Hosted by

Monash Ramaciotti Centre for Cryo EM

©2019 by CRYOZ



1st AMMS Cryo EM Aus/NZ Meeting

June 22, 2018, Monash University, Melbourne

Program Outline
8.30              Registration open
8.50              Start - Introduction
9.00             Session 1 - Small Proteins
10.30            Morning Tea
11.00             Session 2 - Cryo-Tomography & Viruses
12.30             Lunch
13.30             Session 3 - Methods
15.00            Afternoon Tea
15.30             Session 4 - Large Complexes
17.00            Mixer at Sir John Monash Bar (until 19.00)

Keynote 1: Patrick Sexton

Monash Pharmacy

Using cryo-EM for determination of active-state GPCRs; lessons for understanding of receptor activation and biased agonism

Cryo-electron microscopy (cryo-EM) has gained prominence as a method of choice for determination of structure for difficult to crystalize membrane proteins. This is true also for active-state, transducer complexed, G protein-coupled receptors (GPCRs). Our laboratory has applied single particle cryo-EM to determine GPCR structures in complex with the canonical transducer, heterotrimeric G proteins, with an emphasis on class B1 GPCRs. Class B peptide hormone GPCRs bind critically important physiological peptides of ~30-40 amino acids, and are important targets for major disease including diabetes, obesity and osteoporosis. We have applied Volta phase-plate cryo-electron microscopy to derive structures of class B GPCRs as active complexes of peptide agonist, receptor and heterotrimeric G proteins. This methodology has been applied to minimally modified receptors. The human calcitonin (CT) receptor in complex with salmon CT and Gs was solved to a global resolution of 4.1Å. This study revealed significant conformational variance in the position of the N-terminal extracellular domain (ECD), and the a-helical domain of the Gas. Recently, we have solved the structure of the human glucagon-like peptide-1 (GLP-1) receptor, in complex with the G protein-biased agonist, exendin-P5, and Gs at a global resolution of 3.3Å. Common macromolecular changes associated with G protein coupling included a profound kinking of TM6 around the class B GPCR conserved Pro-X-X-Gly motif (required for the outward movement of the base of TM6 to accommodate G protein binding), and outward or lateral movements of the upper segments of TM7 and TM1, bending around conserved Gly residues. Mapping of mutation data and comparison of these structures with the published transmembrane domain structures of inactive class B GPCRs, and the active GLP-1/GLP-1R/Gs structure has enabled novel understanding of the conformational reorganisation accompanying receptor activation, and has provided mechanistic insight into control of biased agonism. For the GLP-1R mapping of data from alanine scanning mutagenesis revealed two distinct domains involved in conformational propagation linked to signalling. One was predominantly associated with reorganisation of extracellular loop (ECL) 2, while the second involved the upper segment of TM1, ECL3 and the membrane proximal segments of TM6 and TM7. This latter domain was critical for peptide activation of ERK, while both domains were required for G protein-dependent signalling. The TM1/ECL3 domain was also the location of the major differences in receptor conformation between the ExP5 and GLP-1 bound GLP-1R structures that was linked to distinct efficacy for Gs signalling. Interestingly, the pattern of mutational effect was remarkably different when comparing ECL2 and ECL3 of the CTR. More recently, we have determined the structure of the GLP-1R bound to oxyntomodulin, an endogenously expressed biased ligand of this receptor, as well as the structure of the class A adenosine A1 receptor bound to its endogenous agonist adenosine and a Gi2 heterotrimeric protein. Collectively this work is providing insight into the complexity of GPCR signalling and the similarity and diversity in modes of receptor activation between members of related subfamilies.


Keynote 2: Alexander Rigort

FEI/Thermo Fisher

In situ cryo-tomography allows unravelling cellular functions

Imaging cells by cryo-electron tomography requires samples that are thin enough to allow electrons with energies of 300 keV to be transmitted - typically not much more than 300 nanometers. Most cells exceed these dimensions and are too thick to study intact in cryo-transmission electron microscopy (cryo-TEM). Therefore, they must be thinned to be transparent to the electron beam before they can be imaged. The method-of-choice for thinning vitreously frozen specimens down to the appropriate thickness of 200-300 nm is cryo-focused-ion-beam (cryo-FIB) milling [1,2].

