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- EMDB-2946: cryo-EM structure of HET-s prion infectious form -

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Basic information

Entry
Database: EMDB / ID: EMD-2946
Titlecryo-EM structure of HET-s prion infectious form
Map dataReconstruction of HET-s prion amyloid assembled at pH3
Sample
  • Sample: HET-s prion domain (218-289) assembled at pH3
  • Protein or peptide: Heterokaryon incompatibility protein s
Keywordsamyloid / prion / cross-beta structure
Function / homologyHet-s prion-forming domain / Het-s prion-forming domain / Prion-inhibition and propagation, HeLo domain / HeLo domain superfamily / Het-s 218-289 / Prion-inhibition and propagation / identical protein binding / cytoplasm / Heterokaryon incompatibility protein s
Function and homology information
Biological speciesPodospora anserina (fungus)
Methodhelical reconstruction / cryo EM / Resolution: 8.5 Å
AuthorsMizuno N / Baxa U / Steven AC
CitationJournal: Proc Natl Acad Sci U S A / Year: 2011
Title: Structural dependence of HET-s amyloid fibril infectivity assessed by cryoelectron microscopy.
Authors: Naoko Mizuno / Ulrich Baxa / Alasdair C Steven /
Abstract: HET-s is a prion protein of the fungus Podospora anserina which, in the prion state, is active in a self/nonself recognition process called heterokaryon incompatibility. Its prionogenic properties ...HET-s is a prion protein of the fungus Podospora anserina which, in the prion state, is active in a self/nonself recognition process called heterokaryon incompatibility. Its prionogenic properties reside in the C-terminal "prion domain." The HET-s prion domain polymerizes in vitro into amyloid fibrils whose properties depend on the pH of assembly; above pH 3, infectious singlet fibrils are produced, and below pH 3, noninfectious triplet fibrils. To investigate the correlation between structure and infectivity, we performed cryo-EM analyses. Singlet fibrils have a helical pitch of approximately 410 Å and a left-handed twist. Triplet fibrils have three protofibrils whose lateral dimensions (36 × 25 Å) and axial packing (one subunit per 9.4 Å) match those of singlets but differ in their supercoiling. At 8.5-Å resolution, the cross-section of the singlet fibril reconstruction is largely consistent with that of a β-solenoid model previously determined by solid-state NMR. Reconstructions of the triplet fibrils show three protofibrils coiling around a common axis and packed less tightly at pH 3 than at pH 2, eventually peeling off. Taken together with the earlier observation that fragmentation of triplet fibrils by sonication does not increase infectivity, these observations suggest a novel mechanism for self-propagation, whereby daughter fibrils nucleate on the lateral surface of singlet fibrils. In triplets, this surface is occluded, blocking nucleation and thereby explaining their lack of infectivity.
History
DepositionMar 23, 2015-
Header (metadata) releaseApr 15, 2015-
Map releaseApr 15, 2015-
UpdateApr 15, 2015-
Current statusApr 15, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0073
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0073
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

2946-HET-s.tif

Downloads & links

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Map

FileDownload / File: emd_2946.map.gz / Format: CCP4 / Size: 708 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of HET-s prion amyloid assembled at pH3
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.84 Å/pix.
x 113 pix.
= 207.92 Å		1.84 Å/pix.
x 41 pix.
= 75.44 Å		1.84 Å/pix.
x 40 pix.
= 73.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0073 / Movie #1: 0.0073
Minimum - Maximum0.0 - 0.02645633
Average (Standard dev.)0.00368884 (±0.00566753)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin1010-1
Dimensions4140113
Spacing4140113
CellA: 73.6 Å / B: 75.44 Å / C: 207.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.841.841.84
M x/y/z4041113
origin x/y/z0.0000.0000.000
length x/y/z73.60075.440207.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-108-108-54
NX/NY/NZ10810854
MAP C/R/S123
start NC/NR/NS1010-1
NC/NR/NS4041113
D min/max/mean0.0000.0260.004

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Supplemental data

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Sample components

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Entire : HET-s prion domain (218-289) assembled at pH3

EntireName: HET-s prion domain (218-289) assembled at pH3
Components
  • Sample: HET-s prion domain (218-289) assembled at pH3
  • Protein or peptide: Heterokaryon incompatibility protein s

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Supramolecule #1000: HET-s prion domain (218-289) assembled at pH3

SupramoleculeName: HET-s prion domain (218-289) assembled at pH3 / type: sample / ID: 1000
Oligomeric state: helical polymer with a rise of 9.4 A per protein unit
Number unique components: 1
Molecular weightExperimental: 8 KDa

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Macromolecule #1: Heterokaryon incompatibility protein s

MacromoleculeName: Heterokaryon incompatibility protein s / type: protein_or_peptide / ID: 1 / Name.synonym: HET-s
Details: HET-s proteins are assembled to make a single strand helix, with a rise of 9.4 A per protein.
Oligomeric state: Helical filament / Recombinant expression: Yes
Source (natural)Organism: Podospora anserina (fungus)
Molecular weightExperimental: 8 KDa / Theoretical: 8 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: pLysS
SequenceUniProtKB: Heterokaryon incompatibility protein s / InterPro: Het-s prion-forming domain

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration1 mg/mL
BufferpH: 3 / Details: 20 mM citrate acid
GridDetails: Glow discharged Quantifoil R2/2 was used.
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK II / Method: Blot for 5 seconds before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
TemperatureMin: 90 K / Max: 100 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
DateApr 1, 2008
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 100 / Average electron dose: 20 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 38000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsThe reconstruction was performed using SPIDER in combination with IHRSR.
Final reconstructionApplied symmetry - Helical parameters - Δz: 18.8 Å
Applied symmetry - Helical parameters - Δ&Phi: 17.1 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: OTHER / Software - Name: SPIder, EMAN, IHRSR, bsoft
CTF correctionDetails: Each micrograph

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