(A) 33-nt ITS2 RNA with 5′-Cy5 and 3′-Cy3 labels. (B) In vitro RNA cleavage assay using 5′-Cy5 and 3′-Cy3-labeled 33-nt RNA. (C) In vitro RNA cleavage assay of unlabeled 33-nt RNA. (D) 81-nt ITS2 …
Original files for the RNA cleavage analysis in Figure 1B (5′-Cy5, 3′-Cy3, Merger).
Original scans of the relevant RNA cleavage analysis in Figure 1B (5′-Cy5, 3′-Cy3, Merger) with band and sample labels.
Original file for the RNA cleavage analysis in Figure 1C.
Original scan of the relevant RNA cleavage analysis in Figure 1C with band and sample labels.
Original file for the RNA cleavage analysis in Figure 1E.
Original scan of the relevant RNA cleavage analysis in Figure 1E with band and sample labels.
Original file for the RNA cleavage analysis in Figure 1G and H.
Original scan of the relevant RNA cleavage analysis in Figure 1G and H with band and sample labels.
Original files for the RNA cleavage analysis in Figure 1—figure supplement 1 (5′-Cy5, 3′-Cy3, Merger).
Original scans of the relevant RNA cleavage analysis in Figure 1—figure supplement 1 (5′-Cy5, 3′-Cy3, Merger) with band and sample labels.
Original files for the RNA cleavage analysis in Figure 1—figure supplement 2 (5′-Cy5, 3′-Cy3, Merger).
Original scans of the relevant RNA cleavage analysis in Figure 1—figure supplement 2 (5′-Cy5, 3′-Cy3, Merger) with band and sample labels.
Original files for the RNA cleavage analysis in Figure 1—figure supplement 3 (5′-Cy5, 3′-Cy3, Merger).
Original scans of the relevant RNA cleavage analysis in Figure 1—figure supplement 3 (5′-Cy5, 3′-Cy3, Merger) with band and sample labels.
Original file for the metal-independent RNA cleavage analysis in Figure 1—figure supplement 4.
Original scan of the relevant metal-independent RNA cleavage analysis in Figure 1—figure supplement 4 with band and sample labels.
(A) Domain organization of ScLas1 and ScGrc3. (B) Ribbon representations of ScLas1-Grc3 complex. Color coding used for Las1 and Grc3 is identical to that used in (A). (C) Surface representations of …
(A) Representative motion-corrected cryo-EM micrograph. (B) Reference-free 2D class averages. (C) Workflow of the data processing. (D) Euler angle distribution of the particle images. (E) Gold …
(A) Domain organization of CjLas1 and CjGrc3. (B) Top: ribbon representations of CjLas1-Grc3 complex. Color coding used for Las1 and Grc3 is identical to that used in (A). Bottom: a diagram showing …
(A) Representative motion-corrected cryo-EM micrograph. (B) Reference-free 2D class averages. (C) Workflow of the data processing. (D) Euler angle distribution of the particle images. (E) Gold …
(A) Superposition of the ScLas1-Grc3 complex crystal structure with the cryo-EM structure. Color coding used for ScLas1 and ScGrc3 in crystal structure is identical to that used in Figure 2A. ScLas1 …
The ScLas1 protein was aligned with its respective homologs. Alignment was performed using the MUSCLE program. The secondary structures of ScLas1 and CjLas1 are shown above and below the sequences, …
(A) Structural comparison between ScLas1-Grc3 complex and CjLas1-Grc3 complex. Color coding used for ScLas1 and ScGrc3 is identical to that used in Figure 2A. The CC domain of CjLas1 is colored in …
Original files for the RNA cleavage and binding analysis in Figure 4C and D.
Original scans of the relevant RNA cleavage and binding analysis in Figure 4C and D with band and sample labels.
(A) The crystal structure shows that ScGrc3 GCT binds at an active channel of ScLas1 HEPN dimer. Two HEPN domains of Las1 are colored in pink and violet, respectively. GCTs of Grc3 are colored in …
Original files for enzymatic assay and GST pull-down analysis in Figure 5D–F.
Original scans of the relevant enzymatic assay and GST pull-down analysis in Figure 5D–F with band and sample labels.
Original files for the RNA cleavage analysis in Figure 5—figure supplement 4.
Original scans of the relevant RNA cleavage analysis in Figure 5—figure supplement 4 with band and sample labels.
(A) The crystal structure shows that ScLas1 LCT binds to the CTD domain of ScGrc3. ScGrc3 is shown as surface, ScLas1 LCT is shown as stick. (B) Detailed interactions between C-terminal residues of …
All cleavage experiments were repeated three times.
Original file for the RNA cleavage analysis in Figure 6—figure supplement 2.
Original scan of the relevant RNA cleavage analysis in Figure 6—figure supplement 2 with band and sample labels.
