Mitigating local over-fitting during single particle reconstruction with SIDESPLITTER

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

doi:10.1016/j.jsb.2020.107545

Cited by 98 publications

(77 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…While this shares the motivation for non-uniform refinement, it relies on manual interaction during refinement, often necessitating a tedious trial and error process that can be difficult to replicate. SIDESPLITTER 31 is a recently proposed method to mitigate over-fitting during refinement. Based on estimates of signal-to-noise ratios from voxel statistics, it smooths local regions with low signal-to-noise more aggressively than indicated by a global FSC-based resolution.…”

Section: Discussionmentioning

confidence: 99%

Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction

2020

View full text Add to dashboard Cite

ingle-particle cryo-EM has transformed rapidly into a mainstream technique in biological research 1. Cryo-EM images individual protein particles, rather than crystals and has therefore been particularly useful for structural studies of integral membrane proteins, which are difficult to crystallize 2. These molecules are critical for drug discovery, targeted by more than half of drugs today 3. Membrane proteins pose challenges in cryo-EM sample preparation, imaging and computational 3D reconstruction, as they are often of small size, appear in multiple conformations, have flexible subunits and are embedded in a detergent micelle or lipid nanodisc 2. These characteristics cause strong spatial variation in structural properties, such as rigidity and disorder, across the target molecule's 3D density. Traditional cryo-EM reconstruction algorithms, however, are based on the simplifying assumption of a uniform, rigid particle. We develop an algorithm that incorporates such domain knowledge in a principled way, improving 3D reconstruction quality and allowing single-particle cryo-EM to achieve higher-resolution structures of membrane proteins. This expands the range of proteins that can be effectively studied and is especially important for structure-based drug design 4,5. We begin by formulating a cross-validation (CV) regularization framework for single-particle cryo-EM refinement and use it to account for the spatial variability in resolution and disorder found in a typical molecular complex. The framework incorporates general domain knowledge about protein molecules, without specific knowledge of any particular molecule and critically, without need for manual user input. Through this framework we derive a new algorithm called non-uniform refinement, which automatically accounts for structural variability, while ensuring that key statistical properties for validation are maintained to mitigate the risk of over-fitting during 3D reconstruction. With a graphics processing unit-accelerated implementation of non-uniform refinement in the cryoSPARC software package 6 , we demonstrate improvements in resolution and map quality for a range of membrane proteins. We show results on a 48-kDa membrane protein in lipid nanodisc with a Fab bound, a 180-kDa membrane protein complex with a large detergent micelle and a 245-kDa sodium channel complex with flexible domains. Non-uniform refinement is reliable and automatic, requiring no change in parameters between datasets and is without reliance on handmade spatial masks or manual labels. Iterative refinement and regularization. In standard cryo-EM 3D structure determination 6-8 , a generative model describes the formation of two-dimensional (2D) electron microscope images from a target 3D protein density (Coulomb potential). According to the model, the target density is rotated, translated and projected along the direction of the electron beam. The 2D projection is modulated by a microscope contrast transfer function (CTF) and corrupted by additive noise. The goal of reconstruction ...

show abstract

Section: Discussionmentioning

confidence: 99%

Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction

2020

View full text Add to dashboard Cite

show abstract

“…To aid de novo model building of cytoplasmic and luminal domains, these parts were subjected to masked focused classification (K = 5, T = 8), 3D autorefinement and post-processing in Relion, yielding improved maps at 3.4 Å and 3.2 Å, respectively. To obtain highest quality maps of the transmembrane domains, the 177560 particles from consensus refinement were processed using Sidesplitter ( Ramlaul et al, 2020 ), producing a 3.3 Å global map after Relion post-processing, where transmembrane helix pitch and side chains were well resolved and allowed for unambiguous sequence assignment. The final particle set was further subjected to 3D variability analysis ( Punjani and Fleet, 2020 ) in cryoSPARC, revealing the presence or absence of the EMC7 lumenal domain between the EMC1 beta-propellers.…”

Section: Methodsmentioning

confidence: 99%

Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients

Miller-Vedam

Bräuning

Popova

et al. 2020

eLife

111

View full text Add to dashboard Cite

Membrane protein biogenesis in the endoplasmic reticulum (ER) is complex and failure-prone. The ER membrane protein complex (EMC), comprising eight conserved subunits, has emerged as a central player in this process. Yet, we have limited understanding of how EMC enables insertion and integrity of diverse clients, from tail-anchored to polytopic transmembrane proteins. Here, yeast and human EMC cryo-EM structures reveal conserved intricate assemblies and human-specific features associated with pathologies. Structure-based functional studies distinguish between two separable EMC activities, as an insertase regulating tail-anchored protein levels and a broader role in polytopic membrane protein biogenesis. These depend on mechanistically coupled yet spatially distinct regions including two lipid-accessible membrane cavities which confer client-specific regulation, and a non-insertase EMC function mediated by the EMC lumenal domain. Our studies illuminate the structural and mechanistic basis of EMC's multifunctionality and point to its role in differentially regulating the biogenesis of distinct client protein classes.

show abstract

“…In the final refinement sequence, half maps were locally filtered between refinement iterations using SIDESPLITTER 63 , an adaptation of the LAFTER algorithm 64 that maintains gold-standard separation between the two half maps. The final model contained 403,991 particles and reached an overall resolution of 3.3 Å with side chains visible for most of the complex (Extended Data Figs.…”

Section: Data Processing and Model Buildingmentioning

confidence: 99%

Molecular basis of β-arrestin coupling to formoterol-bound β1-adrenoceptor

Lee

Warne

Nehmé

et al. 2020

Nature

223

302

View full text Add to dashboard Cite

The β 1-adrenoceptor (β 1 AR) is a G-protein-coupled receptor (GPCR) that couples 1 to the heterotrimeric G protein G s. G-protein-mediated signalling is terminated by phosphorylation of the C terminus of the receptor by GPCR kinases (GRKs) and by coupling of β-arrestin 1 (βarr1, also known as arrestin 2), which displaces G s and induces signalling through the MAP kinase pathway 2. The ability of synthetic agonists to induce signalling preferentially through either G proteins or arrestins-known as biased agonism 3-is important in drug development, because the therapeutic effect may arise from only one signalling cascade, whereas the other pathway may mediate undesirable side effects 4. To understand the molecular basis for arrestin coupling, here we ✉

show abstract

Mitigating local over-fitting during single particle reconstruction with SIDESPLITTER

Abstract: Graphical abstract

Cited by 98 publications

References 44 publications

Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction

Non-uniform refinement: adaptive regularization improves single-particle cryo-EM reconstruction

Structural and mechanistic basis of the EMC-dependent biogenesis of distinct transmembrane clients

Molecular basis of β-arrestin coupling to formoterol-bound β1-adrenoceptor

Contact Info

Product

Resources

About