BEGIN:VCALENDAR VERSION:2.0 PRODID:-//CERN//INDICO//EN BEGIN:VEVENT SUMMARY:Contr. talk - Small-scale expression and purification of the cytok ine receptor\, MPL\, using the ALiCE® cell-free system DTSTART;VALUE=DATE-TIME:20210526T093000Z DTEND;VALUE=DATE-TIME:20210526T095000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1141@lindico453.srv.lu.se DESCRIPTION:Speakers: Julie Tucker (University of York)\nMPL (also called TpoR) is the receptor for the haematopoietic cytokine\, thrombopoietin (TP O). Together\, MPL and TPO control the production of platelets and the mai ntenance of haematopoietic stem cells. Gain of function mutations in MPL c onstitute ˜5-10 % of driver mutations in essential\nthrombocythemia and p rimary myelofibrosis\, whilst loss of function mutations give rise to thro mbocytopenias. MPL comprises an ˜500 aa extracellular domain\, a single t ransmembrane helix\, and an ˜120 aa intracellular domain. The extracellul ar domain contains the thrombopoietin binding site\, whilst the intracellu lar domain provides the binding site for Janus kinase 2\, as well as other downstream signalling molecules\, including the Signal Transducers and Ac tivators of Transcription\n(STAT) proteins. Structural information on MPL is lacking\, in part due to the challenges of producing recombinant protei n. Endogenous expression levels of MPL are very low (< 5000\nreceptors/cel l) and heterologous expression of full-length MPL has not been reported. H ere we report small-scale expression and purification of both full-length and ectodomain of MPL using the ALiCE® cell-free expression system (Lenio Bio GmbH). This plant cell-based system allows for the expression of membr ane and secreted proteins within the microsomal compartment\, ensuring the ir correct folding\, membrane insertion and glycosylation. Optimisation of expression time\, plasmid\nconcentration and downstream processing allowe d for production and purification of glycosylated MPL\, the functional cha racterisation of which is ongoing.\n\nhttps://lindico453.srv.lu.se/event/2 19/contributions/1141/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1141/ END:VEVENT BEGIN:VEVENT SUMMARY:Contr. talk - From expression to purified protein: a P4-ATPase for CryoEM studies DTSTART;VALUE=DATE-TIME:20210526T091000Z DTEND;VALUE=DATE-TIME:20210526T093000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1140@lindico453.srv.lu.se DESCRIPTION:Speakers: LineMarie Christiansen (Aarhus University)\nThe Sacc haromyces cerevisiae P4-ATPase Neo1p is believed to function as a lipid fl ippase\, translocating lipids towards the cytosolic leaflet of both Golgi and endosome membranes. In\norder to screen around both functional and str uctural studies to ascertain the exact role and function of Neo1p an effic ient high-yield expression and purification protocol is vital. By\noverexp ression in S. cerevisiae and purification based on a high-specificity tag we are left with a pure protein sample ideal for both structural and funct ional studies. For CryoEM\ndetergent-exchange into LMNG can be easily perf ormed\, with some attention to detergent concentration\n\nhttps://lindico4 53.srv.lu.se/event/219/contributions/1140/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1140/ END:VEVENT BEGIN:VEVENT SUMMARY:Keynote talk - Whipping GPCRs into shape for structure determinati on DTSTART;VALUE=DATE-TIME:20210526T083000Z DTEND;VALUE=DATE-TIME:20210526T091000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1139@lindico453.srv.lu.se DESCRIPTION:Speakers: Christopher G. Tate (MRC Laboratory of Molecular Bio logy\, UK)\nOver the past 15 years we have determined multiple structures of GPCRs by both X-ray crystallography and cryo-EM. For each GPCR we had t o overcome the difficulties of overexpression\, solubilisation\, stabilisa tion and structure determination\, as you would for any membrane protein. For X-ray crystallography\, extensive protein engineering was required to form well-diffracting crystals\, in parallel with judicious choices of det ergent\, ligand and crystallisation format (vapour diffusion or lipidic cu bic phase). Different strategies also had to be devised depending on which conformational state was required for structure determination. The advent of high-resolution structure determination by single particle cryo-EM has presented new opportunities for GPCR structural biology\, and structures are now being determined that would have been onerous\, if not impossible\ , to determine by X-ray crystallography. I will present the key factors fo r expression\, purification and structure\ndetermination that have made GP CR structures possible.