codon-optimized mouse 5-ht 3a r  (GenScript corporation)

Journal: Nature Communications

Article Title: Molecular mechanism of setron-mediated inhibition of full-length 5-HT 3A receptor

doi: 10.1038/s41467-019-11142-8

Figure Lengend Snippet: Cryogenic electron microscopy (cryo-EM) structure of granisetron-bound full-length serotonin 3A receptor (5-HT 3A R). a A schematic showing three fundamental conformations that constitute the gating cycle in pentameric ligand-gated ion channel (pLGIC) function: a resting state, a transient open state, and a desensitized state. Agonist-binding shifts the equilibrium towards the open state and then to the high-agonist affinity, desensitized state. Orthosteric (competitive) antagonists exert their effect by shifting the equilibrium towards the resting (or inhibited) state. b Trace showing a continuous recording of 5-HT 3A R currents (−60 mV) in oocytes measured by two-electrode voltage clamp (TEVC) in the presence of serotonin (marked by red line) and pre-applied granisetron (marked by orange line). The effect of granisetron inhibition was fully reversible as seen in the third pulse. c Map of full-length 5-HT 3A R-granisetron reconstructed from 46,757 particles at 2.92 Å resolution. Side-view parallel to the membrane and extracellular view are shown in left and right panels, respectively. Each monomer is shown in a different color for clarity. Density corresponding to granisetron (left panel, circle) and glycans (right panel, arrow) are indicated. d Three-dimensional cartoon model of 5-HT 3A R-granisetron structure generated from EM reconstruction (side view). For each subunit, three sets of glycans are shown as stick representation. e Top-view of 5-HT 3A R-granisetron map sliced at the binding site to show all five granisetron molecules, each bound at the interface of two subunits (indicated by arrows)

Article Snippet: Codon-optimized mouse 5-HT 3A R (NCBI Reference Sequence: NM_001099644.1) was purchased from GenScript (Supplementary Table ) and inserted into pFastBac1 vector consisting of four strep-tags (WSHPQFEK) at the N terminus, followed by a linker sequence (GGGSGGGSGGGS) and a TEV-cleavage sequence (ENLYFQG) and a C-terminal 1D4-tag .

Techniques: Electron Microscopy, Cryo-EM Sample Prep, Binding Assay, Inhibition, Generated

Journal: Nature Communications

Article Title: Molecular mechanism of setron-mediated inhibition of full-length 5-HT 3A receptor

doi: 10.1038/s41467-019-11142-8

Figure Lengend Snippet: The granisetron binding site. a The density map of granisetron contoured at 9 σ (left) and map around the residues at the binding site located at the intersubunit interface (right). The residue labels on the principal subunit are marked in black and those on the complementary subunit are marked in brown. b A comparison of the serotonin 3A receptor-apo (5-HT 3A R-apo), 5-HT 3A R-granisetron, and 5-HT 3A R-serotonin structures shows that residues involved in ligand-binding undergo rotameric reorientation. c Alignment of the three structures reveals an inward motion of loop C in 5-HT 3A R-granisetron relative to 5-HT 3A R-apo, which is in the direction toward activation as seen in the 5-HT 3A R-serotonin structure

Article Snippet: Codon-optimized mouse 5-HT 3A R (NCBI Reference Sequence: NM_001099644.1) was purchased from GenScript (Supplementary Table ) and inserted into pFastBac1 vector consisting of four strep-tags (WSHPQFEK) at the N terminus, followed by a linker sequence (GGGSGGGSGGGS) and a TEV-cleavage sequence (ENLYFQG) and a C-terminal 1D4-tag .

Techniques: Binding Assay, Ligand Binding Assay, Activation Assay

Journal: Nature Communications

Article Title: Molecular mechanism of setron-mediated inhibition of full-length 5-HT 3A receptor

