Journal: Nature

Article Title: Structural insights into how Prp5 proofreads the pre-mRNA branch site

doi: 10.1038/s41586-021-03789-5

Figure Lengend Snippet: a , Early assembly of the S. cerevisiae spliceosome. Whereas Prp5 and Tat-SF1 (Cus2 in yeast) are stable components of the human 17S U2 snRNP, they appear to be less-stably associated with the yeast U2 snRNP. The spliceosome undergoes numerous structural and compositional rearrangements during its assembly and catalysis of pre-mRNA splicing , . Conserved DEXH/D-box RNA helicases are important driving forces for these rearrangements, and also ensure the proper recognition of the branch site (BS) and the 5′- and 3′-splice sites (ss) via proofreading mechanisms , . Initially an E complex is formed in an ATP-independent manner. In the yeast E complex (also denoted the commitment complex), the 5′-ss is bound by U1 snRNP, and the BS and 3′-end of the intron are bound by a heterodimer of Msl5 and Mud2. RNP rearrangements that lead to the stable association of U2 snRNP and enable the formation of a U2–BS helix—in which an adenosine is bulged, specifying it as the nucleophile for catalytic step 1 of splicing—require the ATP-dependent action of the DEAD-box RNA helicases Sub2 (refs. , ; UAP56 in humans) and Prp5 (refs. – , ). U2 nucleotides that base pair with the BS are initially sequestered in a stem-loop structure denoted the BSL , . Sub2 may free the BS region by displacing Msl5 (refs. , ), while Prp5 has been proposed to displace U2 snRNP proteins, including Cus2 (TAT–SF1 in humans), from the BSL , . This frees U2 nucleotides to base pair with the BS, and leads to the formation of the A complex with stably bound U2 snRNP. b , Structure of the BSL and U2–BS helices formed on an Act pre-mRNA wild-type (WT) BS (UACUA( A )C, where the BS-A is in bold), ΔBS-A (UACUAC ) or U257A (UACAA( A )C) branch site. Note that the exact conformation of the U257A U2–BS helix is not clear. The U2–BS helix is highlighted in purple, and the extended U2–BS helix, in which the number and nature of base-pairing interactions varies depending on the pre-mRNA intron sequence, is highlighted in yellow. c , Deletion of the BS adenosine from the Act pre-mRNA stalls splicing before the first catalytic step. Splicing was performed in two independent experiments in yeast extract for 30 min at 23 °C with wild-type (lane 1) or ΔBS-A (lane 2) Act pre-mRNA containing MS2 aptamers for affinity purification. *Position of the loading well. **Band artefact not related to pre-mRNA splicing. For gel source data, see Supplementary Fig. . d , RNA (left gels) and protein (right gels) composition of purified yeast pre-A complexes formed on ΔBS-A and U257A Act pre-mRNA. RNA and protein were analysed on NuPAGE gels and visualized by staining with SYBR Gold or Coomassie, respectively, in two independent experiments. Note that fewer picomoles of the U257A pre-A complex were loaded onto the gel and, as a consequence, proteins of lower molecular weight are poorly or not at all visible. e , Prp5 is present in both U257A and ΔBS-A pre-A complexes. Proteins from affinity-purified U257A or ΔBS-A pre-A complexes (as indicated above each lane) were analysed by western blotting in two independent experiments with antibodies against S. cerevisiae Prp5 or Lea1 (used to ensure equal loading). f , Proteins localized in the S. cerevisiae pre-A complex and their human homologues (shown in parentheses). Only U1–70K, U1–A and U1–C have been identified as stable components of human U1 snRNPs. Human homologues of Snu56 and Prp42 have not been identified. g , Residues forming the BS-A-binding pocket. The bulged BS-A is bound in a pocket composed of residues R744, N747, V783 and Y826 of Hsh155 and residue Y36 of Rds3 (refs. , , ). The BS-A ribose and 5′-phosphate are also located near Hsh155 residues K740 and K818, respectively. Most of these residues are evolutionarily highly conserved. A Hsh155 K818A mutation is lethal, as are mutations in residues of Hsh155 that contact the backbone of nucleotides directly adjacent to the bulged BS-A . However, many of the Hsh155 residues forming the BS-A binding pocket are nonessential. That is, single alanine substitutions at K740, R744, N747 and V783 do not affect yeast viability, but they do affect recognition of the branch site . Substitutions with bulkier amino acids decrease the use of nonconsensus branch sites, whereas substitutions with smaller amino acids increase usage .

