[English] 日本語
Yorodumi- PDB-5k2f: Structure of NNQQNY from yeast prion Sup35 with cadmium acetate d... -
+Open data
-Basic information
Entry | Database: PDB / ID: 5k2f | ||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | Structure of NNQQNY from yeast prion Sup35 with cadmium acetate determined by MicroED | ||||||||||||||||||
Components | Eukaryotic peptide chain release factor GTP-binding subunit | ||||||||||||||||||
Keywords | PROTEIN FIBRIL / amyloid / yeast prion | ||||||||||||||||||
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 / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / ribosome binding ...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 / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / ribosome binding / translation / GTPase activity / mRNA binding / GTP binding / identical protein binding / cytosol / cytoplasm Similarity search - Function | ||||||||||||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||||||||
Method | ELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 1 Å | ||||||||||||||||||
Authors | Rodriguez, J.A. / Sawaya, M.R. / Cascio, D. / Eisenberg, D.S. | ||||||||||||||||||
Funding support | United States, 5items
| ||||||||||||||||||
Citation | Journal: 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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5k2f.cif.gz | 18.6 KB | Display | PDBx/mmCIF format |
---|---|---|---|---|
PDB format | pdb5k2f.ent.gz | 8.6 KB | Display | PDB format |
PDBx/mmJSON format | 5k2f.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5k2f_validation.pdf.gz | 324.5 KB | Display | wwPDB validaton report |
---|---|---|---|---|
Full document | 5k2f_full_validation.pdf.gz | 324 KB | Display | |
Data in XML | 5k2f_validation.xml.gz | 1.4 KB | Display | |
Data in CIF | 5k2f_validation.cif.gz | 1.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/k2/5k2f ftp://data.pdbj.org/pub/pdb/validation_reports/k2/5k2f | HTTPS FTP |
-Related structure data
Related structure data | 8197MC 8196C 8198C 8199C 5k2eC 5k2gC 5k2hC M: map data used to model this data C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
-Assembly
Deposited unit |
| ||||||||
---|---|---|---|---|---|---|---|---|---|
1 |
| ||||||||
Unit cell |
| ||||||||
Details | The biological unit is an extended pair of beta sheets comprising peptides at positions X,Y,Z and -X,Y+1/2,-Z extended ad infinitum along the b crystal axis. |
-Components
#1: Protein/peptide | Mass: 779.755 Da / Num. of mol.: 1 / Fragment: UNP residues 8-13 / Source method: obtained synthetically / Source: (synth.) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P05453 |
---|---|
#2: Chemical | ChemComp-CD / |
#3: Chemical | ChemComp-ACT / |
#4: Water | ChemComp-HOH / |
-Experimental details
-Experiment
Experiment | Method: ELECTRON CRYSTALLOGRAPHY |
---|---|
EM experiment | Aggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography |
-Sample preparation
Component | Name: Prion fibril composed of a 6-residue segment of Sup35 and cadmium Type: COMPLEX / Entity ID: #1 / Source: MULTIPLE SOURCES | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Molecular weight | Value: 3.25 kDa/nm / Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) | ||||||||||||||||||||
EM crystal formation | Instrument: 24-well plate Atmosphere: in air, in sealed chamber, in equilibrium with reservoir solution 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. Lipid mixture: none / Temperature: 298 K / Time: 1 DAY | ||||||||||||||||||||
Buffer solution | pH: 7 | ||||||||||||||||||||
Buffer component |
| ||||||||||||||||||||
Specimen | Conc.: 30 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: crystal | ||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Details: Plunged into liquid ethane (FEI VITROBOT MARK IV) | ||||||||||||||||||||
