BEGIN:VCALENDAR VERSION:2.0 PRODID:-//CERN//INDICO//EN BEGIN:VEVENT SUMMARY:Towards more dose efficient cryogenic electron microscopy of biolo gical samples DTSTART;VALUE=DATE-TIME:20230307T140000Z DTEND;VALUE=DATE-TIME:20230307T143000Z DTSTAMP;VALUE=DATE-TIME:20260812T141810Z UID:indico-contribution-42-398@eventi.mlib.ic.cnr.it DESCRIPTION:Speakers: Yue Zhang (M4I)\nCryo-electron microscopy (cryo-EM) has become an indispensable tool for structural biologists studying the re lationship between structure and function of various biomolecules. Recent advancements in transmission electron microscopy (TEM) hardware and data p rocessing software have enabled atomic-level resolution for single particl e cryo-EM. However\, achieving near-atomic resolution for smaller (<100 kD a)\, more heterogeneous\, and non-symmetric samples remains a significant challenge due to the limited scattering information provided by smaller pa rticles\, the high defocus required for image contrast enhancement\, and t he radiation damage that restricts the number of high-energy electrons per surface area\, resulting in low signal-to-noise ratios. To address these challenges\, we simulated single particle data sets using realistic parame ters for ice layer\, dose\, detector performance\, and beam characteristic s for samples that were ideal in terms of homogeneity\, distribution\, and stability. Our simulations could help expand the size limits of cryo-EM. Meanwhile\, we also reviewed alternative TEM techniques such as phase plat e and ptychography that hold promise for providing complementary or additi onal structural information within the limited lifetime of the sample. Fin ally\, we implemented a new event-based electron detector and show experim ental data substantiating some of the promises we could simulate.\n\nhttps ://eventi.mlib.ic.cnr.it/event/42/contributions/398/ LOCATION:Webinar URL:https://eventi.mlib.ic.cnr.it/event/42/contributions/398/ END:VEVENT BEGIN:VEVENT SUMMARY:Structural insight into mycobacterial protein secretion DTSTART;VALUE=DATE-TIME:20230307T150000Z DTEND;VALUE=DATE-TIME:20230307T153000Z DTSTAMP;VALUE=DATE-TIME:20260812T141810Z UID:indico-contribution-42-396@eventi.mlib.ic.cnr.it DESCRIPTION:Speakers: Ye Gao (M4I)\nMycobacteria tuberculosis employs Type 7 Secretion System (T7SS) to secrete effector proteins to help escape fro m the host immune system. There are five gene clusters named ESX-1 to ESX- 5 belonging to T7SS in M. tuberculosis. ESX-1 is the first identified ESX system and has been shown to be essential for phagosome rapture. Secreted proteins have to cross both membranes of the bacterium. ESX-1 is expected to carry out this process\, however\, only the inner-membrane core complex components have been identified thus far.\nESX-1 inner core complex consi sts of five subunits: EccB1\, EccCa1\, EccCb1\, EccD1 and EccE1. It has an estimated molecular mass of ~2 MDa. All five genes were constructed in mu lti-cassette vectors with different tags fused to one of the protein subun its. Presence of all five subunits could be detected after purification (b y both Western Blot and Mass Spectrometry)\, however\, purified complex fr om E. coli did not result in useable cryo-EM data thus far. In contrast\, good success was obtained for purified ESX-1 components as well as substra tes. In here\, I will give a short overview about the TB research work don e at Maastricht University.