The binding mode of epothilone A on a,b-tubulin by electron crystallography
ELECTRON CRYSTALLOGRAPHY
Crystallization
Crystal Properties
Matthews coefficient
Solvent content
66.11
Crystal Data
Unit Cell
Length ( Å )
Angle ( ˚ )
a = 81.2
α = 90
b = 93.5
β = 90
c = 90
γ = 90
Symmetry
Space Group
P 1 21 1
Diffraction
Diffraction Experiment
ID #
Crystal ID
Scattering Type
Data Collection Temperature
Detector
Detector Type
Details
Collection Date
Monochromator
Protocol
1
1
electron
M
SINGLE WAVELENGTH
Refinement
Statistics
Diffraction ID
Structure Solution Method
Cross Validation method
Starting model
Resolution (High)
Resolution (Low)
Number Reflections (All)
Number Reflections (Observed)
Number Reflections (R-Free)
Percent Reflections (Observed)
R-Factor (All)
R-Factor (Observed)
R-Work
R-Free
R-Free Selection Details
Mean Isotropic B
ELECTRON CRYSTALLOGRAPHY
THROUGHOUT
PDB ID 1JFF
2.89
91.29
18321
18321
967
67.03
0.33214
0.33214
0.33275
0.32096
RANDOM
79.8
Temperature Factor Modeling
Anisotropic B[1][1]
Anisotropic B[1][2]
Anisotropic B[1][3]
Anisotropic B[2][2]
Anisotropic B[2][3]
Anisotropic B[3][3]
-5.48
0.41
-3.03
8.5
Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms
Number
Protein Atoms
6578
Nucleic Acid Atoms
Solvent Atoms
Heterogen Atoms
94
Sample
tubulin 2D crystal
Specimen Preparation
Sample Aggregation State
2D ARRAY
3D Reconstruction
Reconstruction Method
CRYSTALLOGRAPHY
Number of Particles
Reported Resolution (Å)
2.89
Resolution Method
Other Details
Refinement Type
Symmetry Type
2D CRYSTAL
Map-Model Fitting and Refinement
Id
1 (1JFF)
Refinement Space
RECIPROCAL
Refinement Protocol
RIGID BODY FIT
Refinement Target
Analysis of comparitive difference densities
Overall B Value
Fitting Procedure
Details
METHOD--annealing, rigid refining REFINEMENT PROTOCOL--rigid body DETAILS--THE
MODEL WAS DERIVED USING HIGH RESOLUTION ELECTRON DIFFRACTIONS FROM TWO ...
METHOD--annealing, rigid refining REFINEMENT PROTOCOL--rigid body DETAILS--THE
MODEL WAS DERIVED USING HIGH RESOLUTION ELECTRON DIFFRACTIONS FROM TWO
DIMENSIONAL CRYSTALS OF TUBULIN INDUCED BY THE PRESENCE OF ZN++ IONS. WHAT
FOLLOWS ARE THE COORDINATES FOR EPOLTHILONE-A BOUND TO AB-TUBULIN DIMER IN THE
ZINC-INDUCED SHEETS. THE LIGAND MODEL WAS FIT INTO A DENSITY MAP FOR WHICH THE
RESOLUTION IN THE PLANE OF THE SHEET WAS 2.89 ANGSTROMS AND THAT PERPENDICULAR
TO THE SHEET WAS ABOUT 4.2 ANGSTROMS AS DESCRIBED IN THE SUPPLEMENTARY
MATERIAL. PHASES WERE DERIVED FROM A PREVIOUS MODEL OF ALPHA/BETA TUBULIN
COMPLEXED WITH TAXOL (1JFF). SHAKING, HIGH TEMPERATURE ANANEALING, AND MODEL
WERE COMBINED TO PRODUCE AN OMIT MAP OF THE BOUND EPOTHILONE THAT SYSTEMATIC
BIAS.A NUMBER OF CONFORMATIONAL MODELS WERE FLEXIBLY FITTED INITIAL MAP AND
TESTED AGAINST THE DIFFRACTIONS BY DIFFERENCE MAP AS DESCRIBED IN THE PRIMARY
REFERENCE. REFINEMENT TO THE FINAL STAGE WAS LIMITED WITHIN AN 8A RADIUS OF THE
LIGAND. ALTHOUGH THE REMAINING PART OF THE PROTEIN SHOWS LITTLE DEVIATION FROM
THAT SEEN IN 1JFF, CER NOT FULLY CONFORM WITH RAMACHANDRAN CHARACTERISTICS. AND
SHOULD B THE FINAL MAPS ASSOCIATED WITH THE PRESENT MODEL WERE DERIVED BY
COMPLEX. AS SUCH, FREE R REPORTED BY THE SOFTWARE BELOW IS NOT RE
Data Acquisition
Detector Type
GENERIC GATAN
Electron Dose (electrons/Å**2)
100
Imaging Experiment
1
Date of Experiment
Temperature (Kelvin)
100
Microscope Model
JEOL 4000EX
Minimum Defocus (nm)
Maximum Defocus (nm)
Minimum Tilt Angle (degrees)
15
Maximum Tilt Angle (degrees)
55
Nominal CS
Imaging Mode
DIFFRACTION
Specimen Holder Model
Nominal Magnification
Calibrated Magnification
Source
Acceleration Voltage (kV)
400
Imaging Details
A weak electron beam and long exposure time (40-60s) were used to minimize the vertical blooming streak in the diffraction pattern recorded with the CCD camera.
