EMD-2238 Experiments and Validation

Cryo-EM Structure of the Mycobacterial Fatty Acid Synthase

Single particle reconstruction
Overview of EMD-2238
Sample name: Mycobacterial Fatty Acid Synthase, FAS I
Organism: Mycobacterium smegmatis
Fitted atomic model: 4v8l

Map parameters

Recommended contour level: 2.70 (author)
Number of grid points: 160 × 160 × 160
Voxel size: 2.45 × 2.45 × 2.45 Å
Minimum density: -7.793
Maximum density: 14.936
Average density: -0.133
Standard deviation: 1.076

Sample information

Sample name: Mycobacterial Fatty Acid Synthase, FAS I
Protein: Mycobacterial Fatty Acid Synthase I

Validation information

Experimental information

 

Image processing

Applied symmetry: D3
Software: Imagic-5, Spider
Number of particles: 106884
Reconstruction protocol: Common lines, projection matching
CTF correction: Each image
Details: Fourier amplitudes of the reconstruction were enhanced using amplitudes from the x-ray structure of the S. cerevisiae FAS; subsequently, the map was filtered using a Butterworth low-pass filter in SPIDER

Fitting

Initial atomic model: 2UV8
Fitting software: Chimera
Fitting protocol: rigid body
Target criteria: Correlation
Refinement space: REAL
Details: Protocol: Domains were separately fitted as rigid bodies and manually adjusted in O. The model was minimized with PHENIX.PDBTOOLS. Domains were separately fitted as rigid bodies and manually adjusted in O.The model was minimized with PHENIX.PDBTOOLS.

Imaging

Session
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.70
Nominal magnification: 59000.0
Defocus max (nm): 5,000.00
Defocus min (nm): 1,500.00
Tilt max (degrees): 0.00
Tilt min (degrees): 0.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Details: Data were collected using the automated image acquisition software FEI EPU.
Acquisition
Number of images: 1556
Details: Data were collected using the automated image acquisition software FEI EPU.

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 1.65
Buffer: 100mM potassium phosphate buffer pH 7.2, 165 mM NaCl, 2mM EDTA, 2mM DTT
Specimen support: Quantifoil R2/1 200 mesh copper grids
Vitrification
Apparatus: HOMEMADE PLUNGER
Cryogen: ETHANE