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- EMDB-28223: CryoEM structure of Resistance to Inhibitors of Cholinesterase-8B... -

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Basic information

Entry
Database: EMDB / ID: EMD-28223
TitleCryoEM structure of Resistance to Inhibitors of Cholinesterase-8B (Ric-8B) in complex with G alpha s
Map dataSharpened cryoEM Map of Ric-8B/G alpha s complex
Sample
  • Complex: Ric-8B complexed with G alpha s
    • Complex: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
      • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Complex: Isoform 1 of Synembryn-B
      • Protein or peptide: Isoform 1 of Synembryn-B
  • Ligand: water
Function / homology
Function and homology information


PKA activation in glucagon signalling / hair follicle placode formation / mu-type opioid receptor binding / developmental growth / corticotropin-releasing hormone receptor 1 binding / G-protein alpha-subunit binding / intracellular transport / D1 dopamine receptor binding / Hedgehog 'off' state / beta-2 adrenergic receptor binding ...PKA activation in glucagon signalling / hair follicle placode formation / mu-type opioid receptor binding / developmental growth / corticotropin-releasing hormone receptor 1 binding / G-protein alpha-subunit binding / intracellular transport / D1 dopamine receptor binding / Hedgehog 'off' state / beta-2 adrenergic receptor binding / adenylate cyclase-activating adrenergic receptor signaling pathway / activation of adenylate cyclase activity / adenylate cyclase activator activity / guanyl-nucleotide exchange factor activity / trans-Golgi network membrane / insulin-like growth factor receptor binding / ionotropic glutamate receptor binding / G-protein beta/gamma-subunit complex binding / bone development / adenylate cyclase-activating G protein-coupled receptor signaling pathway / Prostacyclin signalling through prostacyclin receptor / Glucagon signaling in metabolic regulation / cognition / platelet aggregation / Glucagon-type ligand receptors / Vasopressin regulates renal water homeostasis via Aquaporins / G alpha (z) signalling events / cellular response to catecholamine stimulus / Glucagon-like Peptide-1 (GLP1) regulates insulin secretion / ADORA2B mediated anti-inflammatory cytokines production / adenylate cyclase-activating dopamine receptor signaling pathway / cellular response to prostaglandin E stimulus / GPER1 signaling / heterotrimeric G-protein complex / sensory perception of smell / positive regulation of cold-induced thermogenesis / cell cortex / G alpha (i) signalling events / G alpha (s) signalling events / G protein-coupled receptor signaling pathway / GTPase activity / centrosome / GTP binding / extracellular exosome / membrane / metal ion binding / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Synembryn / Guanine nucleotide exchange factor, Ric8 / Guanine nucleotide exchange factor synembryn / G-protein alpha subunit, group S / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / Armadillo-like helical ...Synembryn / Guanine nucleotide exchange factor, Ric8 / Guanine nucleotide exchange factor synembryn / G-protein alpha subunit, group S / G-alpha domain profile. / Guanine nucleotide binding protein (G-protein), alpha subunit / G protein alpha subunit, helical insertion / G-protein alpha subunit / G protein alpha subunit / Armadillo-like helical / Armadillo-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Guanine nucleotide-binding protein G(s) subunit alpha isoforms short / Synembryn-B
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsPapasergi-Scott MM / Skiniotis G
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM088242 United States
CitationJournal: Structure / Year: 2023
Title: Structures of Ric-8B in complex with Gα protein folding clients reveal isoform specificity mechanisms.
Authors: Makaía M Papasergi-Scott / Frank E Kwarcinski / Maiya Yu / Ouliana Panova / Ann M Ovrutsky / Georgios Skiniotis / Gregory G Tall /
Abstract: Mammalian Ric-8 proteins act as chaperones to regulate the cellular abundance of heterotrimeric G protein α subunits. The Ric-8A isoform chaperones Gαi/o, Gα12/13, and Gαq/11 subunits, while Ric- ...Mammalian Ric-8 proteins act as chaperones to regulate the cellular abundance of heterotrimeric G protein α subunits. The Ric-8A isoform chaperones Gαi/o, Gα12/13, and Gαq/11 subunits, while Ric-8B acts on Gαs/olf subunits. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ric-8B in complex with Gαs and Gαolf, revealing isoform differences in the relative positioning and contacts between the C-terminal α5 helix of Gα within the concave pocket formed by Ric-8 α-helical repeat elements. Despite the overall architectural similarity with our earlier structures of Ric-8A complexed to Gαq and Gαi1, Ric-8B distinctly accommodates an extended loop found only in Gαs/olf proteins. The structures, along with results from Ric-8 protein thermal stability assays and cell-based Gαolf folding assays, support a requirement for the Gα C-terminal region for binding specificity, and highlight that multiple structural elements impart specificity for Ric-8/G protein binding.
History
DepositionSep 23, 2022-
Header (metadata) releaseMar 1, 2023-
Map releaseMar 1, 2023-
UpdateMay 17, 2023-
Current statusMay 17, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28223.png

