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- EMDB-26426: Meprin alpha helix in complex with fetuin-B [subparticle localise... -

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Basic information

Entry
Database: EMDB / ID: EMD-26426
TitleMeprin alpha helix in complex with fetuin-B [subparticle localised reconstruction, masked focused refinement]
Map dataB-factor amplitude corrected sharpened map, filtered by local resolution
Sample
  • Complex: Tetrameric portion of meprin alpha helix in complex with fetuin-B [subparticle localised reconstruction]
    • Protein or peptide: Meprin A subunit alpha
    • Protein or peptide: Fetuin-B
  • Ligand: CALCIUM IONCalcium
  • Ligand: ZINC ION
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


meprin A / meprin A complex / epidermal growth factor receptor ligand maturation / metalloendopeptidase inhibitor activity / metallodipeptidase activity / negative regulation of endopeptidase activity / binding of sperm to zona pellucida / cysteine-type endopeptidase inhibitor activity / endopeptidase inhibitor activity / signaling receptor ligand precursor processing ...meprin A / meprin A complex / epidermal growth factor receptor ligand maturation / metalloendopeptidase inhibitor activity / metallodipeptidase activity / negative regulation of endopeptidase activity / binding of sperm to zona pellucida / cysteine-type endopeptidase inhibitor activity / endopeptidase inhibitor activity / signaling receptor ligand precursor processing / single fertilization / metalloendopeptidase activity / metallopeptidase activity / extracellular space / extracellular exosome / zinc ion binding / extracellular region / plasma membrane
Similarity search - Function
Proteinase inhibitor I25C, fetuin, conserved site / Fetuin-B-type cystatin domain / Fetuin family signature 1. / Fetuin family signature 2. / Fetuin-B-type cystatin domain profile. / Meprin alpha/beta subunit / Meprin peptidase domain / Astacin-like domain profile. / Peptidase M12A / Astacin (Peptidase family M12A) ...Proteinase inhibitor I25C, fetuin, conserved site / Fetuin-B-type cystatin domain / Fetuin family signature 1. / Fetuin family signature 2. / Fetuin-B-type cystatin domain profile. / Meprin alpha/beta subunit / Meprin peptidase domain / Astacin-like domain profile. / Peptidase M12A / Astacin (Peptidase family M12A) / : / TRAF/meprin, MATH domain / MAM domain signature. / Cystatin domain / Cystatin-like domain / Cystatin domain / Domain in meprin, A5, receptor protein tyrosine phosphatase mu (and others) / Cystatin superfamily / MATH/TRAF domain / MATH/TRAF domain profile. / meprin and TRAF homology / MAM domain, meprin/A5/mu / MAM domain / MAM domain profile. / TRAF-like / Peptidase, metallopeptidase / Zinc-dependent metalloprotease / EGF-like domain / Metallopeptidase, catalytic domain superfamily / EGF-like domain profile. / EGF-like domain / Neutral zinc metallopeptidases, zinc-binding region signature. / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Meprin A subunit alpha / Fetuin-B
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsBayly-Jones C / Lupton CJ / Fritz C / Schlenzig D / Whisstock JC
Funding support Germany, Australia, 2 items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research Germany
Australian Research Council (ARC) Australia
CitationJournal: Nat Commun / Year: 2022
Title: Helical ultrastructure of the metalloprotease meprin α in complex with a small molecule inhibitor.
Authors: Charles Bayly-Jones / Christopher J Lupton / Claudia Fritz / Hariprasad Venugopal / Daniel Ramsbeck / Michael Wermann / Christian Jäger / Alex de Marco / Stephan Schilling / Dagmar ...Authors: Charles Bayly-Jones / Christopher J Lupton / Claudia Fritz / Hariprasad Venugopal / Daniel Ramsbeck / Michael Wermann / Christian Jäger / Alex de Marco / Stephan Schilling / Dagmar Schlenzig / James C Whisstock /
Abstract: The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal ...The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acute kidney injury, sepsis, urinary tract infection, metastatic colorectal carcinoma, and inflammatory bowel disease. Accordingly, meprin α is the target of drug discovery programs. In contrast to meprin β, meprin α is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin α, as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin α forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin β reveal unique features of the active site of meprin α, and helical assembly more broadly.
History
DepositionMar 13, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateNov 2, 2022-
Current statusNov 2, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26426.png

Downloads & links

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Map

FileDownload / File: emd_26426.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationB-factor amplitude corrected sharpened map, filtered by local resolution
Voxel sizeX=Y=Z: 1.41333 Å
Density
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-1.1034769 - 2.1551435
Average (Standard dev.)0.0031838627 (±0.05287467)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 305.27927 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26426_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: DeepEMhancer (mask) sharpened final map. Low pass filtered...

Fileemd_26426_additional_1.map
AnnotationDeepEMhancer (mask) sharpened final map. Low pass filtered to global FSC.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: DeepEMhancer (highres) sharpened final map. Low pass filtered...

Fileemd_26426_additional_2.map
AnnotationDeepEMhancer (highres) sharpened final map. Low pass filtered to global FSC.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half-map (1 of 2).

Fileemd_26426_half_map_1.map
AnnotationUnfiltered half-map (1 of 2).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unfiltered half-map (2 of 2).

Fileemd_26426_half_map_2.map
AnnotationUnfiltered half-map (2 of 2).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tetrameric portion of meprin alpha helix in complex with fetuin-B...

