New tools for automated high-resolution cryo-EM structure determination in RELION-3

Zivanov, Jasenko; Nakane, Takanori; Forsberg, Björn; Kimanius, Dari; Hagen, Wim J. H.; Lindahl, Erik; Scheres, Sjors H.W.

doi:10.7554/elife.42166

Cited by 4,633 publications

(3,868 citation statements)

References 66 publications

Supporting

Mentioning

3,861

Contrasting

Unclassified

Order By: Relevance

“…A larger dataset was subsequently collected with the Titan Krios and DE-20 detector, and processed with MotionCor2, Gctf and RELION-2 [18], yielding a final C6 reconstruction at 3.75 Å resolution (FSC 0.143 ) from 74,294 particle images ( Fig 1D-1F, S1 and S2 Figs). A reconstruction of the same data with application of only threefold (C3) symmetry was generated with RELION-3 [19] and reached 4.07 Å resolution ( Fig 1G, S1 and S2 Figs). The baseplate is 298 Å wide and 271 Å tall and consists of six peripheral structures surrounding a core that represents a continuation of the tail (Fig 2A and 2B).…”

Section: Three-dimensional Reconstruction Of the 80α Baseplatementioning

confidence: 99%

“…Generation of the C3 reconstruction used for modeling Tal and Dit required separation of the non-identical orientations of the C3 Tal trimer superimposed as a result of the initial assumption of C6 symmetry. The C6 reconstruction was segmented in UCSF Chimera [43] and a mask generated from all non-Tal density was used in subtracting the non-Tal density from the particle images using RELION-3.0 [19]. The resulting signal-subtracted images were C6 symmetry-expanded using relion_particle_symmetry_expand to create three copies of each particle where the Tal trimer, regardless of initial orientation, was superimposed with the 60r otated non-identical orientation.…”

Section: Three-dimensional Reconstructionmentioning

confidence: 99%

See 1 more Smart Citation

Structure of the host cell recognition and penetration machinery of a Staphylococcus aureus bacteriophage

et al. 2020

View full text Add to dashboard Cite

Staphylococcus aureus is a common cause of infections in humans. The emergence of virulent, antibiotic-resistant strains of S. aureus is a significant public health concern. Most virulence and resistance factors in S. aureus are encoded by mobile genetic elements, and transduction by bacteriophages represents the main mechanism for horizontal gene transfer. The baseplate is a specialized structure at the tip of bacteriophage tails that plays key roles in host recognition, cell wall penetration, and DNA ejection. We have used high-resolution cryo-electron microscopy to determine the structure of the S. aureus bacteriophage 80α baseplate at 3.75 Å resolution, allowing atomic models to be built for most of the major tail and baseplate proteins, including two tail fibers, the receptor binding protein, and part of the tape measure protein. Our structure provides a structural basis for understanding host recognition, cell wall penetration and DNA ejection in viruses infecting Gram-positive bacteria. Comparison to other phages demonstrates the modular design of baseplate proteins, and the adaptations to the host that take place during the evolution of staphylococci and other pathogens. Author summaryThe emergence of virulent strains of Staphylococcus aureus that are resistant to most antibiotics has become a major public health concern. Virulence and resistance determinants in S. aureus are usually carried on mobile genetic elements (MGEs). Transduction by bacteriophages provides the main means by which MGEs are disseminated horizontally through the bacterial population, and is therefore essential to the evolution of pathogenicity of S. aureus and other pathogens. The baseplate is a complex structure at the tip of bacteriophage tails that serves multiple roles, including host recognition and binding, cell wall penetration, and ejection of the phage DNA. We have determined the structure of the baseplate from bacteriophage 80α, a representative of phages involved in host pathogenicity and in the mobilization of MGEs in S. aureus. Our structure provides a basis for understanding host recognition and infection by phages infecting Gram-positive hosts, and the PLOS Pathogens | https://doi.

