mouse 5-ht 3a r gene  (GenScript corporation)

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