[English] 日本語
![](img/lk-miru.gif)
- EMDB-9374: Single particle reconstruction of DARPin and its bound GFP on a s... -
+
Open data
-
Basic information
Entry | Database: EMDB / ID: EMD-9374 | |||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | Single particle reconstruction of DARPin and its bound GFP on a symmetric scaffold | |||||||||||||||||||||
![]() | Refinement of DARPin, GFP, and two adjacent trimers | |||||||||||||||||||||
![]() |
| |||||||||||||||||||||
Function / homology | 5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate isomerase / ![]() ![]() | |||||||||||||||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() | |||||||||||||||||||||
Method | ![]() ![]() | |||||||||||||||||||||
![]() | Liu Y / Huynh D / Yeates TO | |||||||||||||||||||||
Funding support | ![]()
| |||||||||||||||||||||
![]() | ![]() Title: A 3.8 Å resolution cryo-EM structure of a small protein bound to an imaging scaffold. Authors: Yuxi Liu / Duc T Huynh / Todd O Yeates / ![]() Abstract: Proteins smaller than about 50 kDa are currently too small to be imaged at high resolution by cryo-electron microscopy (cryo-EM), leaving most protein molecules in the cell beyond the reach of this ...Proteins smaller than about 50 kDa are currently too small to be imaged at high resolution by cryo-electron microscopy (cryo-EM), leaving most protein molecules in the cell beyond the reach of this powerful structural technique. Here we use a designed protein scaffold to bind and symmetrically display 12 copies of a small 26 kDa protein, green fluorescent protein (GFP). We show that the bound cargo protein is held rigidly enough to visualize it at a resolution of 3.8 Å by cryo-EM, where specific structural features of the protein are visible. The designed scaffold is modular and can be modified through modest changes in its amino acid sequence to bind and display diverse proteins for imaging, thus providing a general method to break through the lower size limitation in cryo-EM. | |||||||||||||||||||||
History |
|
-
Structure visualization
Movie |
![]() |
---|---|
Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
-
Downloads & links
-EMDB archive
Map data | ![]() | 85.5 MB | ![]() | |
---|---|---|---|---|
Header (meta data) | ![]() ![]() | 24.3 KB 24.3 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 10.3 KB | Display | ![]() |
Images | ![]() | 153.8 KB | ||
Masks | ![]() | 91.1 MB | ![]() | |
Others | ![]() ![]() | 59.1 MB 59.1 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6nhvMC ![]() 9373C ![]() 6nhtC C: citing same article ( M: atomic model generated by this map |
---|---|
Similar structure data |
-
Links
EMDB pages | ![]() ![]() |
---|
-
Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Refinement of DARPin, GFP, and two adjacent trimers | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.06 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Mask #1
File | ![]() | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: Half body 1
File | emd_9374_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half body 1 | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: Half body 2
File | emd_9374_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half body 2 | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-
Sample components
-Entire : Subunit A with DARPin + Subunit B + superfolder GFP
Entire | Name: Subunit A with DARPin + Subunit B + superfolder GFP |
---|---|
Components |
|
-Supramolecule #1: Subunit A with DARPin + Subunit B + superfolder GFP
Supramolecule | Name: Subunit A with DARPin + Subunit B + superfolder GFP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
---|
-Supramolecule #2: superfolder GFP
Supramolecule | Name: superfolder GFP / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
---|---|
Source (natural) | Organism: ![]() ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() ![]() |
-Supramolecule #3: Subunit A with DARPin
Supramolecule | Name: Subunit A with DARPin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3 |
---|---|
Source (natural) | Organism: ![]() ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() ![]() |
-Supramolecule #4: Subunit B
Supramolecule | Name: Subunit B / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #2 |
---|---|
Source (natural) | Organism: ![]() ![]() |
Recombinant expression | Organism: ![]() ![]() ![]() |
-Macromolecule #1: superfolder GFP
Macromolecule | Name: superfolder GFP / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: ![]() ![]() ![]() |
Molecular weight | Theoretical: 26.623918 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTL VTTL(CRO)VQCFS RYPDHM KRH DFFKSAMPEG YVQERTISFK DDGTYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNFNSHNVY ITADKQK NG IKANFKIRHN ...String: MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATNG KLTLKFICTT GKLPVPWPTL VTTL(CRO)VQCFS RYPDHM KRH DFFKSAMPEG YVQERTISFK DDGTYKTRAE VKFEGDTLVN RIELKGIDFK EDGNILGHKL EYNFNSHNVY ITADKQK NG IKANFKIRHN VEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD PNEKRDHMVL LEFVTAAGIT HHHHHH |
-Macromolecule #2: DARP14 - Subunit B
Macromolecule | Name: DARP14 - Subunit B / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: ![]() ![]() Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1 |
Molecular weight | Theoretical: 14.346274 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MPHLVIEATA NLRLETSPGE LLEQANKALF ASGQFGEADI KSRFVTLEAY RQGTAAVERA YLHACLSILD GRDIATRTLL GASLCAVLA EAVAGGGEEG VQVSVEVREM ERLSYAKRVV ARQRLEHHHH HH |
-Macromolecule #3: Subunit A-DARPin
Macromolecule | Name: Subunit A-DARPin / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: ![]() ![]() ![]() Strain: ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3 |
Molecular weight | Theoretical: 34.71782 KDa |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MRITTKVGDK GSTRLFGGEE VWKDSPIIEA NGTLDELTSF IGEAKHYVDE EMKGILEEIQ NDIYKIMGEI GSKGKIEGIS EERIAWLLK LILRYMEMVN LKSFVLPGGT LESAKLDVCR TIARRALRKV LTVTREFGIG AEAAAYLLAL SDLLFLLARV I EIEQGKKL ...String: MRITTKVGDK GSTRLFGGEE VWKDSPIIEA NGTLDELTSF IGEAKHYVDE EMKGILEEIQ NDIYKIMGEI GSKGKIEGIS EERIAWLLK LILRYMEMVN LKSFVLPGGT LESAKLDVCR TIARRALRKV LTVTREFGIG AEAAAYLLAL SDLLFLLARV I EIEQGKKL LEAARAGQDD EVRILMANGA DVNAADDVGV TPLHLAAQRG HLEIVEVLLK CGADVNAADL WGQTPLHLAA TA GHLEIVE VLLKNGADVN ARDNIGHTPL HLAAWAGHLE IVEVLLKYGA DVNAQDKFGK TPFDLAIDNG NEDIAEVLQK AA |
-Experimental details
-Structure determination
Method | ![]() |
---|---|
![]() | ![]() |
Aggregation state | particle |
-
Sample preparation
Concentration | 1 mg/mL | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Buffer | pH: 7.5 Component:
| |||||||||||||||
Grid | Details: unspecified | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV Details: 2.5 microliter of sample, 0 sec wait, 0 sec drain, 3 sec blot, -15 blot force, grids pre-treated with 0.1% poly-lysine for 6 hours. |
-
Electron microscopy
Microscope | FEI TITAN KRIOS |
---|---|
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 3-20 / Number grids imaged: 1 / Number real images: 1929 / Average exposure time: 8.0 sec. / Average electron dose: 56.0 e/Å2 |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
-
Image processing
-Atomic model buiding 1
Initial model | (PDB ID: , , ) |
---|---|
Details | Initial local fitting by Chimera and individual residues refined using phenix.real_space_refine for the symmetric core and DARPin, rigid body refinement for GFP |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | ![]() PDB-6nhv: |