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- EMDB-7403: Single-particle reconstruction of DARP14 - A designed protein sca... -

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Basic information

Entry
Database: EMDB / ID: 7403
TitleSingle-particle reconstruction of DARP14 - A designed protein scaffold displaying ~17kDa DARPin proteins - Helical extension
Map data
SampleDARP14
Function/homology5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate delta-isomerase activity / 5-carboxymethyl-2-hydroxymuconate isomerase / Cobalamin adenosyltransferase-like / ATP:cob(I)alamin adenosyltransferase, PduO-type / Cobalamin adenosyltransferase-like superfamily / Cobalamin adenosyltransferase / Tautomerase/MIF superfamily / aromatic compound catabolic process / | ...5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate delta-isomerase activity / 5-carboxymethyl-2-hydroxymuconate isomerase / Cobalamin adenosyltransferase-like / ATP:cob(I)alamin adenosyltransferase, PduO-type / Cobalamin adenosyltransferase-like superfamily / Cobalamin adenosyltransferase / Tautomerase/MIF superfamily / aromatic compound catabolic process / | / Uncharacterized protein / Uncharacterized protein
Function and homology information
SourceSynthetic construct
Pyrococcus horikoshii ot3 / archaea / thermophilic /
Pseudomonas aeruginosa pao1 / bacteria /
Methodsingle particle reconstruction, at 4.03 Å resolution
AuthorsGonen S / Liu Y / Yeates TO / Gonen T
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2018
Title: Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system.
Authors: Yuxi Liu / Shane Gonen / Tamir Gonen / Todd O Yeates
Abstract: Current single-particle cryo-electron microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal ...Current single-particle cryo-electron microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size suitable for cryo-EM. Using a rigid continuous alpha helical linker, we connect a small 17-kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry. We show that the resulting construct is amenable to structural analysis by single-particle cryo-EM, allowing us to identify and solve the structure of the attached small protein at near-atomic detail, ranging from 3.5- to 5-Å resolution. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Furthermore, because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other protein or nucleic acid molecules, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryo-EM to proteins of typical size in the cell.
DateDeposition: Jan 24, 2018 / Header (metadata) release: Feb 28, 2018 / Map release: Mar 21, 2018 / Last update: Apr 11, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0016
  • Imaged by UCSF CHIMERA
  • Download
  • Surface view colored by radius
  • Surface level: 0.0016
  • Imaged by UCSF CHIMERA
  • Download
3D viewer
Supplemental images

Downloads & links

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Map

Fileemd_7403.map.gz (map file in CCP4 format, 131073 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
320 pix
0.66 Å/pix.
= 209.6 Å
320 pix
0.66 Å/pix.
= 209.6 Å
320 pix
0.66 Å/pix.
= 209.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider package.

Voxel sizeX=Y=Z: 0.655 Å
Density
Contour Level:0.0016 (by author), 0.0016 (movie #1):
Minimum - Maximum-0.0024018863 - 0.010321523
Average (Standard dev.)0.00018426872 (0.00093877496)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions320320320
Origin000
Limit319319319
Spacing320320320
CellA=B=C: 209.59999 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6550.6550.655
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z209.600209.600209.600
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0020.0100.000

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Supplemental data

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Sample components

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Entire DARP14

EntireName: DARP14 / Number of components: 4

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Component #1: protein, DARP14

ProteinName: DARP14 / Recombinant expression: No
SourceSpecies: Synthetic construct
Source (engineered)Expression System: Escherichia coli bl21(de3) / bacteria / / image: Escherichia coli

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Component #2: protein, DARP14 - Subunit A

ProteinName: DARP14 - Subunit A / Recombinant expression: No
SourceSpecies: Pyrococcus horikoshii ot3 / archaea / thermophilic /
Source (engineered)Expression System: Escherichia coli bl21(de3) / bacteria / / image: Escherichia coli

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Component #3: protein, DARP14 - Subunit B

ProteinName: DARP14 - Subunit B / Recombinant expression: No
SourceSpecies: Pseudomonas aeruginosa pao1 / bacteria /
Source (engineered)Expression System: Escherichia coli bl21(de3) / bacteria / / image: Escherichia coli

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Component #4: protein, DARP14 - DARPin

ProteinName: DARP14 - DARPin / Recombinant expression: No
SourceSpecies: Synthetic construct
Source (engineered)Expression System: Escherichia coli bl21(de3) / bacteria / / image: Escherichia coli

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Experimental details

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Sample preparation

Specimen stateparticle
Sample solutionpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 3 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: T (tetrahedral) / Number of projections: 18385
3D reconstructionSoftware: RELION / Resolution: 4.03 Å / Resolution method: FSC 0.143 CUT-OFF

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