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- PDB-8uw8: Site-one protease and SPRING -

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Basic information

Entry
Database: PDB / ID: 8uw8
TitleSite-one protease and SPRING
Components
  • Membrane-bound transcription factor site-1 protease
  • SREBP regulating gene protein
KeywordsSIGNALING PROTEIN / serine protease cholesterol metabolism zymogen activation Protein complex glycoprotein secretory pathway
Function / homology
Function and homology information


site-1 protease / CREB3 factors activate genes / positive regulation of SREBP signaling pathway / ATF6-mediated unfolded protein response / regulation of cholesterol biosynthetic process / ATF6 (ATF6-alpha) activates chaperones / ATF6B (ATF6-beta) activates chaperones / Assembly of active LPL and LIPC lipase complexes / membrane protein intracellular domain proteolysis / regulation of vesicle-mediated transport ...site-1 protease / CREB3 factors activate genes / positive regulation of SREBP signaling pathway / ATF6-mediated unfolded protein response / regulation of cholesterol biosynthetic process / ATF6 (ATF6-alpha) activates chaperones / ATF6B (ATF6-beta) activates chaperones / Assembly of active LPL and LIPC lipase complexes / membrane protein intracellular domain proteolysis / regulation of vesicle-mediated transport / Regulation of cholesterol biosynthesis by SREBP (SREBF) / Golgi stack / lysosome organization / mitotic G2 DNA damage checkpoint signaling / protein maturation / endoplasmic reticulum unfolded protein response / response to endoplasmic reticulum stress / cholesterol metabolic process / Post-translational protein phosphorylation / protein processing / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / endoplasmic reticulum lumen / Golgi membrane / serine-type endopeptidase activity / endoplasmic reticulum membrane / Golgi apparatus / proteolysis
Similarity search - Function
SREBP regulating gene protein / SREBP regulating gene protein / Site-1 peptidase catalytic domain / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site / Serine proteases, subtilase domain profile. / Peptidase S8, subtilisin-related / Peptidase S8/S53 domain superfamily ...SREBP regulating gene protein / SREBP regulating gene protein / Site-1 peptidase catalytic domain / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site / Serine proteases, subtilase domain profile. / Peptidase S8, subtilisin-related / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain
Similarity search - Domain/homology
Membrane-bound transcription factor site-1 protease / SREBP regulating gene protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsKober, D.L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM141261 United States
CitationJournal: Nat Commun / Year: 2024
Title: SPRING licenses S1P-mediated cleavage of SREBP2 by displacing an inhibitory pro-domain
Authors: Hendrix, S. / Dartigue, V. / Hall, H. / Bawaria, S. / Kingma, J. / Bajaj, B. / Zelcer, N. / Kober, D.L.
History
DepositionNov 6, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Membrane-bound transcription factor site-1 protease
B: SREBP regulating gene protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)139,0965
Polymers138,2482
Non-polymers8483
Water1,35175
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration, SEC-MALS, assay for oligomerization, Mass Photometry
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein Membrane-bound transcription factor site-1 protease


Mass: 115048.203 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MBTPS1 / Plasmid: BacMAM pEZT / Cell line (production host): HEK 293s GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q14703
#2: Protein SREBP regulating gene protein / SREBF pathway regulator in Golgi 1


Mass: 23200.291 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPRING1, C12orf49, SPRING / Plasmid: BacMAM pEZT / Cell line (production host): HEK 293s GnTI- / Production host: Homo sapiens (human) / References: UniProt: Q9H741

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Sugars , 2 types, 2 molecules

#3: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 76 molecules

#5: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Ca
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 75 / Source method: isolated from a natural source / Formula: H2O

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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

ComponentName: Complex of matured site-1 protease bound to SPRING / Type: COMPLEX
Details: Co-expressed and secreted ectodomains of SPRING-His10 and S1P-FLAG purified using NiNTA chromatography and gel filtration.
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5 / Details: 50 mM HEPES-NaOH and 150 mM NaCl
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
250 mMHEPES buffered with sodium hydroxideHEPES-NaOH1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 30 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingAverage exposure time: 7 sec. / Electron dose: 48 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4885 / Details: CDS mode, 8 electrons per second, 50 frames.
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
10cryoSPARC4.2initial Euler assignment
11cryoSPARC4.2final Euler assignment
12cryoSPARC4.2classification
13cryoSPARC4.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 452453 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 33.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00526750
ELECTRON MICROSCOPYf_angle_d0.76219184
ELECTRON MICROSCOPYf_chiral_restr0.0456992
ELECTRON MICROSCOPYf_plane_restr0.00441192
ELECTRON MICROSCOPYf_dihedral_angle_d37.2596932

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