Article Snippet: For a more detailed explanation, see the Methods section on ‘Image processing’. b , Typical cryo-EM micrograph (out of a total of 74,230) of the S. cerevisiae pre-A complex recorded at ×120,700 magnification with a Titan Krios microscope using a Falcon III direct electron detector operating in integration mode at a calibrated pixel size of 1.16 Å. c , Representative cryo-EM 2D class averages of the yeast pre-A complex reveal considerable flexibility between the U1 snRNP and the U2 snRNP. d , Left, local-resolution estimations of the cryo-EM reconstruction of the U1 snRNP.

Techniques: Stable Transfection, Sequencing, Affinity Purification, Purification, Staining, Molecular Weight, Western Blot, Binding Assay, Mutagenesis

Journal: Nature

Article Title: Structural insights into how Prp5 proofreads the pre-mRNA branch site

doi: 10.1038/s41586-021-03789-5

Figure Lengend Snippet: a , b , EM density map ( a ) and molecular architecture ( b ) of the S. cerevisiae spliceosomal pre-A complex. a , Purple, better-resolved U1 density; grey blue and green, better-resolved densities of the 3′- and 5′-regions of U2 snRNP; translucent grey, cryo-EM map of the pre-A complex.

Article Snippet: For a more detailed explanation, see the Methods section on ‘Image processing’. b , Typical cryo-EM micrograph (out of a total of 74,230) of the S. cerevisiae pre-A complex recorded at ×120,700 magnification with a Titan Krios microscope using a Falcon III direct electron detector operating in integration mode at a calibrated pixel size of 1.16 Å. c , Representative cryo-EM 2D class averages of the yeast pre-A complex reveal considerable flexibility between the U1 snRNP and the U2 snRNP. d , Left, local-resolution estimations of the cryo-EM reconstruction of the U1 snRNP.

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: Structural insights into how Prp5 proofreads the pre-mRNA branch site

doi: 10.1038/s41586-021-03789-5

Figure Lengend Snippet: a , Conformation of the SF3B1 and Hsh155 HEAT domains and position of the U2–BS helix and U2 snRNA SLI and SLIIa in human 17S U2 snRNP (Protein DataBank (PDB) ( https://www.rcsb.org ) accession number 6Y5Q) and in the S. cerevisiae pre-A and A complexes (PDB 6G90). These domains were aligned via Hsh155 heat repeats 19–20, Rse1 BPA and U2 SLIIa. Olive green, SLIIa nucleotides; reddish orange, pre-mRNA branch-site nucleotides; purple, BSL nucleotides that later form the U2–BS helix; yellow, BSL nucleotides forming the extended part of the U2–BS helix; dark green, remaining BSL nucleotides; blue, SLI. b , Fit of Prp5 RecA1 into the pre-A EM density. c , Location of the Prp5 RecA1 and RecA2 domains in the pre-A complex. d , Prp5 RecA1 contacts U2 snRNA nucleotides that connect the U2–BS helix to U2 SLIIa. The positions of Prp5 amino acids (located outside of the SAT motif) that when mutated suppress branch-site mutations are indicated in black.

Article Snippet: For a more detailed explanation, see the Methods section on ‘Image processing’. b , Typical cryo-EM micrograph (out of a total of 74,230) of the S. cerevisiae pre-A complex recorded at ×120,700 magnification with a Titan Krios microscope using a Falcon III direct electron detector operating in integration mode at a calibrated pixel size of 1.16 Å. c , Representative cryo-EM 2D class averages of the yeast pre-A complex reveal considerable flexibility between the U1 snRNP and the U2 snRNP. d , Left, local-resolution estimations of the cryo-EM reconstruction of the U1 snRNP.

