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- PDB-6c9i: Single-Particle reconstruction of DARP14 - A designed protein sca... -

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Basic information

Entry
Database: PDB / ID: 6c9i
TitleSingle-Particle reconstruction of DARP14 - A designed protein scaffold displaying ~17kDa DARPin proteins - Scaffold
Components
  • DARP14 - Subunit A with DARPin
  • DARP14 - Subunit B
KeywordsDE NOVO PROTEIN / Designed Complex / DARPin / Scaffold / Single-Particle Cryo-EM
Function / homology
Function and homology information


5-carboxymethyl-2-hydroxymuconate delta-isomerase activity / catabolic process / transferase activity / ATP binding / metal ion binding
Similarity search - Function
5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate isomerase / Hypothetical Protein Ta1238; Chain: A; / Cobalamin adenosyltransferase-like / Cobalamin adenosyltransferase-like / Corrinoid adenosyltransferase, PduO-type / Cobalamin adenosyltransferase / Cobalamin adenosyltransferase-like superfamily / Macrophage Migration Inhibitory Factor / Macrophage Migration Inhibitory Factor ...5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate isomerase / Hypothetical Protein Ta1238; Chain: A; / Cobalamin adenosyltransferase-like / Cobalamin adenosyltransferase-like / Corrinoid adenosyltransferase, PduO-type / Cobalamin adenosyltransferase / Cobalamin adenosyltransferase-like superfamily / Macrophage Migration Inhibitory Factor / Macrophage Migration Inhibitory Factor / Tautomerase/MIF superfamily / Up-down Bundle / 2-Layer Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Cobalamin adenosyltransferase-like domain-containing protein / 5-carboxymethyl-2-hydroxymuconate isomerase
Similarity search - Component
Biological speciesPyrococcus horikoshii (archaea)
Pseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
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.
History
DepositionJan 26, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 21, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 11, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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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)390,90624
Polymers390,90624
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area62730 Å2
ΔGint-342 kcal/mol
Surface area112370 Å2

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Components

#1: Protein
DARP14 - Subunit A with DARPin


Mass: 18229.264 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (archaea)
Strain: ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3
Gene: PH0671 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O58404
#2: Protein
DARP14 - Subunit B


Mass: 14346.274 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
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) (bacteria) / References: UniProt: Q9I2D8

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1DARP14COMPLEXall0MULTIPLE SOURCES
2DARP14 - Subunit ACOMPLEX#11RECOMBINANT
3DARP14 - Subunit BCOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21synthetic construct (others)32630
32Pyrococcus horikoshii OT3 (archaea)70601
43Pseudomonas aeruginosa PAO1 (bacteria)208964
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Escherichia coli BL21(DE3) (bacteria)469008
32Escherichia coli BL21(DE3) (bacteria)469008
43Escherichia coli BL21(DE3) (bacteria)469008
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
MicroscopyModel: 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 (tetrahedral)
3D reconstructionResolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34650 / Symmetry type: POINT

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