Mitigating Local Over-fitting During Single Particle Reconstruction with SIDESPLITTER

Ramlaul, Kailash; Palmer, Colin M.; Aylett, C.H.S.

doi:10.1101/2019.12.12.874081

Cited by 5 publications

(4 citation statements)

References 36 publications

(54 reference statements)

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“…S3a). This is in line with improvements recently demonstrated using different approaches to local map filtering that do not rely on conventional FSC-based estimates 29,30 .…”

Section: Map Denoising and Local Resolutionsupporting

confidence: 88%

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Multi-particle cryo-EM refinement withMvisualizes ribosome-antibiotic complex at 3.7 Å inside cells

Tegunov

Xue²,

Dienemann

et al. 2020

Preprint

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Cryo-electron microscopy (cryo-EM) enables macromolecular structure determination in vitro and in situ. In addition to aligning individual particles, accurate registration of sample motion and 3D deformation during exposures is crucial for achieving high resolution. Here we describe M, a software tool that establishes a reference-based, multi-particle refinement framework for cryo-EM data and improves the results of structure determination. M provides a unified optimization framework for both in vitro frame series and in situ tomographic tilt series data. We show that tilt series data can provide the same resolution as frame series, indicating that the alignment step no longer limits the resolution obtainable from tomographic data. In combination with Warp and RELION, M improves upon previous methods, and resolves a 70S ribosome bound to an antibiotic inside bacterial cells at a nominal resolution of 3.7 Å. Thus, computational tools are now available to resolve structures from tomographic in situ cryo-EM data at residue level.Correspondence should be addressed to D.T. (dteguno@mpibpc.mpg.de), P.C. (patrick.cramer@mpibpc.mpg.de), and J.M. (julia.mahamid@embl.de). Figure 1| Th e Warp-RELION-M pipeline for frame and tilt series cryo-EM data refi nement Electron microscopy data are pre-processed on-the-fl y in Warp, which then exports particles as images or sub-tomograms. Particles are imported in RELION, where they can be subjected to a multitude of processing strategies, resulting in 3D reference maps, global particle pose alignments, and class assignments. Th e particle population encompassing all classes is then imported in M, where reference-based frame or tilt image alignments are performed simultaneously with further refi nement of particle poses and CTF parameters to improve resolution. Finally, M produces high-resolution reconstructions that can be used for model building. Alternatively, the improved alignments can be used in Warp to re-export particles for further, more accurate classifi cation in RELION.

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“…S3a). This is in line with improvements recently demonstrated using different approaches to local map filtering that do not rely on conventional FSC-based estimates 29,30 .…”

Section: Map Denoising and Local Resolutionsupporting

confidence: 88%

“…Once introduced, the noise pattern can become amplified over multiple iterations, leading to overestimated local resolution and phantom features that can be misinterpreted. More advanced regularization schemes have been proposed 29,30 since to deal with this problem.…”

Section: Map Denoisingmentioning

confidence: 99%

Multi-particle cryo-EM refinement withMvisualizes ribosome-antibiotic complex at 3.7 Å inside cells

Tegunov

Xue²,

Dienemann

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…The initial particle pool was extracted in 524pixel boxes and pruned through a series of 2D and 3D classification rounds. In the case of the ADP-Pdr5 dataset, an improvement in the signal-to-noise ratio of the protein part of the Pdr5/peptidisc assembly was achieved using SIDESPLITTER-1.2 71 which locally de-noises cryo-EM maps 72 and 3D map reconstructions were done externally to RELION-3.1 between the iterative steps of 3D auto-refinement. The overall resolution of the final maps obtained by the combined use of SIDESPLITTER-1.2 and RELION-3.1 was 2.85 Å (ADP-Pdr5) and 3.13 Å (R6G-Pdr5), per the FSC 0.143 criterion.…”

Section: Discussionmentioning

confidence: 99%

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

Harris

Wagner

et al. 2021

Nat Commun

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Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux through uncoupled hydrolysis of nucleotides, enabling organisms such as baker’s yeast and pathogenic fungi to survive in the presence of chemically diverse antifungal agents. Here, we present the molecular structure of Pdr5 solved with single particle cryo-EM, revealing details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens new avenues for the development of effective antifungal compounds.

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“…The initial particle pool was extracted in 524-pixel boxes and pruned through a series of 2D and 3D classification rounds. In the case of the ADP-Pdr5 dataset, an improvement in the signal-to-noise ratio of the protein part of the Pdr5/peptidisc assembly was achieved using SIDESPLITTER ( 62 ) which locally de-noises cryo-EM maps ( 63 ) and 3D map reconstructions were done externally to RELION-3.1 between the iterative steps of 3D auto-refinement. The overall resolution of the final maps obtained by the combined use of SIDESPLITTER and RELION-3.1 was 2.85 Å (ADP-Pdr5) and 3.13 Å (R6G-Pdr5), per the FSC 0.143 criterion.…”

Section: Methodsmentioning

confidence: 99%

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

Harris

Wagner

et al. 2021

Preprint

View full text Add to dashboard Cite

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Through the coupling of nucleotide hydrolysis with drug efflux, Pdr5 homologues enable pathogenic species to survive in the presence of chemically diverse antifungal agents. Our structural and functional results reveal details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens avenues for the development of effective antifungal compounds.

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Mitigating Local Over-fitting During Single Particle Reconstruction with SIDESPLITTER

Cited by 5 publications

References 36 publications

Multi-particle cryo-EM refinement withMvisualizes ribosome-antibiotic complex at 3.7 Å inside cells

Multi-particle cryo-EM refinement withMvisualizes ribosome-antibiotic complex at 3.7 Å inside cells

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5

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