\n\nhttps://eventi.mlib.ic.cnr.it/event/42/cont ributions/396/ LOCATION:Webinar URL:https://eventi.mlib.ic.cnr.it/event/42/contributions/396/ END:VEVENT BEGIN:VEVENT SUMMARY:Insights on the molecular and structural basis of human dystroglyc anopathies DTSTART;VALUE=DATE-TIME:20230307T143000Z DTEND;VALUE=DATE-TIME:20230307T150000Z DTSTAMP;VALUE=DATE-TIME:20260812T141810Z UID:indico-contribution-42-402@eventi.mlib.ic.cnr.it DESCRIPTION:Speakers: Sonia Covaceuszach (CNR-IC (Trieste))\nCassetta A(1) \, Longo F(1)\, Sciandra F(2)\, Bozzi M(2\,3)\, Bigotti MG\,(4) Hübner W( 5)\, Brancaccio A(2)\, Covaceuszach S(1) \n\n1 Istituto di Cristallografia - Trieste \n2 Istituto di Chimica del Riconoscimento Molecolare - Roma\n3 Università Cattolica del S. Cuore\, Roma\n4 University of Bristol\, UK\n 5 University of Bielefeld\, Germany\n\nDystroglycanopathies are neuromuscu lar disorders associated with abnormal neuronal migration and muscular dys trophy. Clinical manifestations are extremely variable\, and include a wid e spectrum of phenotypic severity. Dystroglycanopathies are mainly due to abnormal glycosylation of dystroglycan (DG) which is a cell-surface glycop rotein that links the cytoskeleton with the extracellular matrix acting as a receptor for extracellular matrix proteins containing laminin-G domains . Dystroglycan is composed of two subunits: the extracellular highly glyco sylated α-DG and the transmembrane β-DG. A multistep glycosylation proc ess is necessary to decorate the α-DG subunit with complex glycans that a re crucial for its interaction with the extracellular matrix proteins\, su ch as laminins. Most of the dystroglycanopathies are due to an impaired f unctional state of the enzymes involved in α-DG maturation. Nevertheless\ , a set of missense mutations has been recently identified on the N-termin al region of α-DG (a.a. 50-313 in mouse) that determine the hypoglycosyla tion of the DG complex\, due to the impairment of a key step in α-DG glyc osylation operated by the bifunctional glycosyltransferase LARGE1. \nWith the aim of elucidating the molecular and structural implications of the pa thological mutations leading to dystroglycanopathies\, we have undertaken a multi-technique study\, including synchrotron radiation approaches to ga in insight on the molecular structure of a recently discovered α-DG point pathological mutant (L84F) in both crystals and solution. Indeed\, we det ermined the high-resolution molecular structure of L84F mutant by X-ray cr ystallography. Moreover\, Small Angle X-ray Scattering (SAXS) has been emp loyed as a complementary approach to get low resolution information on the flexibility\, conformation and the structural organization of this α DG mutant at near-physiological conditions. The results of the synchrotron r adiation-based experiments\, combined with biochemical\, cellular and micr oscopic data\, allowed us to shed light on the molecular and structural ba sis of dystroglycanopathies.\n\nhttps://eventi.mlib.ic.cnr.it/event/42/con tributions/402/ LOCATION:Webinar URL:https://eventi.mlib.ic.cnr.it/event/42/contributions/402/ END:VEVENT BEGIN:VEVENT SUMMARY:Beyond the “Amyloid Hypothesis”? Rescuing proteostasis in Alz heimer’s Disease” DTSTART;VALUE=DATE-TIME:20230307T131500Z DTEND;VALUE=DATE-TIME:20230307T134500Z DTSTAMP;VALUE=DATE-TIME:20260812T141810Z UID:indico-contribution-42-403@eventi.mlib.ic.cnr.it DESCRIPTION:Speakers: Danilo Milardi (IC-CNR (Catania))\nDanilo Milardi\n( 1)Institute of Crystallography\, Consiglio Nazionale delle Ricerche\, 9512 5\, Catania\, Italy\n\nAlzheimer’s Disease (AD) is the most common form of dementia in the elderly population. The estimated global prevalence of AD in 2015 was 46 million with an incidence rate in 2050 of 131 million. I n the absence of medical advancements to prevent\, slow down\, or stop the disease\, there will be dramatic effects on society\, global health\, and economy. AD is characterized by an abnormal accumulation of Aβ amyloid p laques in the brain. The idea that amyloid Aβ peptide aggregation into am yloid fibrils is an important factor in AD development (Amyloid Hypothesis ) is supported by a considerable body of evidence. However\, the failure o f clinical trials for molecules targeting Aβ misfolding and self-assembly points to the need for a deeper comprehension of the mechanisms behind th e impaired proteome maintenance occurring in AD.