Techniques:

Journal: Nature

Article Title: Structural insights into how Prp5 proofreads the pre-mRNA branch site

doi: 10.1038/s41586-021-03789-5

Figure Lengend Snippet: a , Left, fit of Hsh155 HEAT and Rds3 to the pre-A EM density; and right, fit of SF3B1, PHF5A and the N-terminal helix of Prp5 to the human 17S U2 density (PDB accession code 6Y5Q). Previous biochemical studies showed that the N-terminal region (NTR) of yeast Prp5 binds to HEAT repeats (HRs) 1–6 and HR 9–12 of Hsh155 (ref. ), and in the human 17S U2 snRNP cryo-EM structure, binding to SF3B1 HR 9–12 involves a long α-helix of the human Prp5 NTR (ref. ). EM density that would accommodate an analogous α-helix of the yeast Prp5 NTR is not apparent in the pre-A complex. However, CXMS indicates that the Prp5 NTR still interacts extensively with Hsh155, albeit solely with HR 1–7 (see Extended Data Fig. ). Base pairing of U2 snRNA with the pre-mRNA branch site (BS) is stabilized by the major scaffolding protein SF3B1 (Hsh155 in S. cerevisiae ) of the SF3b heteromeric complex, whose HEAT domain undergoes a conformational change during early spliceosome assembly. Hsh155 HEAT and SF3B1 HEAT exhibit an open conformation in the pre-A complex and the 17S U2 snRNP, respectively. The closed conformations of the HEAT domains of human SF3B1 and of yeast Hsh155 observed in the A to B act spliceosomal complexes are very similar. Likewise, the open conformation, which we observe here for the first time for Hsh155, also appears to be highly similar in human U2 snRNP and the S. cerevisiae pre-A complex. b , Similar spatial organization of U2 snRNA SLIIa, Prp9 ZnF (SF3A3 in humans) and Cus1 (SF3B2 in humans) in the human 17S U2 snRNP (PDB 6Y5Q) and S. cerevisiae pre-A, A (PDB 6G90) and B (PDB 5NRL) complexes. Aligned via U2 SLIIa and HR 19–20 of SF3B1/Hsh155. In the pre-A complex, SLIIb can be localized downstream of SLIIa and is bound by RRM2 of Hsh49. c , Fit of a modelled 13-base-pair extended U2–BS helix, lacking a bulged A, to EM density adjacent to SLIIa in the pre-A complex. d , Overlay of EM density accommodating the U2–BS helix in the yeast pre-A complex (grey) and EM density accommodating the BSL in human 17S U2 (green) (PDB 6Y5Q). Aligned via U2 SLIIa and HR 19–20 of SF3B1/Hsh155. The sequences of the S. cerevisiae and human U2 BSLs are highly conserved , allowing a meaningful comparison with the fit of the yeast U2–BS helix. Although the BSL in yeast and human is predicted to form a 9-base-pair stem , in the human 17S U2 snRNP, the base of the BSL stem is contacted by a short helix of SF3A3 (designated the separator helix), which ensures that the stem is only 8 base pairs in length . e , Fit in the pre-A EM density of the Prp11 ZnF at the top of the U2–BS helix. f , Protein crosslinks supporting the positioning of the Prp11 ZnF in the pre-A complex. Numbers (colour coded to match protein colours) indicate the positions of crosslinked lysine residues, which are connected by black lines. The Prp11 ZnF appears to act independently of the other SF3a proteins and to dock to the end of the extended U2–BS helix concomitantly with, or soon after, its formation. As the Prp11 ZnF has thus far been observed at this position solely after formation of the U2–BS helix, its location in the pre-A complex is consistent with the conclusion that a U2–BS helix has formed. It is likely that the Prp11 ZnF and the Prp9 separator helix may cooperate in keeping additional intron nucleotides from interacting with the U2 snRNA, and at the same time in stabilizing the end of the U2–BS helix. During clamping of the U2–BS helix by Hsh155 HEAT , Prp11 ZnF moves together with the U2–BS helix and remains associated with the end of the helix in the A, pre-B, B and B act complexes. g , Side view showing that the U2–BS helix is located further away from the C-terminal HRs of Hsh155 HEAT in the pre-A complex compared with its position in the S. cerevisiae A complex and the position of the BSL in human 17S U2 snRNP. Aligned via Hsh155 HR 19–20, Rse1 BPA and U2 SLIIa. Olive green, SLIIa nucleotides; red orange, pre-mRNA BS nucleotides; purple, BSL nucleotides that later form the U2–BS helix; yellow, BSL nucleotides forming the extended part of the U2–BS helix; dark green, the remaining BSL nucleotides; blue, SLI. Movement away from Hsh155 HEAT would be needed to free the SLI-containing 5′-end of U2 snRNA to undergo the rotational movements necessary to generate an extended U2–BS helix. During the transition from the pre-A to the A complex, the U2–BS helix moves back towards the Hsh155 C-terminal HRs such that the corresponding region of the U2 snRNA that contacts the C-terminal HRs in 17S U2 is located in a similar position in the A complex.

