5A9E

Cryo-electron tomography and subtomogram averaging of Rous-Sarcoma- Virus deltaMBD virus-like particles


ELECTRON MICROSCOPY
Sample
IMMATURE ROUS-SARCOMA VIRUS GAG PARTICLES
Specimen Preparation
Sample Aggregation StatePARTICLE
Vitrification InstrumentFEI VITROBOT MARK II
Cryogen NameETHANE
Sample Vitrification DetailsDEGASSED 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 ...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 MethodTOMOGRAPHY
Number of Particles8375
Reported Resolution (Å)7.7
Resolution Method
Other DetailsRECONSTRUCTION CARRIED OUT USING SUBTOMOGRAM AVERAGING. SUBTOMOGRAM AVERAGING WAS PERFORMED USING SCRIPTS DERIVED FROM THE AV3 (FOERSTER, ET AL) TOM ( ...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 TypePOINT
Point SymmetryC6
Map-Model Fitting and Refinement
Id1
Refinement SpaceREAL
Refinement ProtocolFLEXIBLE FIT
Refinement Target
Overall B Value
Fitting Procedure
DetailsMETHOD--MOLECULAR DYNAMICS FLEXIBLE FITTING
Data Acquisition
Detector TypeGATAN MULTISCAN
Electron Dose (electrons/Å**2)34
Imaging Experiment1
Date of Experiment2014-09-10
Temperature (Kelvin)
Microscope ModelFEI TITAN KRIOS
Minimum Defocus (nm)1500
Maximum Defocus (nm)5000
Minimum Tilt Angle (degrees)-45
Maximum Tilt Angle (degrees)45
Nominal CS2.7
Imaging ModeBRIGHT FIELD
Specimen Holder Model
Nominal Magnification81000
Calibrated Magnification
SourceFIELD EMISSION GUN
Acceleration Voltage (kV)200
Imaging Details
EM Software
TaskSoftware PackageVersion
MODEL FITTINGMDFF
MODEL FITTINGUCSF Chimera
MODEL FITTINGVMD
RECONSTRUCTIONAV3
RECONSTRUCTIONDynamo
RECONSTRUCTIONIMOD
RECONSTRUCTIONTOM Toolbox
Image Processing
CTF Correction TypeCTF Correction DetailsNumber of Particles SelectedParticle Selection Details
PHASE-FLIPPING OF INDIVIDUAL MICROGRAPHS