\nHere I propose a brief s urvey on the intertwined biochemical mechanisms that control Aβ homeostas is (proteostasis) by employing an interdisciplinary approach to screen sma ll molecules (e.g. natural compounds\, bioconjugates\, and repurposed drug s) for their ability to restore physiological Aβ homeostasis by a multi-t arget strategy. This research activity spans from fundamental topics relat ed to protein/lipid membrane stability\, amyloid aggregation\, proteasome activation and ligand protein interactions to applications in medicinal ch emistry focusing on the development of bioactive compounds as drug candida tes in AD therapy.\n\nhttps://eventi.mlib.ic.cnr.it/event/42/contributions /403/ LOCATION:Webinar URL:https://eventi.mlib.ic.cnr.it/event/42/contributions/403/ END:VEVENT BEGIN:VEVENT SUMMARY:Machine learning-based prediction of hERG-mediated cardiotoxicity: a structure-based investigation DTSTART;VALUE=DATE-TIME:20230307T121500Z DTEND;VALUE=DATE-TIME:20230307T124500Z DTSTAMP;VALUE=DATE-TIME:20260812T141810Z UID:indico-contribution-42-401@eventi.mlib.ic.cnr.it DESCRIPTION:Speakers: Giuseppe Felice Mangiatordi (IC-CNR (Bari))\nGiusepp e Felice Mangiatordi(1) Pietro Delre(1\,2) Teresa Maria Creanza(3)\, Nicol a Ancona\,(3) Giovanni Lentini\,(4) Michele Saviano(1). \n\n1. Istituto di Cristallografia\, Consiglio Nazionale delle Ricerche\, Via G. Amendola 12 2/O\, 70126 Bari.\n2. Chemistry Department\, University of Bari “Aldo Mo ro”\, via E. Orabona\, 4\, I-70125 Bari\, Italy. \n3. CNR - Institute of Intelligent Industrial Technologies and Systems for Advanced Manufacturin g\, Via Amendola 122/o\, 70126 Bari\, Italy.\n4. Dipartimento di Farmacia —Scienze del Farmaco\, Università̀ degli Studi di Bari “Aldo Moro” \, via E. Orabona\, 4\, I-70125 Bari\, Italy.\n\n\nPrioritizing drug candi dates based on their human ether-à-go-go-related gene potassium channel ( hERG) blocking potential is a mandatory step in the early preclinical stag e of a drug discovery program. The hERG blockade is\, in fact\, considered the main cause of cardiotoxicity in post-marketing surveillance. Several ligand-based approaches were therefore developed in the last years and are currently employed in a drug discovery process for in silico cardiac safe ty assessment of drug candidates. Herein\, the first structure-based class ifiers able to discern hERG binders from non-binders will be presented.(1) LASSO regularized Support Vector Machines were applied to integrate docki ng scores and protein-ligand interaction fingerprints. 396 models were tra ined and validated based on: i) high-quality experimental bioactivity info rmation returned by 8\,337 curated compounds extracted from ChEMBL (versio n 25(2)) and ii) structural predictor data. Molecular docking simulations were performed by using GLIDE and GOLD software programs and different hER G structural models\, namely the recently published structures obtained by cryo-electron microscopy (PDB codes: 5VA1(3 )and 7CN1(4)) and two publish ed homology models selected for comparison. Remarkably\, some models retur n performances comparable to ligand-based classifiers in terms of accuracy (AUCMAX = 0.86±0.01) and negative predictive values (NPVMAX = 0.81±0.01 ) thus putting forward the herein developed computational workflow as a va luable tool for predicting hERG mediated cardiotoxicity without the limita tions of ligand-based models\, typically affected by low interpretability and a limited applicability domain. The study highlights the importance of using hERG structural models accounting for ligand-induced fit effects an d allowed us to select the best performing protein conformation to be empl oyed for a reliable structure-based prediction of hERG-related cardiotoxic ity.