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- PDB-6vbv: Structure of the bovine BBSome:ARL6:GTP complex -

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Basic information

Entry
Database: PDB / ID: 6vbv
TitleStructure of the bovine BBSome:ARL6:GTP complex
Components
  • (Bardet-Biedl syndrome ...Bardet–Biedl syndrome) x 6
  • ADP-ribosylation factor-like protein 6
  • BBS1 domain-containing protein
  • Tetratricopeptide repeat domain 8
KeywordsPROTEIN TRANSPORT / Cilia / ciliopathy / complex / membrane-protein transport
Function / homology
Function and homology information


BBSome binding / protein transport from ciliary membrane to plasma membrane / protein localization to non-motile cilium / primary palate development / regulation of non-motile cilium assembly / multi-ciliated epithelial cell differentiation / negative regulation of appetite by leptin-mediated signaling pathway / renal tubule development / receptor localization to non-motile cilium / photoreceptor cell outer segment organization ...BBSome binding / protein transport from ciliary membrane to plasma membrane / protein localization to non-motile cilium / primary palate development / regulation of non-motile cilium assembly / multi-ciliated epithelial cell differentiation / negative regulation of appetite by leptin-mediated signaling pathway / renal tubule development / receptor localization to non-motile cilium / photoreceptor cell outer segment organization / axonemal microtubule / protein localization to photoreceptor outer segment / regulation of cilium beat frequency involved in ciliary motility / BBSome / camera-type eye photoreceptor cell differentiation / photoreceptor cell morphogenesis / retinal rod cell development / ventricular system development / ciliary transition zone / retina layer formation / smoothened binding / establishment of planar polarity / olfactory behavior / microtubule anchoring at centrosome / positive regulation of cilium assembly / olfactory bulb development / photoreceptor connecting cilium / negative regulation of systemic arterial blood pressure / inner ear receptor cell stereocilium organization / neural precursor cell proliferation / patched binding / striatum development / non-motile cilium / protein localization to cilium / cellular lipid metabolic process / non-motile cilium assembly / maintenance of protein location in nucleus / membrane coat / intracellular transport / negative regulation of actin filament polymerization / hormone metabolic process / brain morphogenesis / eye development / eating behavior / establishment of epithelial cell apical/basal polarity / motile cilium / positive regulation of multicellular organism growth / photoreceptor cell maintenance / centrosome cycle / limb development / phosphatidylinositol-3-phosphate binding / smoothened signaling pathway / regulation of smoothened signaling pathway / protein targeting to membrane / ciliary membrane / fertilization / sensory perception of smell / beta-tubulin binding / fat pad development / fat cell differentiation / dynactin binding / protein localization to centrosome / adult behavior / photoreceptor outer segment / cartilage development / spermatid development / regulation of stress fiber assembly / face development / ciliary basal body / protein polymerization / negative regulation of GTPase activity / pericentriolar material / alpha-tubulin binding / microtubule motor activity / centriolar satellite / cilium assembly / social behavior / dendrite development / RNA polymerase II repressing transcription factor binding / axoneme / mitotic cytokinesis / cilium / centriole / protein localization to plasma membrane / photoreceptor inner segment / microtubule organizing center / vesicle-mediated transport / cerebral cortex development / hippocampus development / protein localization / regulation of cytokinesis / regulation of protein localization / phospholipid binding / neuron migration / neural tube closure / phosphoprotein binding / intracellular protein transport / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / microtubule cytoskeleton organization / protein transport
Ciliary BBSome complex subunit 2, C-terminal domain / Ciliary BBSome complex subunit 2, middle region / Quinoprotein alcohol dehydrogenase-like superfamily / Small GTPase superfamily, ARF/SAR type / Tetratricopeptide repeat / Ciliary BBSome complex subunit 2, middle region / Small GTP-binding protein domain / ADP-ribosylation factor family / Ciliary BBSome complex subunit 2, C-terminal / Tetratricopeptide repeat ...Ciliary BBSome complex subunit 2, C-terminal domain / Ciliary BBSome complex subunit 2, middle region / Quinoprotein alcohol dehydrogenase-like superfamily / Small GTPase superfamily, ARF/SAR type / Tetratricopeptide repeat / Ciliary BBSome complex subunit 2, middle region / Small GTP-binding protein domain / ADP-ribosylation factor family / Ciliary BBSome complex subunit 2, C-terminal / Tetratricopeptide repeat / Ciliary BBSome complex subunit 2, N-terminal / Ciliary BBSome complex subunit 1 / Ciliary BBSome complex subunit 2, N-terminal / Cilia BBSome complex subunit 10 / ADP-ribosylation factor-like protein 6 / WD40-repeat-containing domain superfamily / Bardet-Biedl syndrome 1, N-terminal / TPR repeat / Bardet-Biedl syndrome 5 protein / Bardet-Biedl syndrome 5 protein / Tetratricopeptide repeat / Tetratricopeptide repeat protein 8 / Bardet-Biedl syndrome 1 protein / Cilia BBSome complex subunit 10 / PTHB1, C-terminal domain / PTHB1, N-terminal domain / P-loop containing nucleoside triphosphate hydrolase / Parathyroid hormone-responsive B1 / Tetratricopeptide-like helical domain superfamily / PTHB1 C-terminus / Bardet-Biedl syndrome 5 protein/sex-determination protein fem-3 / Tetratricopeptide repeat / Tetratricopeptide repeat-containing domain / DM16 repeat / Bardet-Biedl syndrome 2 protein / PTHB1 N-terminus / Bardet-Biedl syndrome 7 protein / P-loop containing nucleotide triphosphate hydrolases / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Bardet-Biedl syndrome 5 protein homolog / Bardet-Biedl syndrome 4 protein homolog / Bardet-Biedl syndrome 2 protein homolog / BBS1 domain-containing protein / Bardet-Biedl syndrome 7 protein homolog / Tetratricopeptide repeat domain 8 / Uncharacterized protein / ADP-ribosylation factor-like protein 6 / Uncharacterized protein
Biological speciesBos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsSingh, S.K. / Gui, M. / Koh, F. / Yip, M.C.J. / Brown, A.
Funding support United States, 1items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Elife / Year: 2020
Title: Structure and activation mechanism of the BBSome membrane protein trafficking complex.
Authors: Sandeep K Singh / Miao Gui / Fujiet Koh / Matthew Cj Yip / Alan Brown /
Abstract: Bardet-Biedl syndrome (BBS) is a currently incurable ciliopathy caused by the failure to correctly establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS ...Bardet-Biedl syndrome (BBS) is a currently incurable ciliopathy caused by the failure to correctly establish or maintain cilia-dependent signaling pathways. Eight proteins associated with BBS assemble into the BBSome, a key regulator of the ciliary membrane proteome. We report the electron cryomicroscopy (cryo-EM) structures of the native bovine BBSome in inactive and active states at 3.1 and 3.5 Å resolution, respectively. In the active state, the BBSome is bound to an Arf-family GTPase (ARL6/BBS3) that recruits the BBSome to ciliary membranes. ARL6 recognizes a composite binding site formed by BBS1 and BBS7 that is occluded in the inactive state. Activation requires an unexpected swiveling of the β-propeller domain of BBS1, the subunit most frequently implicated in substrate recognition, which widens a central cavity of the BBSome. Structural mapping of disease-causing mutations suggests that pathogenesis results from folding defects and the disruption of autoinhibition and activation.
Validation Report
SummaryFull reportAbout validation report
History
DepositionDec 19, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 29, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
0: Bardet-Biedl syndrome 18 protein
1: BBS1 domain-containing protein
2: Bardet-Biedl syndrome 2 protein homolog
4: Bardet-Biedl syndrome 4 protein homolog
5: Bardet-Biedl syndrome 5 protein homolog
7: Bardet-Biedl syndrome 7 protein homolog
8: Tetratricopeptide repeat domain 8
9: Bardet-Biedl syndrome 9
3: ADP-ribosylation factor-like protein 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)508,17012
Polymers507,5669
Non-polymers6033
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area52400 Å2
ΔGint-247 kcal/mol
Surface area160510 Å2

