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- EMDB-9277: Single particle cryoEM structure of a DARPin-aldolase platform in... -

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Basic information

Entry
Database: EMDB / ID: 9277
TitleSingle particle cryoEM structure of a DARPin-aldolase platform in complex with GFP
Map dataDARPin-aldolase platform in complex with GFP, Relion Post processed map with B factor -25 angstrom squared
SampleDARPin-aldolase platform in complex with GFP
  • (DARPin, Muscle-type aldolase chimeric ...) x 2
  • (Green fluorescent ...) x 2
Function / homologyFructose-bisphosphate aldolase, class-I / Green fluorescent protein, GFP / Fructose-bisphosphate aldolase class-I active site. / Green fluorescent protein / Fructose-bisphosphate aldolase class-I / Fructose-bisphosphate aldolase class-I active site / Aldolase-type TIM barrel / Green fluorescent protein-related / Green fluorescent protein / negative regulation of Arp2/3 complex-mediated actin nucleation ...Fructose-bisphosphate aldolase, class-I / Green fluorescent protein, GFP / Fructose-bisphosphate aldolase class-I active site. / Green fluorescent protein / Fructose-bisphosphate aldolase class-I / Fructose-bisphosphate aldolase class-I active site / Aldolase-type TIM barrel / Green fluorescent protein-related / Green fluorescent protein / negative regulation of Arp2/3 complex-mediated actin nucleation / fructose-bisphosphate aldolase / fructose-bisphosphate aldolase activity / I band / M band / bioluminescence / generation of precursor metabolites and energy / glycolytic process / protein-chromophore linkage / protein homotetramerization / positive regulation of cell migration / Fructose-bisphosphate aldolase A / Green fluorescent protein
Function and homology information
Sourcesynthetic construct (others) / Aequorea victoria (jellyfish) / Oryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / 3 Å resolution
AuthorsWeaver SJ / Yao Q
CitationJournal: Biorxiv / Year: 2018
Title: Fusion of DARPin to aldolase enables visualization of small protein by cryoEM
Authors: Yao Q / Weaver SJ / Mock JY / Jensen GJ
Validation ReportPDB-ID: 6mwq

SummaryFull reportAbout validation report
DateDeposition: Oct 30, 2018 / Header (metadata) release: Nov 21, 2018 / Map release: Nov 21, 2018 / Last update: Nov 21, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.004
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.004
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6mwq
  • Surface level: 0.004
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_9277.map.gz (map file in CCP4 format, 186625 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
360 pix
0.83 Å/pix.
= 299.34 Å
360 pix
0.83 Å/pix.
= 299.34 Å
360 pix
0.83 Å/pix.
= 299.34 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8315 Å
Density
Contour Level:0.004 (by author), 0.004 (movie #1):
Minimum - Maximum-0.025016628 - 0.056058165
Average (Standard dev.)0.00006114975 (0.001124288)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions360360360
Origin0.00.00.0
Limit359.0359.0359.0
Spacing360360360
CellA=B=C: 299.34 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.83150.83150.8315
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z299.340299.340299.340
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.0250.0560.000

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Supplemental data

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Sample components

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Entire DARPin-aldolase platform in complex with GFP

EntireName: DARPin-aldolase platform in complex with GFP / Number of components: 5

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Component #1: protein, DARPin-aldolase platform in complex with GFP

ProteinName: DARPin-aldolase platform in complex with GFP / Recombinant expression: No
MassTheoretical: 314 kDa
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: Escherichia coli (E. coli) / Strain: BL21 (DE3)

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Component #2: protein, DARPin, Muscle-type aldolase chimeric fusion

ProteinName: DARPin, Muscle-type aldolase chimeric fusion / Recombinant expression: No
SourceSpecies: Aequorea victoria (jellyfish)
Source (engineered)Expression System: Escherichia coli (E. coli) / Strain: BL21 (DE3)

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Component #3: protein, Green fluorescent protein

ProteinName: Green fluorescent protein / Recombinant expression: No

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Component #4: protein, DARPin, Muscle-type aldolase chimeric fusion

ProteinName: DARPin, Muscle-type aldolase chimeric fusion / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 53.113434 kDa
SourceSpecies: Oryctolagus cuniculus (rabbit)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: protein, Green fluorescent protein

ProteinName: Green fluorescent protein / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 25.473697 kDa
SourceSpecies: Aequorea victoria (jellyfish)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 2.5 mg/ml / pH: 8
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 95 %
Details: Grids were frozen on a manual plunger at the Scripps Research Institute Core Microscopy Facility in a 4 degrees C cold room humidified to >95%..

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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: 1.1 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 165000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 10000.0 - 30000.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisition #1Number of digital images: 1133
Details: All three microscope sessions used grids frozen in the same session. In the first microscopy session the stage was untilted (0 degrees).
Image acquisition #2Number of digital images: 548
Details: All three microscope sessions used grids frozen in the same session. In the second microscopy session the stage was untilted (0 degrees).
Image acquisition #3Number of digital images: 1180
Details: All three microscope sessions used grids frozen in the same session. In the third microscopy session the stage was tilted to 26 degrees.

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 236339
Details: Particles from all three microscopy sessions were processed together.
3D reconstructionSoftware: RELION
CTF correction: First whole micrograph correction with CtfFind4. Then per particle CTF Refinement in Relion.
Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: rigid body
Details: PDB models 5vy5 and 5ma6 were used as starting points. These models were mutated to match the sequence of the DARPin-aldolase platform in complex with GFP that were used. The PDB models were docked into the cryoEM density using Chimera fit into map function. No model refinement was used.
Input PDB model: 5VY5, 5MA6, 5MA6
Chain ID: A, A, B
Output model

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