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- EMDB-7437: Single-Particle reconstruction of DARP14 - A designed protein sca... -

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Basic information

Entry
Database: EMDB / ID: EMD-7437
TitleSingle-Particle reconstruction of DARP14 - A designed protein scaffold displaying ~17kDa DARPin proteins
Map dataSingle-Particle reconstruction of DARP14 - A designed protein scaffold displaying ~17kDa DARPin proteins
Sample
  • Complex: DARP14
    • Complex: DARP14 - Subunit A with DARPin
      • Protein or peptide: DARP14 - Subunit A with DARPin
    • Complex: DARP14 - Subunit B
      • Protein or peptide: DARP14 - Subunit B
KeywordsDesigned Complex / DARPin / Scaffold / Single-Particle Cryo-EM / DE NOVO PROTEIN
Function / homology5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate delta-isomerase activity / Tautomerase/MIF superfamily / catabolic process / 5-carboxymethyl-2-hydroxymuconate isomerase
Function and homology information
Biological speciessynthetic construct (others) / Pseudomonas aeruginosa PAO1 (bacteria) / synthetic (others) / Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.49 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 26, 2018-
Header (metadata) releaseFeb 21, 2018-
Map releaseMar 21, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing 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
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  • Surface view with fitted model
  • Atomic models: PDB-6c9k
  • Surface level: 0.0016
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7437.png

Downloads & links

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Map

FileDownload / File: emd_7437.map.gz / Format: CCP4 / Size: 190.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-Particle reconstruction of DARP14 - A designed protein scaffold displaying ~17kDa DARPin proteins
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.66 Å/pix.
x 368 pix.
= 241.04 Å		0.66 Å/pix.
x 368 pix.
= 241.04 Å		0.66 Å/pix.
x 368 pix.
= 241.04 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-0.009832131 - 0.025932336
Average (Standard dev.)0.00025868657 (±0.0013969232)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions368368368
Spacing368368368
CellA=B=C: 241.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6550.6550.655
M x/y/z368368368
origin x/y/z0.0000.0000.000
length x/y/z241.040241.040241.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS368368368
D min/max/mean-0.0100.0260.000

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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 with DARPin
      • Protein or peptide: DARP14 - Subunit A with DARPin
    • Complex: DARP14 - Subunit B
      • Protein or peptide: DARP14 - Subunit B

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Supramolecule #1: DARP14

SupramoleculeName: DARP14 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: synthetic construct (others)

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Supramolecule #2: DARP14 - Subunit A with DARPin

SupramoleculeName: DARP14 - Subunit A with DARPin / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: synthetic construct (others)

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Supramolecule #3: DARP14 - Subunit B

SupramoleculeName: DARP14 - Subunit B / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)

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Macromolecule #1: DARP14 - Subunit A with DARPin

MacromoleculeName: DARP14 - Subunit A with DARPin / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: synthetic (others)
Molecular weightTheoretical: 35.018965 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRITTKVGDK GSTRLFGGEE VWKDSPIIEA NGTLDELTSF IGEAKHYVDE EMKGILEEIQ NDIYKIMGEI GSKGKIEGIS EERIAWLLK LILRYMEMVN LKSFVLPGGT LESAKLDVCR TIARRALRKV LTVTREFGIG AEAAAYLLAL SDLLFLLARV I EIELGKKL ...String:
MRITTKVGDK GSTRLFGGEE VWKDSPIIEA NGTLDELTSF IGEAKHYVDE EMKGILEEIQ NDIYKIMGEI GSKGKIEGIS EERIAWLLK LILRYMEMVN LKSFVLPGGT LESAKLDVCR TIARRALRKV LTVTREFGIG AEAAAYLLAL SDLLFLLARV I EIELGKKL LEAARAGQDD EVRILMANGA DVNAHDDQGS TPLHLAAWIG HPEIVEVLLK HGADVNARDT DGWTPLHLAA DN GHLEIVE VLLKYGADVN AQDAYGLTPL HLAADRGHLE IVEVLLKHGA DVNAQDKFGK TAFDISIDNG NEDLAEILQK LN

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Macromolecule #2: DARP14 - Subunit B

MacromoleculeName: DARP14 - Subunit B / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Molecular weightTheoretical: 14.346274 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MPHLVIEATA NLRLETSPGE LLEQANKALF ASGQFGEADI KSRFVTLEAY RQGTAAVERA YLHACLSILD GRDIATRTLL GASLCAVLA EAVAGGGEEG VQVSVEVREM ERLSYAKRVV ARQRLEHHHH HH

UniProtKB: 5-carboxymethyl-2-hydroxymuconate isomerase

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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

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Resolution.type: BY AUTHOR / Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 183753
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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