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Yorodumi- EMDB-7403: Single-particle reconstruction of DARP14 - A designed protein sca... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-7403 | |||||||||
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Title | Single-particle reconstruction of DARP14 - A designed protein scaffold displaying ~17kDa DARPin proteins - Helical extension | |||||||||
Map data | ||||||||||
Sample |
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Function / homology | Function and homology information 5-carboxymethyl-2-hydroxymuconate delta-isomerase activity / corrinoid adenosyltransferase activity / : / ATP binding / metal ion binding Similarity search - Function | |||||||||
Biological species | synthetic construct (others) / Pyrococcus horikoshii OT3 (archaea) / Pseudomonas aeruginosa PAO1 (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.03 Å | |||||||||
Authors | Gonen S / Liu Y / Yeates TO / Gonen T | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_7403.map.gz | 98.5 MB | EMDB map data format | |
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Header (meta data) | emd-7403-v30.xml emd-7403.xml | 9.1 KB 9.1 KB | Display Display | EMDB header |
Images | emd_7403.png | 176.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-7403 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-7403 | HTTPS FTP |
-Validation report
Summary document | emd_7403_validation.pdf.gz | 78 KB | Display | EMDB validaton report |
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Full document | emd_7403_full_validation.pdf.gz | 77.1 KB | Display | |
Data in XML | emd_7403_validation.xml.gz | 492 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-7403 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-7403 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_7403.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 0.655 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : DARP14
Entire | Name: DARP14 |
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Components |
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-Supramolecule #1: DARP14
Supramolecule | Name: DARP14 / type: complex / ID: 1 / Parent: 0 |
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Source (natural) | Organism: synthetic construct (others) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
-Supramolecule #2: DARP14 - Subunit A
Supramolecule | Name: DARP14 - Subunit A / type: complex / ID: 2 / Parent: 1 |
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Source (natural) | Organism: Pyrococcus horikoshii OT3 (archaea) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
-Supramolecule #3: DARP14 - Subunit B
Supramolecule | Name: DARP14 - Subunit B / type: complex / ID: 3 / Parent: 1 |
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Source (natural) | Organism: Pseudomonas aeruginosa PAO1 (bacteria) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
-Supramolecule #4: DARP14 - DARPin
Supramolecule | Name: DARP14 - DARPin / type: complex / ID: 4 / Parent: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 8 |
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Vitrification | Cryogen name: ETHANE |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 30.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Final reconstruction | Applied 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 |
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Initial angle assignment | Type: ANGULAR RECONSTITUTION |
Final angle assignment | Type: ANGULAR RECONSTITUTION |