ELECTRON MICROSCOPY

Sample

KIF1A R350W bound to microtubules in two-heads-bound state with AMP-PNP

Specimen Preperation
Sample Aggregation State FILAMENT
Vitrification Instrument FEI VITROBOT MARK IV
Cryogen Name ETHANE
Sample Vitrification Details 4uL of a 6uM MT solution in BRB80 (80mM PIPES, 2mM MgCl2, 1mM EGTA pH 6.8) along with 20uM paclitaxel was added onto a plasma-cleaned grid. The MTs were incubated for 1 minute at room temperature, and then the excess liquid was removed from the grid using Whatman #1 paper. Next, 4uL of a solution containing 17.5uM KIF1A-R350W in BRB80 supplemented with 20uM paclitaxel and 5mM AMP-PNP was added to the grid. The grid with the MT and kinesin mixture was then mounted into a Vitrobot, incubated for 1 minute at room temperature, and plunge-frozen into liquid nitrogen-cooled ethane.
3D Reconstruction
Reconstruction Method HELICAL
Number of Particles 556086
Reported Resolution (Å) 3.12
Resolution Method FSC 0.143 CUT-OFF
Other Details ?
Refinement Type
Symmetry Type HELICAL
Map-Model Fitting and Refinement
ID 1
Refinement Space REAL
Refinement Protocol OTHER
Refinement Target ?
Overall B Value ?
Fitting Procedure ?
Details ?
Data Acquisition
Detector Type GATAN K3 (6k x 4k)
Electron Dose (electrons/Å2) 52.14
Imaging Experiment
Date of Experiment ?
Temprature (Kelvin)
Microscope Model TFS KRIOS
Minimum Defocus (nm) 800
Maximum Defocus (nm) 2500
Minimum Tilt Angle (degrees) ?
Maximum Tilt Angle (degrees) ?
Nominal CS 0.001
Imaging Mode BRIGHT FIELD
Specimen Holder Model FEI TITAN KRIOS AUTOGRID HOLDER
Nominal Magnification 64000
Calibrated Magnification ?
Source FIELD EMISSION GUN
Acceleration Voltage (kV) 300
Imaging Details ?
Imaging Experiment
Task Software Package Version
PARTICLE SELECTION cryoSPARC 4.6.0
MODEL FITTING UCSF Chimera ?
MODEL FITTING Rosetta ?
MODEL REFINEMENT PHENIX ?
RECONSTRUCTION cryoSPARC 4.6.0
Image Processing
CTF Correction Type CTF Correction Details Number of Particles Selected Particle Selection Details
PHASE FLIPPING AND AMPLITUDE CORRECTION ?
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