FRISBI
Lionel Imbert, IBS, Grenoble, France
Title: Overview of cell-free protein production & developments for NMR samples
Martin Pelosse, EMBL Grenoble
Title: Eukaryotic expression strategies at the EEF.
Registration: https://us06web.zoom.us/webinar/register/WN_jtzZvMCbRT2cwq3lZWX_PQ
Lionel Imbert, IBS, Grenoble, France
Title: Overview of cell-free protein production & developments for NMR samples
Summary: Cell-free expression is used to produce protein samples for structural biology studies. It uses the translation and transcription machinery of E. coli to synthesize proteins in an open environment, i.e. without the bacterial cell wall. For NMR applications, isotopically-labelled amino acids can be used directly in the reaction to synthesize only the protein of interest. Rapid production and simplified purification processes have enabled many structures to be solved by NMR, but there are a number of limitations. Firstly, the size of the proteins to be studied with uniform labelling cannot exceed 20 kDa. Secondly, for higher molecular weight proteins, a larger quantity of protein needs to be synthesized at reasonable cost. Developments made on the IBS/ISBG cell-free platform to perform perdeuteration in water, selective protonation, therapeutic antibody fragment production and position-specific labelling will be among others presented.
Martin Pelosse, EMBL Grenoble
Title: Eukaryotic expression strategies at the EEF.
Summary: Structural and biochemical characterization of large protein complexes is a prerequisite for better understanding biological functions at a molecular level. However recombinant production of such large samples can sometimes be cumbersome. At the EEF, users have access to modular cloning systems facilitating the assembly of large expression vectors as well as different strategies for the expression of their protein complexes. Additional tools completing traditional baculovirus expression vector systems (BEVS) and developed at the EEF, will be introduced.
Structural Approach to characterize the Metabolic Activation Pathway Of a Nucleotide analogue : From Pro-Drug to Target
Speaker: François Ferron (AFMB, Marseille)
Thursday 17 October 2024 at 01 pm
Registration: https://us06web.zoom.us/meeting/register/tZwoceygrDksEtTQQMT2sikO8CrxQft_sSoN
Summary: Bemnifosbuvir (AT-527) is a guanosine analog currently in clinical trial phase III against SARS-CoV-2, and phase II (with Ruzasvir) against HCV (1,2). This purine nucleotide analogue (NA) is modified at three positions relative to their natural nucleotide counterparts: the purine N6, the 2'-ribose, and the 5'-phosphate.
Here we show this NAs require a minimal set of 5 enzymes, namely CatA/CES1 esterase, HINT1, ADALP1, GUK1, and NDPK for activation to their common 5'-triphosphate active form AT-9010, with an obligate order of reactions prior hitting the main component of the Sars-CoV2 replication complex. AT-9010 inhibits essential viral activities enzymes, accounting for broad spectrum antiviral potency (3). Enzyme assays together with structural data at atomic resolution illuminate N6-purine deamination compatible with metabolic activation by the human ADALP1 enzyme. Crystal structures of human HINT1, ADALP1, GUK1, and NDPK at 2.09, 2.44, 1.76, and 1.9 Å resolution, respectively, with respective precursors of AT-9010 complete the five-steps activation pathway from the orally available drug Bemnifosbuvir to AT-9010, pointing to key drug-protein contacts along the activation pathway as well as chemical positions candidate for further modification on the purine nucleotide.
Our work provides a structural and functional framework to integrate antiviral nucleotide analogue design from drug bio-availability and metabolic activation to antiviral potency.
