Inositol-1,4,5-trisphosphate receptors (InsP3Rs) are ubiquitous ion channels responsible for cytosolic Ca2+ signalling and essential for a broad array of cellular processes ranging from contraction to secretion, and from proliferation to cell death. Despite decades of research on InsP3Rs, a mechanistic understanding of their structure–function relationship is lacking. Here we present the first, to our knowledge, near-atomic (4.7 Å) resolution electron cryomicroscopy structure of the tetrameric mammalian type 1 InsP3R channel in its apo-state. At this resolution, we are able to trace unambiguously ∼85% of the protein backbone, allowing us to identify the structural elements involved in gating and modulation of this 1.3-megadalton channel. Although the central Ca2+-conduction pathway is similar to other ion channels, including the closely related ryanodine receptor, the cytosolic carboxy termini are uniquely arranged in a left-handed α-helical bundle, directly interacting with the amino-terminal domains of adjacent subunits. This configuration suggests a molecular mechanism for allosteric regulation of channel gating by intracellular signals.
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Electron Microscopy Data Bank
Protein Data Bank
Cryo-EM density map of InsP3R1 has been deposited in the Electron Microscopy Data Bank under accession number EMD-6369. The model coordinates have been deposited in the Electron Microscopy Data Bank under accession number 3JAV.
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We thank S. Murray for his assistance with RELION1.3. This work was supported by grants from the National Institutes of Health (R01GM072804, R21AR063255, S10OD016279, P41GM103832, R01GM079429, R01GM080139 and R21GM100229), the American Heart Association (14RNT1980029), the Muscular Dystrophy Association (295138) and National Science Foundation (DBI-1356306). We gratefully acknowledge the assistance and computing resources provided by the Center for Computational and Integrative Biomedical Research of Baylor College of Medicine and the Texas Advanced Computing Center at the University of Texas at Austin in the completion of this work.
The authors declare no competing financial interests.
Extended data figures and tables
a, Representative electron image of ice-embedded InsP3R1 taken at a defocus of 2.1 μm and recorded using the K2 Summit camera. Shown image is motion-corrected. b, Fourier power spectrum of the micrograph shown in a with Thon rings extending to 3.8 Å. c, 2D projections of the InsP3R1 map from the last iteration of refinement by RELION1.3 (rows 1 and 4) and EMAN2.1 (rows 2 and 5) are shown with corresponding 2D class averages (rows 3 and 6).
a, The gold-standard FSC curve for the final cryo-EM 3D reconstruction generated with RELION1.3 (black) and EMAN2.1 (green). The overall resolution is 4.7 Å using the FSC cut-off = 0.143. b, Shown is the FSC curve between the cryo-EM density map generated with RELION1.3 and the molecular model (blue, FSC cut-off is 0.5) and the FSC curve between the 3D reconstructions generated with EMAN2.1 and RELION1.3 (red, FSC cut-off is 0.25). c, The cryo-EM density map of InsP3R1 generated with RELION1.3 is colour-coded based on ResMap (Methods). The map is depicted as viewed along the membrane plane (left) or as a slab of the density (dashed box in left panel) coincident with the four-fold channel axis (right). The cryo-EM density map exhibits local resolution variation ranging from 3.6 Å to 6.5 Å with the most highly resolved densities in the TMD, while parts of the peripheral densities in the cytoplasmic region are of lower resolution. The low resolution peripheral density in the transmembrane region is attributed to detergent bound to the protein. d, Euler angle distribution of all particles used for the final 3D reconstruction. Each view is represented by a sphere, for which the size is proportional to the number of particles for this specific view.
Extended Data Figure 3 3D cryo-EM density map of the tetrameric InsP3R1 visualized in three orthogonal orientations.
InsP3R1 viewed from the cytosol (top), along the membrane plane (middle) and from the lumen (bottom). Four individual subunits are colour-coded. The map is rendered at a threshold level corresponding to a molecular mass of ∼1.3 MDa.
a, Linear representation of a primary structure of InsP3R1 protein (GI accession 17380349). Ten domains identified in the cryo-EM density map are colour-coded. Three sites of alternative splicing (residues 318–332/SI, 918–926/SIII and 1692–1731/SII) are indicated below the sequence bar. Putative binding sites for several channel-specific ligands are indicated above the domains (ATP, ATP-binding CaM/CaBP, calmodulin/Ca2+ binding protein; CGA, chromogranin A; cyt c, cytochrome c; Httexp, huntingtin; PKA, protein kinase A; RIH, RyR/InsP3R homology; yellow circles denote Ca2+-binding20). The panel below shows a linear diagram of the protein sequence colour-coded based on the approach used for modelling different regions in the primary structure. The spaces between the bars correspond to unmodelled sequence (see also Extended Data Fig. 5). b, The structure of the N-terminal domain (NTD) based on cryo-EM density map (red) is shown overlapped with the X-ray crystal structures of NTD (r.m.s.d. values = 1.3–1.4 Å): 1XZZ (blue); 3T8Sa (cyan); 3T8Sb (green); 3UJ4 (yellow); and 3UJ0 (magenta). c, Cryo-EM density map is viewed along the membrane plane. Densities corresponding to the individual domains of one subunit of InsP3R1 are colour-coded as in a. d, Models for solenoid-like α-helical domains are shown superimposed on their corresponding cryo-EM densities. ARM1–ARM3, armadillo repeat domains1–3; HD, α-helical domain. The densities of ARM2 domain are less resolved than those of ARM1 and ARM3 but are sufficient to trace the backbone.
