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- PDB-8vc2: CryoEM structure of insect gustatory receptor BmGr9 in the presen... -

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Basic information

Entry
Database: PDB / ID: 8vc2
TitleCryoEM structure of insect gustatory receptor BmGr9 in the presence of fructose
ComponentsGustatory receptor
KeywordsMEMBRANE PROTEIN / gustatory receptor / homotetramer / ligand-gated ion channel / seven transmembrane
Function / homology7TM chemoreceptor / 7tm Chemosensory receptor / sensory perception of taste / signal transduction / plasma membrane / Chem-PSC / Gustatory receptor
Function and homology information
Biological speciesBombyx mori (domestic silkworm)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.98 Å
AuthorsFrank, H.M. / Walsh Jr., R.M. / Garrity, P.A. / Gaudet, R.
Funding support United States, 3items
OrganizationGrant numberCountry
Other governmentR21DC018497 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM120996 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI157194 United States
CitationJournal: bioRxiv / Year: 2023
Title: Structure of an insect gustatory receptor.
Authors: Heather M Frank / Sanket Walujkar / Richard M Walsh / Willem J Laursen / Douglas L Theobald / Paul A Garrity / Rachelle Gaudet /
Abstract: Gustatory Receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors. However, GR structures have not been experimentally determined. ...Gustatory Receptors (GRs) are critical for insect chemosensation and are potential targets for controlling pests and disease vectors. However, GR structures have not been experimentally determined. We present structures of Gr9 (BmGr9), a fructose-gated cation channel, in agonist-free and fructose-bound states. BmGr9 forms a tetramer similar to distantly related insect Olfactory Receptors (ORs). Upon fructose binding, BmGr9's ion channel gate opens through helix S7b movements. In contrast to ORs, BmGR9's ligand-binding pocket, shaped by a kinked helix S4 and a shorter extracellular S3-S4 loop, is larger and solvent accessible in both agonist-free and fructose-bound states. Also unlike ORs, fructose binding by BmGr9 involves helix S5 and a binding pocket lined with aromatic and polar residues. Structure-based sequence alignments reveal distinct patterns of ligand-binding pocket residue conservation in GR subfamilies associated with distinct ligand classes. These data provide insight into the molecular basis of GR ligand specificity and function.
History
DepositionDec 13, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 10, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 24, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Gustatory receptor
B: Gustatory receptor
C: Gustatory receptor
D: Gustatory receptor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)221,3268
Polymers218,2894
Non-polymers3,0364
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Gustatory receptor


Mass: 54572.320 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bombyx mori (domestic silkworm) / Gene: Gr9 / Production host: Homo sapiens (human) / References: UniProt: B3GTD7
#2: Chemical
ChemComp-PSC / (7R,17E,20E)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSA-17,20-DIEN-1-AMINIUM 4-OXIDE / PHOSPHATIDYLCHOLINE / 2-LINOLEOYL-1-PALMITOYL-SN-GYCEROL-3-PHOSPHOCHOLINE / Phosphatidylcholine


Mass: 759.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C42H81NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Fructose-bound BmGr9 homotetramer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.21805 MDa / Experimental value: NO
Source (natural)Organism: Bombyx mori (domestic silkworm)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8.25
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Calibrated magnification: 60240 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.995 sec. / Electron dose: 80.144 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9002
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1crYOLO1.9.6particle selection
2SerialEM4.1image acquisition
4CTFFIND4.1.14CTF correction
7Coot0.9.8.91model fittingManual rebuilding and refining of the model.
8ISOLDE1.6.1model fittingRemodeling using molecular dynamics.
10PHENIX1.20.1-4487-000model refinementInitial docking of model and real space refinement.
11RELION4.0.1initial Euler assignment
12cryoSPARC3.3.2final Euler assignment
14cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1141024
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 233351 / Symmetry type: POINT
Atomic model buildingB value: 43.02 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation coefficient
Details: Initial fitting was done using DockInMap in PHENIX and refined through cycles of manual rebuilding in Coot, real-space refinement in PHENIX, and remodeling by simulations run in the ISOLDE plugin of ChimeraX.
Atomic model buildingDetails: ColabFold generated / Source name: Other / Type: in silico model
RefinementCross valid method: NONE

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