MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy

475

568

Summary Glucagon-like peptide-1 (GLP-1) is a hormone with essential roles in regulating insulin secretion, carbohydrate metabolism and appetite. GLP-1 effects are mediated through binding to GLP-1R, a family B G protein-coupled receptor (GPCR) signaling primarily through the stimulatory G protein Gs. Family B GPCRs are important therapeutic targets, however our understanding of their mechanism of action is limited by the lack of structural information on activated and full-length receptors. Here we show the electron cryo-microscopy structure of the peptide-activated GLP-1R:Gs complex at near atomic resolution. The peptide is clasped between the N-terminal domain and transmembrane core of the receptor, further stabilized by extracellular loops. Conformational changes in the transmembrane domain result in a sharp kink in the middle of transmembrane helix 6, which pivots its intracellular half outward to accommodate the α5 helix of GαsRas. These results provide a structural framework for understanding family B receptor activation through hormone binding.

Section: Methodsmentioning

confidence: 99%

Cryo-EM structure of the activated GLP-1 receptor in complex with a G protein

Zhang

Sun

Feng

et al. 2017

475

568

“…3056 images were deemed suitable for further data processing. Frames were aligned using MotionCor2 41 ; global and per-particle CTF was calculated using gCTF 42 . Particle picking was done using Gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/) and resulted in 1,206,222 particles.…”

Section: Methodsmentioning

confidence: 99%

“…Images were collected on a Thermo-Fisher Krios equipped with an energy filter and a Gatan K2 Counting camera; the magnification was set to 105,000×, with a calibrated pixelsize of 1.10 Å. b , Representative 2D class averages as calculated with Cryosparc 41 . c , Schematic diagram of the data processing.…”

Section: Extended Datamentioning

confidence: 99%

See 1 more Smart Citation

Structure of the insulin receptor–insulin complex by single-particle cryo-EM analysis

Scapin

Dandey

Zhang

et al. 2018

192

243

Summary The insulin receptor (IR) is a dimeric protein that plays a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels1,2. IR dysfunctions have been associated with many diseases, including diabetes, cancer, and Alzheimer’s1,3,4. The primary sequence has been known since the 1980s5, and is composed of an extracellular portion (ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Insulin binding to the dimeric ECD triggers kinase domain auto-phosphorylation and subsequent activation of downstream signaling molecules. Biochemical and mutagenesis data have identified two putative insulin binding sites (S1 and S2)6. While insulin bound to an ECD fragment containing S1 and the apo ectodomain have been characterized structurally7,8, details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single particle cryoEM reconstructions for the 1:2 (4.3 Å) and 1:1 (7.4 Å) IR ECD dimer:Insulin complexes. The symmetric 4.3 Å structure shows two insulin molecules per dimer, each bound between the Leucine-rich sub domain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the S1 binding interactions and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative sub domain orientations and triggers downstream signal propagation.

“…About 4,000 raw movie micrographs were collected and motion-corrected using the program MotionCorr 2.0 47 . Contrast transfer function parameters of each aligned micrograph were calculated using GCTF 48 .…”

Section: Methodsmentioning

confidence: 99%

The atomic structure of a eukaryotic oligosaccharyltransferase complex

Bai

Wang

Zhao

et al. 2018

101

131

SUMMARY N-glycosylation is a ubiquitous modification of eukaryotic secretory and membrane-bound proteins; about 90% of glycoproteins are N-glycosylated. The reaction is catalyzed by an eight-protein oligosaccharyltransferase complex, OST, embedded in the ER membrane. Our understanding of eukaryotic protein N-glycosylation has been limited due to the lack of high-resolution structures. Here we report a 3.5-Å resolution cryo-EM structure of the Saccharomyces cerevisiae OST, revealing the structures of Ost1–5, Stt3, Wbp1, and Swp1. We found that seven phospholipids mediate many of the inter-subunit interactions, and an Stt3 N-glycan mediates interaction with Wbp1 and Swp1 in the lumen. Ost3 was found to mediate the OST-Sec61 translocon interface, funneling the acceptor peptide towards the OST catalytic site as the nascent peptide emerges from the translocon. The structure provides novel insights into co-translational protein N-glycosylation and may facilitate the development of small-molecule inhibitors targeting this process.