High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

Moriya, Toshio; Saur, Michael; Stabrin, Markus; Merino, Felipe; Voicu, Horatiu; Huang, Zhong; Penczek, Pawel A.; Raunser, Stefan; Gatsogiannis, Christos

doi:10.3791/55448

Cited by 183 publications

(223 citation statements)

References 25 publications

Supporting

Mentioning

223

Contrasting

Order By: Relevance

“…However, ClpXP1/2 dimers ( Supplementary Figure 1a-d) have, to our knowledge, not been described so far. We therefore concentrated our structural analysis first on these intriguing dimers and determined their structure by cryo-EM and single particle analysis using crYOLO (21) and SPHIRE (22) ( Figure 1a-b, Supplementary Fig. 1e-g).…”

Section: Cryo-em Structure Of Clpxp1/2mentioning

confidence: 99%

Cryo-EM structure of the ClpXP protein degradation machinery

Gatsogiannis

Balogh

Merino

et al. 2019

Preprint

View full text Add to dashboard Cite

The ClpXP machinery is a two component protease complex performing targeted protein degradation in bacteria and eukaryotes. The complex consists of the AAA+ chaperone ClpX and the peptidase ClpP. The hexameric ClpX utilizes the energy of ATP binding and hydrolysis to engage, unfold and translocate substrates into the catalytic chamber of tetradecameric ClpP where they are degraded. Formation of the complex involves a symmetry mismatch, since hexameric AAA+ rings bind axially to the opposing stacked heptameric rings of the tetradecameric ClpP. Here we present the first high-resolution cryo-EM structure of ClpXP from Listeria monocytogenes. We unravel the heptamerhexamer binding interface and provide novel insights into the ClpX-ClpP crosstalk and activation mechanism. The comparison with available crystal structures of ClpP and ClpX in different states allows us to understand important aspects of ClpXP's complex mode of action and provides a structural framework for future pharmacological applications.

show abstract

Section: Cryo-em Structure Of Clpxp1/2mentioning

confidence: 99%

Cryo-EM structure of the ClpXP protein degradation machinery

Gatsogiannis

Balogh

Merino

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…TcdA1 assembles in the soluble prepore state in a large bell-like shaped pentamer with a molecular weight of 1.4 MDa 18 . TcdA1 has been our test specimen to develop the software package SPHIRE 23 . We previously used a dataset of 7,475 particles of TcdA1 to obtain a reconstruction at a resolution of 3.5 Å 23 .…”

Section: Test Datasetsmentioning

confidence: 99%

SPHIRE-crYOLO: A fast and accurate fully automated particle picker for cryo-EM

Wagner¹,

Merino²,

Stabrin³

et al. 2018

Preprint

Self Cite

265

315

View full text Add to dashboard Cite

Selecting particles from digital micrographs is an essential step in single particle electron cryomicroscopy (cryo-EM). Since manual selection of complete datasets typically comprising many thousands of particles is a tedious and time-consuming process, many automatic particle pickers have been developed in the past few decades.However, non-ideal datasets pose a challenge to particle picking. Here, we present a novel automated particle picking software called crYOLO, which is based on the deep learning object detection system "You Only Look Once" (YOLO). After training the network with 500 -2,500 particles per dataset, it automatically recognizes particles with high recall and precision reaching a speed of up to five micrographs per second.Importantly, we demonstrate a powerful general network trained on more than 40 datasets to select previously unseen datasets, thus paving the way for completely automated "on-the-fly" cryo-EM data pre-processing during data acquisition. CrYOLO is available as a standalone program under http://sphire.mpg.de/ and will be part of the image processing workflow in SPHIRE.

show abstract

“…We determined the structure of the BBSome core complex by electron cryo microscopy (cryo-EM) and single particle analysis in SPHIRE 27 . A reconstruction of the full data set yielded a resolution of 3.8 Å and besides BBS5 that was only sub-stoichiometrically bound (Figure S1A) all domains were well resolved with only some connecting loops and N-and C-terminal regions missing ( Figure 1A, Figure S1, S3).…”

Section: Architecture Of the Bbsome Corementioning

confidence: 99%

Structure of the human BBSome core complex in the open conformation

Klink

Gatsogiannis

Hofnagel

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

The BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a cargo adapter that recognizes signaling proteins such as GPCRs and links them to the intraflagellar transport machinery. The underlying mechanism is poorly understood.Here we present a high-resolution cryo-EM structure of a human heterohexameric core subcomplex of the BBSome. The structure reveals the architecture of the complex in atomic detail. It explains how the subunits interact with each other and how disease-causing mutations hamper this interaction. The complex adopts a conformation that is open for binding to membrane-associated GTPase Arl6 and a large positively charged patch likely strengthens the interaction with the membrane. A prominent negatively charged cleft at the center of the complex is likely involved in binding of positively charged signaling sequences of cargo proteins.

show abstract

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

Cited by 183 publications

References 25 publications

Cryo-EM structure of the ClpXP protein degradation machinery

Cryo-EM structure of the ClpXP protein degradation machinery

SPHIRE-crYOLO: A fast and accurate fully automated particle picker for cryo-EM

Structure of the human BBSome core complex in the open conformation

Contact Info

Product

Resources

About