Article Snippet: For a more detailed explanation, see the Methods section on ‘Image processing’. b , Typical cryo-EM micrograph (out of a total of 74,230) of the S. cerevisiae pre-A complex recorded at ×120,700 magnification with a Titan Krios microscope using a Falcon III direct electron detector operating in integration mode at a calibrated pixel size of 1.16 Å. c , Representative cryo-EM 2D class averages of the yeast pre-A complex reveal considerable flexibility between the U1 snRNP and the U2 snRNP. d , Left, local-resolution estimations of the cryo-EM reconstruction of the U1 snRNP.

Techniques: Cryo-EM Sample Prep, Binding Assay, Scaffolding

Journal: Nature

Article Title: Structural insights into how Prp5 proofreads the pre-mRNA branch site

doi: 10.1038/s41586-021-03789-5

Figure Lengend Snippet: a , Domain organization of the S. cerevisiae (y) and human (h) DEAD-box helicase Prp5, with the amino-acid boundaries of each domain indicated below. b , Protein crosslinks support the positions of the Prp5 NTR and RecA domains in the pre-A complex. Numbers (colour coded to match protein colours) indicate the positions of crosslinked lysine residues, which are connected by black lines. The proposed path of Prp5 amino acids located more N-terminally of the RecA domains is indicated by a dashed line. That the Prp5 NTR and RecA1 domains, but not RecA2 (and presumably also its C-terminal region), interact with other pre-A components is consistent with previous studies showing that, after destabilization of the U2 BSL, Prp5 NTR and Prp5 RecA1 are sufficient for the subsequent ATP-independent function of Prp5 during A-complex formation . c , Two different views of the fit of the Prp5 RecA1 and RecA2 domains in an open conformation into the pre-A EM density (low-pass filtered to 10 Å). A closed conformation of the Prp5 RecA domain does not fit well to the EM density (not shown). The open conformation of Prp5 found in the pre-A complex indicates that, after ATP hydrolysis, the RecA domains are able to transit spontaneously from the closed conformation back to the open conformation while probably remaining bound to U2. d , e , The positions of the Prp5 RecA domains and the U2 3′-region plus SF3a proteins, relative to SF3b, are different in the human 17S U2 snRNP and the yeast pre-A complex. Aligned via U2 SLIIa and HR 19–20 of SF3B1/Hsh155. A cryo-EM structure of an isolated S. cerevisiae U2 snRNP is currently lacking. However, the high conservation of the sequence of yeast U2 proteins and their human homologues, and the similar structures of their conserved domains, suggests that the molecular architecture of the isolated U2 snRNP is similar in S. cerevisiae and humans. Thus, a comparison of the structures of the human 17S U2 snRNP and yeast pre-A complex reveals structural remodelling that the U2 snRNP most likely undergoes during formation of the pre-A complex. An alignment of the U2 5′-region in both complexes suggests that the U2 3′-region is repositioned after U2 stably interacts during formation of the pre-A complex. Specifically, the U2 3′-domain (that is, the 3′-region minus the SF3a core) and the Prp9 NTR rotate towards the Prp5 RecA domains, whereas the Prp11 β-sandwich and Hsh49 RRM2 move towards Prp9 ZnF . The shifted position of the U2 3′-region is stabilized by different molecular bridges formed between the U2 3′- and 5′-regions. In the pre-A complex, the bridge formed by U2-B′′ RRM2, Prp9 (human SF3A3) and Rse1 BPC (human SF3B3) in the 17S U2 snRNP (denoted bridge B) is disrupted, which allows the 3′-region to move further away from the Rse1 BPC . This then allows Hsh49 RRM2 to dock on top of the Prp9 ZnF , and by binding to Prp9 on one side and the Prp11 β-sandwich domain on the other, a new bridge involving Hsh49 RRM2 is formed. Moreover, in the isolated human 17S U2 snRNP, U2 SLIIb forms a second bridge (denoted bridge A) between the U2 3′- and 5′-regions that is not stabilized and is only poorly resolved. By contrast, in the pre-A complex, Hsh49 RRM2 now binds to the loop of SLIIb and thereby stabilizes the position of SLIIb.

