Membrane protein insertion and quality control by the bacterial holo-translocon and FtsH chaperone/protease complex

Lead Research Organisation: University of Bristol
Department Name: Biochemistry

Abstract

All cells are surrounded by membranes that act as a barrier. Proteins embedded in the membrane are required for the transport of nutrients and information (signals) across this barrier. Translocation systems are required to transport proteins into the membrane or across the membrane to the cellular location where they can fulfil their tasks. Translocation systems recognise the specific proteins to be translocated via signals embedded in the sequence of amino acids from which they are constructed.
The Sec translocation system is well studied and conserved from the bacterium Escherichia coli to humans highlighting its importance. Findings about the mechanism of the SecYEG translocon from bacterial cells can thus inform us how similar systems work in our own bodies. SecYEG is a membrane protein complex that comprises 3 subunits. It contains a central channel for protein translocation through the membrane. The channel can also open up on the side to allow the lateral passage of proteins into the membrane.
Additional accessory proteins were identified in bacteria to help translocating and folding the membrane proteins and to assemble them into larger complexes. Despite the fact that these proteins are essential for cell survival, much less is known about how these proteins work. This is mostly due to the fact that they form a large assembly with the SecYEG translocon and it was difficult to produce this higher-order complex for experimental studies. By using new technology we have succeeded in generating the large holo-translocon complex and suggest here to study how the accessory proteins in the holo-translocon help to fold up membrane proteins that emerge from the ribosome, which is the protein factory of the cell. To this end, we will directly look at these molecular machines consisting of membrane-protein synthesizing ribosomes and active holo-translocon machinery by state-of-the-art electron microscopy and image processing. Due to recent developments in hard and software, this technology now enables to obtain unprecedented high-resolution structures and thus insights into the molecular interplay of translocation proteins with the synthesizing ribosome and with the translocated, to be folded membrane protein.
Proteins that are not correctly folded are recognized by the cellular quality control system. This system first attempts to fold up the protein with the help of energy, and if unsuccessful, degrades the mis-folded protein. Two components of the bacterial membrane are thought to have a key role: YidC and the FtsH-HflKC complex. Both are also present as homologous proteins in a human cellular organelle, the mitochondrion. How they work and recognize the unfolded protein is unknown today. We will elucidate here how these machines work together to ensure the proper folding of membrane proteins and to remove unfolded proteins that could be detrimental to the cell. We will use purified components to biochemically dissect the interplay and the mechanism of the folding/degradation machine, and we will use electron microscopy to visualize at high resolution the relevant complexes which we identify in this work.
Our studies will thus provide essential new insights into the poorly understood process of membrane protein genesis, folding, and concomitant quality control.

Technical Summary

HTL is active in protein secretion and membrane protein integration. We determined the architecture of HTL in an integrated approach combining cryo-EM, SANS, biochemistry and proteomics. We observed intriguing conformational changes in the SecD periplasmic domain in HTL supporting an active role of SecD during substrate translocation. We discovered a lipid-filled cavity in HTL which could serve as a protected folding space for membrane proteins, strikingly mirroring assisted folding of newly synthesized proteins in the cytoplasm. Importantly, SANS indicated that HTL is a dynamic machine, capable of flexibly adapting to accommodate variable sizes of translocating proteins.
We propose now to study the actively translocating HTL in complex with the ribosome. We will isolate native ribosomal assemblies from cell membranes and affinity-purify them. The structure of the ribosome-HTL complexes will be determined by cryo-EM at high resolution.
We will further explore membrane protein biogenesis and quality control by producing the FtsH-HflKC chaperone/protease complex and study its interactions with YidC and HTL. We will use pull-down, crosslinking and in vitro translocation experiments to study protein-protein interactions and interactions with the substrate. Further putative interaction partners will be identified by mass spectrometry. We will determine the stoichiometry of the relevant complexes by size-exclusion chromatography, blue native gel electrophoresis and/or analytical ultracentrifugation. Stable supercomplexes between FtsH-HflKC and YidC or HTL, if confirmed, will be studied by cryo-EM.
Our work seeks to address the fundamental question of how bacterial membrane proteins are inserted into the membrane, folded and assembled to complexes and how aberrant, misfolded membrane proteins are detected and eliminated. What is the role and interplay of HTL, YidC and FtsH-HflKC in these processes, and what is the structural basis of the underlying mechanisms?

