A Bayesian approach to single-particle electron cryo-tomography in RELION-4.0

Figures

Figure 1 with 3 supplements

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Figure 1—figure supplement 1

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Figure 1—figure supplement 2

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Figure 1—figure supplement 3

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Figure 2 with 1 supplement

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Figure 2—figure supplement 1

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Figure 3

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Tables

Appendix 2—table 1

	HIV-1 Gag(EMD-16207 /EMD-16209)	S-layer_inner_domain(EMD-16183)(PDB 8BQE)	COPII inner coat(EMD-15949)(PDB 8BSH)
Data collection
Microscope	Titan Krios	Titan Krios	Titan Krios
Detector	K2 (Gatan)	K2 (Gatan)	K2 (Gatan)
Software	SerialEM (Mastronarde, 2003)	SerialEM (Mastronarde, 2003)	SerialEM (Mastronarde, 2003)
Voltage (kV)	300	300	300
Slit width (eV)	20	20	20
Defocus range ( μm)	–1.5 to –5.0	–1.5 to –5.0	–1.5 to –4.5
Pixel size (Å)	1.35	1.35	1.33
Total exposure (${\displaystyle e^{-}/{\text{AA}}^2}$)	120–145	∼140	∼120
Exposure per tilt ( ${\displaystyle e^{-}/{\text{AA}}^2}$)	3.0–3.5	3.4	2.9
Total number of tilts	41	41	41
Frames per tilt-movie	8–10	10	10
Tilt increment	$\pm 3^{\circ }$	$\pm 3^{\circ }$	$\pm 3^{\circ }$
Tilt-series scheme	Dose-symmetrical	Dose-symmetrical	Dose-symmetrical
Tilt range	$\pm {60}^{\circ }$	$\pm {60}^{\circ }$	$\pm {60}^{\circ }$
Tilt-series (no.)	5/43	110	137
Data processing
Software tilt-series alignment	IMOD (Kremer et al., 1996)	IMOD (Kremer et al., 1996)	Dynamo (Castaño-Díez et al., 2012)
Software CTF estimation	CTFPLOTTER (Xiong et al., 2009)	CTFFIND4 (Rohou and Grigorieff, 2015)	CTFFIND4 (Rohou and Grigorieff, 2015)
Particle images (no.)	12,910/144,275	42,990	106,533
Pre-cropped box-size (pix)	512	600	512
Final box-size (pix)	192	180	196
Pixel size final rec. (Å)	1.35	1.35	1.33
Symmetry imposed	C6	C6	C1
Map resolution (Å)	3.2/3.0	3.5	3.8
FSC threshold	0.143	0.143	0.143
Map resolution range (Å)	3.2–4.3/3.0–3.5	3.5–4.8	3.8–7.2
Map sharpening B factor (Å 2)	–85 / –95	–75	–106
Model refinement
Initial model used (PDB code)		6T72	6GNI
Software		PHENIX (Afonine et al., 2018)	Isolde (Croll, 2018) and PHENIX (Afonine et al., 2018)
Model resolution (Å)		3.6	4.0
FSC threshold		0.5	0.5
Model composition
Non-hydrogen atoms (no.)		11,274	13,635
Protein residues (no.)		1452	1729
R.m.s. deviations
Bond lengths (Å)		0.001	0.004
Bond angles (°)		0.322	0.858
Validation
MolProbity score		1.13	2.01
Clashscore		3.42	14.71
Poor rotamers (%)		0	0.66
C $\beta$ outliers (%)		0	0.00
CABLAM outliers (%)		0.84	2.33
Ramachandran plot
Favoured (%)		98.8	95.1
Allowed (%)		1.2	4.8
Disallowed (%)		0	0.1

Appendix 2—table 2

	HIV-1 Gag	S-layer inner domain	COPII inner coat
Tilt-series (no.)	5/43	110	137
Final particle images (no.)	12,910/144,275	42,990	106,533
Pre-cropped box-size (pix)	512	600	512
Final box-size (pix)	192	180	196
Computational costs
Pseudo-subtomogram
Compute time	21 min/40 min	34 min	67 min
Number of CPU nodes	1/1	1	1
Disk space	343 GB/3.8 TB	777 GB	3.1 TB
Refine3D
Compute time	18 hr*/33 hr	14 hr	57 hr
Number of GPU nodes	1/1	1	1
Ctf refinement
Compute time	15 min*/35 min	2 hr	2 hr
Number of CPU nodes	1/1	1	1
Disk space	32 MB/247 MB	621 MB	673 MB
Frame alignment
Compute time	2 hr*/12 hr	2 hr	6 hr
Number of CPU nodes	1/1	1	1
Disk space	383 MB/4.1 GB	1.9 GB	2.2 GB
Hardware specifics
CPU nodes
CPU model	2x Intel Xeon E5-2698 v4	2x Intel Xeon 6258R	1x AMD EPYC 7H12
CPU memory	512 GB	754 GB	256 GB
GPU nodes
CPU model	2x Intel Xeon Silver 4116	2x Intel Xeon E5-2667 v4	1x AMD EPYC 7H12
CPU memory	384 GB	256 GB	256 GB
GPU model	2x Nvidia Quadro RTX 5000	4x Nvidia GeForce GTX 1080 Ti	4x Nvidia RTX A6000

1. *

   These calculations were performed using the same hardware as for the S-layer inner domain.

Additional files

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