9 ANGSTROM RESOLUTION CRYO-EM RECONSTRUCTION STRUCTURE OF SEMLIKI FOREST VIRUS (SFV) AND FITTING OF THE CAPSID PROTEIN STRUCTURE IN THE EM DENSITY
ELECTRON MICROSCOPY
Sample
SEMLIKI FOREST VIRUS
Specimen Preparation
Sample Aggregation State
PARTICLE
Vitrification Instrument
HOMEMADE PLUNGER
Cryogen Name
ETHANE
Sample Vitrification Details
PLUNGE VITRIFICATION SAMPLES PREPARED AS THIN LAYERS OF VITREOUS ICE MAINTAINED AT NEAR LIQUID NITROGEN TEMPERATURE IN THE ELECTRON MICROSCOPE WITH A ...
PLUNGE VITRIFICATION SAMPLES PREPARED AS THIN LAYERS OF VITREOUS ICE MAINTAINED AT NEAR LIQUID NITROGEN TEMPERATURE IN THE ELECTRON MICROSCOPE WITH A GATAN 626-0300 CRYOTRANSFER HOLDER.
3D Reconstruction
Reconstruction Method
SINGLE PARTICLE
Number of Particles
5267
Reported Resolution (Å)
9
Resolution Method
Other Details
THE ORIENTATIONS WERE REFINED BY THE CROSS COMMON LINES LINES METHOD (SIMPLEX) AND THE POLAR FOURIER TRANSFORM METHOD USING A PARALLEL IMPLEMENTATION ...
THE ORIENTATIONS WERE REFINED BY THE CROSS COMMON LINES LINES METHOD (SIMPLEX) AND THE POLAR FOURIER TRANSFORM METHOD USING A PARALLEL IMPLEMENTATION (PYPFT). THE EFFECTIVE RESOLUTION OF THE FINAL RECONSTRUCTED DENSITY WAS DETERMINED TO BE AT LEAST 9 ANGSTROMS, AS MEASURED BY RANDOMLY SPLITTING THE PARTICLES INTO TWO SETS AND CALCULATING THE FOURIER SHELL CORRELATION OBTAINED FROM SEPARATE RECONSTRUCTIONS (HARAUZ AND VAN HEEL 1986, OPTIK 73, 146-156). THE EIGENVALUE SPECTRUM GAVE AN INDICATION OF THE RANDOMNESS OF THE DATA THAT WAS INCLUDED IN THE RECONSTRUCTION. THE COMPLETENESS OF THE DATA WAS VERIFIED IN THAT ALL EIGENVALUES EXCEEDED 100. THE COORDINATES ARE IN THE P, Q, R FRAME IN ANGSTROM UNITS AND CORRESPOND TO ICOSAHEDRAL SYMMETRY AXES. THE ORIGIN IS CHOSEN AT THE CENTER OF THE VIRUS WITH P, Q AND R ALONG MUTUALLY PERPENDICULAR TWO-FOLD AXES OF THE ICOSAHEDRON. THEY SHOULD REMAIN IN THAT FRAME FOR THE EASE OF THE USER IN CREATING THE BIOLOGICALLY SIGNIFICANT VIRAL COMPLEX PARTICLE USING THE 60 ICOSAHEDRAL SYMMETRY OPERATORS. RESIDUES NOT VISIBLE IN THE ORIGINAL CRYSTAL STRUCTURES ARE NOT INCLUDED IN THE CRYO-EM STRUCTURE MODEL. FOR EXAMPLE, C RESIDUES 1-118, ARE NOT VISIBLE IN THE CRYSTAL STRUCTURE (PDB ENTRY 1VCQ) AND THEREFORE ARE NOT INCLUDED IN THE COORDINATES BELOW.
Refinement Type
Symmetry Type
POINT
Point Symmetry
I
Map-Model Fitting and Refinement
Id
1 (1DYL)
Refinement Space
REAL
Refinement Protocol
RIGID BODY FIT
Refinement Target
R-factor
Overall B Value
Fitting Procedure
Details
METHOD--THE CRYSTAL STRUCTURE OF THE CAPSID PROTEIN FROM CHOI ET AL (1997)
PROTEINS 3 27,345-359 (SUBUNIT A OF PDB FILE 1VCQ) WAS PLACED INTO THE CRY ...
METHOD--THE CRYSTAL STRUCTURE OF THE CAPSID PROTEIN FROM CHOI ET AL (1997)
PROTEINS 3 27,345-359 (SUBUNIT A OF PDB FILE 1VCQ) WAS PLACED INTO THE CRYO-EM
DENSITY MAP. THE CAPSID PROTEIN WAS FIRST MANUALLY POSITIONED INTO THE CRYO-EM
DENSITY CORRESPONDING TO POSITIONS OF THE FOUR INDEPENDENT MONOMER DENSITIES
BETWEEN THE INNER LEAFLET OF THE BILAYER AND THE RNA. THESE POSITIONS WERE THEN
REFINED BY RIGID BODY REFINEMENT IN REAL SPACE WITH THE PROGRAM EMFIT (CHENG ET
AL. 1995, CELL 80, 621-630). THE QUALITY OF THE FIT CAN BE SEEN FROM THE MAP
DENSITY WITHIN THE PROTEIN. ALL 4563 ATOMS ARE IN DENSITY OF AT LEAST 4 SIGMA
(96.73) ABOVE THE AVERAGE (512.04) , 1167 ATOMS ARE IN DENSITY BETWEEN 4 AND 5
SIGMA, 3174 ATOMS ARE IN DENSITY BETWEEN 5 AND 6 SIGMA, AND 222 ATOMS ARE IN
DENSTY OF 6 SIGMA OR ABOVE. THE VARIATION IN DENSITY OVER THE FITTED PROTEIN
CAN BE VISUALIZED WITH THE PSEUDO TEMPERATURE FACTOR. THE DENSITY VALUE AT EACH
ATOM IS GIVEN IN THE 8TH COLUM (USUALLY THE OCCUPANCY) AS THE NUMBER OF
STANDARD DEVIATION ABOVE BACKGROUND. COLUMN NINE (USUALLY THE TEMPERATURE
FACTOR) CONTAINS THE VALUE OF THE RELATIVE DENSITY WITHIN THE FITTED PROTEIN
SCALED LINEARLY SO THAT THE MINIMUM DENSITY IS 100.0 AND THE MAXIMUM DENSITY IS
1.0. THE ATOMS THAT LIE IN THE LOWER DENSITY REGIONS WILL HAVE THE HIGHEST
PSEUDO TEMPERATURE FACTORS. REFINEMENT PROTOCOL--RIGID BODY REFINEMENT