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- PDB-6t1r: Pseudo-atomic model of a 16-mer assembly of reduced recombinant h... -

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Basic information

Entry
Database: PDB / ID: 6t1r
TitlePseudo-atomic model of a 16-mer assembly of reduced recombinant human alphaA-crystallin (non domain swapped configuration)
ComponentsAlpha-crystallin A chain
KeywordsCHAPERONE / sHsp / alphaA-crystallin / domain swapping
Function / homology
Function and homology information


negative regulation of intracellular transport / structural constituent of eye lens / lens development in camera-type eye / visual perception / unfolded protein binding / response to heat / protein refolding / protein stabilization / negative regulation of apoptotic process / structural molecule activity ...negative regulation of intracellular transport / structural constituent of eye lens / lens development in camera-type eye / visual perception / unfolded protein binding / response to heat / protein refolding / protein stabilization / negative regulation of apoptotic process / structural molecule activity / protein-containing complex / nucleoplasm / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
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 A chain
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.8 Å
AuthorsPeters, C. / Kaiser, C.J.O. / Weinkauf, S. / Zacharias, M. / Buchner, J.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research FoundationCRC 1035 Germany
German Research FoundationEXC 114 Germany
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: The structure and oxidation of the eye lens chaperone αA-crystallin.
Authors: Christoph J O Kaiser / Carsten Peters / Philipp W N Schmid / Maria Stavropoulou / Juan Zou / Vinay Dahiya / Evgeny V Mymrikov / Beate Rockel / Sam Asami / Martin Haslbeck / Juri Rappsilber / ...Authors: Christoph J O Kaiser / Carsten Peters / Philipp W N Schmid / Maria Stavropoulou / Juan Zou / Vinay Dahiya / Evgeny V Mymrikov / Beate Rockel / Sam Asami / Martin Haslbeck / Juri Rappsilber / Bernd Reif / Martin Zacharias / Johannes Buchner / Sevil Weinkauf /
Abstract: The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the ...The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human αA-crystallin oligomers by combining cryo-electron microscopy, cross-linking/mass spectrometry, NMR spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of αA-crystallin heterogeneity. Human αA-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced αA-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. The insight into the structure and function of αA-crystallin provides a basis for understanding its role in the eye lens.
History
DepositionOct 5, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 11, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.2May 15, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-4894
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Alpha-crystallin A chain
B: Alpha-crystallin A chain
C: Alpha-crystallin A chain
D: Alpha-crystallin A chain
E: Alpha-crystallin A chain
F: Alpha-crystallin A chain
G: Alpha-crystallin A chain
H: Alpha-crystallin A chain
I: Alpha-crystallin A chain
J: Alpha-crystallin A chain
K: Alpha-crystallin A chain
L: Alpha-crystallin A chain
M: Alpha-crystallin A chain
N: Alpha-crystallin A chain
O: Alpha-crystallin A chain
P: Alpha-crystallin A chain


Theoretical massNumber of molelcules
Total (without water)318,98116
Polymers318,98116
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: cross-linking, microscopy, single particle EM (EMD-4894)
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area31980 Å2
ΔGint-148 kcal/mol
Surface area124490 Å2
MethodPISA

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Components

#1: Protein
Alpha-crystallin A chain / Heat shock protein beta-4 / HspB4


Mass: 19936.314 Da / Num. of mol.: 16
Source method: isolated from a genetically manipulated source
Details: wild type residues 1-166 / Source: (gene. exp.) Homo sapiens (human) / Tissue: lens / Gene: CRYAA, CRYA1, HSPB4 / Organ: eye / Plasmid: pET28 / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): CodonPlus-RIL / References: UniProt: P02489

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Reduced recombinant human alphaA-crystallin / Type: COMPLEX
Details: Recombinant wild type full-length human alphaA-crystallin purified in the presence of reductant.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.319 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human) / Cellular location: cytoplasm / Organ: eye / Tissue: lens
Source (recombinant)Organism: Escherichia coli BL21 (bacteria) / Plasmid: pET28
Buffer solutionpH: 7.4
Details: Buffer was prepared without EDTA and DTT. EDTA stock (500mM) was titrated to pH 8 and added to 1 mM. DTT stock (1M) was added to 1mM.
Buffer component
IDConc.NameFormulaBuffer-ID
1137 mMsodium chlorideNaCl1
22.7 mMpotassium chlorideKCl1
38.1 mMsodium phosphate dibasicNa2HPO41
41.76 mMpotassium phosphate monobasicKH2PO41
51 mMEthylenediaminetetraacetic acidC10H16N2O81
61 mM1,4-DithiothreitC4H10O2S21
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Specimen was thawed, diluted to the final concentration and equilibrated at 310K for 3h.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Chamber temperature: 293 K
Details: Diluted equilibrated specimen was added to glow-discharged (30s) grids. Sample was blotted 30s after sample application and immediately plunged.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 37000 X / Calibrated magnification: 37037 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.2 sec. / Electron dose: 30 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3
EM imaging opticsEnergyfilter slit width: 10 eV
Image scansMovie frames/image: 10 / Used frames/image: 1-10

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Processing

EM software
IDNameVersionCategory
1EMAN22particle selection
2TOMimage acquisition
4BsoftCTF correction
7Amber16model fitting
12IMAGIC53D reconstruction
13Amber16model refinement
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 74068 / Details: particles were picked manually
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 9.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19783 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Details: Structure created by homology modelling using PDB 3N3E and the N-terminal region (1-60) modelled using I-Tasser. The model was refined by flexible fitting into the EM density map EMD-4894. ...Details: Structure created by homology modelling using PDB 3N3E and the N-terminal region (1-60) modelled using I-Tasser. The model was refined by flexible fitting into the EM density map EMD-4894. The N-terminus is modelled only as CA atoms.
Atomic model buildingPDB-ID: 3N3E

3n3e


Accession code: 3N3E / Source name: PDB / Type: experimental model

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