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- EMDB-26424: Meprin alpha helix in complex with fetuin-B [consensus C1 reconst... -

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Basic information

Entry
Database: EMDB / ID: EMD-26424
TitleMeprin alpha helix in complex with fetuin-B [consensus C1 reconstruction]
Map dataB-factor amplitude corrected sharpened map, filtered by local resolution
Sample
  • Complex: Inhibitory complex of meprin alpha helix with fetuin-B [consensus C1 reconstruction]
    • Protein or peptide: Meprin alpha
    • Protein or peptide: fetuin-B
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 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-
UpdateNov 2, 2022-
Current statusNov 2, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26424.png

Downloads & links

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Map

FileDownload / File: emd_26424.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationB-factor amplitude corrected sharpened map, filtered by local resolution
Voxel sizeX=Y=Z: 1.4133 Å
Density
Contour LevelBy AUTHOR: 0.0827
Minimum - Maximum-1.2257886 - 2.0444825
Average (Standard dev.)0.009371695 (±0.053028397)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 423.99002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26424_msk_1.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_26424_additional_1.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_26424_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_26424_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 : Inhibitory complex of meprin alpha helix with fetuin-B [consensus...

EntireName: Inhibitory complex of meprin alpha helix with fetuin-B [consensus C1 reconstruction]
Components
  • Complex: Inhibitory complex of meprin alpha helix with fetuin-B [consensus C1 reconstruction]
    • Protein or peptide: Meprin alpha
    • Protein or peptide: fetuin-B

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Supramolecule #1: Inhibitory complex of meprin alpha helix with fetuin-B [consensus...

SupramoleculeName: Inhibitory complex of meprin alpha helix with fetuin-B [consensus C1 reconstruction]
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Consensus standard reconstruction of full helix of recombinant, secreted helical pro-meprin alpha
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 alpha

MacromoleculeName: Meprin alpha / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Drosophila melanogaster (fruit fly)
SequenceString: WSHPQFEKVP IKYLPEENVH DADFGEQKDI SEINLAAGLD LFQGDIL LQ KSRNGLRDPN TRWTFPIPYI LADNLGLNAK GAILYAFEMF RLKSCVDFKP YEGESSYI I FQQFDGCWSE VGDQHVGQNI SIGQGCAYKA IIEHEILHAL GFYHEQSRTD RDDYVNIWW ...String:
WSHPQFEKVP IKYLPEENVH DADFGEQKDI SEINLAAGLD LFQGDIL LQ KSRNGLRDPN TRWTFPIPYI LADNLGLNAK GAILYAFEMF RLKSCVDFKP YEGESSYI I FQQFDGCWSE VGDQHVGQNI SIGQGCAYKA IIEHEILHAL GFYHEQSRTD RDDYVNIWW DQILSGYQHN FDTYDDSLIT DLNTPYDYES LMHYQPFSFN KNASVPTITA KIPEFNSIIG QRLDFSAID LERLNRMYNC TTTHTLLDHC TFEKANICGM IQGTRDDTDW AHQDSAQAGE V DHTLLGQC TGAGYFMQFS TSSGSAEEAA LLESRILYPK RKQQCLQFFY KMTGSPSDRL VV WVRRDDS TGNVRKLVKV QTFQGDDDHN WKIAHVVLKE EQKFRYLFQG TKGDPQNSTG GIY LDDITL TETPCPTGVW TVRNFSQVLE NTSKGDKLQS PRFYNSEGYG FGVTLYPNSR ESSG YLRLA FHVCSGENDA ILEWPVENRQ VIITILDQEP DVRNRMSSSM VFTTSKSHTS PAIND TVIW DRPSRVGTYH TDCNCFRSID LGWSGFISHQ MLKRRSFLKN DDLIIFVDFE DITHLS

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Macromolecule #2: fetuin-B

MacromoleculeName: fetuin-B / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGLLLPLALC ILVLCCGAMS PPQLALNPSA LLSRGCNDSD VLAVAGFALR DINKDRKDGY VLRLNRVND AQEYRRGGLG SLFYLTLDVL ETDCHVLRKK AWQDCGMRIF FESVYGQCKA I FYMNNPSR VLYLAAYNCT LRPVSKKKIY MTCPDCPSSI PTDSSNHQVL ...String:
MGLLLPLALC ILVLCCGAMS PPQLALNPSA LLSRGCNDSD VLAVAGFALR DINKDRKDGY VLRLNRVND AQEYRRGGLG SLFYLTLDVL ETDCHVLRKK AWQDCGMRIF FESVYGQCKA I FYMNNPSR VLYLAAYNCT LRPVSKKKIY MTCPDCPSSI PTDSSNHQVL EAATESLAKY NN ENTSKQY SLFKVTRASS QWVVGPSYFV EYLIKESPCT KSQASSCSLQ SSDSVPVGLC KGS LTRTHW EKFVSVTCDF FESQAPATGS ENSAVNQKPT NLPKVEESQQ KNTPPTDSPS KAGP RGSVQ YLPDLDDKNS QEKGPQEAFP VHLDLTTNPQ GETLDISFLF LEPMEEKLVV LPFPK EKAR TAECPGPAQN ASPLVLPPHH HHHH

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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 chlorideNaCl
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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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
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: 2.0 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: 132975
Details: Segmented filament particles boxed along the length of the helix
CTF correctionSoftware: (Name: CTFFIND (ver. 4.1.13), Warp (ver. 1.0.7))
Startup modelType of model: OTHER / Details: Ab initio volume
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 103952
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1) / Software - details: Local refinement (non-uniform)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7auw

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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