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- EMDB-4896: Single particle cryo-EM reconstruction of a 20-mer assembly of re... -

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Basic information

Entry
Database: EMDB / ID: EMD-4896
TitleSingle particle cryo-EM reconstruction of a 20-mer assembly of reduced recombinant human alphaA-crystallin.
Map dataSingle particle cryo-EM reconstruction of a 20-mer assembly of reduced recombinant human alphaA-crystallin.
Sample
  • Complex: Reduced recombinant human alphaA-crystallin
    • Protein or peptide: alphaA-crystallin
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 Å
AuthorsPeters C / Kaiser CJO / Buchner J / Weinkauf S
Funding support Germany, 2 items
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
DepositionApr 24, 2019-
Header (metadata) releaseDec 11, 2019-
Map releaseDec 11, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0119
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0119
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4896.png

Downloads & links

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Map

FileDownload / File: emd_4896.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle particle cryo-EM reconstruction of a 20-mer assembly of reduced recombinant human alphaA-crystallin.
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.0119 / Movie #1: 0.0119
Minimum - Maximum-0.00000001640 - 0.124002606
Average (Standard dev.)0.0041572717 (±0.014406236)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 172.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z172.800172.800172.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0000.1240.004

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Supplemental data

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Sample components

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Entire : Reduced recombinant human alphaA-crystallin

EntireName: Reduced recombinant human alphaA-crystallin
Components
  • Complex: Reduced recombinant human alphaA-crystallin
    • Protein or peptide: alphaA-crystallin

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Supramolecule #1: Reduced recombinant human alphaA-crystallin

SupramoleculeName: Reduced recombinant human alphaA-crystallin / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Recombinant wild type full-length human alphaA-crystallin purified in the presence of reductant.
Source (natural)Organism: Homo sapiens (human) / Organ: eye / Tissue: lens / Location in cell: cytoplasm
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant plasmid: pET28
Molecular weightTheoretical: 398 KDa

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Macromolecule #1: alphaA-crystallin

MacromoleculeName: alphaA-crystallin / type: protein_or_peptide / ID: 1 / Details: Wild type full sequence / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human) / Organ: eye / Tissue: lens
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MDVTIQHPWF KRTLGPFYPS RLFDQFFGEG LFEYDLLPFL SSTISPYYRQ SLFRTVLDSG ISEVRSDRD KFVIFLDVKH FSPEDLTVKV QDDFVEIHGK HNERQDDHGY ISREFHRRYR L PSNVDQSA LSCSLSADGM LTFCGPKIQT GLDATHAERA IPVSREEKPT SAPSS

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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

Concentration0.3 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
137.0 mMNaClSodium chloridesodium chloride
2.7 mMKClpotassium chloride
8.1 mMNa2HPO4sodium phosphate dibasic
1.76 mMKH2PO4potassium phosphate monobasic
1.0 mMC10H16N2O8Ethylenediaminetetraacetic acid
1.0 mMC4H10O2S21,4-Dithiothreit

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.
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.007 kPa
VitrificationCryogen name: ETHANE / Chamber temperature: 293 K / Instrument: HOMEMADE PLUNGER
Details: Diluted equilibrated specimen was added to glow-discharged (30s) grids. Sample was blotted 30s after sample application and immediately plunged..
DetailsSpecimen was thawed, diluted to the final concentration and equilibrated at 310K for 3h.

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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 / Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 37000
Specialist opticsEnergy filter - Slit width: 10 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-10 / Number grids imaged: 3 / Average exposure time: 3.2 sec. / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 74068 / Details: particles were picked manually
CTF correctionSoftware - Name: Bsoft
Startup modelType of model: OTHER
Details: simple artificial model of symmetrically arranged pillars
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: IMAGIC (ver. 5)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: IMAGIC (ver. 5)
Final reconstructionApplied symmetry - Point group: D5 (2x5 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: IMAGIC (ver. 5) / Number images used: 14336
FSC plot (resolution estimation)

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