\n\nReferences\n1. Creanza\, T.M.\; Delre\, P.\; Ancona\, N.\; Lentini \, G.\; Saviano\, M.\; Mangiatordi\, G.F. Structure-Based Prediction of hE RG Related Cardiotoxicity: A Benchmark Study. Journal of Chemical Informat ion and Modeling 2021\, 61 (9)\, 4758-4770\n2. Davies\, M.\; Nowotka\, M.\ ; Papadatos\, G.\; Dedman\, N.\; Gaulton\, A.\; Atkinson\, F.\; Bellis\, L .\; Overington\, J. P. ChEMBL Web Services: Streamlining Access to Drug Di scovery Data and Utilities. Nucleic Acids Res 2015\, 43 (W1)\, W612–W620 \n3. Wang\, W.\; MacKinnon\, R. Cryo-EM Structure of the Open Human Ether- à-Go-Go-Related K+ Channel HERG. Cell 2017\, 169 (3)\, 422-430\n4. Asai\, T.\; Adachi\, N.\; Moriya\, T.\; Oki\, H.\; Maru\, T.\; Kawasaki\, M.\; S uzuki\, K.\; Chen\, S.\; Ishii\, R.\; Yonemori\, K.\; Igaki\, S.\; Yasuda\ , S.\; Ogasawara\, S.\; Senda\, T.\; Murata\, T. Cryo-EM Structure of K+-B ound HERG Channel Complexed with the Blocker Astemizole. Structure 2021\, 29 (3)\, 203-212.\n\nhttps://eventi.mlib.ic.cnr.it/event/42/contributions/ 401/ LOCATION:Webinar URL:https://eventi.mlib.ic.cnr.it/event/42/contributions/401/ END:VEVENT BEGIN:VEVENT SUMMARY:Welcome and intro IC-CNR & M4I DTSTART;VALUE=DATE-TIME:20230307T120000Z DTEND;VALUE=DATE-TIME:20230307T121500Z DTSTAMP;VALUE=DATE-TIME:20260812T141810Z UID:indico-contribution-42-400@eventi.mlib.ic.cnr.it DESCRIPTION:Speakers: Cinzia Giannini (IC-CNR)\, Ron Heeren (M4I)\nhttps:/ /eventi.mlib.ic.cnr.it/event/42/contributions/400/ LOCATION:Webinar URL:https://eventi.mlib.ic.cnr.it/event/42/contributions/400/ END:VEVENT BEGIN:VEVENT SUMMARY:Discussion and Conclusions DTSTART;VALUE=DATE-TIME:20230307T153000Z DTEND;VALUE=DATE-TIME:20230307T154000Z DTSTAMP;VALUE=DATE-TIME:20260812T141810Z UID:indico-contribution-42-399@eventi.mlib.ic.cnr.it DESCRIPTION:**Speakers**: Dr. Cinzia Giannini (IC-CNR) and Prof Ron Heeren (M4I)\n\nhttps://eventi.mlib.ic.cnr.it/event/42/contributions/399/ LOCATION:Webinar URL:https://eventi.mlib.ic.cnr.it/event/42/contributions/399/ END:VEVENT BEGIN:VEVENT SUMMARY:Pushing the limits of sample vitrification DTSTART;VALUE=DATE-TIME:20230307T124500Z DTEND;VALUE=DATE-TIME:20230307T131500Z DTSTAMP;VALUE=DATE-TIME:20260812T141810Z UID:indico-contribution-42-397@eventi.mlib.ic.cnr.it DESCRIPTION:Speakers: Navya Premaraj (M4I)\nCryogenic Electron Microscopy (cryo-EM) is a powerful imaging technique for visualizing molecular struct ures with unprecedented detail. To prepare samples for cryo-EM a process c alled vitrification is typically used which involves rapidly freezing a th in layer of sample to create a non-crystalline solid. Plunge freezing is c urrently the most commonly method used method for preparing samples for Si ngle particle analysis and Cryo Electron Tomography (cryo-ET) workflows. T his method works with isolated proteins but is not ideal for thicker sampl es like whole cells which is necessary for Cryo-ET. To address this challe nge\, a Vitrojet for cells (VitroJet4Cells) was developed which utilizes e thane jet to vitrify samples. The power of jet has already been proven eff ective with VitroJet for single particles. A functional prototype of the m achine was successfully developed and is currently being used for testing various samples. The cooling potential of the jet is primarily examined us ing a micro loop set up\, which can accommodate a wide range of samples\, frozen samples are then examined using an X-ray beam to determine their vi trification quality. The machine is also compatible with standard EM grids . Clipped EM grids with adherent cells are\, blotted\, vitrified and subse quently utilized for making lamellas to be used in cryo-ET.\n\nhttps://eve nti.mlib.ic.cnr.it/event/42/contributions/397/ LOCATION:Webinar URL:https://eventi.mlib.ic.cnr.it/event/42/contributions/397/ END:VEVENT END:VCALENDAR