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- EMDB-15223: beta-2-microglobulin DeltaN6 amyloid fibril form 2PFb -

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Basic information

Entry
Database: EMDB / ID: EMD-15223
Titlebeta-2-microglobulin DeltaN6 amyloid fibril form 2PFb
Map dataCryoEM helical symmetrised map of b2m-DN6 Form 2PFb (sharpening b-factor of -37)
Sample
  • Complex: Amyloid fibril polymorph 2PFb of the beta-2-microglobulin deltaN6 variant.
    • Protein or peptide: Beta-2-microglobulin form pI 5.3
Function / homology
Function and homology information


positive regulation of ferrous iron binding / positive regulation of transferrin receptor binding / early endosome lumen / positive regulation of receptor binding / Nef mediated downregulation of MHC class I complex cell surface expression / DAP12 interactions / negative regulation of receptor binding / Endosomal/Vacuolar pathway / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / antigen processing and presentation of exogenous protein antigen via MHC class Ib, TAP-dependent ...positive regulation of ferrous iron binding / positive regulation of transferrin receptor binding / early endosome lumen / positive regulation of receptor binding / Nef mediated downregulation of MHC class I complex cell surface expression / DAP12 interactions / negative regulation of receptor binding / Endosomal/Vacuolar pathway / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / antigen processing and presentation of exogenous protein antigen via MHC class Ib, TAP-dependent / cellular response to iron(III) ion / negative regulation of forebrain neuron differentiation / ER to Golgi transport vesicle membrane / response to molecule of bacterial origin / regulation of erythrocyte differentiation / regulation of iron ion transport / MHC class I peptide loading complex / HFE-transferrin receptor complex / T cell mediated cytotoxicity / cellular response to iron ion / antigen processing and presentation of endogenous peptide antigen via MHC class I / positive regulation of T cell cytokine production / MHC class I protein complex / multicellular organismal-level iron ion homeostasis / positive regulation of T cell mediated cytotoxicity / peptide antigen assembly with MHC class II protein complex / negative regulation of neurogenesis / MHC class II protein complex / positive regulation of receptor-mediated endocytosis / cellular response to nicotine / recycling endosome membrane / phagocytic vesicle membrane / specific granule lumen / peptide antigen binding / positive regulation of cellular senescence / antigen processing and presentation of exogenous peptide antigen via MHC class II / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / Interferon gamma signaling / positive regulation of immune response / negative regulation of epithelial cell proliferation / Modulation by Mtb of host immune system / positive regulation of T cell activation / sensory perception of smell / negative regulation of neuron projection development / tertiary granule lumen / DAP12 signaling / MHC class II protein complex binding / late endosome membrane / T cell differentiation in thymus / positive regulation of protein binding / ER-Phagosome pathway / iron ion transport / protein refolding / early endosome membrane / protein homotetramerization / intracellular iron ion homeostasis / amyloid fibril formation / learning or memory / Amyloid fiber formation / lysosomal membrane / endoplasmic reticulum lumen / external side of plasma membrane / Golgi membrane / focal adhesion / Neutrophil degranulation / SARS-CoV-2 activates/modulates innate and adaptive immune responses / Golgi apparatus / protein homodimerization activity / extracellular space / extracellular exosome / extracellular region / membrane / identical protein binding / plasma membrane / cytosol
Similarity search - Function
Beta-2-Microglobulin / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Beta-2-microglobulin
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodhelical reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsWilkinson M / Gallardo R / Radford SE / Ranson NA
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom) United Kingdom
Wellcome Trust United Kingdom
Royal Society United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: Disease-relevant β-microglobulin variants share a common amyloid fold.
Authors: Martin Wilkinson / Rodrigo U Gallardo / Roberto Maya Martinez / Nicolas Guthertz / Masatomo So / Liam D Aubrey / Sheena E Radford / Neil A Ranson /
Abstract: β-microglobulin (βm) and its truncated variant ΔΝ6 are co-deposited in amyloid fibrils in the joints, causing the disorder dialysis-related amyloidosis (DRA). Point mutations of βm result in ...β-microglobulin (βm) and its truncated variant ΔΝ6 are co-deposited in amyloid fibrils in the joints, causing the disorder dialysis-related amyloidosis (DRA). Point mutations of βm result in diseases with distinct pathologies. βm-D76N causes a rare systemic amyloidosis with protein deposited in the viscera in the absence of renal failure, whilst βm-V27M is associated with renal failure, with amyloid deposits forming predominantly in the tongue. Here we use cryoEM to determine the structures of fibrils formed from these variants under identical conditions in vitro. We show that each fibril sample is polymorphic, with diversity arising from a 'lego-like' assembly of a common amyloid building block. These results suggest a 'many sequences, one amyloid fold' paradigm in contrast with the recently reported 'one sequence, many amyloid folds' behaviour of intrinsically disordered proteins such as tau and Aβ.
History
DepositionJun 21, 2022-
Header (metadata) releaseMar 22, 2023-
Map releaseMar 22, 2023-
UpdateMar 22, 2023-
Current statusMar 22, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15223.png

