TITLE: Structural Analysis of 20S CPs and  Assembly Intermediates by Electron Cryo-Microscopy
AUTHORS: Frank Adolf

[DESCRIPTION]
This protocol details methods for structural determination by transmission electron cryo-microscopy of 20S CPs and assembly intermediates. 

[GUIDELINES]
Please familiarise yourself with the laboratory safety rules and guidelines and follow these while performing the experiment. Please wear appropriate PE while performing the experiment.

[STEPS]
SECTION: Plung  freezing of 20S CPs amd 20S CP assembly intermediates
1. Prepare Vitrobot and grids for plunging
Set up Vitrobot as follows: blot force = 3, blot time = 3 s sec, humidity 100%, temperature 4 °C
Plasma clean Quantifoil R1.2/1.3 Cu 200 grids for 45 s sec , just before plunging


SECTION: Plung  freezing of 20S CPs amd 20S CP assembly intermediates
1.1. Apply 3.5 µL of purified 20S CPs at a concentration of 0.5-0.6 mg/mL or purified and concentrated 20S CP assembly intermediates at a concentration of 4.0-5.0 mg/mL with Fos8-cholin at a finale concentration of 0.25 millimolar (mM) on grids, automatically blot and plunge in ethane/propane mix at -180 °C  with a Vitrobot Mark IV  

SECTION: Plung  freezing of 20S CPs amd 20S CP assembly intermediates
1.2. Clipp and store grids in LN2 until screening/data collection  
SECTION: cryo-EM screening and data acquisition
2. Screen cryoEM grids for particle density and ice quality on a Glacios cryo-TEM (Thermo Fisher
Scientific) or cryo-TEM of your choise
SECTION: cryo-EM screening and data acquisition
3. Data collection was carried out either on a  Glacios cryo-TEM (Thermo Fisher Scientific) operated at 200 kV equipped with a K2 Summit direct electron detector (DED) camera (Gatan) or Titan Krios G2 cryo-TEM (Thermo Fisher Scientific) operated at 300 kV equipped with a Bio Quantum post-column energy filter (Gatan, 10eV) and K3 direct electron detector (DED) camera (Gatan)

Data collection on both on the Glacios and Titan Krios G2 cryo-TEM  and was set up with SerialEM version 4.1 utilizing coma-corrected beam-image shift

 
Glacios datasets where recorded with one movie per hole  in counting mode with a 3x3 or 5x5 multi hole record acquisition scheme at a pixel size of 1.181 Å/pixel with a nominal magnification of 36000x, or at a pixel size of 1.885 Å/pixel with a nominal magnification of 22000x

A total dose of 60 e-/Å2 was fractionated over 40 frames, with a target defocus range of -1.0 μm to -2.6 μm

Krios datasets where recorded with three movie per hole  in counting mode with a 5x5 multi hole record acquisition scheme at a pixel size of 0.8512 Å/pixel with a nominal magnification of 105000x 

A total dose of 68 e-/Å2 was fractionated over 30 frames, with a target defocus range of -1.0 μm to -2.6 μm

SECTION: Processing
4. All data processing steps were performed with cryoSPARC version 4.266

 
Raw movies from Glacios datasets were patch motion corrected in cryoSPARC, and raw movies of the Titan Krios K3 dataset were on the fly motion corrected with FOCUS and subsequently imported into cryoSPARC

 
All subsequent processing steps were preformed in cryoSPARC, for detaied processing shemes see the Extended Data Figures in https://www.biorxiv.org/content/10.1101/2024.01.27.577538v1.full

Final post-processing was preformed with DeepEMhancer

 

SECTION: Model building and refinement
5. AlphaFold2 models of PAC1-4 and POMP along with corresponding chains from a published
model of the 20S CP (PDB 5LE5) were manually docked with ChimeraX version 1.5


 

 

 
Atomic models build in Coot version 0.9.8.7

 

Refinment was carried out in Phenix version 1.19.2 and ISOLDE

 

 

SECTION: Model building and refinement
6.