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- PDB-6c9k: Single-Particle reconstruction of DARP14 - A designed protein sca... -

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Basic information

Entry
Database: PDB / ID: 6c9k
TitleSingle-Particle reconstruction of DARP14 - A designed protein scaffold displaying ~17kDa DARPin proteins
Components
  • DARP14 - Subunit A with DARPin
  • DARP14 - Subunit B
KeywordsDE NOVO PROTEIN / Designed Complex / DARPin / Scaffold / Single-Particle Cryo-EM
Function/homology5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate delta-isomerase activity / 5-carboxymethyl-2-hydroxymuconate isomerase / Tautomerase/MIF superfamily / aromatic compound catabolic process / Uncharacterized protein
Function and homology information
Specimen sourceSynthetic
Pseudomonas aeruginosa / bacteria /
MethodElectron microscopy (3.49 Å resolution / Particle / Single particle) / Transmission electron microscopy
AuthorsGonen, S. / Liu, Y. / Yeates, T.O. / 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jan 26, 2018 / Release: Mar 21, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Mar 21, 2018Structure modelrepositoryInitial release
1.1Apr 11, 2018Structure modelData collection / Database referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: DARP14 - Subunit A with DARPin
B: DARP14 - Subunit A with DARPin
C: DARP14 - Subunit A with DARPin
D: DARP14 - Subunit A with DARPin
E: DARP14 - Subunit B
F: DARP14 - Subunit B
G: DARP14 - Subunit B
H: DARP14 - Subunit B
I: DARP14 - Subunit A with DARPin
J: DARP14 - Subunit A with DARPin
K: DARP14 - Subunit A with DARPin
L: DARP14 - Subunit A with DARPin
M: DARP14 - Subunit B
N: DARP14 - Subunit B
O: DARP14 - Subunit B
P: DARP14 - Subunit B
Q: DARP14 - Subunit A with DARPin
R: DARP14 - Subunit A with DARPin
S: DARP14 - Subunit A with DARPin
T: DARP14 - Subunit A with DARPin
U: DARP14 - Subunit B
V: DARP14 - Subunit B
W: DARP14 - Subunit B
X: DARP14 - Subunit B


Theoretical massNumber of molelcules
Total (without water)592,38324
Polyers592,38324
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)65260
ΔGint (kcal/M)-343
Surface area (Å2)195920
MethodPISA

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Components

#1: Protein/peptide
DARP14 - Subunit A with DARPin


Mass: 35018.965 Da / Num. of mol.: 12 / Source: (gene. exp.) Synthetic / Production host: Escherichia coli BL21(DE3)
#2: Protein/peptide
DARP14 - Subunit B


Mass: 14346.274 Da / Num. of mol.: 12
Source: (gene. exp.) 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
Gene: PA1966 / Production host: Escherichia coli BL21(DE3) / References: UniProt:Q9I2D8

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1DARP14COMPLEX1,20MULTIPLE SOURCES
2DARP14 - Subunit A with DARPinCOMPLEX11RECOMBINANT
3DARP14 - Subunit BCOMPLEX21RECOMBINANT
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
2132630synthetic construct
3232630synthetic construct
43208964Pseudomonas aeruginosa PAO1
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
21469008Escherichia coli BL21(DE3)
32469008Escherichia coli BL21(DE3)
43469008Escherichia coli BL21(DE3)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: T
3D reconstructionResolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 183753 / Symmetry type: POINT

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