Planned Impact

There is an urgent need for novel antibiotics. Interestingly, it has been shown that the FtsH protease complex reinforces the bactericidal effects of antibiotics that inhibit translation elongation, e.g. tetracycline and chloramphenicol. This leads to stalling of co-translational translocation. Such 'jammed' translocation machines are recognized by FtsH - our proposed work should reveal how - and subsequently degraded, a suicidal reaction. Unfortunately, cells have developed mechanisms to counteract: the Cpx envelope stress response induces YccA production, an inhibitor of FtsH. The HTL, FtsH-HflKC and YccA are thus interesting targets for antibiotics. Notably, YccA has a human homologue Bax Inihibitor-1, an anti-apoptotic protein that acts on the tumour suppressor Bax. Bax contributes to apoptosis upon prolonged stress in protein secretion in the endoplasmic reticulum.
I. Collinson (Co-I) is involved in a collaboration with the Dundee Drug Discovery unit who seek to target the bacterial Sec machinery as a target for novel antibiotics. Several compounds have been identified that inhibit the secretion activity of the core-SecY complex. The holo-complex also presents a good target for potential antibiotics. Therefore, new information gained on the mechanism of membrane protein insertion and quality control will be fed into the existing drug discovery platform that may then lead to the development of new and improved drug discovery strategies.
Applications of this work are identified from within the department (through regular discussion with our Impact lead and industrial liaisons) as well as by continuing discussions with our Research and Enterprise Department. Any outcomes of this work that are exploitable, notably in terms of intellectual property or knowledge transfer to the private sector, are handled by the highly experienced team within RED; who engage closely with funders when appropriate. The ACEMBL system, which was specifically developed to enable production of multiprotein complexes such as HTL, has been patented by our previous employer (EMBL) and was successfully commercialized (by Geneva Biotech SARL). We anticipate that similar technological advances may arise from the present project to the benefit of the wider academic and industrial R&D community.

This project includes considerable opportunity to train researchers involved in areas that go beyond the day-to-day research methodology. Examples include our extensive integration with public communication and outreach programmes and the extensive network of University schemes to benefit the training and development of research staff, an area where University of Bristol is very active.

Former research members of my laboratory now work for F. Hoffmann-La Roche Ltd (Basel), AstraZeneca (Cambridge) and as university lecturer (Maitre de conferences, University of Grenoble). Thus, the environment provided by my own lab as well as the University as a whole is highly conducive to career development of our staff beyond academic, basic science research and thus will contribute to the economic development of the nation. Our projects are also very data intensive, and the management and analysis of such large (multi-terabyte) datasets is applicable to many areas of professional life.

This work will lead to significant image data (2D and 3D) that is readily used in both public understanding of science and artistic arenas. Through our public engagement plans, entering competitions, and other outreach activities, this work therefore will contribute to local exhibitions and to communicate science to the public.
 
Description (1) Significant new knowledge generated:
We successfully expressed the FtsH-HflKC protease / chaperone complexes in Escherichia coli and purified it, using improved expression plasmids. We determined the stoichiometry of the complexes and used them for crosslinking/ mass spectrometry to identify protein-protein interactions. We identified interaction partners of FtsH by mass spectroscopy and co-expressed the FtsH-HflKC complexes with one of its binding partners, YidC, to test if they form a stable complex.
We analysed FtsH-HflKC complexes by negative stain electron microscopy which indicated that the complexes are homogenous in shape and size, with minor contamination by 50S large ribosomal subunits and FtsH only. We therefore froze cryo-grids of FtsH-HflKC complexes and optimised sample preparation for cryo-EM. Using the optimised cryo-grids, we collected high-resolution cryo-EM data for the complex at the national facility eBIC at Harwell. Image processing revealed a strong bias towards top and bottom views (which had high resolution) and a lack of side views (which had low resolution), hampering solution of a high resolution 3D structure of FtsH-HflKC, using these data. Because of the problems encountered, we further optimised FtsH-HflKC complex purification to remove remaining impurities and tested different protocols for cryo-grid preparation to obtain thinner ice and achieve a higher amount of FtsH-HflKC complexes per micrograph recorded. As a result of these experiments we stopped using DDM or LMNG as detergent. We collected significantly improved negative stain EM data for the FtsH-HflKC complex prepared according to our new purification protocol. In parallel, we also analyse FtsH and HflKC complexes alone by EM to understand the FtsH-HflKC complex assembly and molecular mechanisms of protease/ chaperone function.
With Ian Collinson we further analysed the lipid-filled cavity of the holotranslocon which we previously discovered (Botte et al., 2016). We used small-angle neutron scattering and molecular modelling (Martin et al., 2019). Current attempts aimed at solving a high-resolution structure of the holotranslocon in complex with the ribosome suffer from the flexibility of the holotranslocon complex: while the ribosome can be solved at high resolution, the holotranslocon part remains low resolution. Focused classification and refinement was tried, but did not improve the quality of the holotranslocon map. We now work to obtain high affinity nanobodies that stabilize the holotranslocon in a single conformation, which would allow us to solve a higher resolution cryo-EM structure.

