ELECTRON MICROSCOPY

Sample

human ASCT2 SLC1A5

Specimen Preperation
Sample Aggregation State PARTICLE
Vitrification Instrument FEI VITROBOT MARK II
Cryogen Name ETHANE
Sample Vitrification Details ?
3D Reconstruction
Reconstruction Method SINGLE PARTICLE
Number of Particles 184080
Reported Resolution (Å) 3.85
Resolution Method FSC 0.143 CUT-OFF
Other Details A total of 6345 dose-fractionated cryo-EM images were recorded and subjected to motion-correction and dose-weighting of frames by MotionCor2. The CTF parameters were estimated on the movie frames by ctffind4.1. Bad images showing contamination, a defocus below or above 0.4 and -3um or a bad CTF estimation were discarded, resulting in 4863 images used for further analysis with the software package RELION2.1. About 3000 particles were picked manually to generate 2D references which where improved in several rounds of autopick. A low threshold was used during the final autopick step to ensure that no particles are missed yielding more than a million particles. Particles were extracted with a box size of 240 pixels, and initial classification steps were performed with three-fold binned data. False positives or bad particles were removed in first rounds of 2D classification, resulting in 628,015 particles that were further sorted in several rounds of 3D classification. A map generated from the GltPh structure (PDB ID 3KBC) was used as reference for the first round, and the best output class was used in subsequent jobs in an iterative way. The best 3D class, comprising 184,080 particles from 4859 images, was subjected to auto-refinement, yielding a map with a resolution of 4.26 A before masking and 3.91 A after masking. Particles were further polished in RELION version 2.1 and subjected to another round of 2D and 3D classification resulting in a final dataset of 133,437 particles. The final polished map had a resolution of 4.26 A before masking and 3.85 A after masking. The map was sharpened using an isotropic B-factor of -171 A2, for manual inspection a B-factor of -225 A2 was used. The approach of focused refinement, where the less-resolved detergent micelle was subtracted from the particle images, did not improve resolution. During 3D classification and auto-refinement jobs a C3-symmetry was imposed. To check for conformational heterogeneity of the data, where single protomers within the trimer might adopt a different conformation, 3D classifications with no symmetry imposed were performed at different stages of image processing. We further performed 3D classification on the individual protomers of a single transporter using symmetry expansion and signal subtraction. Both approaches showed no indication of the existence of a different conformation. Local resolution estimates were estimated by RELION. All resolutions were estimated using the 0.143 cut-off criterion with gold-standard Fourier shell correlation (FSC) between two independently refined half maps. During post-processing, the approach of high-resolution noise substitution was used to correct for convolution effects of real-space masking on the FSC curve.
Refinement Type
Symmetry Type POINT
Map-Model Fitting and Refinement
ID 1
Refinement Space REAL
Refinement Protocol AB INITIO MODEL
Refinement Target ?
Overall B Value ?
Fitting Procedure ?
Details ?
Data Acquisition
Detector Type GATAN K2 SUMMIT (4k x 4k)
Electron Dose (electrons/Å2) 0.87
Imaging Experiment
Date of Experiment ?
Temprature (Kelvin)
Microscope Model FEI TALOS ARCTICA
Minimum Defocus (nm) 0.4
Maximum Defocus (nm) 2.5
Minimum Tilt Angle (degrees) ?
Maximum Tilt Angle (degrees) ?
Nominal CS 2.7
Imaging Mode BRIGHT FIELD
Specimen Holder Model FEI TITAN KRIOS AUTOGRID HOLDER
Nominal Magnification 49407
Calibrated Magnification 49407
Source FIELD EMISSION GUN
Acceleration Voltage (kV) 200
Imaging Details ?
Imaging Experiment
Task Software Package Version
IMAGE ACQUISITION EPU ?
CTF CORRECTION CTFFIND 4.1
MODEL FITTING Coot ?
MODEL REFINEMENT PHENIX ?
INITIAL EULER ASSIGNMENT RELION 2.1
FINAL EULER ASSIGNMENT RELION 2.1
RECONSTRUCTION RELION 2.1
Image Processing
CTF Correction Type CTF Correction Details Number of Particles Selected Particle Selection Details
PHASE FLIPPING AND AMPLITUDE CORRECTION ?
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