in both the native-like oligomer and fibril states. Together, our investigations unveil several features thought to be key mechanistic attributes to sHSPs and point to a critical significance of the NT-IXI motif in αB-crystallin assembly, polydispersity, and chaperone activity.
History
DepositionApr 15, 2024-
Header (metadata) releaseDec 11, 2024-
Map releaseDec 11, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44477.png

Downloads & links

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Map

FileDownload / File: emd_44477.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.79 Å/pix.
x 288 pix.
= 226.944 Å		0.79 Å/pix.
x 288 pix.
= 226.944 Å		0.79 Å/pix.
x 288 pix.
= 226.944 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.788 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.054
Minimum - Maximum-0.2349636 - 0.34744844
Average (Standard dev.)0.0012115794 (±0.010489999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 226.944 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_44477_additional_1.map
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Half map: #2

Fileemd_44477_half_map_1.map
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Half map: #1

Fileemd_44477_half_map_2.map
Projections & Slices
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Sample components

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Entire : Asymmetric dimer of alphaB-crystallin filament state formed by an...

EntireName: Asymmetric dimer of alphaB-crystallin filament state formed by an N-terminal IXI variant
Components
  • Complex: Asymmetric dimer of alphaB-crystallin filament state formed by an N-terminal IXI variant
    • Protein or peptide: Alpha-crystallin B chain
  • Protein or peptide: Alpha-crystallin B chain

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Supramolecule #1: Asymmetric dimer of alphaB-crystallin filament state formed by an...

SupramoleculeName: Asymmetric dimer of alphaB-crystallin filament state formed by an N-terminal IXI variant
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.3 kDa/nm

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Macromolecule #1: Alpha-crystallin B chain

MacromoleculeName: Alpha-crystallin B chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 9.9442 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SVNLDVKHFS PEELKVKVLG DVIEVHGKHE ERQDEHGFIS REFHRKYRIP ADVDPLTITS SLSSDGVLTV NGPRKQVSGP ERTIPITR

UniProtKB: Alpha-crystallin B chain

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Macromolecule #2: Alpha-crystallin B chain

MacromoleculeName: Alpha-crystallin B chain / type: protein_or_peptide / ID: 2
Details: 7 residues are modeled as unknown as they are identifiable in the EM volume with 1 being able to be assigned an identity with intermediate confidence. We cannot clearly identify which ...Details: 7 residues are modeled as unknown as they are identifiable in the EM volume with 1 being able to be assigned an identity with intermediate confidence. We cannot clearly identify which protomer the bound peptide corresponds to though it is believed to play a key role in the oligomerization. We have opted to include the peptide as part of Chain A with numbering based on our best approximation from related protein species and mass spec data published by others. The residues are labelled as unknown to reflect our low confidence of the identity of the peptide.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 12.106734 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)TGL SEMRLEKDRF SVNLDVKHFS PEELKVKVLG DVIEVHGKHE ER QDEHGFI SREFHRKYRI PADVDPLTIT SSLSSDGVLT VNGPRKQVSG PERTIPITR

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
100.0 mMNaClSodium Chloride
1.0 mMC10H16N2O8EDTA
GridModel: Quantifoil / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 10654 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
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

Final reconstructionApplied symmetry - Helical parameters - Δz: 39.9 Å
Applied symmetry - Helical parameters - Δ&Phi: 31.6 °
Applied symmetry - Helical parameters - Axial symmetry: D2 (2x2 fold dihedral)
Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1393386
Segment selectionNumber selected: 2201394 / Software - Name: cryoSPARC
Startup modelType of model: NONE
Final angle assignmentType: NOT APPLICABLE / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial modelPDB ID:

2wj7


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9bee:
alphaB-crystallin N-terminal IXI variant in a fibril state

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