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- EMDB-26421: Meprin alpha helix (activated) - full C1 consensus reconstruction -

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Basic information

Entry
Database: EMDB / ID: EMD-26421
TitleMeprin alpha helix (activated) - full C1 consensus reconstruction
Map dataUnsharpened final map. Low pass filtered to the global FSC.
Sample
  • Complex: Full helical meprin alpha in the activated state
    • Protein or peptide: Human meprin alpha
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 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-
UpdateNov 2, 2022-
Current statusNov 2, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26421.png

Downloads & links

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Map

FileDownload / File: emd_26421.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened final map. Low pass filtered to the global FSC.
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.252
Minimum - Maximum-0.6344477 - 1.5916408
Average (Standard dev.)0.013302645 (±0.07894397)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 423.99997 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26421_msk_1.map
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_26421_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_26421_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 : Full helical meprin alpha in the activated state

EntireName: Full helical meprin alpha in the activated state
Components
  • Complex: Full helical meprin alpha in the activated state
    • Protein or peptide: Human meprin alpha

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Supramolecule #1: Full helical meprin alpha in the activated state

SupramoleculeName: Full helical meprin alpha in the activated state / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Full C1 reconstruction of the filamentous helix of recombinant, secreted helical meprin alpha in the activated state
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly) / Recombinant cell: Schneider-2 / Recombinant plasmid: pMT/BiP/V5
Molecular weightTheoretical: 85 kDa/nm

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Macromolecule #1: Human meprin alpha

MacromoleculeName: Human meprin alpha / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: meprin A
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString: WSHPQFEKVP IKYLPEENVH DADFGEQKDI SEINLAAGLD LFQGDILLQK SR NGLRDPN TRWTFPIPYI LADNLGLNAK GAILYAFEMF RLKSCVDFKP YEGESSYIIF QQF DGCWSE VGDQHVGQNI SIGQGCAYKA IIEHEILHAL GFYHEQSRTD RDDYVNIWWD QILS ...String:
WSHPQFEKVP IKYLPEENVH DADFGEQKDI SEINLAAGLD LFQGDILLQK SR NGLRDPN TRWTFPIPYI LADNLGLNAK GAILYAFEMF RLKSCVDFKP YEGESSYIIF QQF DGCWSE VGDQHVGQNI SIGQGCAYKA IIEHEILHAL GFYHEQSRTD RDDYVNIWWD QILS GYQHN FDTYDDSLIT DLNTPYDYES LMHYQPFSFN KNASVPTITA KIPEFNSIIG QRLDF SAID LERLNRMYNC TTTHTLLDHC TFEKANICGM IQGTRDDTDW AHQDSAQAGE VDHTLL GQC TGAGYFMQFS TSSGSAEEAA LLESRILYPK RKQQCLQFFY KMTGSPSDRL VVWVRRD DS TGNVRKLVKV QTFQGDDDHN WKIAHVVLKE EQKFRYLFQG TKGDPQNSTG GIYLDDIT L TETPCPTGVW TVRNFSQVLE NTSKGDKLQS PRFYNSEGYG FGVTLYPNSR ESSGYLRLA FHVCSGENDA ILEWPVENRQ VIITILDQEP DVRNRMSSSM VFTTSKSHTS PAINDTVIWD RPSRVGTYH TDCNCFRSID LGWSGFISHQ MLKRRSFLKN DDLIIFVDFE DITHLS

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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 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
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
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: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 147823 / Details: Standard C1 reconstruction
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 - 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 reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 111312
DetailsCompressed to LZW TIFF. Motion corrected by MotionCor.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7uab

DetailsRigid body fit only
RefinementSpace: REAL / Protocol: OTHER

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