Article Snippet: For a more detailed explanation, see the Methods section on ‘Image processing’. b , Typical cryo-EM micrograph (out of a total of 74,230) of the S. cerevisiae pre-A complex recorded at ×120,700 magnification with a Titan Krios microscope using a Falcon III direct electron detector operating in integration mode at a calibrated pixel size of 1.16 Å. c , Representative cryo-EM 2D class averages of the yeast pre-A complex reveal considerable flexibility between the U1 snRNP and the U2 snRNP. d , Left, local-resolution estimations of the cryo-EM reconstruction of the U1 snRNP.

Techniques: Cryo-EM Sample Prep, Isolation, Sequencing, Stable Transfection, Binding Assay

Journal: Nature

Article Title: Structural insights into how Prp5 proofreads the pre-mRNA branch site

doi: 10.1038/s41586-021-03789-5

Figure Lengend Snippet: a , Two different views of the spatial organization of the yeast U1 snRNP, with the density shown on the left and the molecular model on the right. b , The U1–5′ss helix is stabilized in the pre-A complex by Luc-7 and Yhc1, in the same manner as in the yeast E and A complexes. Top, fit of the U1–5′ss helix plus Luc7 and Yhc1 to the pre-A EM density. Bottom, the U1–5′ss helix and adjacent proteins. c , Fit of the Prp40 FF1–6 domains in the pre-A EM density. Top, domain organization of the S. cerevisiae Prp40 protein; below, amino-acid boundaries of each domain. WW, domain containing two conserved tryptophans that are spaced 20–22 amino acids apart; FF, domain containing two conserved phenylalanines at its N and C termini. d , Protein crosslinks between Prp40, Prp5 NTR and other pre-A-complex proteins. Numbers (colour coded to match protein colours) indicate the positions of crosslinked lysine residues, which are connected by black lines. Prp40, Snu71 and Luc7 form a stable trimer that in the cryo-EM structure of the yeast E complex bridges the U1 snRNP to the branch site, and we show here that they also help to bridge U1 snRNP to U2 during the early stages of prespliceosome formation. In humans and in Schizosaccharomyces pombe , Prp5 facilitates formation of the A complex by bridging the U1 and U2 snRNPs , with the Prp5 N-terminal RS domain interacting with proteins of the SF3b complex . Although S. cerevisiae Prp5 lacks an N-terminal RS domain, CXMS data indicate that its N terminus also interacts with Snu71 and Rse1 BPB . Therefore, the bridge formed by Rse1 and the Prp40–Luc7–Snu71 trimer in the S. cerevisiae pre-A complex probably serves as an anchoring point for Prp5’s N terminus.

