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- PDB-6xtb: Subunit BBS 5 of the human core BBSome complex -

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Basic information

Entry
Database: PDB / ID: 6xtb
TitleSubunit BBS 5 of the human core BBSome complex
ComponentsBardet-Biedl syndrome 5 proteinBardet–Biedl syndrome
KeywordsPROTEIN TRANSPORT / ciliary transport / Arl6 effector / adaptor protein / complex
Function / homology
Function and homology information


RNA polymerase II-specific DNA-binding transcription factor binding => GO:0061629 / BBSome / motile cilium assembly / melanosome transport / BBSome-mediated cargo-targeting to cilium / phosphatidylinositol-3-phosphate binding / response to stimulus / ciliary membrane / heart looping / axoneme ...RNA polymerase II-specific DNA-binding transcription factor binding => GO:0061629 / BBSome / motile cilium assembly / melanosome transport / BBSome-mediated cargo-targeting to cilium / phosphatidylinositol-3-phosphate binding / response to stimulus / ciliary membrane / heart looping / axoneme / intracellular transport / cilium assembly / centriolar satellite / visual perception / ciliary basal body / protein transport / cytosol
Similarity search - Function
Bardet-Biedl syndrome 5 protein / DM16 repeat / Bardet-Biedl syndrome 5 protein/sex-determination protein fem-3 / Bardet-Biedl syndrome 5 protein / Repeats in sea squirt COS41.4, worm R01H10.6, fly CG1126 etc.
Similarity search - Domain/homology
Bardet-Biedl syndrome 5 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsKlink, B.U. / Raunser, S. / Gatsogiannis, C.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Elife / Year: 2020
Title: Structure of the human BBSome core complex.
Authors: Björn Udo Klink / Christos Gatsogiannis / Oliver Hofnagel / Alfred Wittinghofer / Stefan Raunser /
Abstract: The BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a ...The BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a cargo adapter that recognizes signaling proteins such as GPCRs and links them to the intraflagellar transport machinery. The underlying mechanism is poorly understood. Here we present a high-resolution cryo-EM structure of a human heterohexameric core subcomplex of the BBSome. The structure reveals the architecture of the complex in atomic detail. It explains how the subunits interact with each other and how disease-causing mutations hamper this interaction. The complex adopts a conformation that is open for binding to membrane-associated GTPase Arl6 and a large positively charged patch likely strengthens the interaction with the membrane. A prominent negatively charged cleft at the center of the complex is likely involved in binding of positively charged signaling sequences of cargo proteins.
History
DepositionJan 15, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 19, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 29, 2020Group: Data collection / Category: em_imaging_optics / Item: _em_imaging_optics.phase_plate

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Structure visualization

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Assembly

Deposited unit
E: Bardet-Biedl syndrome 5 protein


Theoretical massNumber of molelcules
Total (without water)38,7981
Polymers38,7981
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Bardet-Biedl syndrome 5 protein / Bardet–Biedl syndrome


Mass: 38797.926 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BBS5 / Cell line (production host): Hi5 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q8N3I7

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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: Human BBSome core complex / Type: COMPLEX
Details: BBSome core complex containing BBS1,4,5,8,9 and 18. Only BBS5 was modelled into this map since we obtained another map with higher resolution for the other subunits (see related entry)
Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: Hi5 / Plasmid: ACEMBL
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMTRIS-HCLTris1
2150 mMNaClSodium chloride1
30.1 mMTCEP1
SpecimenConc.: 0.08 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was cross linked with 0.5% glutaraldehyde
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K
Details: double blot with 2 minutes incubation after first sample application

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated defocus min: 300 nm / Calibrated defocus max: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 15 sec. / Electron dose: 67 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 15266
EM imaging opticsEnergyfilter slit width: 20 eV / Phase plate: VOLTA PHASE PLATE
Image scansMovie frames/image: 50 / Used frames/image: 1-50

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Processing

EM software
IDNameVersionCategoryDetails
1SPHIRE1.2-1.3particle selectionCryolo
2EPUimage acquisition
3SPHIREimage acquisitionTransphire used to aid data collection
5CTFFIND4.1.10CTF correction
11SPHIRE1.2-1.3initial Euler assignment
12SPHIRE1.2-1.3final Euler assignmentMeridien
13SPHIRE1.2-1.3classificationSort3D
14SPHIRE1.2-1.33D reconstructionMeridien
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2831329
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 180654 / Num. of class averages: 1 / Symmetry type: POINT

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