6T72
Structure of the RsaA N-terminal domain bound to LPS
ELECTRON MICROSCOPY
Sample |
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Structure of RsaA N-terminal domain bound to LPS |
Specimen Preparation | |
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Sample Aggregation State | PARTICLE |
Vitrification Instrument | FEI VITROBOT MARK IV |
Cryogen Name | ETHANE |
Sample Vitrification Details | Vitrobot options: Blot time 3 seconds, Blot force -13,1, Wait time 10 seconds, Drain time 0.5 seconds, |
3D Reconstruction | |
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Reconstruction Method | SINGLE PARTICLE |
Number of Particles | 115776 |
Reported Resolution (Å) | 3.7 |
Resolution Method | FSC 0.143 CUT-OFF |
Other Details | Particles from two main 3D classes containing 21 or 20 RsaA subunits were combined for a focused 3D auto refinement on the central 14 subunits using t ... |
Refinement Type | |
Symmetry Type | POINT |
Point Symmetry | C1 |
Map-Model Fitting and Refinement | |||||
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Id | 1 | ||||
Refinement Space | RECIPROCAL | ||||
Refinement Protocol | BACKBONE TRACE | ||||
Refinement Target | Best fit | ||||
Overall B Value | 85.819 | ||||
Fitting Procedure | |||||
Details | The carbon backbone of the RsaA protein was manually traced through a single subunit of the cryo-EM density using Coot (Emsley et al., 2010). Initiall ... |
Data Acquisition | |||||||||
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Detector Type | GATAN K2 SUMMIT (4k x 4k) | ||||||||
Electron Dose (electrons/Å**2) | 43 |
Imaging Experiment | 1 |
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Date of Experiment | |
Temperature (Kelvin) | |
Microscope Model | FEI TITAN KRIOS |
Minimum Defocus (nm) | -1000 |
Maximum Defocus (nm) | -4000 |
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 | 130000 |
Calibrated Magnification | 130000 |
Source | FIELD EMISSION GUN |
Acceleration Voltage (kV) | 300 |
Imaging Details | EPU software |
EM Software | ||
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Task | Software Package | Version |
PARTICLE SELECTION | RELION | 3.0 |
IMAGE ACQUISITION | EPU | |
CTF CORRECTION | CTFFIND | 4.1.13 |
MODEL FITTING | Coot | 0.9-pre |
INITIAL EULER ASSIGNMENT | RELION | 3.0 |
FINAL EULER ASSIGNMENT | RELION | 3.0 |
CLASSIFICATION | RELION | 3.0 |
RECONSTRUCTION | RELION | 3.0 |
MODEL REFINEMENT | REFMAC | 5 |
Image Processing | ||||
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CTF Correction Type | CTF Correction Details | Number of Particles Selected | Particle Selection Details | |
PHASE FLIPPING AND AMPLITUDE CORRECTION | RELION refinement with in-built CTF correction. The function is similar to a Wiener filter, so amplitude correction included. | 129633 | Particles were automatically picked from the motion and CTF corrected micrographs using the AutoPick function in Relion 3.0 (Zivanov et al., 2018). As particle reference a 3 dimensional reconstruction from an earlier dataset with different pixelsize was used which was reconstructed using an unbiased subtomogram average structure of the same sample. |