(2) New or improved research methods or skills developed:
The techniques we develop to overcome the problems associated with these membrane protein complexes (holotranslocon and FtsH-HflKC complex) are of general interest in the field as evidenced by invitations to contribute methods papers and book chapters in Current Opinion in Structural Biology (2015), Methods Enzymology (2015, 2019), Methods Mol. Biol. (2017, 2020 in press) and Adv. Exp. Med. Biol. (2020 in press) and invitations to seminars and international conferences (see narrative impact and engagement activities). Using the modular approach to produce membrane protein complexes by co-expression of all membrane protein subunits (published first in Nature Methods 2008), Imre Berger and Christiane Schaffitzel supported collaborators to produce and purify human gamma secretase from insect cells (Khan et al., Sci. Reports 2018).
Exploitation Route We have shared the holotranslocon expression construct with numerous laboratories worldwide, thus enabling research on the Escherichia coli translocation machinery in those laboratories. We further have entered collaborations to support others to produce membrane complexes, including gamma secretase (Sci. Reports 2018). The holotranslocon construct is currently also used for co-expression with difficult to produce membrane proteins from other species to test the idea that co-expression of the holotranslocon might improve membrane protein complex production for biotechnological or medical research by assisting their integration into the membrane, protein folding and complex assembly.
Sectors Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description (1) Training highly skilled individuals for the UK workforce: We are constantly engaging in staff training: Dr. Burak Kabasakal (PDRA employed by this grant) and Lucy Troman (PhD student) from the Schaffitzel and Collinson labs visited a 1-day Relion image processing workshop in Bristol, organised by GW4 and CCPEM in May 2018. In March 2019, Dr. Burak Kabasakal participated in a course organised by CCPEM on 'Introduction to model building and refinement using cryo-EM maps' in Madrid. Dr. George Orriss, currently working on the BBSRC-funded project, is going to participate in the GW4/CCPEM Relion 3.1 image processing workshop in June 2020. Dr Burak Kabasakal left the Schaffitzel team in September 2019 to become an independent PI/ Lecturer at Bilkent University of Ankara, Turkey and to start his research team as PI there. He recently gave seminar presentations about the BBSRC-funded membrane protein quality control project at Universities of Ankara and Koc (see engagement activities). Teaching undergraduates: We have implemented single-particle cryo-EM and image processing in the curriculum of the Biochemistry students in Year 3 as a 2 hours lecture and in the Masters of Science programme 'Biophysics and Molecular Life Sciences' at University of Bristol. More recently, Schaffitzel implemented cryo-EM training as 3 hours lecture and a 2 hours hands-on initial training workshop, with ribosomal complexes in co-translational targeting and translocation as examples in the Masters of Science module 'Protein Assemblies and Molecular Machines'. In addition, the Schaffitzel lab trained two internship students from University of Bristol working on this BBSRC-funded project (see secondments, internship and placements section for details). Moreover, we regularly invite outstanding seminar speakers from the membrane protein and cryo-EM fields to give seminars at the school of Biochemistry. In September 2017, Christiane Schaffitzel organised the first Cryo-EM Symposium