\n\nhttps://lindico453.srv.lu.se/event/219/contribu tions/1139/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1139/ END:VEVENT BEGIN:VEVENT SUMMARY:Contr. talk - Production of Bcl-2 proteins involved in regulation of mitochondrial apoptosis DTSTART;VALUE=DATE-TIME:20210526T080000Z DTEND;VALUE=DATE-TIME:20210526T082000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1138@lindico453.srv.lu.se DESCRIPTION:Speakers: Jörgen Ådén (Umeå University)\nProgrammed cell d eath (apoptosis) is essential for human life. In its intrinsic pathway\, t he Bcl-2 (B-cell lymphoma 2) protein family regulates cell life and death by controlling permeability of the mitochondrial outer membrane (MOM). How ever\, the molecular basis of cell protection by its anti-apoptotic Bcl-2 members remains elusive due to the lack of detailed structural insight int o their action at the MOM to ensure its integrity. To provide atomic-level insight into their functioning\, we will use the founding member of this family\, the human antiapoptotic Bcl-2 protein itself\, whose involvment i n p53 regulation and its overexpression\nplays a notorious role in many ca ncers and their treatment resistance. However\, for a long time\, obtainin g sufficient protein was cumbersome due to its insolubility as a membrane\ nprotein and low yields. Therefore\, we establishes an expression and puri fication protocol [1] to produce routinely mg amounts of the fully functio nal full-length human isoform 2 of Bcl-2\n(Bcl-2(2)) which can also be iso topically labelled as 15N/13C/2H versions\, ideally suited for neutron and NMR studies. The protocol even allows to generate residue specific Bcl-2\ nmutants and various constructs [2].\n\n1. Aden\, A.U. Mushtaq\, A. Dingel dein\, M. Wallgren\, G. Gröbner. A novel recombinant expression and purif ication approach for the full-length anti-apoptotic membrane protein Bcl-2 . Protein expression and purification. 172 (2020) 105628.\n2. A. Ul Mushta q\, J. Aden\, T. Sparrman\, M. Hedenstrom\, G. Gröbner. Insight into Func tional Membrane Proteins by Solution NMR: The Human Bcl-2 Protein-A Promis ing Cancer Drug Target. Molecules 26 (2021) 1467\, 1-14\n\nhttps://lindico 453.srv.lu.se/event/219/contributions/1138/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1138/ END:VEVENT BEGIN:VEVENT SUMMARY:Contr. talk - Towards Neutron crystallography of membrane proteins : Insights into production of deuterium-labelled OmpF DTSTART;VALUE=DATE-TIME:20210526T074000Z DTEND;VALUE=DATE-TIME:20210526T080000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1137@lindico453.srv.lu.se DESCRIPTION:Speakers: Swati Aggarwal (European Spallation Source)\nHydroge n bonds play a crucial role for protein function and involved in almost ev ery mechanism from foundation of protein structure to enzyme catalysis. Hy drogen (1H) atoms form the basis of hydrogen bond that is not scattered by X-ray crystallography due to its poor scattering power. Neutron protein c rystallography (NPX) is a powerful tool that is capable of locating hydrog ens and study the significance of hydrogen bonding interactions in biomacr omolecules. However\, due to\nthe requirement of large crystals very few n eutron structures have been deposited in PDB with no membrane protein stru cture determined yet. Additionally\, 1H has a negative scattering length a nd large incoherent cross-section giving rise to a significant background noise in neutron data collection.\nThis effect can be minimized by isotopi c substitution of 1H with its heavier isotope deuterium (2H or D) leading to less ambiguous data analysis and better structure interpretation. Overa ll ˜25% H atoms in a protein are solvent exchangeable and can be exchange d by dissolving in heavy water. However\, complete deuterium labelling (pe rdeuteration) is required for the remaining 75% H atoms. In this work\, an optimized methodology for large scale production of perdeuterated bacteri al outer membrane protein F (OmpF) has been designed. OmpF was produced in deuterated minimal medium with different carbon sources. Mass spectrometr y and thermal stability experiments\nverified the purity and level of deut eration of OmpF protein. Perdeuterated OmpF crystals also diffracted X-ray s to 9 Å resolution emphasising the need of fine tuning of perdeuterated crystallisation conditions.