EMD-9876 Experiments and Validation

Structure of PaaZ, a bifunctional enzyme in complex with NADP+ and CCoA

Single particle reconstruction
Overview of EMD-9876
Sample name: PaaZ
Organism: Escherichia coli K-12
Fitted atomic model: 6jqo
Related EM entries by publication: EMD-9873, EMD-9874, EMD-9875

Map parameters

Recommended contour level: 0.085 (author)
Number of grid points: 412 × 412 × 412
Voxel size: 1.04 × 1.04 × 1.04 Å
Minimum density: -0.308
Maximum density: 0.587
Average density: 0.00
Standard deviation: 0.014

Sample information

Sample name: PaaZ
Ligands: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE, CROTONYL COENZYME A
Proteins: PaaZ, Bifunctional protein PaaZ

Validation information

Experimental information

 

Image processing

Applied symmetry: C3
Software: RELION
Number of particles: 120968
Reconstruction protocol: BACK PROJECTION
CTF correction: CTF was corrected per particle within relion during processing

Fitting

Overall B value: 44.399999999999999
Refinement space: REAL
Details: Real space refinement with secondary structure enabled, minimization and adp

Imaging

Session
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 27.0
Nominal CS (mm): 2.70
Nominal magnification: 75000.0
Defocus max (nm): 3,200.00
Defocus min (nm): 2,200.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Details: Data was collected with EPU software
Acquisition
Number of images: 563
Sampling interval (μm): 14.0
Details: The 60 second exposure was saved into 75 frames with each frame ~0.36 e-. The frames were then grouped into 3 for alignment and summed images were used for data processing

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.015
Buffer: Protein was purified and kept in 25mM Hepes buffer and 50 mM NaCl
Specimen support: Grids were glow discharged for 5 minutes and graphene oxide was applied. This was followed by 3 times wash with water and dried.
Vitrification
Apparatus: FEI VITROBOT MARK IV
Cryogen: ETHANE
Humidity: 100
Details: blotting force 10, blotting time 4 sec, waiting time 15 sec, drying time 0, blotting time 1..