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- EMDB-45892: The Ectodomains of SPRING and S1P with the inhibitor PF-429242 -

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Basic information

Entry
Database: EMDB / ID: EMD-45892
TitleThe Ectodomains of SPRING and S1P with the inhibitor PF-429242
Map datamap of SPRING/S1P/PF-429242
Sample
  • Complex: Complex of the ectodomains of S1P and SPRING in the presence of the small molecule PF-429242
    • Protein or peptide: Membrane-bound transcription factor site-1 protease
    • Protein or peptide: SREBP regulating gene protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 4-[(diethylamino)methyl]-N-[2-(2-methoxyphenyl)ethyl]-N-[(3R)-pyrrolidin-3-yl]benzamide
  • Ligand: CALCIUM ION
  • Ligand: water
Keywordsprotease / complex / inhibitor / MEMBRANE PROTEIN / MEMBRANE PROTEIN-INHIBITOR complex
Function / homology
Function and homology information


site-1 protease / CREB3 factors activate genes / positive regulation of SREBP signaling pathway / ATF6-mediated unfolded protein response / ATF6 (ATF6-alpha) activates chaperones / ATF6B (ATF6-beta) activates chaperones / regulation of cholesterol biosynthetic process / 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 / ATF6 (ATF6-alpha) activates chaperones / ATF6B (ATF6-beta) activates chaperones / regulation of cholesterol biosynthetic process / 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 / endoplasmic reticulum unfolded protein response / cholesterol metabolic process / protein maturation / response to endoplasmic reticulum stress / Post-translational protein phosphorylation / protein processing / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / protein import into nucleus / 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 / : / Membrane-bound Site-1 Protease Family N-terminal domain / MBTPS1, fourth GATase-like domain / MBTPS1, third Ig-like domain / : / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. ...SREBP regulating gene protein / SREBP regulating gene protein / Site-1 peptidase catalytic domain / : / Membrane-bound Site-1 Protease Family N-terminal domain / MBTPS1, fourth GATase-like domain / MBTPS1, third Ig-like 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 / Peptidase S8, subtilisin-related / Serine proteases, subtilase domain profile. / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsKober DL
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM155152 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural basis for substrate selectivity by site-one protease revealed by studies with a small-molecule inhibitor.
Authors: Ashley V Bullington / Ilaria Micallo / Bilkish Bajaj / Pankaj Kumar / Netanya Schlamowitz / Aurora Silva / Sebastian Hendrix / Noam Zelcer / Daniel L Kober /
Abstract: Site-one protease (S1P) carries out the first proteolytic step to activate membrane-bound effector proteins in the Golgi. S1P matures through an autocatalytic process that begins in the endoplasmic ...Site-one protease (S1P) carries out the first proteolytic step to activate membrane-bound effector proteins in the Golgi. S1P matures through an autocatalytic process that begins in the endoplasmic reticulum (ER) and culminates with the displacement of its inhibitory pro-domain by its cofactor, sterol regulatory element binding protein-regulating gene (SPRING). Spatial control of S1P activity and substrate localization underpins signaling pathways governing, among others, lipogenesis, ER stress, and lysosome biogenesis. The factors governing these pathways are activated by S1P-mediated proteolysis upon their regulated transport from the ER to the Golgi. S1P cleaves substrates with the recognition sequence RX(L/I/V)Z, where X is any residue other than Cys or Pro and Z is preferably Leu or Lys. However, the structural basis for substrate recognition by S1P has remained unknown. Here, we used the small molecule PF-429242, a competitive inhibitor of S1P, to investigate substrate recognition by the S1P/SPRING complex. We determined the structure of S1P/SPRING bound to PF-429242 and found that PF-429242 binds S1P in the same pocket that recognizes the substrate's conserved P Arg. Further structural analysis suggests that S1P requires a conformation change to accommodate the substrate's P (L/I/V) residue. We designed an S1P mutation (I308A) to reduce the steric clash at the P position and generated an S1P that was resistant to PF-429242 in biochemical and cell culture assays. Our findings reveal selectivity in the recognition of substrates by S1P and provide a roadmap for the rational design of improved S1P inhibitors.
History
DepositionJul 23, 2024-
Header (metadata) releaseApr 23, 2025-
Map releaseApr 23, 2025-
UpdateMay 21, 2025-
Current statusMay 21, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45892.png

Downloads & links

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Map

FileDownload / File: emd_45892.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap of SPRING/S1P/PF-429242
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.74 Å/pix.
x 300 pix.
= 221.4 Å		0.74 Å/pix.
x 300 pix.
= 221.4 Å		0.74 Å/pix.
x 300 pix.
= 221.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.738 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.10373428 - 0.20659499
Average (Standard dev.)-0.000083232226 (±0.0040134257)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 221.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: deepemhancer map of SPRING/S1P/PF-429242 for model building and...

Fileemd_45892_additional_1.map
Annotationdeepemhancer map of SPRING/S1P/PF-429242 for model building and illustrations
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A for S1P/SPRING/PF-429242

Fileemd_45892_half_map_1.map
Annotationhalf map A for S1P/SPRING/PF-429242
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B for S1P/SPRING/PF-429242

Fileemd_45892_half_map_2.map
Annotationhalf map B for S1P/SPRING/PF-429242
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of the ectodomains of S1P and SPRING in the presence of t...

