CM01: a facility for cryo-electron microscopy at the European Synchrotron

Kandiah, Eaazhisai; Giraud, Thierry; Antolinos, Alejandro De María; Dobias, Fabien; Effantin, Grégory; Flot, David; Hons, Michael; Schoehn, Guy; Susini, J.; Svensson, Olof; Leonard, Gordon A.; Mueller-Dieckmann, Christoph

doi:10.1107/s2059798319006880

Cited by 89 publications

(73 citation statements)

References 32 publications

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“…A total of 3.5 µL of sample were applied to glow discharged 1.2/1.3 Quantifoil holey carbon grids (Quantifoil Micro Tools GmbH, Großlöbichau, Germany) and they were plunged frozen in liquid ethane with a Vitrobot Mark IV (Thermo Fisher Scientific) (100% humidity, 20 °C, 6 s blot time, blot force 0). The sample was observed at the beamline CM01 of the ESRF (European Synchrotron Radiation Facility, Grenoble, France) [ 36 ] with a Titan Krios G3 (Thermo Fischer Scientific) at 300 kV equipped with an energy filter (Bioquantum LS/967, Gatan, Inc., Pleasanton, CA, USA) (slit width of 20 eV) and a Volta phase plate. Moreover, 2376 images were recorded automatically on a K2 summit direct electron detector (Gatan, Inc., USA) in super resolution mode with EPU software (Thermo Fisher Scientific).…”

Section: Methodsmentioning

confidence: 99%

Binding Mechanism Elucidation of the Acute Respiratory Disease Causing Agent Adenovirus of Serotype 7 to Desmoglein-2

Hograindleur

Effantin

Fenel

et al. 2020

Viruses

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The study of viruses causing acute respiratory distress syndromes (ARDS) is more essential than ever at a time when a virus can create a global pandemic in a matter of weeks. Among human adenoviruses, adenovirus of serotype 7 (HAdV7) is one of the most virulent serotypes. This virus regularly re-emerges in Asia and has just been the cause of several deaths in the United States. A critical step of the virus life cycle is the attachment of the knob domain of the fiber (HAd7K) to the cellular receptor desmoglein-2 (DSG2). Complexes between the fiber knob and two extracellular domains of DSG2 have been produced. Their characterization by biochemical and biophysical methods show that these two domains are sufficient for the interaction and that the trimeric HAd7K could accommodate up to three DSG2 receptor molecules. The cryo-electron microscopy (cryo-EM) structure of these complexes at 3.1 Å resolution confirmed the biochemical data, and allowed the identification of the critical amino acid residues for this interaction, which shows similarities with other DSG2 interacting adenoviruses, despite a low homology in the primary sequences.

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Section: Methodsmentioning

confidence: 99%

Binding Mechanism Elucidation of the Acute Respiratory Disease Causing Agent Adenovirus of Serotype 7 to Desmoglein-2

Hograindleur

Effantin

Fenel

et al. 2020

Viruses

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“…We acknowledge Diamond for access and support of the Cryo-EM facilities at the UK national electron bio-imaging centre (eBIC), (proposal EM 19716-1), funded by the Wellcome Trust, MRC and BBSRC, and Sarah Neumann, Andrew Howe and James Gilchrist for assistance. We also acknowledge the European Synchrotron Radiation Facility for provision of microscope time on CM01 30 and we thank Michael Hons for his assistance. This work has been supported by iNEXT, grant number 3901, funded by the Horizon 2020 program of the European Union.…”

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confidence: 99%

Ornithine capture by a translating ribosome controls bacterial polyamine synthesis

Valle

Seip

Cervera-Marzal

et al. 2019

Preprint

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Polyamines are essential metabolites that play an important role in cell growth, stress adaptation, and microbial virulence 1-3 . In order to survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues 2 . Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in γ-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF 4 , via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryo-EM structure of an Escherichia coli (E. coli) ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor RF1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.

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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.…”

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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CM01: a facility for cryo-electron microscopy at the European Synchrotron

Cited by 89 publications

References 32 publications

Binding Mechanism Elucidation of the Acute Respiratory Disease Causing Agent Adenovirus of Serotype 7 to Desmoglein-2

Binding Mechanism Elucidation of the Acute Respiratory Disease Causing Agent Adenovirus of Serotype 7 to Desmoglein-2

Ornithine capture by a translating ribosome controls bacterial polyamine synthesis

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

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