Downloads & links

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Map

FileDownload / File: emd_28223.map.gz / Format: CCP4 / Size: 58.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened cryoEM Map of Ric-8B/G alpha s complex
Voxel sizeX=Y=Z: 0.8521 Å
Density
Contour LevelBy AUTHOR: 0.752
Minimum - Maximum-4.615237 - 6.610058
Average (Standard dev.)-0.0006304767 (±0.19827615)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions248248248
Spacing248248248
CellA=B=C: 211.3208 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28223_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 1 of Ric-8B/G alpha s complex

Fileemd_28223_half_map_1.map
AnnotationHalf Map 1 of Ric-8B/G alpha s complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2 of Ric-8B/G alpha s complex

Fileemd_28223_half_map_2.map
AnnotationHalf Map 2 of Ric-8B/G alpha s complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ric-8B complexed with G alpha s

EntireName: Ric-8B complexed with G alpha s
Components
  • Complex: Ric-8B complexed with G alpha s
    • Complex: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
      • Protein or peptide: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
    • Complex: Isoform 1 of Synembryn-B
      • Protein or peptide: Isoform 1 of Synembryn-B
  • Ligand: water

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Supramolecule #1: Ric-8B complexed with G alpha s

SupramoleculeName: Ric-8B complexed with G alpha s / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

SupramoleculeName: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
type: complex / ID: 2 / Chimera: Yes / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Isoform 1 of Synembryn-B

SupramoleculeName: Isoform 1 of Synembryn-B / type: complex / ID: 3 / Chimera: Yes / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short

MacromoleculeName: Guanine nucleotide-binding protein G(s) subunit alpha isoforms short
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.32616 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKSTIVKQM RILHVNGFNG DSEKATKVQD IKNNLKEAI ETIVAAMSNL VPPVELANPE NQFRVDYILS VMNVPDFDFP PEFYEHAKAL WEDEGVRACY ERSNEYQLID C AQYFLDKI ...String:
MGCLGNSKTE DQRNEEKAQR EANKKIEKQL QKDKQVYRAT HRLLLLGAGE SGKSTIVKQM RILHVNGFNG DSEKATKVQD IKNNLKEAI ETIVAAMSNL VPPVELANPE NQFRVDYILS VMNVPDFDFP PEFYEHAKAL WEDEGVRACY ERSNEYQLID C AQYFLDKI DVIKQADYVP SDQDLLRCRV LTSGIFETKF QVDKVNFHMF DVGGQRDERR KWIQCFNDVT AIIFVVASSS YN MVIREDN QTNRLQEALN LFKSIWNNRW LRTISVILFL NKQDLLAEKV LAGKSKIEDY FPEFARYTTP EDATPEPGED PRV TRAKYF IRDEFLRIST ASGDGRHYCY PHFTCAVDTE NIRRVFNDCR DIIQRMHLRQ YELL

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Macromolecule #2: Isoform 1 of Synembryn-B

MacromoleculeName: Isoform 1 of Synembryn-B / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 63.928043 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: GEFMDEERAL YIVRAGEAGA IERVLRDYSD KHRATFKFES ADEDKRKKLC EGIFKVLVKE VPTTCQVSCL EVLRILSRDK KILVPVTTK ENMQILLRLA KLHESDDSLE KVSEFPVIVE SLKCLCNIVF NSQMAQQLSL ELNLAAKLCN LLRKCKDRKF I NDIKCFDL ...String:
GEFMDEERAL YIVRAGEAGA IERVLRDYSD KHRATFKFES ADEDKRKKLC EGIFKVLVKE VPTTCQVSCL EVLRILSRDK KILVPVTTK ENMQILLRLA KLHESDDSLE KVSEFPVIVE SLKCLCNIVF NSQMAQQLSL ELNLAAKLCN LLRKCKDRKF I NDIKCFDL RLLFVLSLLH TDIRSQLRYE LQGLPLLTQI LESAFSIKWT DEYESAIDHN GPPLSPQETD CAIEALKALF NV TVDSWKV HKESDSHQFR VMAAVLRHCL LIVGPTEDKT EELHSNAVNL LSNVPVSCLD VLICPLTHEE TAQEAATLDE LPS DKTTEK DTALKNSTMV YNGMNMEAIH VLLNFMEKRI DKGSSYREGL TPVLSLLTEC SRAHRNIRKF LKDQVLPPLR DVTN RPEVG STVRNKLVRL MTHVDLGVKQ IAAEFLFVLC KERVDSLLKY TGYGNAAGLL AARGLLAGGR GDNWY(SEP)EDED (TPO)DTEEYKNA KPNINLITGH LEEPMPNPID EMTEEQKEYE AMKLVNMLDK LSREELLKPM GLKPDGTITP LEEALSQ YS VIEETSSDTD

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 5 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.3 mg/mL
BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 9416 / Average exposure time: 2.497 sec. / Average electron dose: 60.43 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 57050 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 9597316
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 349857
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6vu8

RefinementProtocol: AB INITIO MODEL
Output model

PDB-8el7:
CryoEM structure of Resistance to Inhibitors of Cholinesterase-8B (Ric-8B) in complex with G alpha s

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