EntireName: Complex of the ectodomains of S1P and SPRING in the presence of the small molecule PF-429242
Components
  • Complex: Complex of the ectodomains of S1P and SPRING in the presence of the small molecule PF-429242
    • Protein or peptide: Membrane-bound transcription factor site-1 protease
    • Protein or peptide: SREBP regulating gene protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: 4-[(diethylamino)methyl]-N-[2-(2-methoxyphenyl)ethyl]-N-[(3R)-pyrrolidin-3-yl]benzamide
  • Ligand: CALCIUM ION
  • Ligand: water

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Supramolecule #1: Complex of the ectodomains of S1P and SPRING in the presence of t...

SupramoleculeName: Complex of the ectodomains of S1P and SPRING in the presence of the small molecule PF-429242
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 120 KDa

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

MacromoleculeName: Membrane-bound transcription factor site-1 protease / type: protein_or_peptide / ID: 1
Details: Construct contains 1-998 of human site one protease followed by a 3xFLAG affinity tag
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 115.048203 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKLVNIWLLL LVVLLCGKKH LGDRLEKKSF EKAPCPGCSH LTLKVEFSST VVEYEYIVAF NGYFTAKARN SFISSALKSS EVDNWRIIP RNNPSSDYPS DFEVIQIKEK QKAGLLTLED HPNIKRVTPQ RKVFRSLKYA ESDPTVPCNE TRWSQKWQSS R PLRRASLS ...String:
MKLVNIWLLL LVVLLCGKKH LGDRLEKKSF EKAPCPGCSH LTLKVEFSST VVEYEYIVAF NGYFTAKARN SFISSALKSS EVDNWRIIP RNNPSSDYPS DFEVIQIKEK QKAGLLTLED HPNIKRVTPQ RKVFRSLKYA ESDPTVPCNE TRWSQKWQSS R PLRRASLS LGSGFWHATG RHSSRRLLRA IPRQVAQTLQ ADVLWQMGYT GANVRVAVFD TGLSEKHPHF KNVKERTNWT NE RTLDDGL GHGTFVAGVI ASMRECQGFA PDAELHIFRV FTNNQVSYTS WFLDAFNYAI LKKIDVLNLS IGGPDFMDHP FVD KVWELT ANNVIMVSAI GNDGPLYGTL NNPADQMDVI GVGGIDFEDN IARFSSRGMT TWELPGGYGR MKPDIVTYGA GVRG SGVKG GCRALSGTSV ASPVVAGAVT LLVSTVQKRE LVNPASMKQA LIASARRLPG VNMFEQGHGK LDLLRAYQIL NSYKP QASL SPSYIDLTEC PYMWPYCSQP IYYGGMPTVV NVTILNGMGV TGRIVDKPDW QPYLPQNGDN IEVAFSYSSV LWPWSG YLA ISISVTKKAA SWEGIAQGHV MITVASPAET ESKNGAEQTS TVKLPIKVKI IPTPPRSKRV LWDQYHNLRY PPGYFPR DN LRMKNDPLDW NGDHIHTNFR DMYQHLRSMG YFVEVLGAPF TCFDASQYGT LLMVDSEEEY FPEEIAKLRR DVDNGLSL V IFSDWYNTSV MRKVKFYDEN TRQWWMPDTG GANIPALNEL LSVWNMGFSD GLYEGEFTLA NHDMYYASGC SIAKFPEDG VVITQTFKDQ GLEVLKQETA VVENVPILGL YQIPAEGGGR IVLYGDSNCL DDSHRQKDCF WLLDALLQYT SYGVTPPSLS HSGNRQRPP SGAGSVTPER MEGNHLHRYS KVLEAHLGDP KPRPLPACPR LSWAKPQPLN ETAPSNLWKH QKLLSIDLDK V VLPNFRSN RPQVRPLSPG ESGAWDIPGG IMPGRYNQEV DYKDDDDKGS DYKDDDDKGS DYKDDDDK

UniProtKB: Membrane-bound transcription factor site-1 protease

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Macromolecule #2: SREBP regulating gene protein

MacromoleculeName: SREBP regulating gene protein / type: protein_or_peptide / ID: 2
Details: Construct contains an IgK leader peptide followed by human SPRING residues 35-205 followed by a 10-His tag
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.200291 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: METDTLLLWV LLLWVPGSTG DKQEERAVRD RNLLQVHDHN QPIPWKVQFN LGNSSRPSNQ CRNSIQGKHL ITDELGYVCE RKDLLVNGC CNVNVPSTKQ YCCDGCWPNG CCSAYEYCVS CCLQPNKQLL LERFLNRAAV AFQNLFMAVE DHFELCLAKC R TSSQSVQH ...String:
METDTLLLWV LLLWVPGSTG DKQEERAVRD RNLLQVHDHN QPIPWKVQFN LGNSSRPSNQ CRNSIQGKHL ITDELGYVCE RKDLLVNGC CNVNVPSTKQ YCCDGCWPNG CCSAYEYCVS CCLQPNKQLL LERFLNRAAV AFQNLFMAVE DHFELCLAKC R TSSQSVQH ENTYRDPIAK YCYGESPPEL FPAHHHHHHH HHH

UniProtKB: SREBP regulating gene protein

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #5: 4-[(diethylamino)methyl]-N-[2-(2-methoxyphenyl)ethyl]-N-[(3R)-pyr...

MacromoleculeName: 4-[(diethylamino)methyl]-N-[2-(2-methoxyphenyl)ethyl]-N-[(3R)-pyrrolidin-3-yl]benzamide
type: ligand / ID: 5 / Number of copies: 1 / Formula: A1AZU
Molecular weightTheoretical: 409.564 Da

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Macromolecule #6: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #7: water

MacromoleculeName: water / type: ligand / ID: 7 / Number of copies: 42 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Average electron dose: 64.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Number images used: 122723
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4)
FSC plot (resolution estimation)

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