\n\nhttps://lindico453.srv.lu.se/event/219/cont ributions/1137/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1137/ END:VEVENT BEGIN:VEVENT SUMMARY:Keynote talk - Membrane protein purification and application in dr ug discovery DTSTART;VALUE=DATE-TIME:20210526T070000Z DTEND;VALUE=DATE-TIME:20210526T074000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1136@lindico453.srv.lu.se DESCRIPTION:Speakers: Arjan Snijder (Astra Zeneca)\nIn the presentation I will discuss our capabilities\, processes and methods for membrane protein expression and purification\, including our recently developed teabag pur ification method. The teabag method allows rapid purification of membrane proteins with reduced hands-on and processing time. Application of membran e proteins in drug discovery will be illustrated by our work on the protea se-activated receptor 2\, where extensive protein engineering allowed DNA- encoded library screening on purified receptor\, establishment of biophysi cal assays\, and structure assisted drug design.\n\nhttps://lindico453.srv .lu.se/event/219/contributions/1136/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1136/ END:VEVENT BEGIN:VEVENT SUMMARY:Contr. talk - Production and purification of the K+-dependent P-T ype ATPase KdpFABC for crystallization and cryo-EM DTSTART;VALUE=DATE-TIME:20210525T141000Z DTEND;VALUE=DATE-TIME:20210525T143000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1135@lindico453.srv.lu.se DESCRIPTION:Speakers: Charlott Stock (Aarhus University)\nAt very low extr acellular K+ concentrations (<100 μM)\, KdpFABC rescues bacterial cells b y taking up K+ against an up to 105-fold concentration gradient. To unders tand the mechanism of this heterotetrameric complex\, KdpFABC was\npurifie d for functional and structural investigations. A new expression system fo r KdpFABC was generated\, a 3-step chromatographic purification implemente d and protein functionality in different detergents as well as in the pres ence of\ninhibitors was tested. The work was performed in the lab of Prof. Inga Hänelt at the University of Frankfurt from 2014-2020.\n\nhttps://li ndico453.srv.lu.se/event/219/contributions/1135/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1135/ END:VEVENT BEGIN:VEVENT SUMMARY:Keynote talk - Preparation of lipid flippases for cryo-EM studies DTSTART;VALUE=DATE-TIME:20210525T133000Z DTEND;VALUE=DATE-TIME:20210525T141000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1134@lindico453.srv.lu.se DESCRIPTION:Speakers: Joseph Lyons (Aarhus University\, Denmark)\nP4-ATPas es are lipid flippases that drive the ATP-dependent inward translocation ( flipping) of lipids within the membrane. They are members of the P-type AT Pase superfamily and largely function as binary complexes with an auxiliar y protein from the CDC50 family. Select lipid flippases are auto-regulated by conserved motifs in their termini and require activation by an externa l stimulus\, i.e.\, binding of an activating lipid or regulatory protein\, and/or specific phosphorylation of the termini. To elucidate the auto-re gulation and transport mechanisms\, we determined the structures of yeast and mammalian lipid flippases by cryo-electron microscopy. \n\nIn this tal k\, I will give an overview of the tour de force we embarked on\, from exp ression through purification to cryo-EM imaging of these lipid flippases.\ n\nhttps://lindico453.srv.lu.se/event/219/contributions/1134/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1134/ END:VEVENT BEGIN:VEVENT SUMMARY:Contr. talk - Production of insect odorant receptors in yeast for structural studies DTSTART;VALUE=DATE-TIME:20210525T130000Z DTEND;VALUE=DATE-TIME:20210525T132000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1133@lindico453.srv.lu.se DESCRIPTION:Speakers: Cassie Sims (Lund University)\nInsect olfactory rece ptors (ORs) are proteins involved in olfaction in insects. Insect ORs are 7-transmembrane domain proteins that possess a unique structure\, forming a heteromeric complex with a highly conserved co-receptor (ORCO). The hete romeric complex constitutes an ion channel\, which opens allowing movement of ions upon activation by a ligand. Specificity of ORs is variable\, and little is understood about where this specificity arises and how the bind ing of an active ligand subsequently opens the ion channel. Despite signif icant functional studies of insect ORs\, only two structures\, ORCO\nfrom the fig wasp Apocrypta bakeri\, and a homomeric complex of OR5 from the ju mping bristletail\, Machilis hrabei\, have been determined. This structura l work relied on a well-established\nexpression system\, HEK293 cells\, wh ich are commonly used for functional screening of ORs. However\, HEK cells are not an optimal system for protein production for structural analysis. In this project\, we begin to develop a more efficient protein production system for structural investigation of insect ORORCO\ncomplexes\, focusin g on ORs of Lepidoptera and Culicidae (moths and mosquitoes). Initial work has focused on the expression of insect OR-ORCO complexes with a fusion e GFP in a Pichia pastoris yeast.