rInsP3R1, Rattus norvegicus (GI accession 17380349); hInsP3R1, Homo sapiens (GI accession 519668682); hInsP3R2, Homo sapiens (GI accession 259016258); hInsP3R3, Homo sapiens (GI accession 209572633); the primary sequence numbering includes the first methionine. The numbering of residues is given below the sequences, secondary structure elements are indicated above the sequences and colour-coded in correspondence to the domains shown in Fig. 1b; dashed lines indicate regions that were not sufficiently resolved to be modelled. Given the enormous size of InsP3R proteins, the full-length sequence alignment was divided into two panels: sequence alignment for the transmembrane domains is shown in Extended Data Fig. 7a (note, overlap at the loop between the helices α90 and α91).
a–c, The cryo-EM densities for some regions are shown overlaid with the corresponding model: cytosolic helices (a); transmembrane helices (b); the constriction point at Phe2586 within the ion conduction pathway (c), view from the cytosol along the four-fold axis, colour-coded by subunit.
a, Alignment of the channel-forming domains; residues discussed in the text are labelled as following: blue circles, negatively charged residues; red circles, positively charged His2541; yellow highlight, N-glycosylation sites (Asn2476 and Asn2504); green highlight, selectivity filter; blue highlight, conserved Gly2587; green box, hydrophobic constriction region and Phe2586 shown in green; dark blue, Zn2+-finger-like residues Cys2611/Cys2614 and His2631/His2636; blue box, tetramerization region. b, Structural comparison of pore-forming elements of InsP3R1, RyR1, Kv1.2–2.1, NavRh, TRPV1 (PDB accessions: 3J8H, 2R9R, 4DXW and 3J5P, respectively). Note substantial overlap between structures.
a, Two opposing subunits of InsP3R1 and RyR1 (PDB accession 3J8H) are viewed along the membrane plane. One InsP3R1 subunit is colour-coded by domain (left). Structurally consistent domains in one RyR1 subunit are colour-coded using InsP3R1 domain architecture. Domains of RyR1 not in common are shown in grey. b, TMDs of RyR1 (tan) and InsP3R1 (coloured by subunit) are superimposed using Chimera’s Matchmaker and viewed from the cytosol (left) and lumen (right). The r.m.s.d. between 80 atom pairs is 2.0 Å. For clarity, P-helices are not shown. c, Structural comparison of the selectivity filter (red) in InsP3R1 with that in some tetrameric cation channels: RyR1, TRPV1, TRPA1, CavAb (PDB accessions: 3J8H, 3J5Q, 3J9P and 4MS2, respectively). Two opposing subunits are shown; TM5 and TM6 helices are coloured tan, P-helices are in green.
a, Cut-open side view of the cryo-EM density map of InsP3R1 is shown with the structures of the Ca2+ sensor region for InsP3R1 (residues 1952–2270) and RyR1 (residues 3877–4251; PDB accession 3J8H); the EF-hand of RyR1 includes residues 4071–4130. b, Close-up view of the overlapped Ca2+ sensor structures for InsP3R1 (colour-coded by domain as in Fig. 1) and RyR1 (tan). The conserved Glu2101/InsP3R1 and Glu4032/RyR1 are shown in red. c, Sequence alignment of the predicted Ca2+ sensor regions comprising the conserved Glu2101/InsP3R1 and Glu4032/RyR1 (red) (rInsP3R1/GI code:17380349; hInsP3R1/GI code: 519668682; hInsP3R2/GI code: 259016258; hInsP3R3/GI code: 209572633; RyR1/GI code: 134134; RyR2/GI code: 308153559; RyR3/GI code: 75074791); green highlight denotes completely conserved residues; yellow highlight denotes identical residues.
a, A bundle of the CTD helices is viewed perpendicular to the channel axis at the position of a predicted inter-subunit saltbridge between residues Gln2700 and Lys2701. The right panel shows the predicted salt bridge normal to the channel axis. Helices are colour-coded per subunit. b, Structures of the CTD and β-TF2 domains are superimposed on their corresponding cryo-EM densities and the surfaces are colour-coded according to electrostatic charges calculated for the model: blue denotes positive charges; red denotes negative charges. Shown is a side view with the cytoplasmic side facing up. c, Close-up view of the interface between the CTD and β-TF2 domains of two neighbouring subunits; viewed from cytosol perpendicular to the membrane plane.
This file contains a Supplementary Discussion and Supplementary References. (PDF 141 kb)
This animation shows the overall structure of the tetrameric IP3R1 and individual channel subunits. (MOV 12663 kb)
This animation shows the domain organization of the individual IP3R1 subunit and their arrangement within the tetrameric IP3R1 assembly. (MOV 26048 kb)
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Fan, G., Baker, M., Wang, Z. et al. Gating machinery of InsP3R channels revealed by electron cryomicroscopy. Nature 527, 336–341 (2015). https://doi.org/10.1038/nature15249
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