in Bristol; speakers included John Briggs and Sjors Scheres from LMB Cambridge, as well as Vicki Gold and Bertram Daum from MPI Frankfurt now University of Exeter. Further, we invited e.g. Tom Rapoport (Harvard), Bonnie Murphy (MPI Frankfurt) and Thomas Meier (Imperial College London) to visit and talk at the Biochemistry Seminar series about their research on large membrane protein complexes, including cryo-EM structures of translocation machineries and ATP synthase. (2) Societal Benefit: Public Engagement: We frequently have visitors in the laboratory, e.g. placements of students and of school pupils in the lab and visitors at Open Days in the School of Biochemistry. We use the membrane protein projects and cryo-EM to showcase cutting-edge technology in Biochemistry and Bioimaging to the public both from an information perspective as well as engaging young people in future science careers. (3) Further Communication and Engagement: Schaffitzel has given a many presentations during the course of this grant, including EMBO practical courses on cryo-EM, small angle neutron scattering and macromolecular complexes (see engagement activities). Further, CS was invited speaker to present the biochemical and structural work on the holotranslocon at the Zing conference on 'bacterial protein secretion' (St Pete Beach USA 2016), on the Biochemical Society meeting on 'New Approaches for Investigating Nascent Peptide Folding' (Cambridge, UK 2018) and on the 'Protein Folding on the ribosome meeting' (Stockholm 2018). Dr. Remy Martin (PDRA, Schaffizel lab) presented the holotranslocon project on the '3RD CCPBIOSIM/CCP5 conference on multi-scale modelling' in Leeds (July 2017). Dr. Burak Kabasakal presented his progress on the project on the GW4 cryo-EM user meetings in February 2019 and more recently as invited seminar speaker at the University of Ankara and University of Koc, Turkey. Co-I Imre Berger presented the project at the EMBO course 'integrated structural biology' at the Institute Pasteur, Paris (2019), the EMBO course 'high-throughput methods in structural biology' in Oxford (2019) and at the symposium 'new horizons in membrane transport and communication' in Frankfurt (2017). CS was invited to contribute book chapters on membrane protein production (Kabasakal, B.V., Jiang, Q. & Schaffitzel C., Meth. Mol. Biol. 2020 In Press) and single particle electron cryo-microscopy (Deniaud, A., Kabasakal., B., & Schaffitzel, C., Adv. Exp. Med. Biol. 2020 In Press). (4) Economic Impact, Exploitation and Application: Progress of this projects is at a "pre-competitive" stage in terms of commercial exploitation. The bacterial holo-translocon project aiming at a high resolution cryo-EM structure of the active translocation machinery is accompanied by an on-going drug discovery programme (by Ian Collinson and Dr A. Woodland, Drug Discovery Unit, Dundee) aimed at the identification of small molecule inhibitors of the bacterial translocation machinery. High-resolution cryo-EM structures of the FtsH-HflKC complex will be the starting point for developing inhibitors to be used as antibiotics against bacterial infection.
First Year Of Impact 2017
Sector Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description 'Academic Lead' of the South West Regional Facility for High-Resolution Electron Cryo-Microscopy, Bristol (2018-present)
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Membership of a guideline committee
Impact We made significant changes in the organisation of the facility. We are in the process of improving visibility, efficiency and effectiveness of usage of microscope time and computing resources.
 