\n\nhttps://lindico453.srv.lu.se/event/219/ contributions/1133/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1133/ END:VEVENT BEGIN:VEVENT SUMMARY:Contr. talk - Study of anti-cancer effects of TTA-A2 and paclitaxe l due to antagonistic interactions with T-type calcium channels DTSTART;VALUE=DATE-TIME:20210525T124000Z DTEND;VALUE=DATE-TIME:20210525T130000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1132@lindico453.srv.lu.se DESCRIPTION:Speakers: Vikram Dalal (Washington University in St. Louis)\nS tudies have shown that in cancer cells\, there is an increased T-type calc ium channel (TTCC) expression compared to healthy cells. Therefore\, the s tudies targeting TTCC for cancer therapy\nhave shown many positive outcome s. Here\, we have used TTA-A2- a potent TTCC inhibitor as a test drug\, an d paclitaxel (PTX)- a tubule-binding anti-cancer agent as a positive contr ol. Blocking\nTTCC has shown to overcome resistance in cancer cells toward s anti-cancer drugs by reducing calcium influx\, and some studies have sho wn that PTX treatment also reduces the intracellular calcium signaling in cells. So\, there is a possibility that PTX might be interacting with calc ium channels. Since\, drug-drug interaction can cause severe side-effects\ , or alter the actions of each other\; we aim to study the interactions am ong TTA-A2\, PTX\, and TTCC. Therefore\, in this study we have analyzed th e binding of of TTA-A2 and PTX with TTCC. Our results showed that both the drugs\, TTA-A2 and PTX\, could interact at the same site of TTCC to form a higher stable complex as compared to the TTCC-native. The result indicat ed that sequential treatment could help to overcome the antagonistic inter action between the two drugs.\n\nhttps://lindico453.srv.lu.se/event/219/co ntributions/1132/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1132/ END:VEVENT BEGIN:VEVENT SUMMARY:Keynote talk - An yeast GFP-based platform for functional and stru ctures investigation of SLC transporters DTSTART;VALUE=DATE-TIME:20210525T120000Z DTEND;VALUE=DATE-TIME:20210525T124000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1131@lindico453.srv.lu.se DESCRIPTION:Speakers: David Drew (Stockholm University\, Sweden)\nRecombin ant expression screening of eukaryotic membrane proteins for functional an d structural investigation is still a trial-and-error process. We have fou nd that cloning gene-strings by homologuous recombination into a 2u vector S. cerevisiae expression vector and then detecting membrane protein expre ssion and stability by working with GFP-fusions\, enables many constructs to be rapidly tested. We have found an excellent correlation between the b est-behaving membrane proteins in yeast to those produced by transient tra nsfection in mammalian HEK293 cells. Here\, I will outline these GFP-based methods with a focus on ion and sugar SLC transporters.\n\nhttps://lindic o453.srv.lu.se/event/219/contributions/1131/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1131/ END:VEVENT BEGIN:VEVENT SUMMARY:Contr. talk - Aquaporins – expression\, purification and charact erization DTSTART;VALUE=DATE-TIME:20210525T113500Z DTEND;VALUE=DATE-TIME:20210525T115500Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1130@lindico453.srv.lu.se DESCRIPTION:Speakers: Kristina Hedfalk ()\nAquaporin water channels facili tate the bi-directional flow of water and small\, neutral solutes down an osmotic gradient in all kingdoms of life. Over the last two decades\, the availability of\nhigh-quality protein has underpinned progress in the stru ctural and functional characterization of these water channels. In particu lar\, recombinant protein technology has guaranteed the supply of aquapori n samples that were of sufficient quality and quantity for further study. Here we review the features of successful expression\, purification and ch aracterization strategies that have underpinned these successes and that w ill drive further breakthroughs in the field. Summarizing\nthe production processes which has resulted in high resolution aquaporin structures revea ls that Escherichia coli is a suitable host for prokaryotic isoforms\, whi le Pichia pastoris is the most\ncommonly used recombinant host for eukaryo tic variants. Generally\, a two-step purification procedure is applied aft er solubilization in glucopyranosides and most structures are determined b y X-ray following crystallization.