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- EMDB-3712: Human BBsome core complex with subunits BBS1, BBS4, BBS5, BBS8, B... -

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Basic information

Entry
Database: EMDB / ID: EMD-3712
TitleHuman BBsome core complex with subunits BBS1, BBS4, BBS5, BBS8, BBS9, BBS18
Map dataNegative Stain Reconstruction:BBsome Complex
Sample
  • Complex: A 6subunit, BBsome complex.
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsKlink BU / Zent E / Juneja P / Kuhlee A / Raunser S / Wittinghofer A
CitationJournal: Elife / Year: 2017
Title: A recombinant BBSome core complex and how it interacts with ciliary cargo.
Authors: Björn Udo Klink / Eldar Zent / Puneet Juneja / Anne Kuhlee / Stefan Raunser / Alfred Wittinghofer /
Abstract: Cilia are small, antenna-like structures on the surface of eukaryotic cells that harbor a unique set of sensory proteins, including GPCRs and other membrane proteins. The transport of these proteins ...Cilia are small, antenna-like structures on the surface of eukaryotic cells that harbor a unique set of sensory proteins, including GPCRs and other membrane proteins. The transport of these proteins involves the BBSome, an eight-membered protein complex that is recruited to ciliary membranes by the G-protein Arl6. BBSome malfunction leads to Bardet-Biedl syndrome, a ciliopathy with severe consequences. Short ciliary targeting sequences (CTS) have been identified that trigger the transport of ciliary proteins. However, mechanistic studies that relate ciliary targeting to BBSome binding are missing. Here we used heterologously expressed BBSome subcomplexes to analyze the complex architecture and to investigate the binding of GPCRs and other receptors to the BBSome. A stable heterohexameric complex was identified that binds to GPCRs with interactions that only partially overlap with previously described CTS, indicating a more complex recognition than anticipated. Arl6•GTP does not affect these interactions, suggesting no direct involvement in cargo loading/unloading.
History
DepositionMay 8, 2017-
Header (metadata) releaseJun 14, 2017-
Map releaseDec 6, 2017-
UpdateDec 6, 2017-
Current statusDec 6, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.7
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3712_1.png

emd_3712_2.png

Downloads & links

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Map

FileDownload / File: emd_3712.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative Stain Reconstruction:BBsome Complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.89 Å/pix.
x 180 pix.
= 340.2 Å		1.89 Å/pix.
x 180 pix.
= 340.2 Å		1.89 Å/pix.
x 180 pix.
= 340.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.89 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.7 / Movie #1: 0.7
Minimum - Maximum0. - 7.2863293
Average (Standard dev.)0.022554556 (±0.22782631)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 340.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.891.891.89
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z340.200340.200340.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean0.0007.2860.023

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Supplemental data

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Sample components

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Entire : A 6subunit, BBsome complex.

EntireName: A 6subunit, BBsome complex.
Components
  • Complex: A 6subunit, BBsome complex.

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Supramolecule #1: A 6subunit, BBsome complex.

SupramoleculeName: A 6subunit, BBsome complex. / type: complex / ID: 1 / Parent: 0
Details: Subunits: BBsome subunit 1 BBsome subunit 4 BBsome subunit 5 BBsome subunit 8 BBsome subunit 9 BBsome subunit18
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper) / Recombinant cell: High Five cells
Molecular weightTheoretical: 330 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
Details: 50 mM Hepes pH 8.0, 150 mM NaCl, 5 mM MgCl2, 10% glycerol and 1 mM TCEP
StainingType: NEGATIVE / Material: Uranyl formate
GridPretreatment - Type: GLOW DISCHARGE
Details: Glow discharged 400 mesh copper grids with thin carbon support.

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Electron microscopy

MicroscopeJEOL 1400
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 197 / Average electron dose: 35.0 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 3.4 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 60000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC

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Image processing

Final reconstructionNumber classes used: 140 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPARX / Number images used: 8339
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING

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