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

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Basic information

Entry
Database: PDB / ID: 9bee
TitlealphaB-crystallin N-terminal IXI variant in a fibril state
Components(Alpha-crystallin B chain) x 2
KeywordsCHAPERONE / small heat-shock protein fibril proteostasis cataract
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 / tubulin complex assembly / cardiac myofibril / 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 / tubulin complex assembly / cardiac myofibril / 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 / cellular response to gamma radiation / negative regulation of cell growth / response to hydrogen peroxide / Z disc / unfolded protein binding / protein folding / response to estradiol / amyloid-beta binding / response to heat / protein refolding / microtubule binding / dendritic spine / perikaryon / response to hypoxia / lysosome / protein stabilization / axon / negative regulation of gene expression / negative regulation of DNA-templated transcription / protein-containing complex binding / negative regulation of apoptotic process / 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)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsMcFarland, R. / Reichow, S.L.
Funding support United States, 4items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Alpha-crystallin B chain
A: Alpha-crystallin B chain


Theoretical massNumber of molelcules
Total (without water)22,0512
Polymers22,0512
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Alpha-crystallin B chain / Alpha(B)-crystallin / Heat shock protein beta-5 / HspB5 / Heat shock protein family B member 5 / ...Alpha(B)-crystallin / Heat shock protein beta-5 / HspB5 / Heat shock protein family B member 5 / Renal carcinoma antigen NY-REN-27 / Rosenthal fiber component


Mass: 9944.200 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CRYAB, CRYA2, HSPB5 / Production host: Escherichia coli (E. coli) / References: UniProt: P02511
#2: Protein Alpha-crystallin B chain / Alpha(B)-crystallin / Heat shock protein beta-5 / HspB5 / Heat shock protein family B member 5 / ...Alpha(B)-crystallin / Heat shock protein beta-5 / HspB5 / Heat shock protein family B member 5 / Renal carcinoma antigen NY-REN-27 / Rosenthal fiber component


Mass: 12106.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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.
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
Has protein modificationN
Sequence detailsThe complete sample sequence is MDAAAHHPWIRRPFFPFHSPSRLFDQFFGEHLLESDLFPTSTSLSPFYLRPPSFLRAPSW ...The complete sample sequence is MDAAAHHPWIRRPFFPFHSPSRLFDQFFGEHLLESDLFPTSTSLSPFYLRPPSFLRAPSW FDTGLSEMRLEKDRFSVNLDVKHFSPEELKVKVLGDVIEVHGKHEERQDEHGFISREFHR KYRIPADVDPLTITSSLSSDGVLTVNGPRKQVSGPERTIPITREEKPAVTAAPKK

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Asymmetric dimer of alphaB-crystallin filament state formed by an N-terminal IXI variant
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 40.3 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2100 mMSodium ChlorideNaCl1
31 mMEDTAC10H16N2O81
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.3 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10654

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
13PHENIXmodel refinement
14Cootmodel refinement
15ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 31.6 ° / Axial rise/subunit: 39.9 Å / Axial symmetry: D2
Particle selectionNum. of particles selected: 2201394
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1393386 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 2WJ7

2wj7


Accession code: 2WJ7 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0131574
ELECTRON MICROSCOPYf_angle_d1.8312125
ELECTRON MICROSCOPYf_dihedral_angle_d11.954606
ELECTRON MICROSCOPYf_chiral_restr0.099240
ELECTRON MICROSCOPYf_plane_restr0.019281

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