EMDB-14467: 2.1A T20S Proteosome from 200kV Glacios with Selectris and Falcon 4

Basic information

• Entry: Database: EMDB / ID: EMD-14467
• Title: 2.1A T20S Proteosome from 200kV Glacios with Selectris and Falcon 4

Sample

• Complex: T20S proteosome

• Keywords: D7 symmetry / Glacios / Falcon 4 Selectris / HYDROLASE

Function / homology

Function:

proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / proteasomal protein catabolic process / ubiquitin-dependent protein catabolic process / endopeptidase activity / cytoplasm

Domain/homology:

Peptidase T1A, proteasome beta-subunit, archaeal / Proteasome alpha subunit, archaeal / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal

Component:

Proteasome subunit alpha / Proteasome subunit beta

• Biological species: Thermoplasma acidophilum (acidophilic)
• Method: single particle reconstruction / cryo EM / Resolution: 2.1 Å
• Authors: Khavnekar S / Koh FA / Kotecha A

Funding support

Netherlands, 1 items

Organization	Grant number	Country
Other private		Netherlands

• Citation: Journal: J Vis Exp / Year: 2022; Title: Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope.; Authors: Adrian Koh / Sagar Khavnekar / Wen Yang / Dimple Karia / Dennis Cats / Rob van der Ploeg / Fanis Grollios / Oliver Raschdorf / Abhay Kotecha / Daniel Němeček / ; Abstract: Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 Å resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 Å, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-Å resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

History

Deposition	Feb 28, 2022	-
Header (metadata) release	Mar 9, 2022	-
Map release	Mar 9, 2022	-
Update	Dec 13, 2023	-
Current status	Dec 13, 2023	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_14467.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Voxel size: X=Y=Z: 0.9123 Å

Density

Contour Level	By AUTHOR: 0.035 / Movie #1: 0.04
Minimum - Maximum	-0.15233749 - 0.27323306
Average (Standard dev.)	0.000051420386 (±0.012270275)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 300 300 300 Spacing 300 300 300
Cell	A=B=C: 273.69 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	0.9123	0.9123	0.9123
M x/y/z	300	300	300
origin x/y/z	0.000	0.000	0.000
length x/y/z	273.690	273.690	273.690
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	0	0	0
NX/NY/NZ	640	640	640
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	300	300	300
D min/max/mean	-0.152	0.273	0.000

Supplemental data

Mask #1

• File: emd_14467_msk_1.map

Sample components

Entire : T20S proteosome

• Entire: Name: T20S proteosome

Components

• Complex: T20S proteosome

Supramolecule #1: T20S proteosome

• Supramolecule: Name: T20S proteosome / type: complex / ID: 1 / Parent: 0
• Source (natural): Organism: Thermoplasma acidophilum (acidophilic)

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Buffer: pH: 8 / Details: 20 mM Tris pH 8.0, 50 mM NaCl, 0.1 mM EDTA.
• Vitrification: Cryogen name: ETHANE

Electron microscopy

• Microscope: TFS GLACIOS
• Electron beam: Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 0.8 µm / Nominal defocus min: 0.4 µm
• Image recording: Film or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 40.0 e/Ų

Image processing

Startup model

Type of model: EMDB MAP
EMDB ID:

6287

• Initial angle assignment: Type: MAXIMUM LIKELIHOOD
• Final angle assignment: Type: MAXIMUM LIKELIHOOD
• Final reconstruction: Applied symmetry - Point group: D₇ (2x7 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 140795