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- EMDB-26420: Human pro-meprin alpha (zymogen state)[Focused classification and... -

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Basic information

Entry
Database: EMDB / ID: EMD-26420
TitleHuman pro-meprin alpha (zymogen state)[Focused classification and refinement]
Map dataDeepEMhancer (mask) sharpened final map. Low pass filtered to global FSC.
Sample
  • Complex: Single subunit of helical meprin alpha in the zymogen state
    • Protein or peptide: Meprin A subunit alpha
  • Ligand: CALCIUM ION
  • Ligand: ZINC ION
KeywordsMetalloprotease / complex / helical / extracellular / ONCOPROTEIN
Function / homology
Function and homology information


meprin A / meprin A complex / epidermal growth factor receptor ligand maturation / metallodipeptidase activity / signaling receptor ligand precursor processing / metalloendopeptidase activity / metallopeptidase activity / extracellular space / extracellular exosome / zinc ion binding / plasma membrane
Similarity search - Function
Meprin alpha/beta subunit / Meprin peptidase domain / Astacin-like domain profile. / Peptidase M12A / Astacin (Peptidase family M12A) / MAM domain signature. / Domain in meprin, A5, receptor protein tyrosine phosphatase mu (and others) / MATH domain / MAM domain, meprin/A5/mu / MAM domain ...Meprin alpha/beta subunit / Meprin peptidase domain / Astacin-like domain profile. / Peptidase M12A / Astacin (Peptidase family M12A) / MAM domain signature. / Domain in meprin, A5, receptor protein tyrosine phosphatase mu (and others) / MATH domain / MAM domain, meprin/A5/mu / MAM domain / MAM domain profile. / MATH/TRAF domain / MATH/TRAF domain profile. / meprin and TRAF homology / 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
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 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 12, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26420.png

Downloads & links

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Map

FileDownload / File: emd_26420.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDeepEMhancer (mask) sharpened final map. Low pass filtered to global FSC.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 224 pix.
= 237.44 Å		1.06 Å/pix.
x 224 pix.
= 237.44 Å		1.06 Å/pix.
x 224 pix.
= 237.44 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.207
Minimum - Maximum-0.0011741364 - 2.0487995
Average (Standard dev.)0.0029851268 (±0.044916987)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 237.43999 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26420_msk_1.map
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_26420_additional_1.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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Additional map: B-factor amplitude corrected sharpened map.

Fileemd_26420_additional_2.map
AnnotationB-factor amplitude corrected sharpened map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened final map. Low pass filtered to the global FSC.

Fileemd_26420_additional_3.map
AnnotationUnsharpened final map. Low pass filtered to the 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_26420_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_26420_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 : Single subunit of helical meprin alpha in the zymogen state

EntireName: Single subunit of helical meprin alpha in the zymogen state
Components
  • Complex: Single subunit of helical meprin alpha in the zymogen state
    • Protein or peptide: Meprin A subunit alpha
  • Ligand: CALCIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: Single subunit of helical meprin alpha in the zymogen state

SupramoleculeName: Single subunit of helical meprin alpha in the zymogen state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Focused classification and local refinement reconstruction of single subunit of recombinant, secreted helical pro-meprin alpha
Source (natural)Organism: Homo sapiens (human)
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: 1 / 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

UniProtKB: Meprin A subunit alpha

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration0.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
30.0 mMTRIS
100.0 mMSodium chlorideNaCl
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: 3 s blot, -3 force.
DetailsPolydisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 2648 / Average electron dose: 44.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsCompressed to LZW TIFF. Motion corrected by MotionCor.
Particle selectionNumber selected: 905660
Details: Subparticles extracted after particle expansion about pseudo-helical symmetry
Startup modelType of model: OTHER / Details: Ab initio volume
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 338429
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1, 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Software:
Namedetails
cryoSPARC (ver. 3.3.1)Local refinement (non-uniform)
RELION (ver. 3.1)Global alignment + SIDESPLITTER
Final 3D classificationNumber classes: 10 / Avg.num./class: 50000 / Details: Angles fixed, classification only
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4gwn


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-7uac:
Human pro-meprin alpha (zymogen state)

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