doi: 10.1038/s41467-019-11142-8

Figure Lengend Snippet: Conformational differences between the apo and ligand-bound states. a An extracellular view of the extracellular domain (ECD) upon global alignment of serotonin 3A receptor-apo (5-HT 3A R-apo) structure with 5-HT 3A R-granisetron (left) and 5-HT 3A R-serotonin (right). Only ECDs from two non-adjacent subunits is shown for clarity. A counter-clockwise motion of the ECD is observed as indicated by the arrows. The serotonin-induced motion is of larger magnitude compared to that of granisetron, highlighted by the solid and dotted arrows, respectively. b A comparison of the transmembrane domains (TMDs) (viewed from the extracellular side) in 5-HT 3A R-granisetron structure (left) and 5-HT 3A R-serotonin (right) when aligned with respect to 5-HT 3A R-apo. Only two non-adjacent TMD subunits are shown for clarity. In both panels, a clockwise rotation of the TMD is observed with 5-HT 3A R-serotonin revealing a larger change. c Pathway of ion permeation of 5-HT 3A R-apo and 5-HT 3A R-granisetron generated with HOLE . The cartoon representation of two subunits are shown for clarity. The locations of pore constrictions are shown as sticks. The pore radius is plotted as a function of distance along the pore axis. The dotted line indicates the approximate radius of a hydrated Na + ion, which is estimated at 2.76 Å (right)

Article Snippet: Codon-optimized mouse 5-HT 3A R (NCBI Reference Sequence: NM_001099644.1) was purchased from GenScript (Supplementary Table ) and inserted into pFastBac1 vector consisting of four strep-tags (WSHPQFEK) at the N terminus, followed by a linker sequence (GGGSGGGSGGGS) and a TEV-cleavage sequence (ENLYFQG) and a C-terminal 1D4-tag .

Techniques: Generated

Journal: Nature Communications

Article Title: Molecular mechanism of setron-mediated inhibition of full-length 5-HT 3A receptor

doi: 10.1038/s41467-019-11142-8

Figure Lengend Snippet: Assessment of the overall stability of the granisetron-serotonin 3A receptor (5-HT 3A R) structure. a Time evolution of the root mean squared deviations (RMSD) of Cα atoms of secondary structure elements of the extracellular domain (ECD) and all Cα atoms of the 5-HT 3A R pentamer (left panel). The RMSD of each of the granisetron molecules (labeled CWB) in each subunit A to E (right panel) calculated with respect to the cryo-EM-derived structure during 100 ns production simulations. b Two possible granisetron poses with the bicyclic ring in boat/chair or chair/chair conformation and the N -methyl group in axial or equatorial positions in the piperidine chair conformation. These two poses were used as input for metadynamics-based ranking. c Ranking of the two granisetron poses shown in b using metadynamics. Error bars represent the standard error of the mean of RMSD estimates from 10 metadynamics simulations. Source data are provided as a

Article Snippet: Codon-optimized mouse 5-HT 3A R (NCBI Reference Sequence: NM_001099644.1) was purchased from GenScript (Supplementary Table ) and inserted into pFastBac1 vector consisting of four strep-tags (WSHPQFEK) at the N terminus, followed by a linker sequence (GGGSGGGSGGGS) and a TEV-cleavage sequence (ENLYFQG) and a C-terminal 1D4-tag .

Techniques: Labeling, Cryo-EM Sample Prep, Derivative Assay

Journal: Nature Communications

Article Title: Molecular mechanism of setron-mediated inhibition of full-length 5-HT 3A receptor

doi: 10.1038/s41467-019-11142-8

Figure Lengend Snippet: Effects of mutations at the ligand-binding pocket on granisetron inhibition. a Granisetron interactions with Trp156 and Tyr207 from the principal subunit and Trp63, Arg65, Tyr126 from the complementary subunit are depicted as stick representation. b Serotonin dose response measured by two-electrode voltage clamp (TEVC) recordings (at −60 mV) for wild-type (WT) 5-HT 3A R, W63Y, R65A, Y126F, W156Y, and Y207F mutants, expressed in oocytes. The half-maximal effective concentration (EC 50 ), the Hill coefficient (nH), and the number of independent oocyte experiments for WT and mutants are: WT (EC 50 : 2.70 + 0.09 μM; nH: 2.3 + 0.17; n: 3), W63Y (EC 50 : 9.93 + 0.77 μM; nH: 3.1 + 0.72; n: 4), R65A (EC 50 : 13.79 + 0.50 μM; nH: 4.4 + 0.59; n: 4), Y126F (EC 50 : 42.8 + 4.4 μM; nH: 2.6 + 0.71; n: 4), W156Y (EC 50 : 306 + 44 μM; nH: 1.58 + 0.24; n: 4), and Y207F (EC 50 : 20.35 + 1.7 μM; nH: 1.9 + 0.27; n: 5). c Currents were elicited in response to serotonin (concentrations used near EC 50 values of WT and mutants). The following concentrations of serotonin were used: WT-1 μM, W63Y-10 μM, R65A-10 μM; Y126F-40 μM, W156Y-200 μM, and Y207F-20 μM. Currents were measured in response to serotonin (marked by red line) and pre-application of granisetron (marked by orange line). Dotted arrows show the extent of granisetron inhibition. d A plot of the ratio of peak current in the presence of granisetron to peak current in the absence of granisetron is shown for WT and mutants. Data are shown as mean ± s.d. (n is indicated within parentheses). Significance at p = 0.001 (***) and p = 0.05 (**) calculated by two-sample t test for WT and mutants. Source data are provided as a

