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- EMDB-8196: Structure of NNQQNY from yeast prion Sup35 with zinc acetate dete... -

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Basic information

Entry
Database: EMDB / ID: EMD-8196
TitleStructure of NNQQNY from yeast prion Sup35 with zinc acetate determined by MicroED
Map dataNNQQNY from yeast prion Sup35 with zinc acetate
Sample
  • Complex: Prion fibril composed of a 6-residue segment of Sup35 and zinc
    • Protein or peptide: Eukaryotic peptide chain release factor GTP-binding subunit
  • Ligand: ZINC ION
  • Ligand: ACETIC ACID
  • Ligand: water
Keywordsamyloid / yeast prion / PROTEIN FIBRIL
Function / homology
Function and homology information


Eukaryotic Translation Termination / translation release factor complex / translation release factor activity / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / translational termination / cytoplasmic stress granule / ribosome binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement ...Eukaryotic Translation Termination / translation release factor complex / translation release factor activity / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / translational termination / cytoplasmic stress granule / ribosome binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / translation / GTPase activity / mRNA binding / GTP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Eukaryotic peptide chain release factor GTP-binding subunit / GTP-eEF1A C-terminal domain-like / : / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain ...Eukaryotic peptide chain release factor GTP-binding subunit / GTP-eEF1A C-terminal domain-like / : / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Translation protein, beta-barrel domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Eukaryotic peptide chain release factor GTP-binding subunit
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron crystallography / cryo EM
AuthorsRodriguez JA / Sawaya MR
Funding support United States, 6 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)MCB-0445429 United States
National Institutes of Health/National Institute on Aging (NIH/NIA)1R01-AG029430 United States
Alzheimer's Disease Reasearch Center United States
Howard Hughes Medical Institute (HHMI) United States
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
Giannini Foundation United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2016
Title: Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED.
Authors: Michael R Sawaya / Jose Rodriguez / Duilio Cascio / Michael J Collazo / Dan Shi / Francis E Reyes / Johan Hattne / Tamir Gonen / David S Eisenberg /
Abstract: Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This ...Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.
History
DepositionMay 18, 2016-
Header (metadata) releaseSep 14, 2016-
Map releaseSep 14, 2016-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5k2e
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5k2e
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8196.png

Downloads & links

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Map

FileDownload / File: emd_8196.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNNQQNY from yeast prion Sup35 with zinc acetate
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesY (Sec.)X (Row.)Z (Col.)
0.31 Å/pix.
x 23 pix.
= 4.95 Å		0.34 Å/pix.
x 116 pix.
= 21.48 Å		0.33 Å/pix.
x 123 pix.
= 23.87 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 0.33563 Å / Y: 0.30938 Å / Z: 0.33153 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.4 / Movie #1: 1
Minimum - Maximum-1.1949614 - 5.9052873
Average (Standard dev.)-0.000060885202 (±0.3415682)
SymmetrySpace group: 4
Details

EMDB XML:

Map geometry
Axis orderZXY
Origin-51-72-3
Dimensions11612323
Spacing641672
CellA: 21.48 Å / B: 4.95 Å / C: 23.869999 Å
α: 90.0 ° / β: 103.952 ° / γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.3356250.3093750.33152777777778
M x/y/z641672
origin x/y/z0.0000.0000.000
length x/y/z21.4804.95023.870
α/β/γ90.000103.95290.000
start NX/NY/NZ-51-3-72
NX/NY/NZ11623123
MAP C/R/S312
start NC/NR/NS-72-51-3
NC/NR/NS12311623
D min/max/mean-1.1955.905-0.000

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Supplemental data

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Sample components

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Entire : Prion fibril composed of a 6-residue segment of Sup35 and zinc

EntireName: Prion fibril composed of a 6-residue segment of Sup35 and zinc
Components
  • Complex: Prion fibril composed of a 6-residue segment of Sup35 and zinc
    • Protein or peptide: Eukaryotic peptide chain release factor GTP-binding subunit
  • Ligand: ZINC ION
  • Ligand: ACETIC ACID
  • Ligand: water

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Supramolecule #1: Prion fibril composed of a 6-residue segment of Sup35 and zinc

SupramoleculeName: Prion fibril composed of a 6-residue segment of Sup35 and zinc
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 3.25 kDa/nm

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Macromolecule #1: Eukaryotic peptide chain release factor GTP-binding subunit

MacromoleculeName: Eukaryotic peptide chain release factor GTP-binding subunit
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 779.755 Da
SequenceString:
NNQQNY

UniProtKB: Eukaryotic peptide chain release factor GTP-binding subunit

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: ACETIC ACID

MacromoleculeName: ACETIC ACID / type: ligand / ID: 3 / Number of copies: 1 / Formula: ACY
Molecular weightTheoretical: 60.052 Da
Chemical component information

ChemComp-ACY:
ACETIC ACID

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state3D array

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Sample preparation

Concentration30 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
0.1 MC8H18N2O4SHEPES
1.0 MNaC2H3O2sodium acetate
0.01 MZnSO4zinc sulfate
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 30 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
Details: Plunged into liquid ethane (FEI VITROBOT MARK IV).
Detailscrystal
Crystal formationLipid mixture: none / Instrument: 24-well plate
Atmosphere: in air, in sealed chamber, in equilibrium with reservoir solution
Temperature: 298.0 K / Time: 1.0 DAY
Details: Crystals were grown by hanging-drop vapor diffusion at ~20 degrees C. The reservoir solution contained 100 mM HEPES, pH 7.0, and 1 M sodium acetate (pH not adjusted). Drops were prepared by ...Details: Crystals were grown by hanging-drop vapor diffusion at ~20 degrees C. The reservoir solution contained 100 mM HEPES, pH 7.0, and 1 M sodium acetate (pH not adjusted). Drops were prepared by pipetting 5 microliters of 30 mg/mL aqueous peptide solution, 4 microliters of reservoir solution, and 1 microliter of 0.1 M zinc sulfate onto a glass coverslip. Crystals grew within a day.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 100.0 K / Max: 100.0 K
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 2 / Number diffraction images: 879 / Average exposure time: 2.0 sec. / Average electron dose: 0.01 e/Å2
Details: The detector was operated in rolling shutter mode with 2x2 pixel binning.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 1350 mm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: SHELXD (ver. 2013/2) / Software - details: direct methods
Details: The density map was obtained using measured diffraction intensities and phases acquired from crystallographic direct methods program SHELXD.
Merging software listSoftware - Name: SCALEPACK (ver. 1.98.7)
Crystallography statisticsNumber intensities measured: 16753 / Number structure factors: 2399 / Fourier space coverage: 82.7 / R sym: 15.1 / R merge: 15.1 / Overall phase error: 0.1 / Overall phase residual: 0.1 / Phase error rejection criteria: 0 / High resolution: 1.0 Å
Details: Phase statistics are not applicable. No imaging was used. The phases were obtained by a crystallographic direct methods program, SHELXD.
Shell:
Shell IDHigh resolutionLow resolutionNumber structure factorsPhase residualFourier space coverageMultiplicity
11.71 Å90.0 Å5970.185.7999999999999975.3
21.36 Å1.71 Å5810.189.55.9
31.19 Å1.36 Å5330.187.55.0
41.08 Å1.19 Å5410.185.9000000000000064.8
51.0 Å1.08 Å4600.179.9000000000000063.0

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 4.4 / Target criteria: maximum likelihood
Output model

PDB-5k2e:
Structure of NNQQNY from yeast prion Sup35 with zinc acetate determined by MicroED

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