EntireName: Tetrameric portion of meprin alpha helix in complex with fetuin-B [subparticle localised reconstruction]
Components
  • Complex: Tetrameric portion of meprin alpha helix in complex with fetuin-B [subparticle localised reconstruction]
    • Protein or peptide: Meprin A subunit alpha
    • Protein or peptide: Fetuin-B
  • Ligand: CALCIUM IONCalcium
  • Ligand: ZINC ION
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Tetrameric portion of meprin alpha helix in complex with fetuin-B...

SupramoleculeName: Tetrameric portion of meprin alpha helix in complex with fetuin-B [subparticle localised reconstruction]
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Subparticle localised reconstruction of tetrameric region of recombinant, secreted helical meprin alpha in complex with fetuin-B
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Drosophila (fruit flies) / Recombinant cell: Schneider-2 / Recombinant plasmid: pMT/BiP/V5
Molecular weightTheoretical: 85 kDa/nm

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Macromolecule #1: Meprin A subunit alpha

MacromoleculeName: Meprin A subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: meprin A
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 67.28582 KDa
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString: WSHPQFEKVP IKYLPEENVH DADFGEQKDI SEINLAAGLD LFQGDILLQK SRNGLRDPNT RWTFPIPYIL ADNLGLNAKG AILYAFEMF RLKSCVDFKP YEGESSYIIF QQFDGCWSEV GDQHVGQNIS IGQGCAYKAI IEHEILHALG FYHEQSRTDR D DYVNIWWD ...String:
WSHPQFEKVP IKYLPEENVH DADFGEQKDI SEINLAAGLD LFQGDILLQK SRNGLRDPNT RWTFPIPYIL ADNLGLNAKG AILYAFEMF RLKSCVDFKP YEGESSYIIF QQFDGCWSEV GDQHVGQNIS IGQGCAYKAI IEHEILHALG FYHEQSRTDR D DYVNIWWD QILSGYQHNF DTYDDSLITD LNTPYDYESL MHYQPFSFNK NASVPTITAK IPEFNSIIGQ RLDFSAIDLE RL NRMYNCT TTHTLLDHCT FEKANICGMI QGTRDDTDWA HQDSAQAGEV DHTLLGQCTG AGYFMQFSTS SGSAEEAALL ESR ILYPKR KQQCLQFFYK MTGSPSDRLV VWVRRDDSTG NVRKLVKVQT FQGDDDHNWK IAHVVLKEEQ KFRYLFQGTK GDPQ NSTGG IYLDDITLTE TPCPTGVWTV RNFSQVLENT SKGDKLQSPR FYNSEGYGFG VTLYPNSRES SGYLRLAFHV CSGEN DAIL EWPVENRQVI ITILDQEPDV RNRMSSSMVF TTSKSHTSPA INDTVIWDRP SRVGTYHTDC NCFRSIDLGW SGFISH QML KRRSFLKNDD LIIFVDFEDI THLS

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Macromolecule #2: Fetuin-B

MacromoleculeName: Fetuin-B / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 42.926691 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGLLLPLALC ILVLCCGAMS PPQLALNPSA LLSRGCNDSD VLAVAGFALR DINKDRKDGY VLRLNRVNDA QEYRRGGLGS LFYLTLDVL ETDCHVLRKK AWQDCGMRIF FESVYGQCKA IFYMNNPSRV LYLAAYNCTL RPVSKKKIYM TCPDCPSSIP T DSSNHQVL ...String:
MGLLLPLALC ILVLCCGAMS PPQLALNPSA LLSRGCNDSD VLAVAGFALR DINKDRKDGY VLRLNRVNDA QEYRRGGLGS LFYLTLDVL ETDCHVLRKK AWQDCGMRIF FESVYGQCKA IFYMNNPSRV LYLAAYNCTL RPVSKKKIYM TCPDCPSSIP T DSSNHQVL EAATESLAKY NNENTSKQYS LFKVTRASSQ WVVGPSYFVE YLIKESPCTK SQASSCSLQS SDSVPVGLCK GS LTRTHWE KFVSVTCDFF ESQAPATGSE NSAVNQKPTN LPKVEESQQK NTPPTDSPSK AGPRGSVQYL PDLDDKNSQE KGP QEAFPV HLDLTTNPQG ETLDISFLFL EPMEEKLVVL PFPKEKARTA ECPGPAQNAS PLVLPPHHHH HH

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Macromolecule #6: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 8 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #8: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 8 / Number of copies: 4 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration1.0 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
30.0 mMTRIS
100.0 mMSodium chlorideNaClSodium chloride
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: Pelco EasyGlow
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: 3 s blot, -3 force.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-25 / Number grids imaged: 1 / Number real images: 4068 / Average electron dose: 44.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 519760
Details: Localised subparticles extrated after expansion of pseudo-helical symmetry
CTF correctionSoftware: (Name: CTFFIND (ver. 4.1.13), Warp (ver. 1.0.7))
Startup modelType of model: OTHER / Details: Ab initio volume
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Software:
Namedetails
cryoSPARC (ver. 3.3.1)Global alignment consensus map
RELION (ver. 3.1)Global alignment localised reconstruction
Final 3D classificationNumber classes: 8 / Avg.num./class: 100000 / Software - Name: RELION (ver. 3.1) / Details: Focused classification without changes to angles
Final angle assignmentType: MAXIMUM LIKELIHOOD
Software:
Namedetails
RELION (ver. 3.1)Global alignment localised reconstruction
cryoSPARC (ver. 3.3.1)Local refinement of localised reconstruction (non-uniform)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 115951
DetailsCompressed to LZW TIFF. Motion corrected by MotionCor.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

7uab

,

7auw

)
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7uai:
Meprin alpha helix in complex with fetuin-B

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