CCEMMP Seminar Series

The CCeMMP are running a monthly research seminar series for interested researchers, research staff, students and enthusiasts. The seminar will be based mainly around cryo-electron microscopy of/and membrane proteins with a mixture of domestic and international speakers in this field. Speakers will be announced monthly with a calendar invitation with speaker and virtual details.
The seminar will be held at 10:00-11:00am AEDT/AEST on every second Tuesday of each month. If you would like to be invited to the seminars, please register to this event and the Centre Manager will send you calendar invitations.

 

Seminar dates for 2022

The CCeMMP also run special seminars outside of the regular seminar series. See below for the presenters and registration details. 

Past seminars

13 September 2022

Dr. David Thal

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville)

Cryo-EM has revolutionised the determination of protein structures and holds great promise for structure-based drug discovery. Recent structural studies from their group, utilising cryo-EM to determine the structures of therapeutically relevant protein targets in complex with novel chemical ligands, will be presented. This will include examples from different types of proteins, including G protein-coupled receptors (GPCRs), ion channels, and soluble enzymes. Specific examples will include cases where the cryo-EM structures revealed ligands in expected binding sites, ligands in unexpected binding sites, unexpected ligands in expected binding sites, no ligand at any site, and examples in between. Validating these ligand binding sites and current limitations, will also be discussed.

Bringing life into frozen proteins to elucidate their molecular mechanisms
Prof. Raunser will focus on membrane proteins and filamentous protein complexes and present examples where interactions with small molecules have been fully characterized at atomic resolution. In particular, he will present his team’s structural work on the canonical TRPC4 ion channel and its regulation by calmodulin and pharmacological agents as well as the small molecule modulation of the Drosophila Slo channel. In addition, Prof. Raunser will highlight several F-actin structures in complex with toxins and small molecules. In the second half, he will concentrate on Tc toxins from insect-pathogenic bacteria. Tc toxins form a special protein complex that stores a “killer enzyme” in a cocoon and injects it into the cell only after contact with the host via a novel, syringe-like mechanism. There, the killer enzyme unfolds its toxic effect and leads to the aggregation of the cytoskeleton and ultimately to cell death. The team have structurally and functionally elucidated this mechanism which is of fundamental importance for the general understanding of the transport of active substances through membranes and could even be used for specific medical applications.

6 September 2022

Professor Stefan Raunser

Department of Structural Biochemistry

Max Planck Institute of Molecular Physiology

9 August 2022

Associate Professor Megan Maher

Bio21 Molecular Science and Biotechnology Institute

The University of Melbourne

Structural insights into manganese transport across membranes

Metal ions are essential for all forms of life. In prokaryotes, ATP-binding cassette (ABC) permeases serve as the primary import pathway for many micronutrients including the first-row transition metal manganese. However, the structural features of ionic metal transporting ABC permeases have remained undefined. This presentation will describe the crystal structure of the manganese transporter PsaBC from Streptococcus pneumoniae in an open-inward conformation. The Type II transporter has a tightly closed transmembrane channel due to ‘extracellular gating’ residues that prevent water permeation or ion reflux. Below these residues, the channel contains a hitherto unreported metal coordination site, which is essential for manganese translocation. These structural features are highly conserved in metal-specific ABC transporters and are represented throughout the kingdoms of life. Collectively, our results define the structure of PsaBC and reveal the features required for divalent cation transport.

Structural insights into the cytotoxic peptides ATP-driven exporter and drug resistance

Staphylococcus aureus is a major human pathogen that has acquired an alarming broad-spectrum resistance to many of the commonly used antibiotics including beta-lactams such as penicillin. S. au\reus often causes hospital- and community- associated infections responsible for significant morbidity and death. Staphylococci infections are mediated trough a large array of secreted toxins including the phenol-soluble modulins (PSMs). PSMs are amphipathic, α-helical peptides with pronounced surfactant-like properties that have multiple key roles in pathogenesis. A specialized ATP-binding cassette (ABC) transport system exports PSMs to the extracellular environment and is essential for bacterial growth by providing an immunity against self-expressed PSMs. Here, we present the structural characterization of the PSM transporter determined by high-resolution single-particle cryo-EM and X-ray crystallography accompanied with functional characterization in vivo. 

