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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: EMD-7403
TitleSingle-particle reconstruction of DARP14 - A designed protein scaffold displaying ~17kDa DARPin proteins - Helical extension
Map data
Sample
  • Complex: DARP14
    • Complex: DARP14 - Subunit A
    • Complex: DARP14 - Subunit B
    • Complex: DARP14 - DARPin
Function / homology
Function and homology information


5-carboxymethyl-2-hydroxymuconate delta-isomerase activity / corrinoid adenosyltransferase activity / catabolic process / ATP binding / metal ion binding
Similarity search - Function
5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate isomerase / Cobalamin adenosyltransferase-like / Corrinoid adenosyltransferase, PduO-type / Cobalamin adenosyltransferase / Cobalamin adenosyltransferase-like superfamily / Tautomerase/MIF superfamily
Similarity search - Domain/homology
Cobalamin adenosyltransferase-like domain-containing protein / 5-carboxymethyl-2-hydroxymuconate isomerase
Similarity search - Component
Biological speciessynthetic construct (others) / Pyrococcus horikoshii OT3 (archaea) / Pseudomonas aeruginosa PAO1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.03 Å
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.
History
DepositionJan 24, 2018-
Header (metadata) releaseFeb 28, 2018-
Map releaseMar 21, 2018-
UpdateApr 11, 2018-
Current statusApr 11, 2018Processing site: RCSB / Status: Released

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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
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7403.png

Downloads & links

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Map

FileDownload / File: emd_7403.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.655 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0016 / Movie #1: 0.0016
Minimum - Maximum-0.0024018863 - 0.010321523
Average (Standard dev.)0.00018426872 (±0.00093877496)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 209.59999 Å
α=β=γ: 90.0 °

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
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
Components
  • Complex: DARP14
    • Complex: DARP14 - Subunit A
    • Complex: DARP14 - Subunit B
    • Complex: DARP14 - DARPin

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Supramolecule #1: DARP14

SupramoleculeName: DARP14 / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #2: DARP14 - Subunit A

SupramoleculeName: DARP14 - Subunit A / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Pyrococcus horikoshii OT3 (archaea)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #3: DARP14 - Subunit B

SupramoleculeName: DARP14 - Subunit B / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Supramolecule #4: DARP14 - DARPin

SupramoleculeName: DARP14 - DARPin / type: complex / ID: 4 / Parent: 1
Source (natural)Organism: synthetic construct (others)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Resolution.type: BY AUTHOR / Resolution: 4.03 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 18385
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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