Ref : Chazot A, Zimberger C, Feracci M, Moussa A, Good S, Sommadossi JP, Alvarez K, Ferron F, Canard B. The activation cascade of the broad-spectrum antiviral bemnifosbuvir characterized at atomic resolution. PLoS Biol. 2024 Aug 27;22(8):e3002743. doi: 10.1371/journal.pbio.3002743
The three national infrastructures ProFi, FranceBioImaging and FRISBI along with the GIS IBiSA are pleased to welcome you to Strasbourg for the 1st edition of a feedback meeting in relation to a funded access call to IBiSA-labelled facilities. This meeting will be prefaced by the 8th FRISBI annual users meeting November 18th https://frisbi.eu/ and followed by the FBI annual meeting November 20-21th https://france-bioimaging.org/fbi-special-events/fbi-annual-meeting-2024/. The organizing committee
8h30 ARRIVAL
9h-10h: Welcome: IBiSA – FRISBI-ProFI-FBI and Laureate presentation
10h-10h20: X. MANIVAL : Insights into the R2SP quaternary chaperone by XL-MS and cryo-EM.” (Protéomique / Biologie structurale)
10h20-10h40: S. MERABET Samir (Lyon) : A nanobody based approach to capture interactomes of dimeric protein complexes in living cells". (Protéomique/Imagerie)
10h40-11h20: BREAK
11h20-11h40: I.KRIMM : “Understanding the role of protein kinase CK2 using inhibitors: the contribution of biophysical techniques and proteomic studies “ (Protéomique / Biologie structurale)
11h-40-12h: M. SUSKIEWICZ “Higher-order multimerisation: insights from ZBTB and SIAH/SINA families” (Biologie structurale/Imagerie)
12h-12h20: G. LABESSE: “Dynamic structural biology of MgtC virulence factor to assess the regulatory role of magnesium and phosphate” (Biologie structurale/Protéomique).
12h20-14h: LUNCH
14h-14h20: M. BETERMIER: Proximity labeling identifies a developmental SMC complex required for programmed DNA elimination in Paramecium (Protéomique/Biologie structurale/Imagerie)
14h20-14h40: S. QUEVILLON-CHERUEL: DciA, the Bacterial Replicative Helicase Loader, promotes LLPS in the presence of ssDNA (Biologie structurale/Imagerie)
14h40-15h20: Round table: feedback from laureates & attendees / Q&A
15h30: Closing
Meeting registration: Registration is free of charge but you must be registered to attend the meeting. Deadline for registration: October 27th, 2024 Venue: IGBMC auditorium, Ilkirch, https://www.igbmc.fr/en/igbmc/life-at-igbmc/visiting-the-igbmc
REGISTRATION is CLOSED for on site but you can still register for remote.
We offer the meeting in remote, please register here https://us06web.zoom.us/webinar/register/WN_dtfGpNwrQyyB007Y-gZ2cA
Thursday 12 September 2024 at 01 pm, we will a have a FRISBI/ReNaFoBis webinar with 2 speakers:
Alicja Razew (IBS, Grenoble)
Tittle: Fighting Antibiotic Resistance: How to Monitor Drug-Protein Interactions in the Bacterial Periplasm
Christina Sizun (ICSN, Gif sur Yvette)
Tittle: A dual binding mode between RSV NS1 and MED25 contributes to reshaping of antiviral responses
Please register here: https://us06web.zoom.us/webinar/register/WN_z6OlN_U9S-S2V92TlwD8Dg
A dual binding mode between RSV NS1 and MED25 contributes to reshaping of antiviral responses Christina Sizun (ICSN, Gif sur Yvette)
Respiratory syncytial virus (RSV), responsible for pneumonia in infants, elicits a weak innate immune response. This is partially mediated by the non-structural NS1 protein of RSV. We and others found that NS1 interacts with the MED25 subunit of the Mediator complex. NS1 binds to the MED25-ACID domain, targeted by transcription activators. This suggests that NS1 could modulate host transcription, by an unknown mechanism, as NS1 lacks a DNA binding domain. NS1 consists of a globular core domain and a C-terminal helix α3. α3 was reported to be involved in modulation of host gene expression. The α3 peptide binds to the “H2” transactivation domain (TAD)-binding face of MED25-ACID, with micromolar affinity. However, the affinity of full-length NS1 is 15 nM. Structural predictions for a heterodimer by AlphaFold2 hint at a dual binding mode, involving both TAD-binding faces of MED25-ACID, “H1” and “H2”, as well as α3 and the core domain of NS1. This rationalizes binding observed for the NS1Δα3 deletion mutant to MED25-ACID. Structural analysis of NS1 by NMR revealed significant conformational plasticity that could promote interactions with partner proteins versus self-assembly. To investigate the individual roles of NS1 subdomains, we designed mutations in the NS1 core domain. These NS1 mutants displayed significantly weaker interaction with MED25-ACID than WT NS1. Using recombinant RSV, we observed that rRSV mutants were attenuated and induced increased production of several interferon-stimulated genes, as compared to wild-type rRSV. Taken together, our results suggest that a strong interaction with MED25-ACID, achieved by cooperative binding of α3 and the core domain, is correlated to antiviral response antagonism
