Software tools for automated transmission electron microscopy

Schorb, Martin; Haberbosch, Isabella; Hagen, Wim J. H.; Schwab, Yannick; Mastronarde, David N.

doi:10.1038/s41592-019-0396-9

Cited by 464 publications

(345 citation statements)

References 46 publications

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“…Fluorescence microscopy maps were loaded using a point-based registration to low mag tiled acquisitions (2,300x) of the corresponding grid squares. High-magnification tilt series (15,500x) were then acquired at the selected fluorescent spot locations, using a Serial EM script (Schorb et al, 2019). Tomograms were then reconstructed using the IMOD software package (Kremer et al, 1996).…”

Section: Correlative Light and Electron Microscopy (Clem)mentioning

confidence: 99%

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

Choudhary

Atab

Mizzon

et al. 2020

Preprint

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Lipid droplets (LDs) are fat storage organelles that originate from the endoplasmic reticulum (ER).Relatively little is known about how sites of LD formation are selected, and which proteins/lipids are necessary for the process. Here, we show that LDs induced by the yeast triacylglycerol (TAG)synthases Lro1 and Dga1 are formed at discrete ER subdomains defined by seipin (Fld1), and a regulator of diacylglycerol (DAG) production, Nem1. Fld1 and Nem1 colocalize to ER-LD contact sites. We find that Fld1 and Nem1 localize to ER subdomains independently of each other and of LDs, but both are required for the subdomains to recruit the TAG synthases and additional LD biogeneiss factors: Yft2, Pex30, Pet10, and Erg6. These subdomains become enriched in DAG.We conclude that Fld1 and Nem1 are both necessary to recruit proteins to ER subdomains where LD biogenesis occurs.10-, 30-60-, 90-, 120-min ( Fig S4F), at 10-min, 2 h, 4 h, 21 h (Fig. 1C), and at 21 h (Fig. 1D, 4D, S1G). Cells were lysed using a Precellys 24 homogenizier, lipids were extracted as described and equal aliquots were brought to dryness. To quantify TAG and SE, lipids were spotted onto silica gel 60 thin layer chromatography (TLC) plates (Merck, Darmstadt, Germany) and developed with petroleum ether/diethylether/acetic acid (70:30:2; per vol).Glycerophospholipids were separated by TLC as described (Vaden et al., 2005). TLC plates were exposed to a tritium-sensitive screen, visualized and quantified using a phosphorimager (GE Healthcare). Brightness and contrast of the TIFF images were adjusted using Image J (NIH). Western blot analysisCells were grown over night in SC raffinose media, diluted to 1 OD600 ml in SC galactose media and cells corresponding to 3 OD600 units were harvested at 0-, 2-, 4-, 6-, 21-h time points.Samples were extracted by NaOH, and proteins were precipitated using trichloroacetic acid (TCA), and resuspended into sample buffer (Horvath and Riezman, 1994). Proteins were separated using SDS-PAGE and detected using a monoclonal antibody against GFP (Roche Diagnostics, Rotkreuz, Switzerland, dilution 1:2000), or Kar2 (Santa Cruz Biotechnology, Dallas, TX, dilution 1:5000) and using horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, #2302, dilution 1:10000). Western blot experiments were performed at least twice with similar results. Quantification and statistical analysisUnless otherwise stated, data were obtained from at least three independent experiments.The images presented are representative of the results obtained. Quantitative results are expressed

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Section: Correlative Light and Electron Microscopy (Clem)mentioning

confidence: 99%

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

Choudhary

Atab

Mizzon

et al. 2020

Preprint

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“…Grids were plunged into liquid nitrogen cooled ethane. Movies were collected using SerialEM (Schorb et al, 2019) on a Titan Krios (Thermo Fisher) operation at 300 kV, equipped with BioQuantum Energy Filter (20 eV slit width, Gatan) and a K3 direct electron detector (Gatan). SIgA movies were collected at 130,000 magnification in super resolution mode with calibrated pixel size of 0.326 Å/pixel, 40 frames per movie, 0.03 s per frame, and total dose of ~60 electrons/Å 2 .…”

