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- PDB-6mwq: Single particle cryoEM structure of a DARPin-aldolase platform in... -

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Basic information

Entry
Database: PDB / ID: 6mwq
TitleSingle particle cryoEM structure of a DARPin-aldolase platform in complex with GFP
Components
  • DARPin, Muscle-type aldolase chimeric fusion
  • Green fluorescent protein
KeywordsLYASE/FLUORESCENT PROTEIN / synthetic construct / platform / single particle cryoEM / small protein display / LYASE-FLUORESCENT PROTEIN complex
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
Specimen sourcesynthetic construct (others)
Oryctolagus cuniculus (rabbit)
Aequorea victoria (jellyfish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3 Å resolution
AuthorsWeaver, S.J. / Yao, Q.
CitationJournal: Biorxiv / Year: 2018
Title: Fusion of DARPin to aldolase enables visualization of small protein by cryoEM
Authors: Yao, Q. / Weaver, S.J. / Mock, J.Y. / Jensen, G.J.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 30, 2018 / Release: Nov 21, 2018

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Structure visualization

Movie
  • Biological unit as author_defined_assembly
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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-9277
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DARPin, Muscle-type aldolase chimeric fusion
G: Green fluorescent protein
B: DARPin, Muscle-type aldolase chimeric fusion
J: Green fluorescent protein
C: DARPin, Muscle-type aldolase chimeric fusion
I: Green fluorescent protein
D: DARPin, Muscle-type aldolase chimeric fusion
H: Green fluorescent protein


Theoretical massNumber of molelcules
Total (without water)314,3498
Polyers314,3498
Non-polymers00
Water0
1
A: DARPin, Muscle-type aldolase chimeric fusion
G: Green fluorescent protein


Theoretical massNumber of molelcules
Total (without water)78,5872
Polyers78,5872
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
2
B: DARPin, Muscle-type aldolase chimeric fusion
J: Green fluorescent protein


Theoretical massNumber of molelcules
Total (without water)78,5872
Polyers78,5872
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
3
C: DARPin, Muscle-type aldolase chimeric fusion
I: Green fluorescent protein


Theoretical massNumber of molelcules
Total (without water)78,5872
Polyers78,5872
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
4
D: DARPin, Muscle-type aldolase chimeric fusion
H: Green fluorescent protein


Theoretical massNumber of molelcules
Total (without water)78,5872
Polyers78,5872
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide
DARPin, Muscle-type aldolase chimeric fusion / / Fructose-bisphosphate aldolase A


Mass: 53113.434 Da / Num. of mol.: 4
Source: (gene. exp.) synthetic construct (others), (gene. exp.) Oryctolagus cuniculus (rabbit)
Details: Rabbit (Oryctolagus cuniculus) muscle aldolase and a synthetic DARPin were fused to create this synthetic construct
Plasmid name: pET21b / Gene: ALDOA
Details (production host): C-terminal His-tag of DARPin-aldolase chimeric fusion
Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P00883, fructose-bisphosphate aldolase
#2: Protein/peptide
Green fluorescent protein /


Mass: 25473.697 Da / Num. of mol.: 4 / Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: GFP / Plasmid name: pACYCDuet / Details (production host): no tag / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P42212

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1DARPin-aldolase platform in complex with GFPCOMPLEX1, 20RECOMBINANT
2DARPin, Muscle-type aldolase chimeric fusionCOMPLEX1RECOMBINANT
3Green fluorescent proteinCOMPLEX1RECOMBINANT
Molecular weightValue: 0.314 MDa / Experimental value: NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
1132630synthetic construct (others)
226100Aequorea victoria (jellyfish)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganismStrain
11469008Escherichia coli (E. coli)BL21 (DE3)
22469008Escherichia coli (E. coli)BL21 (DE3)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer ID
125 mMTris HCl1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 2.5 mg/ml
Details: The protein complex was then purified with Ni-NTA affinity chromatography (Qiagen), and Superdex 200 chromatography (GE healthcare). The purified GFP-DARPin-aldolase complex was concentrated to 2.5mg/ml in a buffer containing 25 mM Tris-HCl pH 8.0 and 150 mM NaCl.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 / Grid type: UltrAuFoil
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 kelvins
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
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 / Nominal defocus max: 30000 nm / Nominal defocus min: 10000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 microns
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recording

Imaging ID: 1 / Average exposure time: 4 sec. / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 1

IDElectron doseDetailsNumber of real images
12.3All three microscope sessions used grids frozen in the same session. In the first microscopy session the stage was untilted (0 degrees).1133
22.3All three microscope sessions used grids frozen in the same session. In the second microscopy session the stage was untilted (0 degrees).548
31.1All three microscope sessions used grids frozen in the same session. In the third microscopy session the stage was tilted to 26 degrees.1180
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
3EPUimage acquisition
5CTFFIND4.1.10CTF correctionwhole micrographs
6RELION3.0 beta 2CTF correctionPer particle CtfRefine
9UCSF Chimera1.12model fittingFit into map
11RELION3.0 beta 2initial Euler assignment
12RELION3.0 beta 2final Euler assignment
14RELION3.0 beta 23D reconstruction
Image processingDetails: Particles from all three microscopy sessions were processed together.
CTF correctionDetails: First whole micrograph correction with CtfFind4. Then per particle CTF Refinement in Relion.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: Manually picked particles were used to generate references for autopicking. Particles from all three microscopy sessions were processed together. Microscope session 1 - 358,381 particles Microscope session 2 - 64,368 particles Microscope session 3 - 402,427 particles
Number of particles selected: 851776
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 236339 / Symmetry type: POINT
Atomic model buildingDetails: 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.
Ref protocol: RIGID BODY FIT
Atomic model building
IDPDB-IDPdb chain ID 3D fitting ID
15VY5A1
25MA6A1
35MA6B1

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