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- EMDB-9374: Single particle reconstruction of DARPin and its bound GFP on a s... -

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Basic information

Entry
Database: EMDB / ID: EMD-9374
TitleSingle particle reconstruction of DARPin and its bound GFP on a symmetric scaffold
Map data
SampleSubunit A with DARPin + Subunit B + superfolder GFP:
(superfolder GFP) x 2 / Subunit A with DARPin / Subunit B / DARP14 - Subunit B / Subunit A-DARPin
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 speciesAequorea victoria (jellyfish) / Pyrococcus horikoshii OT3 (archaea) / Pseudomonas aeruginosa PAO1 (bacteria) / Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) (bacteria) / Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsLiu Y / Huynh D / Yeates TO
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.
Validation ReportPDB-ID: 6nhv

SummaryFull reportAbout validation report
History
DepositionDec 24, 2018-
Header (metadata) releaseJan 23, 2019-
Map releaseMay 8, 2019-
UpdateMay 8, 2019-
Current statusMay 8, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 7.26
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 7.26
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6nhv
  • Surface level: 7.26
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9374.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.06 Å/pix.
x 288 pix.
= 305.28 Å
1.06 Å/pix.
x 288 pix.
= 305.28 Å
1.06 Å/pix.
x 288 pix.
= 305.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy EMDB: 7.26 / Movie #1: 7.26
Minimum - Maximum-28.381074999999999 - 42.377045000000003
Average (Standard dev.)-0.0012176203 (±0.93447405)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
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
start NX/NY/NZ000
NX/NY/NZ288288288
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-28.38142.377-0.001

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Supplemental data

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Segmentation: #1

Fileemd_9374_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Subunit A with DARPin + Subunit B + superfolder GFP

EntireName: Subunit A with DARPin + Subunit B + superfolder GFP / Number of components: 7

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Component #1: protein, Subunit A with DARPin + Subunit B + superfolder GFP

ProteinName: Subunit A with DARPin + Subunit B + superfolder GFP / Recombinant expression: No

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Component #2: protein, superfolder GFP

ProteinName: superfolder GFP / Recombinant expression: No
SourceSpecies: Aequorea victoria (jellyfish)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Subunit A with DARPin

ProteinName: Subunit A with DARPin / Recombinant expression: No
SourceSpecies: Pyrococcus horikoshii OT3 (archaea)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, Subunit B

ProteinName: Subunit B / Recombinant expression: No
SourceSpecies: Pseudomonas aeruginosa PAO1 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, superfolder GFP

ProteinName: superfolder GFP / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.623918 kDa
SourceSpecies: Aequorea victoria (jellyfish)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, DARP14 - Subunit B

ProteinName: DARP14 - Subunit B / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 14.346274 kDa
SourceSpecies: 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
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: protein, Subunit A-DARPin

ProteinName: Subunit A-DARPin / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 34.71782 kDa
SourceSpecies: 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
Source (engineered)Expression System: Escherichia coli (E. coli)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/mL / pH: 7.5
Support filmunspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %
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 imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 56 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 1929

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 91211
3D reconstructionSoftware: RELION / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
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
Input PDB model: 6C9K, 5MA8, 4W6B
Output model

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