Downloads & links

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Map

FileDownload / File: emd_15223.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM helical symmetrised map of b2m-DN6 Form 2PFb (sharpening b-factor of -37)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.006
Minimum - Maximum-0.012230094 - 0.027232416
Average (Standard dev.)0.000107659864 (±0.0012243333)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 332.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: halfmap2

Fileemd_15223_half_map_1.map
Annotationhalfmap2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap1

Fileemd_15223_half_map_2.map
Annotationhalfmap1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Amyloid fibril polymorph 2PFb of the beta-2-microglobulin deltaN6...

EntireName: Amyloid fibril polymorph 2PFb of the beta-2-microglobulin deltaN6 variant.
Components
  • Complex: Amyloid fibril polymorph 2PFb of the beta-2-microglobulin deltaN6 variant.
    • Protein or peptide: Beta-2-microglobulin form pI 5.3

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Supramolecule #1: Amyloid fibril polymorph 2PFb of the beta-2-microglobulin deltaN6...

SupramoleculeName: Amyloid fibril polymorph 2PFb of the beta-2-microglobulin deltaN6 variant.
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Details: Recombinantly expressed and fibrillated in vitro at pH 6.2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Beta-2-microglobulin form pI 5.3

MacromoleculeName: Beta-2-microglobulin form pI 5.3 / type: protein_or_peptide / ID: 1
Details: Natural variant deltaN6 with additional methionine added prior to peptide sequence for bacterial expression
Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.153478 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
MIQVYSRHPA ENGKSNFLNC YVSGFHPSDI EVDLLKNGER IEKVEHSDLS FSKDWSFYLL YYTEFTPTEK DEYACRVNHV TLSQPKIVK WDRDM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 6.2
Component:
ConcentrationFormulaName
115.0 mMNaClSodium chloridesodium chloride
25.0 mMsodium phosphate
GridModel: EMS Lacey Carbon / Material: COPPER / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec.
Details: The grid was plasma cleaned prior to 2x application of graphene oxide-DDM mixture, then grid was used immediately for sample application and vitrification
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 6s blot.
DetailsFibrillation conditions: 20 uM monomeric b2m-DN6 at 37C with shaking at 600 rpm for 2-3 weeks

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 4095 / Average exposure time: 7.0 sec. / Average electron dose: 43.0 e/Å2
Details: 1687 raw EER frames were collected per image and combined into 40 fractions for processing
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 612949 / Software - Name: crYOLO
Details: Manually picked a subset of images to train a model for automatic fibril segment picking in crYOLO
Startup modelType of model: INSILICO MODEL
Details: Model generated from 2D class averages using relion_helix_inimodel2d
Final angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 4.0)
Final reconstructionApplied symmetry - Helical parameters - Δz: 4.86 Å
Applied symmetry - Helical parameters - Δ&Phi: -0.6 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 10594
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 81
Output model

PDB-8a7p:
beta-2-microglobulin DeltaN6 amyloid fibril form 2PFb

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