12 July 2022

Assistant Professor Natalie Zeytuni

Department of Anatomy and Cell Biology

McGill University

14 June 2022

Assistant Professor Jianping Wu

School of Life Sciences

Westlake University

Structural elucidation of CatSper by cryo-EM

The cation channel of sperm (CatSper) is essential for sperm motility and fertility. CatSper is exclusively expressed in testis and dysfunction of CatSper can lead to male infertility. It is therefore an ideal target for the treatment of male infertility and the development of novel non-hormonal contraceptives. CatSper is the most complicated ion channel known, and its structural study has been extremely challenging. I will discuss our recent progress in purifying the native CatSper from the sperm of transgenic knock-in mice. We further using cryo-EM to elucidate its high-resolution structure. Aside from the exciting unique assembly features of CatSper, the structure also reveals several previously uncharacterized components of CatSper, exemplified by an organic anion transporter SLCO6C1. CatSper thus is a channel–transporter ultracomplex that we termed the CatSpermasome. Our study showcases the power of structural biology in making new discoveries.

Structural studies of heteromeric AMPA Glutamate receptors
AMPA Glutamate Receptors (AMPARs) are ion channels located at post-synaptic neurons, where they mediate fast-excitatory neurotransmission. AMPARs are tetrameric receptors composed of different combinations of four subunits, GluA1 to GluA4, which also interact with auxiliary subunits, such as TARPs, cornichons or GSG1L. Here I will summarize the recent advances in the structural biology of AMPAR complexes, focusing on the gating cycle of the GluA1/2-TARP-g8 complex. I will describe the structural features of the receptor in different functional states and dissect the impact of TARP-g8 in the modulation of the channel properties. Combining structural data with electrophysiology and molecular dynamics simulations we show how TARP-g8 extracellular domains interact with the receptor ligand binding domain to modulate AMPAR function. We also analyze how receptor gating is coupled to changes in the selectivity filter, which determines cation selectivity, as well the role of TARP-g8 in the modulation of receptor rectification.

7 June 2022

Doctor Beatriz Herguedas

“Ramón y Cajal” fellow

Institute for Biocomputation and Physics of Complex systems (BIFI)

University of Zaragoza (Spain)

10 May 2022

Doctor Raphael Trenker

Postdoctoral Fellow

Natalia Jura Lab

Cardiovascular Research Institute

University of California San Francisco

Structures of the HER2-HER3-NRG1b receptor complex reveal a dynamic dimer interface induced

The Human Epidermal Growth Factor Receptor 3 (HER3) and its close homolog, the orphan receptor HER2, are single pass transmembrane receptor tyrosine kinases that form a pro-oncogenic signaling complex upon binding to the HER3 ligand neuregulin-1b (NRG1b). Until recently, there were no structural insights into the HER2/HER3 heterodimer owing to the difficulties in its reconstitution in vitro. We isolated near full-length HER2/HER3/NRG1b heterocomplex and obtained a 2.9 Å cryo-electron microscopy (cryo-EM) reconstruction of the extracellular domain module, which revealed a surprisingly dynamic dimerization interface. Based on additional structures of this heterocomplex in which HER2 harbors its most frequently observed oncogenic mutation, S310F, and of this complex bound to the therapeutic antibody trastuzumab, it will be discussed how oncogenic mutations and therapeutics appear to exploit the intrinsic dynamics of the HER2/HER3 heterodimer.

Structural characterization of conserved neutralizing epitopes on the SARS-CoV-2 spike glycoprotein

SARS-CoV-2 infection or vaccination produces neutralizing antibody responses that contribute to better clinical outcomes. The receptor binding domain (RBD) and the N-terminal domain (NTD) of the spike trimer (S) constitute major neutralizing targets for the antibody system. Here we describe structures of donor-derived broadly-neutralizing antibodies bound to RBD and NTD epitopes that are conserved across the major SARS-CoV-2 variants of concern. We conclude SARS-CoV-2 infection and/or Wuhan-Hu-1 mRNA vaccination produces a diverse collection of memory B cells that produce anti-spike antibodies, some of which can neutralize variants of concern and likely contributes to the relatively benign course of subsequent infections with SARS-CoV-2 variants including omicron.