Fighting Antibiotic Resistance: How to Monitor Drug-Protein Interactions in the Bacterial Periplasm Alicja Razew (IBS, Grenoble)
Tuesday, June the 25th at 11 am, we will a have a FRISBI/ReNaFoBis webinar by Florian FAESSLER (IGBMC, Illkirch)
Title: Cryo-electron tomography of filament assemblies and their organizers
Please register here: https://us06web.zoom.us/webinar/register/WN_XP6fismwQQCFNXtjA1wEUg
Summary: Cryo-electron tomography (cryo-ET) combined with subtomogram averaging allows structure determination in complex environments, such as cells, often recalcitrant to other structural biology approaches. While the achievable resolutions in most cases remain lower than possible with single particle analysis cryo-electron microscopy, the contextual insight provided by the tomography approach delivers a unique flavor of insights. Such insights can be crucial for understanding the architecture of filament assemblies (e.g., the actin-rich protrusions of migrating cells) and the role of their organizers. To make the best use of the structural and contextual information contained in tomograms, we assembled workflows and software pipelines that allow for rapid ultrastructural analysis of parallel and meshwork-like filament assemblies, as well as structure determination of proteins organizing such macromolecular constructions.
During this presentation, I will give a short overview of the pipelines and showcase their application, mainly in the context of actin filament networks.
Speaker: Agathe Urvoas, Institute for Integrative Biology of the Cell (I2BC),
Tittle: AlphaRep: a new platform dedicated to the selection of artificial protein binders
May 14, 2024 at 11 am
Summary: We have designed and assembled a large library of artificial repeat proteins named alphaRep. AlphaRep proteins binding tightly and specifically any predefined folded protein can be selected out from this library. AlphaRep generation does not require animal immunization and gives rise to renewable, sequence defined, binders. Specific alphaReps are soluble, efficiently produced, disulfide-free and extremely stable. These artificial proteins can be easily modified (fusion, dimerisation, incorporation of fluorophores, tracers, etc.) and can be used as protein tools in various contexts including Structural Biology and Cellular Biology.
The platform offers generation of alphaReps that bind with high affinity and specificity any chosen protein target. Information and contacts: https://www.i2bc.paris-saclay.fr/structural-biology/alpha-rep/
Please register at: https://us06web.zoom.us/webinar/register/WN_ldZ2FglTSRKoA9_XHvA9aQ
The three national infrastructures ProFi, FranceBioImaging and FRISBI along with the GIS IBiSA are pleased to announce a third call for a funded access to IBiSA-labelled facilities. Our aim is to promote IBiSA facilities networking through transdisciplinary research projects.
Applications should request access to at least two different IBiSA facilities from two disciplines (structural biology, Biological imaging and proteomics, see below a non-exhaustive list). The call is open to any academic laboratory.
For application:
Applications should be submitted to Call-IBISA-FBI-FRISBI-PROFI@i2bc.paris-saclay.fr
using the template document 
Télécharger Template-Call-2023-IBISA-FBI-FRISBI-PROFI.docx (15 KB)
Deadline May 31, 2024
Preparation and biophysical/MS characterization of multiprotein complexes for cryo-EM analysis
The rise in popularity of structural approaches, in particular the advent of the cryo-EM “resolution revolution” has resulted in a dire need for improved strategies for sample production and characterization to prevent congestion in the workflow. This advanced EMBO course will train participants (mainly PhD students and postdocs) with the state-of-the-art approaches for the preparation of large macromolecular assemblies in view of structural studies.
https://meetings.embo.org/event/23-cryo-em
The three national infrastructures ProFi, FranceBioImaging and FRISBI along with the GIS IBiSA
are pleased to announce a second call for a funded access to IBiSA-labelled facilities. Our aim
is to promote IBiSA facilities networking through transdisciplinary research projects.