Description Appointed Member of Diamond Peer Review Panel, panel 12 covering eBIC and B24, Harwell, UK (05/2019-11/2021)
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
Impact I am appointed member of the peer review panel 12 responsible for distribution of access to the national facility for Electron Bio-Imaging Centre (eBIC) and beamline 24 (including X-ray tomography). The panel ensures fair and transparent distribution of access to the high-end research infrastructure available at Diamond, Harwell, enabling cutting-edge research.
 
Description Appointed Member of the 'Wellcome Trust Dynamic Molecular Cell Doctoral Training Program Management Group'
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Membership of a guideline committee
 
Description Appointed Member of the Advisory Network of the Schering Stiftung, Berlin, Germany (Since 05/209)
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
URL https://scheringstiftung.de/en/stiftung/advisorynetwork/
 
Description Appointed Member of the BBSRC-funded SWBio Doctoral Training Program Management Group
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Membership of a guideline committee
 
Description Biochemistry Management Group, School of Biochemistry, Univ. of Bristol
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Membership of a guideline committee
 
Description Chair of Steering Committee, GW4 Regional Facility for High-Resolution Electron Cryo-Microscopy, Bristol (2017-2018)
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Participation in a advisory committee
 
Description Teaching postgraduate students, EMBO practical courses (as tutor and speaker) (since 2008)
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
 
Description BBSRC/EPSRC funded BrisSynBio Centre Bristol, Big Ideas in Biodesign
Amount £70,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2018 
End 03/2019
 
Description Biotechnology and Biological Sciences Research Council (BBSRC)-funded SWBio doctoral training programme studentship
Amount £100,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2020 
End 08/2024
 
Description South West Regional Facility for High-Resolution Electron Cryo-Microscopy
Amount £1,000,000 (GBP)
Funding ID 202904/Z/16/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2016 
End 01/2021
 
Description University of Bristol PhD Research Partnership Programme Studentship
Amount £100,000 (GBP)
Organisation University of Bristol 
Sector Academic/University
Country United Kingdom
Start 07/2020 
End 05/2024
 
Description Wellcome Trust Multi-User Equipment Grant, 'Expanding the capabilities and use of the South West Regional Facility for High-Resolution Electron Cryo-microscopy' (Co-I)
Amount £1,000,000 (GBP)
Funding ID (206181/Z/17/Z) 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2016 
End 11/2021
 
Description Collaboration with Prof Ian Collinson on membrane protein complexes 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution In this collaboration with the Collinson laboratory we share our expertise in membrane protein expression, purification, biochemical/ biophysical characterisation and structural biology. We share data, know-how, equipment and the cryo-EM & image processing facilities. We generated all constructs for production of membrane protein complexes: holo-translocon with different affinity tag combination and FtsH-HflKC complexes. We use the purified complexes for cryo-EM and image processing as well as for biophysical characterisation.
Collaborator Contribution In this collaboration, the Collinson laboratory provides expertise in membrane protein solubilisation and purification, expertise in reconstitution into proteoliposomes and establishment of activity assays as well as structural characterisation of membrane protein complexes. On a daily basis, we share data, expertise and equipment. The current project builds on the successes of work through a continuation of ongoing collaborations between Schaffitzel, Berger and Collinson laboratories which led to nine publications already, including Mol Cell, 2009, Nat Methods 2009, PNAS 2014 and Sci Rep 2016. Recent work on the lipid filled central cavity in the holotranslocon is under review and deposited in the Biorxiv preprint server.
Impact This collaboration is multidisciplinary, involving advanced Molecular Biology, Biochemistry, Biophysics and Structural Biology. To date, the ongoing collaboration between our laboratories yielded 10 publications: (1) Multiprotein Complex Production in E. coli: The SecYEG-SecDFYajC-YidC Holotranslocon. doi: 10.1007/978-1-4939-6887-9_18. (2) The structure of the SecYEG-SecDFYajC-YidC holo-translocon complex reveals a central lipid-filled cavity. doi:10.1038/srep38399. (3) Membrane protein insertion and assembly by the bacterial holo-translocon SecYEG-SecDF-YajC-YidC. DOI: 10.1042/BCJ20160545 (4) ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts. doi: 10.1007/978-3-319-27216-0_3. (5) Advances and challenges of membrane-protein complex production. doi: 10.1016/j.sbi.2015.03.010. (6) ACEMBLing a Multiprotein Transmembrane Complex: The Functional SecYEG-SecDFYajC-YidC Holotranslocon Protein Secretase/Insertase. doi: 10.1016/bs.mie.2014.12.027. (7) Membrane protein insertion and proton motive force-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC. doi: 10.1073/pnas.1315901111. (8) YidC and Oxa1 form dimeric insertion pores on the translating ribosome. doi: 10.1016/j.molcel.2009.04.019. (9) Automated unrestricted multigene recombineering for multiprotein complex production. doi: 10.1038/nmeth.1326. Schaffitzel has been invited to more than 20 conferences, meetings and universities to present outcomes from this collaboration (listed in engagement activities). Together with Ian Collinson, Imre Berger and other members of the SW structural biology community, Schaffitzel successfully applied for funding of a GW4 Facility for high-resolution cryo-EM (funded by two Wellcome Trust Multi User Equipment Grants and shared by Universities of Bath, Bristol, Cardiff and Exeter) and for the BBSRC Alert-funded BlueCryo image processing cluster. Our collaborative membrane protein projects profit from this new infrastructure available at University of Bristol.
Start Year 2006
 
