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- EMDB-28224: CryoEM structure of Resistance to Inhibitors of Cholinesterase-8B... -

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Basic information

Entry
Database: EMDB / ID: EMD-28224
TitleCryoEM structure of Resistance to Inhibitors of Cholinesterase-8B (Ric-8B) in complex with olfactory G protein alpha olf
Map dataSharpened map
Sample
  • Complex: Ric-8B in complex with G protein subunit alpha olf
    • Protein or peptide: Guanine nucleotide-binding protein G(olf) subunit alpha
    • Protein or peptide: Isoform 1 of Synembryn-B
Function / homology
Function and homology information


Adenylate cyclase activating pathway / sensory perception of chemical stimulus / G-protein alpha-subunit binding / Adenylate cyclase inhibitory pathway / guanyl-nucleotide exchange factor activity / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / adenylate cyclase-activating dopamine receptor signaling pathway / heterotrimeric G-protein complex ...Adenylate cyclase activating pathway / sensory perception of chemical stimulus / G-protein alpha-subunit binding / Adenylate cyclase inhibitory pathway / guanyl-nucleotide exchange factor activity / G protein-coupled receptor binding / G-protein beta/gamma-subunit complex binding / Olfactory Signaling Pathway / adenylate cyclase-activating dopamine receptor signaling pathway / heterotrimeric G-protein complex / cell cortex / G protein-coupled receptor signaling pathway / GTPase activity / centrosome / GTP binding / signal transduction / extracellular exosome / metal ion binding / plasma membrane / cytosol / cytoplasm
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(olf) subunit alpha / Synembryn-B
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 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_28224.png

Downloads & links

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Map

FileDownload / File: emd_28224.map.gz / Format: CCP4 / Size: 58.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 0.8521 Å
Density
Contour LevelBy AUTHOR: 0.453
Minimum - Maximum-2.2634022 - 3.720395
Average (Standard dev.)-0.00087175524 (±0.108213335)
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_28224_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_28224_half_map_1.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_28224_half_map_2.map
AnnotationHalf map 1
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 in complex with G protein subunit alpha olf

EntireName: Ric-8B in complex with G protein subunit alpha olf
Components
  • Complex: Ric-8B in complex with G protein subunit alpha olf
    • Protein or peptide: Guanine nucleotide-binding protein G(olf) subunit alpha
    • Protein or peptide: Isoform 1 of Synembryn-B

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Supramolecule #1: Ric-8B in complex with G protein subunit alpha olf

SupramoleculeName: Ric-8B in complex with G protein subunit alpha olf / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Guanine nucleotide-binding protein G(olf) subunit alpha

MacromoleculeName: Guanine nucleotide-binding protein G(olf) subunit alpha
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.37043 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGCLGGNSKT TEDQGVDEKE RREANKKIEK QLQKERLAYK ATHRLLLLGA GESGKSTIVK QMRILHVNGF NPEEKKQKIL DIRKNVKDA IVTIVSAMST IIPPVPLANP ENQFRSDYIK SIAPITDFEY SQEFFDHVKK LWDDEGVKAC FERSNEYQLI D CAQYFLER ...String:
MGCLGGNSKT TEDQGVDEKE RREANKKIEK QLQKERLAYK ATHRLLLLGA GESGKSTIVK QMRILHVNGF NPEEKKQKIL DIRKNVKDA IVTIVSAMST IIPPVPLANP ENQFRSDYIK SIAPITDFEY SQEFFDHVKK LWDDEGVKAC FERSNEYQLI D CAQYFLER IDSVSLVDYT PTDQDLLRCR VLTSGIFETR FQVDKVNFHM FDVGGQRDER RKWIQCFNDV TAIIYVAACS SY NMVIRED NNTNRLRESL DLFESIWNNR WLRTISIILF LNKQDMLAEK VLAGKSKIED YFPEYANYTV PEDATPDAGE DPK VTRAKF FIRDLFLRIS TATGDGKHYC YPHFTCAVDT ENIRRVFNDC RDIIQRMHLK QYELL

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.5 mg/mL
BufferpH: 8
GridModel: UltrAuFoil / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 57050 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 4670 / Average exposure time: 2.49 sec. / Average electron dose: 68.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4695512
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 247416
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8el7

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 171.1
Output model

PDB-8el8:
CryoEM structure of Resistance to Inhibitors of Cholinesterase-8B (Ric-8B) in complex with olfactory G protein alpha olf

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