The cryo-FIB microscope brings the structural cell biologist a preparation tool which allows preparing large, distortion-free windows into the cell’s interior and site-specific regions from vitrified cells. Such electron-transparent windows are referred to as ‘in situ lamellas’. A thin in situ lamella is prepared from frozen-hydrated cellular specimens by removing material above and below the target section with the ion beam while observing the operation with the scanning electron beam. Notably, the ‘in-situ lamella milling’ or ‘on-the-grid-thinning’ approach [3] does not require any subsequent lift-out of the lamella and its placement on a cooled TEM lift-out grid with a micromanipulation device. Instead, the electron-transparent lamella remains on the EM grid, which afterwards is transferred to a cryo-TEM, where a tomographic image series is acquired as the lamella sample is tilted incrementally. Finally, the images are combined computationally to reconstruct the final 3D tomogram.

Recent technical advancements in cryo-FIB instrumentation and cryo-electron tomography have made it possible to study native complexes in situ with molecular resolution [4] and to discover hitherto unknown ultrastructural changes inside cells with unprecedented level of detail [5,6]. The cryo-FIB is on its way to become a key technique which is able to prepare optimal samples for cryo-electron tomography. Inside cryo-lamellas, proteins and their functional relationships and interactions with other components in the cellular environment can be studied in their native context.


[1] Rigort and Plitzko, Arch Biochem Biophys. 2015; 581:122-30.

[2] Schaffer et al., J Struct Biol. 2017, 197(2):73-82.

[3] Rigort et al., Proc Natl Acad Sci U S A, 2012, 109(12):4449-54.

[4] Bykov et al., Elife, 2017, Nov 17;6 pii: e32493.

[5] Baeuerlein et al., Cell, 2017, 171, 179–187.

[6] Guo et al., Cell, 2018, 172, 1–10.

Session 1:  9am

Small Proteins

Chairs: Georg Ramm (Monash)/Eric Hanssen (UoM)

Keynote Lecture:  Patrick Sexton (Monash) - Using cryo-EM for determination of active-state GPCRs; lessons for understanding of receptor activation and biased agonism

Ben Gully (Berry lab, Monash) - The cryo-EM structure of the cell-surface receptor DEC205 reveals a conformational control-mechanism for antigen detection by dendritic cells

Nick Liau (Babon lab, WEHI) - Cryo-EM of a JAK complex

Wilson Wong (WEHI) - Structural studies of the Plasmodium falciparum Rh5/CyRPA/Ripr invasion complex by cryo-electron microscopy

Hari Venugopal (Monash Ramaciotti Centre) - using the Volta Phase Plate in a high-throughput Cryo EM facility


Session 2:  11am    Viruses, larger proteins & Cryo-Tomography

Chairs: James Bouwer (UoW)/Andrew Leis (UoM)

Alastair Steward (Victor Cheng) - Cryo-EM studies of E. coli ATP synthase

Mihnea Bostina (Otago) - Cryo-EM Structure of Seneca Valley Virus Procapsid

Josh Hardy (Coulibaly lab, Monash) - Investigating the architecture of an assembled non-contractile bacteriophage tail using cryo-EM and helical reconstruction

Kevin Chen (Collins lab, UQ) - Molecular architecture of the membrane-assembled retromer coat by cryo-electron tomography

Georg Ramm (Monash) - using multiple EM approaches to study Mitochondria 

Session 3:  1.30pm   Methods and Instrumentation

Chairs: Roger Wepf (UQ)/Nick Ariotti (UNSW)

Alexander Rigort (FEI, Keynote) - In situ cryo-tomography allows unravelling cellular functions

Roger Wepf (UQ) Cryo-Connectivity: closing the gap for sample manipulation & transfer in cryo electron microscopy & cryo characterisation workflows.

Alex de Marco (Monash) -  Oxygen Plasma FIB

Hans Elmlund (Monash) - Algorithms for accelerated near-atomic resolution single- and multi-particle 3D reconstruction

Chris Lupton (Whisstock lab, Monash) - Establishing micro electron diffraction as a new tool for structural biology

Company Presentations


Session 4:  3.30pm    Large Complexes (Infection & Immunity)

Chairs: Matthias Floetenmeyer (UQ)/Hari Venugopal (Monash)

Bostjan Kobe (UQ) - Using cryo-electron microscopy and helical reconstruction to characterize cytoplasmic signalling by Toll-like receptors

Leann Tilley (UoM) - Structure and function of the proteasome activator PA28 of the malaria parasite Plasmodium falciparum

Bradley Spicer (Dunstone lab, Monash) - Protein conformation of C9 controls the final membrane complex assembly

Isabelle Rouiller (UoM) - Cryo-EM structure of the Triclosan Efflux pump TriAxBC from pseudomonas aeruginosa

Michael Landsberg (UQ) - ABC toxins from pathogenic bacteria

Matt Belousoff (Lithgow lab, Monash) - Tackling antimicrobial resistance (AMR) using single particle cryoEM