show abstract

Section: Three-dimensional Reconstruction Of the 80α Baseplatementioning

confidence: 99%

Section: Three-dimensional Reconstructionmentioning

confidence: 99%

Structure of the host cell recognition and penetration machinery of a Staphylococcus aureus bacteriophage

et al. 2020

View full text Add to dashboard Cite

show abstract

“…All following processing steps were performed in Relion-2-beta or Relion-3 (JASP aged 520 2) (Scheres, 2012;Zivanov et al, 2018). Particles were extracted from dose weighted, aligned 521 micrographs for all data sets using a box size of 256 px.…”

Section: Image Processing Of Cryo-em Data Sets 500mentioning

confidence: 99%

Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

Pospich

Merino

Raunser

2019

Preprint

View full text Add to dashboard Cite

Actin undergoes structural transitions during polymerization, ATP hydrolysis and subsequent release of inorganic phosphate. Several actin binding proteins sense specific states during this transition and can thus target different regions of the actin filament. Here we show in atomic detail that phalloidin, a mushroom toxin that is routinely used to stabilize and label actin filaments, suspends the structural changes in actin, likely influencing its interaction with actin binding proteins. Furthermore, high-resolution cryo-EM structures reveal structural rearrangements in F-actin upon inorganic phosphate release in phalloidin-stabilized filaments. We find that the effect of the sponge toxin jasplakinolide differs from the one of phalloidin, despite their overlapping binding site and similar interactions with the actin filament. Analysis of structural conformations of F-actin suggests that stabilizing agents trap states within the natural conformational space of actin.

show abstract

“…Interaction of such presequences (N-terminal cleavable 49 sequences that target ~60-70% mitochondrial precursor proteins) is a key step in translocation 11,[24][25][26] . A 50 cryo-electron microscopy (cryo-EM) structure of an apo form of the core TOM complex from Neurospora 51 crassa was reported 27 , but its relatively low resolution (~7-Å) offered only limited insight and precluded 52 building of an atomic model. In addition, the oligomeric architecture of the TOM complex remained a 53 puzzle.…”

Section: Introduction 22mentioning

confidence: 99%

Cryo-EM structure of the mitochondrial protein-import channel TOM complex from Saccharomyces cerevisiae

Tucker¹,

Park

2019

Preprint

View full text Add to dashboard Cite

9Nearly all mitochondrial proteins are encoded by the nuclear genome and imported into mitochondria 10 following synthesis on cytosolic ribosomes. These precursor proteins are translocated into mitochondria 11 by the TOM complex, a protein-conducting channel in the mitochondrial outer membrane. Using cryo-EM, 12we have obtained high-resolution structures of both apo and presequence-bound core TOM complexes 13from Saccharomyces cerevisiae in dimeric and tetrameric forms. Dimeric TOM consists of two copies each of 14 five proteins arranged in two-fold symmetry-Tom40, a pore-forming β-barrel with an overall negatively-15 charged inner surface, and four auxiliary α-helical transmembrane proteins. The structure suggests that 16 presequences for mitochondrial targeting insert into the Tom40 channel mainly by electrostatic and polar 17interactions. The tetrameric complex is essentially a dimer of dimeric TOM, which may be capable of 18forming higher-order oligomers. Our study reveals the molecular organization of the TOM complex and 19provides new insights about the mechanism of protein translocation into mitochondria. 20 21

show abstract

New tools for automated high-resolution cryo-EM structure determination in RELION-3

Cited by 4,633 publications

References 66 publications

Structure of the host cell recognition and penetration machinery of a Staphylococcus aureus bacteriophage

Structure of the host cell recognition and penetration machinery of a Staphylococcus aureus bacteriophage

Structural effects and functional implications of phalloidin and jasplakinolide binding to actin filaments

Cryo-EM structure of the mitochondrial protein-import channel TOM complex from Saccharomyces cerevisiae

Contact Info

Product

Resources

About