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3JCI
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ELECTRON MICROSCOPY
Sample
Porcine circovirus PCV2 virus-like particles
Specimen Preperation
Sample Aggregation State
PARTICLE
Vitrification Instrument
GATAN CRYOPLUNGE 3
Cryogen Name
ETHANE
Sample Vitrification Details
Blot for 5 seconds before plunging into liquid ethane (GATAN CRYOPLUNGE 3).
3D Reconstruction
Reconstruction Method
SINGLE PARTICLE
Number of Particles
50352
Reported Resolution (Å)
2.9
Resolution Method
FSC 0.143 CUT-OFF
Other Details
For 3D reconstruction, whole datasets were divided into even and odd halves and the initial de novo models and subsequent iterative refinements were all independently performed for each half dataset. Particles were selected from scanned micrograph images using e2boxer.py in EMAN2. The TEM instrument contrast transfer function parameters were determined automatically using fitctf2.py in JSPR and were then visually validated using the EMAN ctfit program. The datasets were then divided into two subsets (even and odd) and processed completely independently, including both de novo initial models and refinements. The images were first binned 4x to obtain initial models and particle parameters assuming icosahedral symmetry. De novo initial models were built using the random model approach. Random subsets of particles were assigned random initial orientations and iteratively refined until convergence. Multi-model competitive refinements were used to choose the winning model (with most assigned particles) as corrective initial models for subsequent refinement. Particles with inconsistent/unstable view parameters in the initial refinements were excluded in further image processing. The orientation and center parameters were then transferred to the un-binned images for high-resolution refinements which included Simplex method-based orientation/center optimization and grid search-based refinement of defocus, astigmatism, beam tilt, and overall and anisotropic magnification of the images. All image refinement and reconstructions were performed with JSPR software that was built on EMAN2 and EMAN library functions and programs. (Single particle details: The particles were selected using the e2boxer.py program in EMAN2. CTF parameters were determined using fitctf2.py in JSPR.) (Single particle--Applied symmetry: I)
Refinement Type
Symmetry Type
POINT
Map-Model Fitting and Refinement
ID
1
Refinement Space
?
Refinement Protocol
?
Refinement Target
?
Overall B Value
?
Fitting Procedure
?
Details
?
Data Acquisition
Detector Type
KODAK SO-163 FILM
Electron Dose (electrons/Å
2
)
25
Imaging Experiment
Date of Experiment
2011-02-22
Temprature (Kelvin)
Microscope Model
FEI TITAN KRIOS
Minimum Defocus (nm)
200
Maximum Defocus (nm)
2500
Minimum Tilt Angle (degrees)
?
Maximum Tilt Angle (degrees)
?
Nominal CS
2.7
Imaging Mode
BRIGHT FIELD
Specimen Holder Model
FEI TITAN KRIOS AUTOGRID HOLDER
Nominal Magnification
59000
Calibrated Magnification
587963
Source
FIELD EMISSION GUN
Acceleration Voltage (kV)
300
Imaging Details
?
Imaging Experiment
Task
Software Package
Version
RECONSTRUCTION
EMAN
2
RECONSTRUCTION
jspr
?
Image Processing
CTF Correction Type
CTF Correction Details
Number of Particles Selected
Particle Selection Details
.
Each particle
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