5 April 2022

Assistant Professor Christopher Barnes

Biology and ChEM-H Institute Scholar

Stanford University

8 March 2022

Assistant Professor Oliver Clarke

Physiology and Cellular Biophysics 

Anesthesiology & the Irving Institute for Clinical and Translational Research 

Columbia University

Architecture of the erythrocyte ankyrin-1 complex elucidated by Cryo-EM
The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes, and sub-tomogram averages of the same complexes from native membrane vesicles. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.2 and glycophorin A. The distinct T-shaped conformation of membrane-bound ankyrin-1 facilitates recognition of RhCE and unexpectedly, the water channel aquaporin-1. Together, our results uncover the molecular details of ankyrin-1 association with the erythrocyte membrane, and illustrate the mechanism of ankyrin-mediated membrane protein clustering.
Discovering how pore forming proteins evolve different assembly and targeting mechanisms

Pore forming proteins are proteins that can literally punch holes (pores) into target cell membranes. There are over 30 different types of pore forming proteins that have evolved independently but one of the most fascinating is the MACPF/CDC superfamily. The MACPF/CDC proteins can oligomerise into a ring-shaped transmembrane beta-barrel pore capable of either direct cell lysis or the passive transport other large protein toxins. Members of the MACPF/CDC superfamily are found in all kingdoms of life with a range of functions including as immune effectors, pathogenicity factors, parasite egress, fungal defence and marine toxins. Whilst current structural biology research on the MACPF/CDC family suggest there is a common domain and a common pore structure for the family, there is a wide variation in the assembly pathways observed. Recent research using combinations of structure and single molecule imaging methods explains how and why different members have evolved different assembly pathways to suit their evolved function.

15 February 2022

Associate Professor Michelle Dunstone

Department of Biochemistry and Molecular Biology

Biomedical Discovery Institute

Monash University

 

14 December 2021

Doctor Doreen Matthies

Earl Stadtman tenure track investigator

Head, Unit on Structural Biology

Division of Basic and Translational Biophysics

Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)

Cryo-EM of membrane proteins:
What have we learned in the last decades and what are the challenges?

In the last decade the field of Structural Biology has made great advances in using electron microscopy to solve structures of protein complexes including membrane proteins to high resolution. Best practices of how to use Single Particle Cryo-EM and more importantly how to optimize a membrane protein sample such as an ion channel or a transporter will be discussed. Most membrane protein structures are currently resolved in a detergent micelle, but cryo-EM also makes it possible to look at membrane protein complexes in a lipid bilayer, such as synthetic or native lipid nanodiscs, liposomes, or even inside cells now. A brief introduction to each of these techniques will be discussed with examples of the conformational landscape of magnesium channel CorA, voltage-gated potassium channel Kv1.2-2.1, a human excitatory amino acid transporter and more.

Using cryo-EM to interrogate the structure and dynamics of GPCRs

Cryo-electron microscopy (cryo-EM) continues to grow as a powerful method for structural studies of biomolecules and their complexes. Nowadays, it can routinely determine molecular structures with resolutions in the 2.5 – 3.5 Å range. Such results are adequate for modelling of the protein but lack fidelity for confident localization of water molecules and hydrogen atoms. Unambiguous elucidation of the biochemistry behind protein function and pharmacology of drugs would require atomic resolution structures, at levels below 1.5 Å. Last year, several groups worldwide demonstrated atomic resolution cryo-EM with a test sample comprising the “easy” soluble protein apoferritin. This was an important technological milestone showcasing the best-case-scenario capabilities of cryo-EM. However, membrane proteins, and other real-world samples, impose numerous experimental challenges, such as small size, heterogeneity, flexibility, preferential orientation, etc. 

9 November 2021

Professor Radostin Danev

Advanced Structural Studies

Graduate School of Medicine, The University of Tokyo

12 October 2021

Professor Patrick Sexton

Director, ARC Centre for Cryo-electron Microscopy of Membrane Proteins; 

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University

Using cryo-EM to interrogate the structure and dynamics of GPCRs

G protein-coupled receptors (GPCRs) are the largest superfamily of cell surface receptor proteins and a major target class for drug development. GPCRs are inherently flexible proteins that have evolved to allosterically communicate external signals to modulation of cellular function through recruitment and activation of transducer proteins, particularly G proteins. Technological evolution in cryo-EM combined with continuing advances in biochemical approaches for the stabilisation of active-state complexes of GPCRs with different transducer proteins is now enabling structural interrogation of receptor activation and transducer engagement. Moreover, cryo-EM can access conformational ensembles of GPCR complexes that are present during vitrification, which can provide a window into the dynamics of these complexes.