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- EMDB-8199: Structure of GNNQQNY from yeast prion Sup35 in space group P21212... -

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Basic information

Entry
Database: EMDB / ID: EMD-8199
TitleStructure of GNNQQNY from yeast prion Sup35 in space group P212121 determined by MicroED
Map dataGNNQQNY from yeast prion Sup35
Sample
  • Complex: Prion fibril composed of a 7-residue segment of Sup35
    • Protein or peptide: Eukaryotic peptide chain release factor GTP-binding subunit
  • 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 / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / cytoplasmic stress granule / translation ...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 / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / cytoplasmic stress granule / translation / mRNA binding / GTPase activity / GTP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Eukaryotic peptide chain release factor GTP-binding subunit / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / 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 / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / 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: 0.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8199.png

Downloads & links

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Map

FileDownload / File: emd_8199.map.gz / Format: CCP4 / Size: 2.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGNNQQNY from yeast prion Sup35
Voxel sizeX: 0.34059 Å / Y: 0.30813 Å / Z: 0.33758 Å
Density
Contour LevelBy EMDB: 0.4 / Movie #1: 0.8
Minimum - Maximum-0.67714727 - 1.4965872
Average (Standard dev.)0.0015150023 (±0.23481336)
SymmetrySpace group: 19
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-54-3-120
Dimensions12323205
Spacing6816120
CellA: 23.16 Å / B: 4.93 Å / C: 40.51 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.340588235294120.3081250.33758333333333
M x/y/z6816120
origin x/y/z0.0000.0000.000
length x/y/z23.1604.93040.510
α/β/γ90.00090.00090.000
start NX/NY/NZ-54-3-120
NX/NY/NZ12323205
MAP C/R/S213
start NC/NR/NS-3-54-120
NC/NR/NS23123205
D min/max/mean-0.6771.4970.002

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Supplemental data

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Sample components

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Entire : Prion fibril composed of a 7-residue segment of Sup35

EntireName: Prion fibril composed of a 7-residue segment of Sup35
Components
  • Complex: Prion fibril composed of a 7-residue segment of Sup35
    • Protein or peptide: Eukaryotic peptide chain release factor GTP-binding subunit
  • Ligand: water

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Supramolecule #1: Prion fibril composed of a 7-residue segment of Sup35

SupramoleculeName: Prion fibril composed of a 7-residue segment of Sup35 / 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: 836.807 Da
SequenceString:
GNNQQNY

UniProtKB: Eukaryotic peptide chain release factor GTP-binding subunit

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / 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

Concentration10 mg/mL
BufferpH: 7 / Details: water
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: Grown in batch at ~20 degrees C in a microcentrifuge tube. Crystals grew within a day after seeding with NNQQNY-Zn crystals.

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Electron microscopy

MicroscopeFEI TECNAI F20
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
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: 127 / 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.
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

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 Å4270.167.53.5
21.36 Å1.71 Å4410.174.7000000000000033.8
31.19 Å1.36 Å3960.173.2000000000000033.6
41.05 Å1.19 Å5720.175.0999999999999943.9
51.05 Å90.0 Å18360.172.7000000000000033.7
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)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 4.7 / Target criteria: maximum likelihood
Output model

PDB-5k2h:
Structure of GNNQQNY from yeast prion Sup35 in space group P212121 determined by MicroED

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