ISDA: 9S34

ELECTRON MICROSCOPY

Sample

20S Proteasome from P. abyssi in complex with the heptameric protein APA1 (Q9UYJ3)

Specimen Preperation

Sample Aggregation State	PARTICLE
Vitrification Instrument	FEI VITROBOT MARK IV
Cryogen Name	ETHANE-PROPANE
Sample Vitrification Details	?

3D Reconstruction

Reconstruction Method	SINGLE PARTICLE
Number of Particles	50092
Reported Resolution (Å)	3.15
Resolution Method	FSC 0.143 CUT-OFF
Other Details	50,092 particles aligning to the complex were used for Non Uniform refinement with an imposed C7 symmetry. This yielded the consensus alignment at 3.23 Angstrom, which served as a base for the focused refinements used to make this composite map. Upon reconstruction with imposed C7 symmetry, the two proteasome Beta1-2 subunits of the 20S became merged together. We noticed that the same was happening when the 20S proteasome was reconstructed without an imposed symmetry, suggesting a random distribution of the two subunit types in the Beta rings. Due to the high degree of similarity between the two proteins (>70 percent sequence identity) and the small number of particles, we opted for not continuing with further 3D classification.
Refinement Type
Symmetry Type	POINT

Map-Model Fitting and Refinement

ID	1
Refinement Space	REAL
Refinement Protocol	RIGID BODY FIT
Refinement Target	Cross correlation
Overall B Value	?
Fitting Procedure	?
Details	Model building done in coot, refinement in Phenix. No models built for the merged Beta 1-2 chains of the 20S proteasome, due to the identical folding of the two proteins and the very small (single side chains) differences between the two subunits, which prevented effective 3D classification with the small number of particles left. IMPORTANT: the outlier Y204 nonplanar bond was modelled as such based on a higher resolution X-ray model, to be deposited soon.

Data Acquisition

Detector Type	GATAN K2 SUMMIT (4k x 4k)
Electron Dose (electrons/Å²)	40

Imaging Experiment

Date of Experiment	?
Temprature (Kelvin)
Microscope Model	TFS GLACIOS
Minimum Defocus (nm)	1000
Maximum Defocus (nm)	2500
Minimum Tilt Angle (degrees)	?
Maximum Tilt Angle (degrees)	?
Nominal CS	2.7
Imaging Mode	BRIGHT FIELD
Specimen Holder Model	?
Nominal Magnification	?
Calibrated Magnification	?
Source	FIELD EMISSION GUN
Acceleration Voltage (kV)	200
Imaging Details	?

Imaging Experiment

Task	Software Package	Version
PARTICLE SELECTION	cryoSPARC	4.5
IMAGE ACQUISITION	SerialEM	?
MODEL FITTING	UCSF ChimeraX	1.8
INITIAL EULER ASSIGNMENT	cryoSPARC	?
FINAL EULER ASSIGNMENT	cryoSPARC	?
RECONSTRUCTION	cryoSPARC	4.5
MODEL REFINEMENT	PHENIX	1.21.2

Image Processing

CTF Correction Type	CTF Correction Details	Number of Particles Selected	Particle Selection Details
PHASE FLIPPING AND AMPLITUDE CORRECTION	?