Description Collaboration with Prof Imre Berger on expression technologies, membrane protein complexes and cryoEM 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution The Berger and Schaffitzel laboratory share data, information, expertise, training of staff, methods and equipment. In the FtsH project the Schaffitzel laboratory generates the plasmids for membrane protein expression and purifies the complexes. Purified protein complexes (from both labs) are used for electron microscopy and image processing and for biophysical, biochemical characterisation.
Collaborator Contribution The Berger laboratory provides training and expertise in state-of-the-art cloning and expression technology: e.g. Gibson assembly, CreLox recombineering and ACEMBL system. In collaboration with the Berger Lab we adapted the MultiBac system for multiprotein expression in insect cells for production of bacterial protein assemblies such as the holo-translocon complex. The ACEMBL system for expression in Escherichia coli has been published in Nat Methods (doi: 10.1038/nmeth.1326) and is patented (CA2754161A1) by Berger.
Impact This collaboration is multidisciplinary, involving advanced molecular biology, biochemistry, biophysics and structural biology. The collaboration with the Berger laboratory resulted in more than 24 publications to date; ten related to expression technologies or membrane protein complexes: (1) Multiprotein Complex Production in E. coli: The SecYEG-SecDFYajC-YidC Holotranslocon doi: 10.1007/978-1-4939-6887-9_18.; (2) A central cavity within the holo-translocon suggests a mechanism for membrane protein insertion. doi: 10.1038/srep38399. (3) Membrane protein insertion and assembly by the bacterial holo-translocon SecYEG-SecDF-YajC-YidC. doi: 10.1042/BCJ20160545. (4) ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts. doi: 10.1007/978-3-319-27216-0_3. (5) ACEMBLing a multiprotein transmembrane complex: the functional SecYEG-SecDF-YajC-YidC Holotranslocon protein secretase/insertase. doi: 10.1016/bs.mie.2014.12.027. (6) Membrane protein insertion and proton-motive-force-dependent secretion through the bacterial holo-translocon SecYEG-SecDF-YajC-YidC. doi: 10.1073/pnas.1315901111. (7) Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells. doi: 10.1016/j.jsb.2011.03.007. (8) Automated unrestricted multigene recombineering for multiprotein complex production. doi: 10.1038/nmeth.1326. (9) Multiprotein expression strategy for structural biology of eukaryotic complexes. PMID: 17355863. (10) Protein complex expression by using multigene baculoviral vectors. PMID: 17117155. This collaborative work has been presented at many conferences, meetings and seminars, highlights are listed above. Moreover, together with Imre Berger, we successfully applied for funding of a state-of-the-art cryo microscope (funded by two Wellcome Trust Multi-User Equipment grants) and the BlueCryo Image Processing Cluster (BBSRC funded). This new equipment is shared in a GW4 facility and helps many researchers in the South West of UK and in Wales to advance their research.
 
Description AFMB, University Aix-Marseille, France, Seminar Speaker (22.06.2020) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact I am invited to present my laboratory's current research activity and inform about our cryo-EM research infrastructure.
Year(s) Of Engagement Activity 2020
 
Description Biochemical Society Meeting "New Approaches for Investigating Nascent Peptide Folding", Downing College, Cambridge, invited speaker and session chair 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I was invited to present our research at the Biochemical Society Meeting at Downing College in Cambridge "New Approaches for Investigating Nascent Peptide Folding" , 11-13. December 2017. The audience was primarily academic, i.e. postgraduate students. The scientific discussions were excellent and new collaborations were discussed.
Year(s) Of Engagement Activity 2017
URL https://www.biochemistry.org/Events/tabid/379/MeetingNo/SA193/view/Conference/Default.aspx
 
Description CHEM Seminar: "Structural Studies of Membrane Proteins Involved in Quality Control" by Burak V. Kabasakal (now Ankara University), 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact 80 students attended the CHEM Seminar, entitled 'Structural Studies of Membrane Proteins Involved in Quality Control' by Dr Burak V. Kabasakal (Bristol University, now Ankara University), where Burak presented his work on the FtsH project. The presentation sparked questions and discussion afterwards and several undergraduate students were interested in joining the lab.
Year(s) Of Engagement Activity 2020
URL https://w3.bilkent.edu.tr/bilkent/chem-seminar-structural-studies-of-membrane-proteins-involved-in-q...
 
