Structural Analysis of 20S CPs and Assembly Intermediates by Electron Cryo-Microscopy

Frank Adolf

Published: 2024-02-07 DOI: 10.17504/protocols.io.x54v9px14g3e/v1

Abstract

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

Steps

Plung freezing of 20S CPs amd 20S CP assembly intermediates

Prepare Vitrobot and grids for plunging

Set up Vitrobot as follows: blot force = 3, blot time = 0h 0m 3s sec, humidity 100%, temperature 4°C
Plasma clean Quantifoil R1.2/1.3 Cu 200 grids for 0h 0m 45s sec , just before plunging

1.1.

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

1.2.

Clipp and store grids in LN2 until screening/data collection

cryo-EM screening and data acquisition

Screen cryoEM grids for particle density and ice quality on a Glacios cryo-TEM (Thermo Fisher

Scientific) or cryo-TEM of your choise

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

Citation

Mastronarde DN 2005 Automated electron microscope tomography using robust prediction of specimen movements. https://doi.org/

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

Processing

All data processing steps were performed with cryoSPARC version 4.266

Citation

Punjani A, Rubinstein JL, Fleet DJ, Brubaker MA 2017 cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. https://doi.org/10.1038/nmeth.4169

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

Citation

Biyani N, Righetto RD, McLeod R, Caujolle-Bert D, Castano-Diez D, Goldie KN, Stahlberg H 2017 Focus: The interface between data collection and data processing in cryo-EM. https://doi.org/10.1016/j.jsb.2017.03.007

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

Citation

Sanchez-Garcia R, Gomez-Blanco J, Cuervo A, Carazo JM, Sorzano COS, Vargas J 2021 DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. https://doi.org/10.1038/s42003-021-02399-1

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

Citation

Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, Bridgland A, Meyer C, Kohl SAA, Ballard AJ, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D 2021 Highly accurate protein structure prediction with AlphaFold. https://doi.org/10.1038/s41586-021-03819-2

Citation

Jumper J, Hassabis D 2022 Protein structure predictions to atomic accuracy with AlphaFold. https://doi.org/10.1038/s41592-021-01362-6

Citation

Goddard TD, Huang CC, Meng EC, Pettersen EF, Couch GS, Morris JH, Ferrin TE 2018 UCSF ChimeraX: Meeting modern challenges in visualization and analysis. https://doi.org/10.1002/pro.3235

Atomic models build in Coot version 0.9.8.7

Citation

Emsley P, Lohkamp B, Scott WG, Cowtan K 2010 Features and development of Coot. https://doi.org/10.1107/S0907444910007493

Refinment was carried out in Phenix version 1.19.2 and ISOLDE

Citation

Liebschner D, Afonine PV, Baker ML, Bunkóczi G, Chen VB, Croll TI, Hintze B, Hung LW, Jain S, McCoy AJ, Moriarty NW, Oeffner RD, Poon BK, Prisant MG, Read RJ, Richardson JS, Richardson DC, Sammito MD, Sobolev OV, Stockwell DH, Terwilliger TC, Urzhumtsev AG, Videau LL, Williams CJ, Adams PD 2019 Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. https://doi.org/10.1107/S2059798319011471

Citation

Croll TI 2018 ISOLDE: a physically realistic environment for model building into low-resolution electron-density maps. https://doi.org/10.1107/S2059798318002425