All experiments were repeated three times.
Original file for the GST pull-down analysis in Figure 6—figure supplement 3.
Original scan of the relevant GST pull-down analysis in Figure 6—figure supplement 3 with band and sample labels.
(A) Crystal structure of CjLas1 HEPN domain. (B) The HEPN3 molecule (in slate) and its symmetry-related molecule (in blue white) in Las1 HEPN domain structure. (C) The HEPN1 (in pink) and HEPN2 (in …
Inset: a magnified view of the comparison of the catalytic site in the two structures.
(A) Structural comparison of catalytic sites between ScLas1-Grc3 complex (Las1A HEPN in pink, Las1B HEPN in violet) and CtLas1-Grc3 complex in state 1 (in gray, PDB code: 6of3). (B) Structural …
Prior to assembly with Grc3, Las1 shows weak processing activity for ITS2 precursor RNA. When combined with Grc3 to form a tetramer complex, Las1 shows high processing activity for ITS2 precursor …
ScLas1-Grc3 | CjLas1-Grc3 | CjLas1 | |
---|---|---|---|
Data collection* | |||
Space group | C2 | C2221 | P212121 |
Cell dimensions | |||
a, b, c (Å) | 233.6, 116.1, 159.3 | 152.6, 240.0, 237.0 | 51.5, 59.0, 158.7 |
α,β,γ (°) | 90.0, 96.4, 90.0 | 90.0, 90.0, 90.0 | 90.0, 90.0, 90.0 |
Resolution (Å) | 50.00–3.50 (3.56–3.50) | 50.00–3.23 (3.29–3.23) | 50.00–1.80 (1.83–1.80) |
Rmerge | 0.298 (0.980) | 0.344 (0.958) | 0.103 (0.929) |
I/σI | 4.8 (1.1) | 4.3 (1.6) | 22.0 (2.5) |
Completeness (%) | 98.8 (96.5) | 99.9 (99.9) | 97.7 (95.8) |
Redundancy | 4.5 (3.5) | 7.5 (6.4) | 10.1 (9.5) |
Refinement | |||
Resolution (Å) | 3.69 | 3.39 | 1.80 |
No. reflections | 36,773 | 41,321 | 44,296 |
Rwork/Rfree | 0.2798/0.3151 | 0.3041/0.3281 | 0.2120/0.2334 |
No. atoms | |||
Protein | 22,873 | 18,763 | 3657 |
Water | 180 | 386 | 212 |
B-factors (Å2) | |||
Protein | 95.5 | 125.6 | 23.3 |
Water | 33.2 | 55.9 | 28.5 |
R.m.s. deviations | |||
Bond length (Å) | 0.008 | 0.011 | 0.015 |
Bond angles (°) | 1.516 | 1.785 | 1.500 |
Ramachandran plot | |||
Favored region | 94.96 | 95.72 | 97.98 |
Allowed region | 4.86 | 4.28 | 2.02 |
Outlier region | 0.18 | 0.00 | 0.00 |
Highest resolution shell is shown in parentheses.
ScLas1-Grc3 | CjLas1-Grc3 | |
---|---|---|
Data collection and processing | ||
Microscope | Titan Krios | Titan Krios |
Voltage (kV) | 300 | 300 |
Camera | Gatan K3 | Gatan K3 |
Magnification | 105,000× | 105,000× |
Pixel size (Å) | 0.82 | 0.82 |
Total exposure (e-/Å2) | 50 | 50 |
Exposure time (s) | 3 | 3 |
Number of frames per exposure | 30 | 30 |
Energy filter slit width (keV) | 20 | 20 |
Data collection software | EPU | EPU |
Defocus range (μm) | –1.3 to –2.7 | –1.2 to –3 |
Number of micrographs | 2520 | 8616 |
Number of initial particles | 525,213 | 2,215,555 |
Symmetry | C2 | C2 |
Number of final particles | 264,341 | 523,843 |
Resolution (0.143 gold standard FSC, Å) | 3.07 | 3.39 |
Local resolution range (Å) | 2.8–4.8 | 2.8–4.8 |
Microscope | Titan Krios | Titan Krios |
Refinement | ||
Model composition | ||
Nonhydrogen atoms | 11,212 | 10,013 |
Protein residues | 1426 | 1339 |
B-factors (Å2) | ||
Protein | 92.58 | 85.58 |
R.m.s. deviations | ||
Bond length (Å) | 0.009 | 0.004 |
Bond angles (°) | 1.055 | 0.849 |
Validation | ||
MolProbity score | 2.90 | 2.69 |
Clashscore | 20.56 | 18.60 |
Rotamer outliers (%) | 8.75 | 7.86 |
Ramachandran plot | ||
Favored region | 94.46 | 96.38 |
Allowed region | 4.83 | 3.31 |
Outlier region | 0.71 | 0.31 |