Applications should request access to at least two different IBiSA facilities from two
disciplines (structural biology, Biological imaging and proteomics, see below a non-exhaustive
list). The call is open to any academic laboratory.
media/Access_call_inter-infra_IBiSA_2023.pdf
This is a workshop distributed over several participating centres of the iNEXT-Discovery infrastructure https://inext-discovery.eu.
Practicals will be run in parallel sessions on each site and shared morning lectures on the background and technical aspects will be followed jointly by all the participants.
It comprises sample vitrification, focused ion beam milling to obtain lamella and cryo-ET data acquisition & evaluation.
If you are interested to join please register here: https://inextfibet.sciencesconf.org/
This call aims to encourage teams to obtain initial access to the services offered by the Research Infrastructures to help their research project.
The objective is to demonstrate the significant impact of these services to improve the quality of their results (resolution, reproducibility, change of scale, etc.) or to help remove technological barriers. We also aim to make our teams aware of the cutting-edge technologies, methods and expertise offered by these national infrastructures.
For more info and application https://www.insb.cnrs.fr/fr/appel-projets-insb-acces-aux-infrastructures-nationales-de-recherche
The RéNaFoBIS network is organising its 10th national school from 1 to 9 June 2023 in Oléron. This school offers theoretical and practical training to the different approaches used in structural biology (diffraction and diffusion of X-rays, NMR, electron microscopy, sample preparations for structural studies, macromolecular interactions. It will focus on the integration of several of these experimental methods to answer the major questions of functional biology at the atomic scale.
https://ecolebios2023.sciencesconf.org/
FRISBI co-finances training in France that is open to national and European structural biology communities.
FRISBI will particularly consider training co-organised with a centre from an another infrastructure written in the “Feuille de route Nationale des Infrastructures” https://www.enseignementsup-recherche.gouv.fr/sites/default/files/2022-03/feuille-de-route-nationale-des-infrastructures-de-recherche---2021-v2--17318.pdf
Deadline for submission: 11 September 2022 for a course held between the 1st of January and 31st of December 2023.
Formular for application 42.3 KB
The 8th FRISBI call for funded access to national structural biology platforms is now open.
Access to FRISBI infrastructure is possible for all scientists working in French laboratories. Scientists working at FRISBI centres cannot request funding for access to their local centre.
· Depending on project size 250-2000 € of support is available as a contribution towards the access costs of each project (consumables, reagents and travel for one person). The number of awards is limited. The user is expected to cover any additional expenses beyond the award price.
· Scientists may visit single or multiple FRISBI platforms for their experiments.
· The deadline for this round of proposal submission is 30 June 2022. Proposals should be submitted via the FRISBI online proposal submission system at http://frisbi.eu/submit-a-proposal/. Once selected, projects should be performed within 12 months.
FRISBI is listed as a National Research Infrastructure in Biology and Health in the new "Feuille de route Nationale des Infrastuctures de Recherche" on the MESRI site
Interdisciplinary access call to Structural biology, Biological imaging and Proteomics facilities
The three national infrastructures ProFi, FranceBioImaging and FRISBI along with the GIS IBiSA are pleased to announce a call for a funded access to IBiSA-labelled facilities. Our aim is to promote IBiSA facilities networking through transdisciplinary research projects.
Applications should request access to at least two different IBiSA facilities from two disciplines (structural biology, Biological imaging and proteomics). The call is open to any academic laboratory.
The amount of the financial support will be up to 5000 € per application to cover facility costs.
Retrouver FRISBI aux Rendez-vous Carnot
https://www.rdv-carnot.com/ et au rendez-vous sur le thème "Research Infrastructures and Health"