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- EMDB-9373: Single particle reconstruction of the symmetric core an engineere... -

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Basic information

Entry
Database: EMDB / ID: EMD-9373
TitleSingle particle reconstruction of the symmetric core an engineered protein scaffold
Map dataSingle particle reconstruction of the symmetric core of an engineered protein scaffold (DARP14)
Sample
  • Complex: Subunit A with DARPin Subunit B
    • Complex: Subunit B
      • Protein or peptide: DARP14 - Subunit B
    • Complex: Subunit A with DARPin
      • Protein or peptide: DARP14 - Subunit A with DARPin
Keywordsprotein engineering / symmetric scaffold / small protein cryo-EM / display platform / BIOSYNTHETIC PROTEIN
Function / homology5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate isomerase / 5-carboxymethyl-2-hydroxymuconate delta-isomerase activity / Tautomerase/MIF superfamily / aromatic compound catabolic process / 5-carboxymethyl-2-hydroxymuconate isomerase
Function and homology information
Biological speciesPseudomonas aeruginosa PAO1 (bacteria) / Pyrococcus horikoshii OT3 (archaea) / Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (archaea) / Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsLiu Y / Huynh D
Funding support United States, 6 items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)S10RR23057 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)S10OD018111 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)U24GM11679 United States
National Science Foundation (NSF, United States)DBI-1338135 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
CitationJournal: Nat Commun / Year: 2019
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
DepositionDec 23, 2018-
Header (metadata) releaseJan 23, 2019-
Map releaseMay 8, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.9
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.9
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nht
  • Surface level: 0.9
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9373.png

Downloads & links

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Map

FileDownload / File: emd_9373.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle particle reconstruction of the symmetric core of an engineered protein scaffold (DARP14)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.9 / Movie #1: 0.9
Minimum - Maximum-4.0524282 - 6.067027
Average (Standard dev.)0.013889648 (±0.20456113)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 305.27997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z305.280305.280305.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-4.0526.0670.014

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Supplemental data

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Mask #1

Fileemd_9373_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_9373_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_9373_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Subunit A with DARPin Subunit B

EntireName: Subunit A with DARPin Subunit B
Components
  • Complex: Subunit A with DARPin Subunit B
    • Complex: Subunit B
      • Protein or peptide: DARP14 - Subunit B
    • Complex: Subunit A with DARPin
      • Protein or peptide: DARP14 - Subunit A with DARPin

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Supramolecule #1: Subunit A with DARPin Subunit B

SupramoleculeName: Subunit A with DARPin Subunit B / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: Subunit B

SupramoleculeName: Subunit B / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)

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Supramolecule #3: Subunit A with DARPin

SupramoleculeName: Subunit A with DARPin / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Pyrococcus horikoshii OT3 (archaea)

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Macromolecule #1: DARP14 - Subunit A with DARPin

MacromoleculeName: DARP14 - Subunit A with DARPin / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: 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
Molecular weightTheoretical: 34.71782 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: DARP14 - Subunit B

MacromoleculeName: DARP14 - Subunit B / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: 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
Molecular weightTheoretical: 14.346274 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MPHLVIEATA NLRLETSPGE LLEQANKALF ASGQFGEADI KSRFVTLEAY RQGTAAVERA YLHACLSILD GRDIATRTLL GASLCAVLA EAVAGGGEEG VQVSVEVREM ERLSYAKRVV ARQRLEHHHH HH

UniProtKB: 5-carboxymethyl-2-hydroxymuconate isomerase

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
10.0 mMC4H11NO3Tris
500.0 mMNaClSodium chloridesodium chloride
1.0 mMC4H10O2S2DTT
0.5 %C3H8O3glycerol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200
VitrificationCryogen 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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm 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

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Image processing

Particle selectionNumber selected: 81319
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. v.2.4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v.2.4.0)
Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v.2.4.0) / Number images used: 80253

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Atomic model buiding 1

Initial modelPDB ID:

6c9k


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial local fitting by Chimera and individual residues refined using phenix.real_space_refine
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6nht:
Single particle reconstruction of the symmetric core an engineered protein scaffold

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