[English] 日本語
Yorodumi
- PDB-6c9k: Single-Particle reconstruction of DARP14 - A designed protein sca... -

+
Open data


ID or keywords:

Loading...

-
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 / Tautomerase/MIF superfamily / 5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate delta-isomerase activity / aromatic compound catabolic process / Uncharacterized protein
Function and homology information
Biological speciessynthetic (others)
Pseudomonas aeruginosa (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.49 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.
Validation Report
SummaryFull reportAbout validation report
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

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-7437
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
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
Polymers592,38324
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area65260 Å2
ΔGint-343 kcal/mol
Surface area195920 Å2
MethodPISA

-
Components

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


Mass: 35018.965 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein/peptide
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

-
Experimental details

-
Experiment

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

-
Sample preparation

Component

Type: COMPLEX

IDNameEntity IDParent-IDSource
1DARP141,20MULTIPLE SOURCES
2DARP14 - Subunit A with DARPin11RECOMBINANT
3DARP14 - Subunit B21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21synthetic construct (others)32630
32synthetic construct (others)32630
43Pseudomonas aeruginosa PAO1 (bacteria)208964
Source (recombinant)

Ncbi tax-ID: 469008 / Organism: Escherichia coli BL21(DE3) (bacteria)

IDEntity assembly-ID
21
32
43
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

-
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 FIELDBright-field microscopy
Image recordingElectron dose: 30 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

-
Processing

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

+
About Yorodumi

-
News

-
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force. (see PDBe EMDB page)
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.: Q: What is "EMD"? / ID/Accession-code notation in Yorodumi/EM Navigator

External links: EMDB at PDBe / Contact to PDBj

-
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary. This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated. See below links for details.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software). Now, EM Navigator and Yorodumi are based on the updated data.

External links: wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

+
Jun 16, 2017. Omokage search with filter

Omokage search with filter

  • Result of Omokage search can be filtered by keywords and the database types

Related info.: Omokage search

+
Sep 15, 2016. EM Navigator & Yorodumi renewed

EM Navigator & Yorodumi renewed

  • New versions of EM Navigator and Yorodumi started

Related info.: Changes in new EM Navigator and Yorodumi

+
Aug 31, 2016. New EM Navigator & Yorodumi

New EM Navigator & Yorodumi

  • In 15th Sep 2016, the development versions of EM Navigator and Yorodumi will replace the official versions.
  • Current version will continue as 'legacy version' for some time.

Related info.: Changes in new EM Navigator and Yorodumi / EM Navigator / Yorodumi

Read more

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.

Related info.: EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more