\n\nhttps://lindico453.srv.lu.se/event/2 19/contributions/1130/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1130/ END:VEVENT BEGIN:VEVENT SUMMARY:Contr. talk - The baculovirus/insect cells system DTSTART;VALUE=DATE-TIME:20210525T111500Z DTEND;VALUE=DATE-TIME:20210525T113500Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1129@lindico453.srv.lu.se DESCRIPTION:Speakers: Jan H Driller (Aarhus University)\nMammalian membran e proteins are notoriously difficult to express. In contrast to bacteria o r yeast\, the baculovirus/insect cells system provides a good alternative to express these proteins in higher eukaryotes. Insect cells provide post- translational modifications\, such as disulfide bonds or glycosylation\, a s well as a more akin membrane lipid composition compared to bacterial or yeast expression systems. Insect cells grow to high cell densities and usi ng the baculovirus system even multi-protein complexes can be co-expressed .\n\nhttps://lindico453.srv.lu.se/event/219/contributions/1129/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1129/ END:VEVENT BEGIN:VEVENT SUMMARY:Keynote talk - Recombinant membrane protein production in microbia l hosts DTSTART;VALUE=DATE-TIME:20210525T103500Z DTEND;VALUE=DATE-TIME:20210525T111500Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1128@lindico453.srv.lu.se DESCRIPTION:Speakers: Roslyn Bill (Aston University\, UK)\nDespite many hi gh-profile successes\, recombinant membrane protein production remains a t echnical challenge\; it is still the case that many fewer membrane protein structures have\nbeen published than those of soluble proteins. However\, progress is being made because empirical methods have been developed to p roduce the required quantity and quality of\nthese challenging targets. Mi crobial expression systems are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. A combination of rat ional construct design and host cell choice can dramatically improve membr ane protein yields suitable for structural and functional characterization . In addition to traditional detergents\, polymer-based systems are availa ble for solubilization and biophysical characterization of these proteins\ , with polymers providing opportunities to investigate protein-lipid inter actions.\n\nhttps://lindico453.srv.lu.se/event/219/contributions/1128/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1128/ END:VEVENT BEGIN:VEVENT SUMMARY:Closing remarks\, introducing the autumn workshop DTSTART;VALUE=DATE-TIME:20210526T102000Z DTEND;VALUE=DATE-TIME:20210526T102500Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1043@lindico453.srv.lu.se DESCRIPTION:https://lindico453.srv.lu.se/event/219/contributions/1043/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1043/ END:VEVENT BEGIN:VEVENT SUMMARY:General Discussion (Chair: Susanna Horsefield) DTSTART;VALUE=DATE-TIME:20210525T143500Z DTEND;VALUE=DATE-TIME:20210525T150000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1124@lindico453.srv.lu.se DESCRIPTION:https://lindico453.srv.lu.se/event/219/contributions/1124/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1124/ END:VEVENT BEGIN:VEVENT SUMMARY:General Discussion (Chair: Urban Johanson) DTSTART;VALUE=DATE-TIME:20210526T095500Z DTEND;VALUE=DATE-TIME:20210526T102000Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1123@lindico453.srv.lu.se DESCRIPTION:https://lindico453.srv.lu.se/event/219/contributions/1123/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1123/ END:VEVENT BEGIN:VEVENT SUMMARY:Welcome and purpose of the workshop DTSTART;VALUE=DATE-TIME:20210525T103000Z DTEND;VALUE=DATE-TIME:20210525T103500Z DTSTAMP;VALUE=DATE-TIME:20260528T141720Z UID:indico-contribution-219-1028@lindico453.srv.lu.se DESCRIPTION:https://lindico453.srv.lu.se/event/219/contributions/1028/ LOCATION: URL:https://lindico453.srv.lu.se/event/219/contributions/1028/ END:VEVENT END:VCALENDAR