Jeudi 08 juillet
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Jeudi 08 juillet

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SDV1
16:00 - 18:55

Symposium Sciences de la Vie 1
Cryométhodes : Particules isolées : applications et développements
sdv1

Modérateurs : Irina GUTSCHE (IBS, Grenoble), Célia PLISSON-CHASTANG (LBME, Toulouse)
La cryo-microscopie électronique a vécu une véritable révolution ces dernières années puisqu’elle permet maintenant couramment de déterminer la structure 3D de divers complexes biologiques à une résolution atomique. La taille des complexes macromoléculaires accessibles aux études par cryo-MET ne cesse de baisser, la flexibilité de ces objets devient un atout pour des analyses dynamiques plutôt qu’une entrave à la résolution, et l’hétérogénéité compositionnelle n’est plus une véritable contrainte puisque les images correspondant à chaque type objet peuvent être distinguées et traitées séparément. Au travers de cette session, il est proposé un éventail de ce qui se fait actuellement en cryo-MET, des développements récents de la cryo-MET 3D à l’apport de la cryo-MET à des problématiques biologiques diverses.
16:00 - 16:30 Novel algorithms for cryo-electron tomography. Dimitry TEGUNOV (MPIBPC, Göttingen, Allemagne)
While single-particle analysis of in vitro samples made major advances over the past decade to achieve atomic resolution, the analysis pipeline for tomographic in situ data has lagged behind. With the recent development of Warp and M, we were able to close the gap in processing, obtaining residue-level resolution for ribosomes images inside cells for the first time. In my talk, I will highlight our recent results and the underlying ideas, and discuss how new deep learning-based algorithms allow to further make sense of in situ data.
16:30 - 16:40 Discussion.
16:40 - 17:10 Phage revenge: allosteric inhibition of CRISPR-Cas9 by the anti-CRISPR protein AcrIIA6. Adeline GOULET (AFMB, Marseille)
Bacteriophages (phages) and their preys are engaged in an endless evolutionary arms race. Bacteria have evolved sophisticated defense mechanisms to thrive in virus-rich ecosystems, including the well-known CRISPR-Cas9 immune system. In parallel, phages have developed counter-attack strategies to overcome their host’s defenses, including anti-CRISPR proteins. The striking sequence diversity of anti-CRISPR proteins and the lack of homology with proteins of known function raise questions relating to their modes of action. Moreover, anti-CRISPR proteins, as natural CRISPR-Cas9 inhibitors, hold great promise as biotechnological tools to fine-tune CRISPR-Cas9-based gene edition, and as useful addition to phage therapy.
In this context, we embarked on the structural and functional characterization of the anti-CRISPR protein AcrIIA6, discovered in virulent phages infecting Streptococcus thermophilus, combining cryo-electron microscopy and single particle analysis, in vitro analyses of macromolecular interactions, and functional cellular assays. The AcrIIA6 molecular mechanism is unique: we showed that AcrIIA6 acts as an allosteric inhibitor and induces St1Cas9 dimerization. AcrIIA6 affects St1Cas9 conformational dynamics, which reduces St1Cas9 binding affinity for DNA and prevents St1Cas9 binding to its target within cells. Interestingly, these findings led us to identify a natural variant of St1Cas9 resistant to AcrIIA6, illustrating anti-CRISPR-driven mutational escape and molecular diversification of Cas9 proteins.
17:10 - 17:20 Discussion.
17:20 - 17:35 #26369 - How to get the maximum out of your cryo-EM data collection session.
How to get the maximum out of your cryo-EM data collection session.

Single-particle cryogenic transmission electron microscopy (cryo-EM) can be used to elucidate the 3D structure of macromolecular complexes. The sample is embedded in a thin layer of vitreous ice and maintained at liquid nitrogen temperature. It is then imaged directly in the microscope and a 3D reconstruction may be calculated from projections of individual macromolecular complexes by determining the orientations of the projections. For many years cryo-EM was limited to low-resolution, but driven by technological breakthroughs in electron microscope and electron detector developments coupled with improved image processing procedures,  cryo-EM is now a method of choice for the determination of high-resolution structures, termed as “The Resolution Revolution” [1].

These developments deeply modified the way of collecting cryo-EM data. Currently, after only a few hours of setup, data collection can run in a fully unattended and automated way for several days, producing high quality dataset of thousands of micrographs. These improvements led to new challenges in term of session setup, microscope alignments and acquisition parameters and new strategies were required to tackle these questions. This communication will present how high-end data collection is performed at the EMBL Heidelberg cryo-EM platform using SerialEM (Fig. 1) [3], insisting on some of the key steps of the workflow: Microscope alignments allowing a small electron beam and aberration-free beam shift/image shift [2]; pixel size choice for optimized data collection; alternative collection strategies for samples with very low concentration and uneven spreading on the grid [4]; ice thickness characterisation for optimal holes selection.

References :

[1] W Kühlbrandt, Science 343 (2014), p. 1443.

[2] F Weis and WJH Hagen, Acta Crystallogr D Struct Biol 76 (2020), p. 724.

[3] DN Mastronarde, J Struct Biol 152 (2005), p. 36.