Article Snippet: For a more detailed explanation, see the Methods section on ‘Image processing’. b , Typical cryo-EM micrograph (out of a total of 74,230) of the S. cerevisiae pre-A complex recorded at ×120,700 magnification with a Titan Krios microscope using a Falcon III direct electron detector operating in integration mode at a calibrated pixel size of 1.16 Å. c , Representative cryo-EM 2D class averages of the yeast pre-A complex reveal considerable flexibility between the U1 snRNP and the U2 snRNP. d , Left, local-resolution estimations of the cryo-EM reconstruction of the U1 snRNP.

Techniques: Cryo-EM Sample Prep

Journal: Nature

Article Title: Structural insights into how Prp5 proofreads the pre-mRNA branch site

doi: 10.1038/s41586-021-03789-5

Figure Lengend Snippet: a , Computation sorting scheme, with all major image-processing steps depicted. For a more detailed explanation, see the Methods section on ‘Image processing’. b , Typical cryo-EM micrograph (out of a total of 27,502) of the S. cerevisiae U257A pre-A complex recorded at ×120,700 magnification with a Titan Krios microscope using a Falcon III direct electron detector operating in integration mode at a calibrated pixel size of 1.16 Å. c , Representative cryo-EM 2D class averages of the yeast U257A pre-A complex. d , FSC calculated using the ‘Post-processing’ routine in RELION indicates a global resolution of 10.4 Å for the entire yeast U257A pre-A complex, and resolutions of 7.5 Å and 13 Å for the multibody-refined U1 and U2 regions, respectively. The global resolution was lower than that of the ΔBS-A pre-A complex, mainly because of the lower number of particles analysed. e , Overlay of the EM densities of the ΔBS-A (purple) and U257A (grey) pre-A complexes. f , Fit of the 3D model of the ΔBS-A pre-A complex into the EM density of the U257A pre-A complex. Note that, for both complexes, density encompassing Prp5 is first observed at a lower threshold. An extended U2–BS helix has also formed in complexes formed on the U257A mutant. However, the precise conformation of the helix cannot be discerned. The Hsh155 HEAT domain is in an open conformation and Prp5 is still bound at the same position, and the same U1–U2 bridges are also observed, indicating that the U257A complexes are also stalled at the same pre-A stage. g , Fit of the extended U2–BS helix from the ΔBS-A pre-A complex into the U257A pre-A EM density. h , Fit of the Prp5 RecA domains and U2–BS helix from the ΔBS-A pre-A model into the EM density of the U257A pre-A complex. i , Fit of the Prp40 FF domains and Rse1 BPB (which comprise bridge 1) from the ΔBS-A pre-A model into the EM density of the U257A pre-A complex.

Article Snippet: For a more detailed explanation, see the Methods section on ‘Image processing’. b , Typical cryo-EM micrograph (out of a total of 74,230) of the S. cerevisiae pre-A complex recorded at ×120,700 magnification with a Titan Krios microscope using a Falcon III direct electron detector operating in integration mode at a calibrated pixel size of 1.16 Å. c , Representative cryo-EM 2D class averages of the yeast pre-A complex reveal considerable flexibility between the U1 snRNP and the U2 snRNP. d , Left, local-resolution estimations of the cryo-EM reconstruction of the U1 snRNP.

Techniques: Cryo-EM Sample Prep, Microscopy, Mutagenesis

Journal: Nature

Article Title: Structural insights into how Prp5 proofreads the pre-mRNA branch site

doi: 10.1038/s41586-021-03789-5

Figure Lengend Snippet: Molecular organization of U1 and U2 snRNPs in S. cerevisiae pre-A, A (PDB 6G90) and pre-B complexes (PDB 5ZWM and PDB 5ZWN). Movements of U1 and U2 snRNPs during the pre-A to A transition are indicated by curved arrows. All structures were aligned as in Fig. . For simplicity, the U1 snRNA stem-loops in the poorly resolved region of the U1 snRNP are not shown in the pre-A, A and pre-B complexes.