Article Snippet: Codon-optimized mouse 5-HT 3A R (NCBI Reference Sequence: NM_001099644.1) was purchased from GenScript (Supplementary Table ) and inserted into pFastBac1 vector consisting of four strep-tags (WSHPQFEK) at the N terminus, followed by a linker sequence (GGGSGGGSGGGS) and a TEV-cleavage sequence (ENLYFQG) and a C-terminal 1D4-tag .

Techniques: Ligand Binding Assay, Inhibition, Concentration Assay

Journal: Nature

Article Title: Cryo-EM reveals two distinct serotonin-bound conformations of full-length 5-HT 3A receptor

doi: 10.1038/s41586-018-0660-7

Figure Lengend Snippet: Sequence of mouse 5-HT 3A R used in the cryo-EM study and the data on cryo-EM and refinement. a, Full length mouse 5-HT 3A R sequence used in the cryo-EM study. Regions in the sequence highlighted in green, blue, gray, and yellow represent strep-tag, linker, TEV cleavage site, and 1D4-tag, respectively. Secondary structural elements as seen in State 1 are plotted above the sequence. Loops in gray color are not seen in the final refined structure. All the important loops, sheets, and helices are labeled. Glycosylation sites are marked as blue arrows. Key residues within the serotonin-binding sites are highlighted in brown color. Cysteines present in the cys-loop are shown as cyan color. Pore-facing residues in M2 are shown in green color. Arg416 in the ICD is shown in red. b, Cryo-EM data collection, refinement and validation statistics.

Article Snippet: Codon optimized mouse 5-HT 3A R gene (NCBI Reference Sequence: NM_001099644.1 ) was purchased from GenScript USA Inc and subcloned into pFastBac1 vector.

Techniques: Sequencing, Strep-tag, Labeling, Glycoproteomics, Biomarker Discovery

Journal: Nature Communications

Article Title: Cryo-EM structure of 5-HT 3A receptor in its resting conformation

doi: 10.1038/s41467-018-02997-4

Figure Lengend Snippet: Data collection and processing

Article Snippet: Codon-optimized mouse 5-HT 3A R gene (NCBI Reference Sequence: NM_001099644.1) was purchased from GenScript USA Inc.

Techniques: Microscopy

Journal: Nature Communications

Article Title: Cryo-EM structure of 5-HT 3A receptor in its resting conformation

doi: 10.1038/s41467-018-02997-4

Figure Lengend Snippet: Cryo-EM structure of apo-5-HT 3A R. a The 3D reconstruction map from the full-length 5-HT 3A R at 4.3 Å resolution. The views, going from left to right, are parallel to the membrane (side view), from the extracellular side (top view), and from the intracellular side (bottom view). Individual subunits are depicted in different colors, and the three domains are labeled. The solid lines denote putative membrane limits. b Cartoon representations of the 5-HT 3A R structural model based on the EM reconstruction. The views correspond to the orientations shown in a . For each subunit, three sets of glycans (green) and one lipid (brown) molecule are shown as stick representation

Article Snippet: Codon-optimized mouse 5-HT 3A R gene (NCBI Reference Sequence: NM_001099644.1) was purchased from GenScript USA Inc.