Description Cystic Fibrosis Foundation Workshop on Nonsense Readthrough, Bethesda, Maryland, USA, invited speaker (22-23 January 2019) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Supporters
Results and Impact Research Seminar and workshop, reporting about our recent work on protein quality control mechanisms, presenting and advertising the new GW4 Cryo-EM infrastructure in Bristol. Presentation sparked questions and discussion afterwards, invitation to future meetings and plans for future collaborations. The workshop was organised by the Cystic Fibrosis Foundation; the ca 60 participants were from charities, industry and academia (about one third each).
Year(s) Of Engagement Activity 2019
 
Description EMBO Practical Course "High throughput methods for protein production and structural analysis" at Diamond and the Research Complex at Harwell, invited speaker (10.-19.6.2019) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I am going to present our work on membrane protein complex production and new cryo-EM structures. I will represent the GW4 Facility for High-Resolution Cryo-EM and advertise our Cryo-EM infrastructure and image processing infrastructure, with the intent to spark interest in this research area and the techniques.
Year(s) Of Engagement Activity 2019
URL http://meetings.embo.org/event/19-protein-production
 
Description EMBO Practical Course "Small angle neutron and X-ray scattering from proteins in solution", Grenoble, France, invited speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I presented the use of cryo-EM and image processing to address biological questions and advertised the new cryo-EM and BlueCryo image processing capabilities in Bristol. I presented the cryo-EM structure of the holo-translocon and discussed its functions in membrane protein integration, folding and protein complex assembly. The presentation sparked excellent discussions with the students about membrane protein complexes, cryo-EM and image processing.
Year(s) Of Engagement Activity 2017
URL http://meetings.embo.org/event/17-small-angle-scattering
 
Description EMBO Practical Course "Small angle neutron and X-ray scattering from proteins in solution", Grenoble, France, invited speaker (24.09.2019) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I am going to present our work on membrane protein complex production, characterisation and new cryo-EM structures. I will represent the GW4 Facility for High-Resolution Cryo-EM and advertise our Cryo-EM infrastructure and image processing infrastructure, with the intent to spark interest in this research area and the techniques.
Year(s) Of Engagement Activity 2019
URL http://meetings.embo.org/event/19-small-angle-scattering
 
Description Faculty of Biology, Medicine and Health, University of Manchester, invited seminar speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Research Seminar. I presented our recent cryo-EM structure of the human 48S initiation complex and our holotranslocon project.
I represented the GW4 Facility for High-Resolution cryo-EM and advertised our cryo-EM and image processing infrastructure. I sparked interest in this research area and the techniques, we had good discussions and made plans for future collaboration.
Year(s) Of Engagement Activity 2018
URL http://events.manchester.ac.uk/event/event:o1hn-jnlm6bty-ynvpil/protein-and-rna-fate-seminar-christi...
 
Description MBGE Seminar: 'Structural Studies of Membrane Proteins Involved in Quality Control' by Burak V. Kabasakal (Bristol University, now Ankara University) at Koc University, Istambul, Turkey 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact About 70 researchers and students attened the seminar given by Dr. Burak Kabasakal reporting on his project 'Structural Studies of Membrane Proteins Involved in Quality Control'. His presentation sparked questions and discussion afterwards, and students asked for possible internships in Burak's laboratory.
Year(s) Of Engagement Activity 2020
URL https://zambak.ku.edu.tr/detail.php?i=21484
 
Description Novozymes Prize Symposium "Protein Folding on the Ribosome", Stockholm, Sweden, invited speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Research Seminar, reporting about our recent work on protein quality control mechanisms, presenting and advertising the new GW4 Cryo-EM infrastructure in Bristol. Presentation sparked questions and discussion afterwards, and invitation to future meetings.
Year(s) Of Engagement Activity 2018
URL https://cotranslationalfolding.wordpress.com/programme/
 