Section: Cryoem Grid Preparation and Data Collectionmentioning

confidence: 99%

The Structures of Secretory and Dimeric Immunoglobulin A

Bharathkar

Parker

Malyutin

et al. 2020

Preprint

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Secretory (S) Immunoglobulin (I) A is the predominant mucosal antibody, which binds pathogens and commensal microbes. SIgA is a polymeric antibody, typically containing two copies of IgA that assemble with one joiningchain (JC) to form dimeric (d) IgA that is bound by the polymeric Ig-receptor ectodomain, called secretory component (SC). Here we report the cryo-electron microscopy structures of murine SIgA and dIgA. Structures reveal two IgAs conjoined through four heavy-chain tailpieces and the JC that together form a β-sandwich-like fold. The two IgAs are bent and tilted with respect to each other, forming distinct concave and convex surfaces.In SIgA, SC is bound to one face, asymmetrically contacting both IgAs and JC. The bent and tilted arrangement of complex components limits the possible positions of both sets of antigen binding fragments (Fabs) and preserves steric accessibility to receptor binding sites, likely influencing antigen binding and effector functions.

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“…A high quality cryo-EM grid pre-screened on a 200kV Thermofischer Glacios microscope was used to collect data on a Thermofischer Titan Krios G3 operated at 300 kV equipped with a Gatan Bioquantum LS/967 energy filter (slit width of 20 eV) coupled to a Gatan K2 direct electron detector camera 23 . Automated data collection was performed with SerialEM using a beam-tilt data collection scheme 24 , acquiring one image per hole from 9 holes before moving the stage. Micrographs were recorded in super-resolution mode at a 165,000x magnification giving a pixel size of 0.4135 Å with defocus ranging from −0.8 to −3.5 µm.…”

Section: Methodsmentioning

confidence: 99%

Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes

Arragain

Effantin

Gerlach

et al. 2020

Preprint

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18Bunyavirales is an order of segmented negative stranded RNA viruses comprising several life-19 threatening pathogens such as Lassa fever virus (Arenaviridae), Rift Valley Fever virus 20 (Phenuiviridae) and La Crosse virus (LACV, Peribunyaviridae) against which neither specific 21 treatment nor licenced vaccine is available. Replication and transcription of Bunyavirales 22 genome constitute essential reactions of their viral cycle that are catalysed by the virally 23 encoded RNA-dependent RNA polymerase or L protein. Here we describe the complete high-24 2 resolution cryo-EM structure of the full-length (FL) LACV-L protein. It reveals the presence of 25 key C-terminal domains, notably the cap-binding domain that undergoes large movements 26 related to its role in transcription initiation and a zinc-binding domain that displays a fold not 27 previously observed. We capture the structure of LACV-L FL in two functionally relevant states, 28 pre-initiation and elongation, that reveal large conformational changes inherent to its 29 function. We uncover the coordinated movement of the polymerase priming loop, lid domain 30 and C-terminal region required for the establishment of a ten-base-pair template-product 31 RNA duplex before strand separation into respective exit tunnels. The revealed structural 32 details and dynamics of functional elements will be instrumental for structure-based 33 development of compounds that inhibit RNA synthesis by the polymerase. 34 35 INTRODUCTION 36Segmented negative stranded RNA viruses (sNSV) are divided into two major orders: 37Bunyavirales, that comprises more than 500 species classified in twelve families 1 , and 38Articulavirales, that include influenza virus from the Orthomyxoviridae family. Replication and 39 transcription of sNSV viral genomic segments are performed by the virally encoded RNA-40 dependent RNA polymerase or L protein 2 . These processes are performed in the cytoplasm of 41 infected cells for Bunyaviruses, whereas they occur in the nucleus for influenza virus 3,4 . 42Replication generates full-length genome or antigenome copies (vRNA and cRNA 43 respectively), whereas transcription produces capped viral mRNA that are recognized by the 44 cellular translation machinery to produce viral proteins. Transcription is initiated by a "cap-45 snatching" mechanism, whereby host 5ʹ capped RNAs are bound by the L cap-binding domain 46 (CBD), cleaved by the L endonuclease domain several nucleotides downstream, and then used 47 to prime synthesis of mRNA 2,5,6 . Although the overall mechanism of transcription initiation is 48 3 likely conserved between sNSVs, several elements suggest some divergences between viral 49 families. First, the source of capped RNA differs. Whereas influenza polymerase interacts 50 directly with the host polymerase II to snatch the caps of nascent transcripts in the nucleus 7 , 51 it is currently unclear which and in what context cytoplasmic capped RNAs are accessed by 52 bunyavirus polymerases and if the polymerase contains specific domains that w...

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Software tools for automated transmission electron microscopy

Cited by 464 publications

References 46 publications

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

Seipin and Nem1 establish discrete ER subdomains to initiate yeast lipid droplet biogenesis

The Structures of Secretory and Dimeric Immunoglobulin A

Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes

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