Cell Symposia S4 Notes

Visualization of protein biogenesis at the ER membrane

Friedrich Forster, Utrecht University

imaging in native context complements structures of isolated complexes

cryo-ET, multi-particle approach resolves ER-bound ribosomes at high resolution

processing pipeline yields high-resolution reconstructions of ribosomes caught in the act

classification reveals different states of ribosome

Hibernating ribosomes reside at ER membrane

A zoo of protein(complexes) interacts with ER translocon co-translationally

different types of ER translocons co-exist at the ER membrane

Ribosome 3d distribution of cytosolic and ER-bound polysomes differ

TRAP-OSTA-Sec61 translocon is most abundant in HEK cells

AlphaFold fascilitates complete atomic model building of TRAP-OSTA-Sec61

AlphaFold multimer indicates association

Multipass translocon, consists of BOS, GEL, PAT subcomplexes

ER signal peptides have 3 distinct regions, SPs with h-region around 20 residues are not cleaved

the short h-region is a binding determinant

High-resolution structure of SP-engaged SPC reveals interactions

ribosome intermediates change on anisomysin stress

methane-oxidising enzymes

Yanan Zhu, University of Oxford, UK

pMMO, important membrane protein

Hexagonally pMMO arrays in ICMs

summary

• We show by serial cryoFIB/SEM volume imaging and lamellae-based cellular cryoET that these ICMs are derived from the inner cell membrane
• The pMMO trimer, resolved by cryoET and STA to 4.8A in the ICM, forms higher-order hexagonal arrays in intact cells
• Array formation correlates with increased enzymatic activity, providing a new perspective for understanding pMMPO function
• MDH and pMMO are concentrated in small compartment for their equential reaction in the cell
• These findings also demonstrate the power of cryoET to structurally characterize native membrane enzymes in the cellular context

cryoET studies of coat complexes

Glulia Zanetti, Birkbeck University of London, UK

secretory pathway

the COPII coat mediates ER to Golgi transport

induce membrane remodeling and recruit cargo in budding carrier

the molecular determinants of COPII assembly

the outer COPII coat: the Sec13-31 complex

outer coat structure and interactions

the inner coat layer and interactions between inner and out coat. two interesting sites of interaction that are not detected in the crystal structure

the inner coat arranges in a ‘patchy’ lattice

cargo is bound to the inner coat

human COPII versus yeast COPII

in situ analysis of structures and dynamics inside neuronal synapses

Guoqiang Bi, University of Science and Technology of China Synapse

postsynaptic receptors and scaffold

visualize native synaptic ultrastructure with cryoET

cryoCLEM to distinguish different synapses

distinct morphological features of excitatory and inhibitory synapses

$GABA_{A}$ receptor organization, semi-ordered, mesophasic,

distinct size distribution of vesiicles near the presynaptic membrane, vesicle status: contacting, pore, closed vesicles

distinct states of contacting vesicles following optogenetic stimulation

summary

• With cryo-ET and CLEM, previously unattainable ultrastructural features of identified synapses are resolved at molecular resolution in their native state
• In an inhibitory neuronal synapse, $GABA_{A}$ receptors and scaffolding proteins self-organize into a distinct mesophasic supramolecular assembly
• With time-resolved cryoET and optogenetic stimulation, a series of dynamic states of synaptic vesicles undergoing exocytosis are captured
• The existence of small contacting vesicles indicates quasi-stable intermediate states during synaptic exocytosis

IgG and COVID-19

Sai Li, Tsinghua University

Avidity matters for antibody neutralization potency against enveloped viruses

IgG acquires its avidity by encoding 2 copies of Fab and a flexible hinge region

SARS-CoV-2 spike protein has exceptional mobility for efficient receptor binding

SARS-CoV-2 became hairy after IgG-incubation

IgG induces an RBD to shift from down to up conformation

IgG induces S1 and S shedding over time

Bivalent binding of IgG on S-trimers, an S-IgG dimer-of-trimers coupled by bivalent binding

IgG actively recruit S-trimers for bivalent binding, couple S into S-IgG Gemini complexes

IgGs cluster S-trimers on the virion surface

More ways of S-oligomerization by IgG bivalent binding

IgGs cluster S on the virion surface

Viral envelope provides a native context for bivalent IgG binding

axonemal structure of ependymal cilia

Yao Cong, CAS

Motile cilia/Flagella drive extracellular fluid flow or rapid cell movement

Dysfunction in motile cilia/flagella lead to PCD

RS transmits mechanochemical signals between central pair and dynein arms to coordinate ciliary motility

distinct RS-head morphologies between protozoa and metazoan

conformational dynamics of RS head-neck dimer

cryo-ET axonemal structure of ependymal cilia, auto-picking method

stepwise mammalian RS assembly mechanism

summary

• Near atomic resolution cryoEM of mammalian RS head-neck complex
• Mapping of PCD causing mutations, and possible causes of related PCD
• Revealed dynamic motions of RS head-neck dimer
• The first in situ cryoET structure of ependymal cilia
• Lack of IDA-b/c/e on and Tektin filaments within DMT in ependymal cilia
• Proposed an assembly mechanism of mammalian RS head-neck
• Coordinated rigid and elastic RS-CP interaction modes beneficial for the regulation of asymmetric ciliary beating

in-cell structure of sperm microtubule doublets

Yun Zhu, Institute of Biophysics, CAS

structure of motile cilia/flagella

sperm axoneme

axoneme basic element: DMT, using SPA to resolve isolated DMT structure

the intact mouse sperm axoneme, cryo FIB milling, intact 96nm axoneme structure

AI facilitated modeling of 16nm repeats MIPs

tektin 1/2/3/4/5 arranges in offset manner

unique 5-3-3-4-4-4 pattern

Disease-associated mutations mapped on sperm DMT structure