ELECTRON MICROSCOPY

Sample

IMMATURE ROUS-SARCOMA VIRUS GAG PARTICLES

Specimen Preperation
Sample Aggregation State PARTICLE
Vitrification Instrument FEI VITROBOT MARK II
Cryogen Name ETHANE
Sample Vitrification Details DEGASSED C-FLAT 2 2-3C GRIDS WERE GLOW DISCHARGED FOR 30 SECONDS AT 20 MA. VIRUS SOLUTION WAS DILUTED IN OBS CONTAINING 10NM COLLOIDAL GOLD. 2.5UL OF THIS MIXTURE WAS APPLIED TO A GRID. BLOTTING TIME 2.5 SECONDS.
3D Reconstruction
Reconstruction Method TOMOGRAPHY
Number of Particles 8375
Reported Resolution (Å) 7.7
Resolution Method ?
Other Details RECONSTRUCTION CARRIED OUT USING SUBTOMOGRAM AVERAGING. SUBTOMOGRAM AVERAGING WAS PERFORMED USING SCRIPTS DERIVED FROM THE AV3 (FOERSTER, ET AL) TOM (NICKELL, ET AL) AND DYNAMO (CASTANO-DIEZ, ET AL) PACKAGES. STRUCTURES FOR P10 AND CA-NTD (PDB 1P7N) AND CA-CTD (PDB 3G1I, ONE MONOMER) WERE RIGID BODY DOCKED INTO THE EM-MAP USING THE FIT-IN-MAP OPTION IN CHIMERA. REDUNDANT RESIDUES OF THE ANTIPARALLEL DIMER OF PDB 1P7N WERE REMOVED. MISSING RESIDUES IN THE HELIX 7-HELIX 8 LINKER REGION OF CA, THE CONNECTION LOOP BETWEEN P10 AND CA-NTD AND RESIDUES UPSTREAM OF THE HELIX IN P10 WERE MANUALLY MODELED USING COOT. THE FIT WAS FURTHER REFINED USING MOLECULAR DYNAMICS FLEXIBLE FITTING. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3101. (DEPOSITION ID: 13603).
Refinement Type
Symmetry Type POINT
Map-Model Fitting and Refinement
ID 1
Refinement Space REAL
Refinement Protocol FLEXIBLE FIT
Refinement Target ?
Overall B Value ?
Fitting Procedure ?
Details METHOD--MOLECULAR DYNAMICS FLEXIBLE FITTING
Data Acquisition
Detector Type GATAN MULTISCAN
Electron Dose (electrons/Å2) 34
Imaging Experiment
Date of Experiment 2014-09-10
Temprature (Kelvin)
Microscope Model FEI TITAN KRIOS
Minimum Defocus (nm) 1500
Maximum Defocus (nm) 5000
Minimum Tilt Angle (degrees) -45
Maximum Tilt Angle (degrees) 45
Nominal CS 2.7
Imaging Mode BRIGHT FIELD
Specimen Holder Model .
Nominal Magnification 81000
Calibrated Magnification ?
Source FIELD EMISSION GUN
Acceleration Voltage (kV) 200
Imaging Details ?
Imaging Experiment
Task Software Package Version
MODEL FITTING MDFF ?
MODEL FITTING UCSF Chimera ?
MODEL FITTING VMD ?
RECONSTRUCTION AV3 ?
RECONSTRUCTION Dynamo ?
RECONSTRUCTION IMOD ?
RECONSTRUCTION TOM Toolbox ?
Image Processing
CTF Correction Type CTF Correction Details Number of Particles Selected Particle Selection Details
? PHASE-FLIPPING OF INDIVIDUAL MICROGRAPHS
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