Techniques: Cryo-EM Sample Prep, Labeling

Journal: Nature Communications

Article Title: Cryo-EM structure of 5-HT 3A receptor in its resting conformation

doi: 10.1038/s41467-018-02997-4

Figure Lengend Snippet: Alignment of the apo-5-HT 3A R with the crystal structure of nanobody-bound 5-HT 3A R. a A view of the ECDs from the extracellular end when aligned with respect to the TMDs (left). The view of the TMDs from the intracellular end when aligned with respect to the ECDs (right). The apo-5-HT 3A R structure and the 5-HT 3A R crystal structure are shown in salmon red and pale green, respectively. The arrows show the putative direction of displacements between the two structures. b A comparison of the ECD of the apo-structure with the crystal structure when aligned with respect to the TMD of the (−) subunit (left). A comparison of the TMD between the two structures when aligned with the ECD of the (−) subunit. The relative tilt of the axis parallel to the TM helices between the two structures are indicated. The dotted lines highlight the differences in the intrasubunit cavity volume. The spheres indicate the position of residues M1 (Leu227), M2 (Leu266), M3 (Met291), and M4 (Trp456). The arrows show the putative direction of displacements between the two structures (right). The alignment in b highlights the relative changes in the two structures, both with respect to the neighboring subunit, as well as with respect to the other domain

Article Snippet: Codon-optimized mouse 5-HT 3A R gene (NCBI Reference Sequence: NM_001099644.1) was purchased from GenScript USA Inc.

Techniques:

Journal: Nature Communications

Article Title: Cryo-EM structure of 5-HT 3A receptor in its resting conformation

doi: 10.1038/s41467-018-02997-4

Figure Lengend Snippet: Profile of ion permeation pathway. a The pore profile generated by the HOLE program depicts an ion permeation pathway of ~165 Å encompassing the ECD, TMD, and the ICD. Only two subunits are shown for clarity. Sidechains of residues that line the constricted areas are shown as sticks. b A comparison of the pore radii along the pore axis for the 5-HT 3A R cryo-EM structure (salmon red) with that of the crystal structure (pale green). The dashed line indicates an approximate radius of a hydrated Na + ion . The pore is constricted below 3 Å radius at three sites: L260 and E250 along M2 and R416 in the ICD. c Non-protein densities along the pore axis were modeled as water (red), Na + (magenta), and Cl − (green). The map around the ions is shown as a mesh representation calculated at various σ values (outer water ring: 4 σ ; inner water ring:5 σ ; Na + ion: 6 σ ; Cl − ion: 7 σ )

Article Snippet: Codon-optimized mouse 5-HT 3A R gene (NCBI Reference Sequence: NM_001099644.1) was purchased from GenScript USA Inc.

Techniques: Generated, Cryo-EM Sample Prep

Journal: Nature Communications

Article Title: Cryo-EM structure of 5-HT 3A receptor in its resting conformation

doi: 10.1038/s41467-018-02997-4

Figure Lengend Snippet: The neurotransmitter binding site. a The map around the aromatic residues at the subunit interface that constitutes the neurotransmitter binding site (top). The map for the residues in Loop F that are involved in ligand binding (bottom). b Alignment of 5-HT 3A R apo (salmon red) and crystal (pale green) structures reveals a twist and an expansion at the region lined Loop C, Loop B, and Loop F. The arrows indicate the direction of movement. c A comparison of the orientations of the residues that are involved in neurotransmitter binding

Article Snippet: Codon-optimized mouse 5-HT 3A R gene (NCBI Reference Sequence: NM_001099644.1) was purchased from GenScript USA Inc.

Techniques: Binding Assay, Ligand Binding Assay

Journal: Nature Communications

Article Title: Cryo-EM structure of 5-HT 3A receptor in its resting conformation

doi: 10.1038/s41467-018-02997-4

Figure Lengend Snippet: The Intracellular domain. a The ICD is comprised of the post-M3 loop, the MX, helix, a stretch of unstructured region, followed by the MA helix. The density from the MX helix is bent downward to the intracellular end of the MA helix, but the unstructured region is not resolved. Superposition of the 5-HT 3A R apo and crystal structures reveals differences in the conformation of ICD in the two structures. The (−) subunit of the two structures are aligned. The expansion of the ICD resulting from an outward displacement of MA and MX helices are indicated by arrows. b The residues within the ICD involved in several potential intra and inter-subunit interactions. c The solvent-accessible electrostatic potential map generated using the APBS tool. The inset shows a zoomed-in view of the lipid-binding pocket within the dotted green box. The lipid (partially built phospholipid) and the interacting residues (R306 and R435) are shown in stick. The map around the lipid is shown as blue mesh

Article Snippet: Codon-optimized mouse 5-HT 3A R gene (NCBI Reference Sequence: NM_001099644.1) was purchased from GenScript USA Inc.

Techniques: Generated, Binding Assay