Article Snippet: For a more detailed explanation, see the Methods section on ‘Image processing’. b , Typical cryo-EM micrograph (out of a total of 74,230) of the S. cerevisiae pre-A complex recorded at ×120,700 magnification with a Titan Krios microscope using a Falcon III direct electron detector operating in integration mode at a calibrated pixel size of 1.16 Å. c , Representative cryo-EM 2D class averages of the yeast pre-A complex reveal considerable flexibility between the U1 snRNP and the U2 snRNP. d , Left, local-resolution estimations of the cryo-EM reconstruction of the U1 snRNP.

Techniques:

Journal: Nature

Article Title: Structural insights into how Prp5 proofreads the pre-mRNA branch site

doi: 10.1038/s41586-021-03789-5

Figure Lengend Snippet: a , Close-up of the rotation of the U2 3′-region after the release of Prp5. The 3′-region rotates around the indicated axis by roughly 55°. To better show the movement of the 3′-region, the SmD2 protein is in yellow. For simplicity, only the 3′-region of U2 plus U2 SLII and the U2–BS helix are shown in the pre-A complex and the yeast A complex (PDB 6G90). The pre-A and A complexes are aligned via U2 SLIIa and HR 19–20 of Hsh155. b , Close-up of the movement of the U1 snRNP and 3′-region of U2. A top view is shown, with the black dot indicating the pivot point of the U2 3′-region, which rotates by roughly 55° in the plane of the paper. For simplicity, only the region of U1 snRNP that contains Prp39 is shown. The U1 snRNP rotates around the indicated axis by roughly 45°. In the pre-A complex, Prp39 and Lea1 are separated by roughly 130 Å, but the movements of U1 and U2 bring them into close proximity in the A complex. Even though Lea1 is not essential in S. cerevisiae , its depletion prevents formation of the A complex, and adding back Lea1 restores A-complex assembly . The Prp39–Lea1 interaction is a structural marker for the formation of a mature A complex, and as such its absence in the pre-A complex is a clear indication that our complex has stalled at an earlier assembly stage. This interaction is also maintained in the pre-B complex and is therefore also a structural marker for the conformation that allows joining of the tri-snRNP.

Article Snippet: For a more detailed explanation, see the Methods section on ‘Image processing’. b , Typical cryo-EM micrograph (out of a total of 74,230) of the S. cerevisiae pre-A complex recorded at ×120,700 magnification with a Titan Krios microscope using a Falcon III direct electron detector operating in integration mode at a calibrated pixel size of 1.16 Å. c , Representative cryo-EM 2D class averages of the yeast pre-A complex reveal considerable flexibility between the U1 snRNP and the U2 snRNP. d , Left, local-resolution estimations of the cryo-EM reconstruction of the U1 snRNP.

Techniques: Marker

Journal: Frontiers in photonics

Article Title: Incoherent color holography lattice light-sheet for subcellular imaging of dynamic structures