[4] M Schorb et al, Nat Methods 16 (2019), p. 471.


Felix WEIS (Heidelberg, Allemagne)
17:35 - 17:45 Discussion.
17:45 - 18:00 #26370 - Structural characterization of a human Kir potassium channel and its involvement in Andersen syndrome.
Structural characterization of a human Kir potassium channel and its involvement in Andersen syndrome.

Inward rectifier potassium (Kir) channels belong to a family of integral membrane proteins that selectively control the K+ ions permeation at the cell membranes of various tissues and regulate the membrane electrical excitability [1]. The Kir channels gating is essentially modulated by phosphatidylinositol-4,5-bisphosphate (PIP2), although a full description of PIP2 role requires more details [1]. Genetically-inherited defects in Kir channels are responsible for several rare human diseases, including Andersen-Tawil syndrome (AS), a muscular disease that causes episodes of muscle weakness, arrhythmia, and developmental abnormalities, which the available treatments are inefficient [2]. Two-third of the mutations that causes this syndrome are situated on the PIP2 binding site [2]. 

Here, we present a 3.5 Å resolution cryo-EM map of the human Kir2.1 channel (Fig. 1). The data were collected on a Titan Krios TEM (FEI) operating at 300 kV equipped with a Gatan K2 Summit DED and Cs corrector. After micrographs selection based on visual quality inspection, it was obtained 7,188 images and 837,808 particles. All data was processed using Relion 3.0.

This is the first structural data of the whole human Kir2.1 channel, which can reveal useful insights about selectivity filter gate. The near-atomic resolution of the obtained cryo-EM map can provide a complete description of the PIP2 binding site in the Kir2.1 channel and the understanding of the clinically-relevant disease-causing mutations impact on the Kir channels function and dynamics. Molecular dynamis simulations performed with the final cryo-EM structure can provide the understanding of the full gating mechanism of human Kir2.1 channel and the PIP2 role on the dynamics of this channel.

References

[1] Suh et al. Curr. Opin.Neurobiol. v.15 (2005), p. 370-378.

[2] Andersen et al. Acta Paediatr. Scand. v.60 (1971), p. 559-564.

[3] Donaldson et al. Neurology v.60 (2003), p.1811-1816.

Figure 1: Structure of the human Kir2.1 channel. (A) Representative cryo-electron micrograph; (B) Representative 2D class averages of the particles; (C) Fourier Shell Correlation (FSC) curve indicating the map overall resolution; (D) Cryo-EM map obtained at 3.5 Å resolution highlighting the PIP2 binding site between cytoplasmic and transmembrane domains and including a map detailed view at the transmembrane domain. 


Carlos A. H. FERNANDES (Bioinformatique et BioPhysique (BIBIP))
18:00 - 18:10 Discussion.
18:10 - 18:25 #26372 - A hybrid approach to analyze continuous conformational variability of single particles based on deep learning.
A hybrid approach to analyze continuous conformational variability of single particles based on deep learning.

Cryo-electron microscopy (cryo-EM) allows high-resolution 3D reconstruction of biomolecular structures from highly noisy 2D parallel-beam projection images containing tens of thousands of copies of the same macromolecular complex but at different random orientations and positions. However, biomolecular complexes are not rigid but flexible entities that change their conformations gradually (continuous transition with many intermediate states) to accomplish biological functions (e.g., DNA replication, protein synthesis, etc.). The determination of the full distribution of conformations (conformational space or landscape) from cryo-EM images is challenging but could provide insights into working mechanisms of the complexes. In this presentation, we describe a method for conformational space determination, which uses deep learning in combination with cryo-EM image analysis and normal mode analysis [1,2] (Fig. 1), where the amplitudes of normal modes are the parameters of the elastic conformations of complexes. We show the performance of this new method

using synthetic cryo-EM data (Fig. 2). The conformational space predicted using the proposed method (Fig. 2(a)) allows obtaining molecular movies of the conformational transition (Fig. 2(b)).

Acknowledgment:

We acknowledge the support of the French National Research Agency — ANR (ANR-19-CE11-0008-01 and ANR-20-CE11-0020-03 to SJ) and the access to the HPC resources of CINES and IDRIS granted by GENCI (2019-A0070710998, AP010712190, AD011012188 to S.J.).

References :

[1] Harastani M, Sorzano COS, Jonic S, Hybrid Electron Microscopy Normal Mode Analysis with Scipion. Protein Sci 29 (2020), p. 223-236.

[2] Jin Q, Sorzano CO, de la Rosa-Trevin JM, Bilbao-Castro JR, Nunez-Ramirez R, Llorca O, Tama F, Jonic S, Iterative Elastic 3D-to-2D Alignment Method Using Normal Modes for Studying Structural Dynamics of Large Macromolecular Complexes. Structure 22 (2014), p. 496-506.


Ilyes HAMITOUCHE (Paris)
18:25 - 18:35 Discussion.
18:35 - 18:45 CRYO TEM: Latest improvements on throughput & resolution - JEOL. Guillaume BRUNETTI (Responsable Marketing) (JEOL EUROPE)
18:45 - 18:55 Discussion.
Room 1