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Components

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Bardet-Biedl syndrome ... , 6 types, 6 molecules 024579

#1: Protein Bardet-Biedl syndrome 18 protein / Bardet–Biedl syndrome


Mass: 8070.502 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Eye / Tissue: Retina / References: UniProt: G3N2W1
#3: Protein Bardet-Biedl syndrome 2 protein homolog / Bardet–Biedl syndrome


Mass: 79911.484 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Eye / Tissue: Retina / References: UniProt: Q32L13
#4: Protein Bardet-Biedl syndrome 4 protein homolog / Bardet–Biedl syndrome


Mass: 58289.133 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Eye / Tissue: Retina / References: UniProt: Q1JQ97
#5: Protein Bardet-Biedl syndrome 5 protein homolog / Bardet–Biedl syndrome


Mass: 38880.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Eye / Tissue: Retina / References: UniProt: A6QLF9
#6: Protein Bardet-Biedl syndrome 7 protein homolog / Bardet–Biedl syndrome


Mass: 80471.375 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Eye / Tissue: Retina / References: UniProt: F1MB52
#8: Protein Bardet-Biedl syndrome 9 / Bardet–Biedl syndrome


Mass: 99230.914 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Eye / Tissue: Retina / References: UniProt: E1BHJ5

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Protein , 3 types, 3 molecules 183

#2: Protein BBS1 domain-containing protein


Mass: 64939.141 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Eye / Tissue: Retina / References: UniProt: E1BN34
#7: Protein Tetratricopeptide repeat domain 8


Mass: 56686.406 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Bos taurus (cattle) / Organ: Eye / Tissue: Retina / References: UniProt: F1N4X0
#9: Protein ADP-ribosylation factor-like protein 6


Mass: 21086.486 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: ARL6 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: Q0IIM2

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Non-polymers , 2 types, 3 molecules

#10: Chemical ChemComp-CA / CALCIUM ION / Calcium


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#11: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM

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Details

Has ligand of interestN

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Bovine BBSome:ARL6:GTP complexCOMPLEXNative BBSome complex isolated from bovine retina and incubated with recombinant bovine ARL6 in the presence of GTP.#1-#90MULTIPLE SOURCES
2BBSome complexCOMPLEX#1-#81NATURAL
3ADP-ribosylation factor-like protein 6COMPLEX#91RECOMBINANT
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDOrganTissue
22Bos taurus (cattle)9913EyeRetina
33Bos taurus (cattle)9913
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepes1
2220 mMNaClSodium chloride1
35 mMMgCl21
44 mMbeta-mercaptoethanol1
51 mMGTP1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: Grids were blotted for 2 s with a -2 offset.

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1100 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 56 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 9408
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 25 eV

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Processing

EM software
IDNameVersionCategoryDetails
1SPHIREparticle selectioncrYOLO was used to pick particles
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7Coot0.9model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
19PHENIX1.17.1-3660model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 75201 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 43.6 / Protocol: OTHER / Space: REAL / Target criteria: Correlation coefficient
Details: During refinement, the resolution limit was set to 3.5 Angstrom. Secondary structure, Ramachandran and rotamer restraints were applied during refinement.

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