Description Organisation of the Opening Symposium of the GW4 Regional Facility for High-Resolution Electron Cryo-Microscopy (September 1st, 2017) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact Around 200 researchers, students, officials (Deans, Vice-Chancellors) from the GW4 universities, representatives from BBSRC, Wellcome Trust, and industry (GSK) joined the opening symposium of the GW4 Regional Facility for High-Resolution Electron Cryo-Microscopy in Bristol. The aim of the event was to make the researchers and the public aware of the new cryo-EM facility and image processing capabilities and the exciting possibilities offered by this new technology. There is clearly increased interest in the use of cryo-EM and image processing to answer biological questions. -- https://twitter.com/hashtag/GW4cryo?src=hash. http://gw4.ac.uk/news/gw4-alliance-unveil-cutting-edge-microscopy-facility/
Year(s) Of Engagement Activity 2017
URL http://gw4.ac.uk/shared-facilities/
 
Description Organisation of the SW Structural Biology/CCPEM Relion 3.1 Image Processing Workshop, University of Bristol (25th June 2020) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact I organise a Relion image processing workshop for early stage career researchers in collaboration with CCPEM and GW4 to train new users of cryo-EM and image processing , i.e. to help familiarise PhD students and postdocs with the new technique and software packages. We meet with this workshop the increased demand of hands-on training in this technology. The workshop will further provide networking opportunities enabling new/ outside users to access the equipment, and bring together scientists with diverse background sparking interesting discussions and increased interest in cryo-EM usage.
Year(s) Of Engagement Activity 2020
 
Description Organisation of the first GW4/CCPEM Relion Image Processing Workshop, University of Bristol (18th May 2018) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact I organised this workshop in collaboration with CCPEM and GW4 to train new users of cryo-EM and image processing , i.e. to help familiarise with the new technique and software packages. We meet with this workshop the increased demand of training in this technology (evidenced by 10-fold over-subscription of these type of training events). We accepted 32 participants , 30 from UK, 2 from EU countries, 4 from UK industry (GSK , Heptares, UCB Pharma).
The workshop provided a networking opportunity enabling new/ outside users to access the equipment, and brought together scientists with diverse background sparking interesting discussions and increased interest in cryo-EM usage.
Year(s) Of Engagement Activity 2018
URL http://www.ccpem.ac.uk/courses.php
 
Description Presentation, Japanese Society for Microscopy 'Frontiers in cellular, viral and molecular microscopy' meeting, University of Bristol, (16.09.2019) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact About 65 researchers attended the presentation, which sparked interest in our research and a vivid discussion afterwards. I received several emails asking for technical advice and collaboration.
Year(s) Of Engagement Activity 2019
URL https://www.yamauchilab.com/post/2019/09/17/two-days-of-great-science-ends-in-success
 
Description South West Structural Biology Annual Meeting, Cardiff, Plenary Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact I gave a Plenary Lecture about the new, shared Cryo-EM and BlueCryo Image Processing capabilities at the South West Structural Biology Annual Meeting in Cardiff. I thus raised awareness of the new facilities and increased the interest in applying electron cryo-microscopy to address biological questions. In addition, I presented our holotranslocon project. The presentation sparked excellent scientific discussions about membrane protein expression in bacteria versus vertebrates as well as the limits of X-ray and cryo-EM in structural biology.
Year(s) Of Engagement Activity 2017
URL https://www.cardiff.ac.uk/conferences/south-west-structural-biology-consortium-2017
 
Description UCL London, UK, invited seminar speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact I presented our research at the Institute of Molecular and Structural Biology, UCL (24.05.2017). It sparked excellent scientific discussions and led to an invitation to present our research at the Biochemical Society Meeting December 2017.
Year(s) Of Engagement Activity 2017
 
Description University of Glasgow, UK, invited seminar speaker 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact I presented our holo-translocon project at the Institute of Molecular, Cell and Systems Biology, University of Glasgow (15.02.2017). It sparked excellent scientific discussions and led to an invitation to present our research at the Biochemical Society Meeting December 2017.
Year(s) Of Engagement Activity 2017