Crystal grow | Temperature: 273 K / Method: vapor diffusion, hanging drop / pH: 7 Details: 1.0 M sodium acetate, 0.1 M HEPES, pH 7.0, 0.01 M cadmium sulfate |
-Data collection
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company | ||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Microscopy | Model: FEI TECNAI F20 | ||||||||||||||||||||||||||||||||||||||||||
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM | ||||||||||||||||||||||||||||||||||||||||||
Electron lens | Mode: DIFFRACTION / Alignment procedure: BASIC | ||||||||||||||||||||||||||||||||||||||||||
Specimen holder | Cryogen: NITROGEN Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER Temperature (max): 100 K / Temperature (min): 100 K | ||||||||||||||||||||||||||||||||||||||||||
Image recording | Average exposure time: 2 sec. / Electron dose: 0.01 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Num. of diffraction images: 650 / Num. of grids imaged: 2 Details: The detector was operated in rolling shutter mode with 2x2 pixel binning. | ||||||||||||||||||||||||||||||||||||||||||
Image scans | Sampling size: 15.6 µm / Width: 4096 / Height: 4096 | ||||||||||||||||||||||||||||||||||||||||||
EM diffraction | Camera length: 1350 mm | ||||||||||||||||||||||||||||||||||||||||||
EM diffraction shell |
| ||||||||||||||||||||||||||||||||||||||||||
EM diffraction stats | Details: Phase statistics are not applicable. No imaging was used. The phases were obtained by a crystallographic direct methods program, SHELXD. Fourier space coverage: 82.7 % / High resolution: 1 Å / Num. of intensities measured: 16753 / Num. of structure factors: 2399 / Phase error: 0.1 ° / Phase residual: 0.1 ° / Phase error rejection criteria: 0 / Rmerge: 15.1 / Rsym: 15.1 | ||||||||||||||||||||||||||||||||||||||||||
Diffraction | Mean temperature: 100 K | ||||||||||||||||||||||||||||||||||||||||||
Diffraction source | Source: ELECTRON MICROSCOPE / Type: TECNAI F20 TEM / Wavelength: 0.0251 Å | ||||||||||||||||||||||||||||||||||||||||||
Detector | Type: TVIPS F416 CMOS CAMERA / Detector: CMOS / Date: Feb 3, 2016 | ||||||||||||||||||||||||||||||||||||||||||
Radiation wavelength | Wavelength: 0.0251 Å / Relative weight: 1 | ||||||||||||||||||||||||||||||||||||||||||
Reflection | Resolution: 1→90 Å / Num. obs: 2564 / % possible obs: 84.5 % / Redundancy: 4.6 % / Rmerge(I) obs: 0.18 / Χ2: 1.222 / Net I/av σ(I): 7.346 / Net I/σ(I): 5.1 / Num. measured all: 11810 | ||||||||||||||||||||||||||||||||||||||||||
Reflection shell | Diffraction-ID: 1 / Rejects: _
|
-Processing
Software |
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
EM software |
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
EM 3D crystal entity | ∠α: 90 ° / ∠β: 104.29 ° / ∠γ: 90 ° / A: 22.074 Å / B: 4.932 Å / C: 23.51 Å / Space group name: P1211 / Space group num: 4 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: NONE | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution method: DIFFRACTION PATTERN/LAYERLINES Details: The density map was obtained using measured diffraction intensities and phases acquired from crystallographic direct methods program SHELXD. Symmetry type: 3D CRYSTAL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 6.44 / Protocol: OTHER / Space: RECIPROCAL / Target criteria: maximum likelihood | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 1→22.79 Å / Cor.coef. Fo:Fc: 0.922 / Cor.coef. Fo:Fc free: 0.934 / SU B: 2.367 / SU ML: 0.048 / SU R Cruickshank DPI: 0.0475 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.047 / ESU R Free: 0.045 / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso max: 22.93 Å2 / Biso mean: 6.444 Å2 / Biso min: 3.46 Å2
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement step | Cycle: final / Resolution: 0.98→22.79 Å
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
LS refinement shell | Resolution: 0.978→1.004 Å / Total num. of bins used: 20
|