doi: 10.3389/fphot.2023.1096294

Figure Lengend Snippet: The ICHLLS system. (A,B) Schematics of the ICHLLS systems with (C) one diffractive lens (ICHLLS 1L) of focal length f SLM,488nm = 400 mm or f SLM,561nm = 415 mm at the phase shift θ = 0; (D) The axis orientation in the ICHLLS 1L system; (E) The top-view of the lattice beams inside the ICHLLS 1L; (F) The OpticStudio schematics used to calculate the focal distances for the ICHLLS 1L; (G) Schematics of the ICHLLS systems with two diffractive lenses (ICHLLS 2L) with focal lengths f d1,488nm = 220 mm , f d2,488nm = 2356 mm, f d1,561nm = 228 mm, and f d2,561nm = 2444 mm, at the phase shift θ = 0, superposed with a slight defocus to bring the objects in focus in the middle of the camera FOV; (H) The z-galvo positions in the ICHLLS 2L system; (I) The top-view of the lattice beams superposed with Fresnel diffractive lenses inside the ICHLLS 2L; (J) The OpticStudio schematics used to calculate the focal distances for the ICHLLS 2L; The system consists of a water immersed microscope objective MO (Nikon ×25, NA1.1, WD 2 mm), lenses L 1 = L 4 with focal lengths 175 mm, L 2 = L 3 with focal lengths 100 mm; mirrors M 1 , M 2 , M 3 ; polarizer P oriented at a 45° angle ( ) to the active axis of the SLM ( ); band pass filters BPF 1 centered at 519 nm (Chroma Tech, 26 nm bandpass width) for the excitation wavelength λ = 488 nm, and BPF 2 centered at 575 nm (Chroma Tech, 23 nm bandpass width) for the excitation wavelength λ = 561 nm; phase spatial light modulator SLM (Meadowlark Inc.). The light propagates through either pathway LLS emission (dotted black line in A) for the original LLS or pathway ICHLLS (red and green line in A) for ICHLLS, depending on the orientation of sliding mirror. A collimated 30 Bessel beam is focused by an excitation objective lens (A,B) which generates a lattice light sheet. While the z-galvo (light sheet) and z-piezo (detection objective) are moved along the z-axis to acquire stacks in LLS and IHLLS1L, (D,E) , in IHLLS 2L only the z-galvo is moved at various z positions (B,H,I) . For IHLLS, the size of the beam coming out the objective is diminished in half by the relay lens system, L 1 and L 2 , to fit the size of the SLM. The SLM plane is optically conjugated with the objective back-focal-plane. The diffraction mask in the original LLS system was positioned for all experiments on the anulus of 0.55 outer NA and 0.48 inner NA. The CMOS camera, tube lens, filter, and detection objective lens are used for fluorescence detection. The detection magnification is 62.5. The width of the light sheet in the center of the FOV is about 400 nm. x -axis is the direction of the x-galvo mirror motion, z -axis is the direction of the z-piezo mirror motion, and s-axis is the direction of excitation light propagation. For values of distances d1 to d8, see .

Article Snippet: Each hologram processing can be sub-divided as follows: 1) Image pre-processing in which a background correction is performed by subtracting an average background level obtained by measuring the mean intensity of each stain outside the cells; 2) Hologram reconstruction in which the complex hologram is propagated and reconstructed at the best focal plane using a custom diffraction method routine in MATLAB (MathWorks, Inc.); and 3) Object feature extraction performed on the reconstructed amplitude and phase images using FIJI (ImageJ).

Techniques: Microscopy, Fluorescence

Journal: Frontiers in photonics

Article Title: Incoherent color holography lattice light-sheet for subcellular imaging of dynamic structures

doi: 10.3389/fphot.2023.1096294

Figure Lengend Snippet: The ICHLLS system diffraction. (A) The 9 z-galvo position hologram intensity images: z galvo = ± 40μm± 30μm; ± 20 μm; ± 10 μm,; 0 μm; (B) Hologram intensity images at θ = 0, for each z-galvo level; (C) Example of hologram reconstruction at different z-galvo positions; (D) The reconstructed amplitude and phase at two z-galvo positions; z galvo = −40 μm and 0μm. The FOV is 208 μm 2 × 208 μm 2 and pixel size 0.101 μm.

Article Snippet: Each hologram processing can be sub-divided as follows: 1) Image pre-processing in which a background correction is performed by subtracting an average background level obtained by measuring the mean intensity of each stain outside the cells; 2) Hologram reconstruction in which the complex hologram is propagated and reconstructed at the best focal plane using a custom diffraction method routine in MATLAB (MathWorks, Inc.); and 3) Object feature extraction performed on the reconstructed amplitude and phase images using FIJI (ImageJ).

Techniques: