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- EMDB-36579: A cryo-EM structure of the bovine chromogranin B dimer at a nomin... -

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Basic information

Entry
Database: EMDB / ID: EMD-36579
TitleA cryo-EM structure of the bovine chromogranin B dimer at a nominal resolution of ~0.35 nm
Map dataChromogranin B dimers purified from bovine pancreatic granules studied by single particle cryo-EM.
Sample
  • Organelle or cellular component: chromogranin B
KeywordsCHGB dimer C2 symmetry anion shunt pathway secretory granule regulated secretory pathways / TRANSPORT PROTEIN
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsJiang Q-X / Zhu MX / Yadav GP
Funding support United States, 12 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM093271 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM111367 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R21GM131231 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM116788 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM116789 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)C06RR30414 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)U24GM129539 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RP120474 United States
American Heart Association12IRG9400019 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01GM088745 United States
Cystic Fibrosis FoundationJIANG15G0 United States
Welch FoundationI-1684 United States
CitationJournal: Front Mol Neurosci / Year: 2023
Title: Chromogranin B (CHGB) is dimorphic and responsible for dominant anion channels delivered to cell surface via regulated secretion.
Authors: Gaya P Yadav / Haiyuan Wang / Joke Ouwendijk / Stephen Cross / Qiaochu Wang / Feng Qin / Paul Verkade / Michael X Zhu / Qiu-Xing Jiang /
Abstract: Regulated secretion is conserved in all eukaryotes. In vertebrates granin family proteins function in all key steps of regulated secretion. Phase separation and amyloid-based storage of proteins and ...Regulated secretion is conserved in all eukaryotes. In vertebrates granin family proteins function in all key steps of regulated secretion. Phase separation and amyloid-based storage of proteins and small molecules in secretory granules require ion homeostasis to maintain their steady states, and thus need ion conductances in granule membranes. But granular ion channels are still elusive. Here we show that granule exocytosis in neuroendocrine cells delivers to cell surface dominant anion channels, to which chromogranin B (CHGB) is critical. Biochemical fractionation shows that native CHGB distributes nearly equally in soluble and membrane-bound forms, and both reconstitute highly selective anion channels in membrane. Confocal imaging resolves granular membrane components including proton pumps and CHGB in puncta on the cell surface after stimulated exocytosis. High pressure freezing immuno-EM reveals a major fraction of CHGB at granule membranes in rat pancreatic β-cells. A cryo-EM structure of bCHGB dimer of a nominal 3.5 Å resolution delineates a central pore with end openings, physically sufficient for membrane-spanning and large single channel conductance. Together our data support that CHGB-containing (CHGB+) channels are characteristic of regulated secretion, and function in granule ion homeostasis near the plasma membrane or possibly in other intracellular processes.
History
DepositionJun 15, 2023-
Header (metadata) releaseJul 26, 2023-
Map releaseJul 26, 2023-
UpdateJul 26, 2023-
Current statusJul 26, 2023Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_36579.png

Downloads & links

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Map

FileDownload / File: emd_36579.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationChromogranin B dimers purified from bovine pancreatic granules studied by single particle cryo-EM.
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-2.224178 - 3.4532552
Average (Standard dev.)0.0022367241 (±0.09655509)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 233.28001 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: first half map

Fileemd_36579_half_map_1.map
Annotationfirst half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 2nd half map

Fileemd_36579_half_map_2.map
Annotation2nd half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : chromogranin B

EntireName: chromogranin B
Components
  • Organelle or cellular component: chromogranin B

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Supramolecule #1: chromogranin B

SupramoleculeName: chromogranin B / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Details: bovine proteins purified from pancreatic secretory granules
Source (natural)Organism: Bos taurus (cattle) / Organ: pancreas
Molecular weightTheoretical: 190 MDa

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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

BufferpH: 6.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Details: Ab initio model from cisTEM and cryoSPARC, and a reference model from angular reconstitution of a negative stain dataset.
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Details: Initial assignment was based on the reference model.
Final angle assignmentType: MAXIMUM LIKELIHOOD
Details: Refinement was done in cryoSPARC and relion separately.
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2)
Details: CryoSPARC and relion were used for independent analyses to generate similar results.
Number images used: 70000

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