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- EMDB-43646: Human calpain-3 forms a homohexamer -

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Basic information

Entry
Database: EMDB / ID: EMD-43646
TitleHuman calpain-3 forms a homohexamer
Map data
Sample
  • Complex: Calpain-3 hexamer particle
    • Protein or peptide: Human calpain-3
KeywordsCalpain-3 / cysteine protease / Ca2+-dependent / Autoproteolytic / Tissue specific calpain. / METAL BINDING PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.85 Å
AuthorsYe Q / Serrao VHB / Davies PL
Funding support Canada, 1 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)FRN 148422 Canada
CitationJournal: J Biol Chem / Year: 2025
Title: Human calpain-3 and its structural plasticity: Dissociation of a homohexamer into dimers on binding titin.
Authors: Qilu Ye / Amy Henrickson / Borries Demeler / Vitor Hugo Balasco Serrão / Peter L Davies /
Abstract: Calpain-3 is an intracellular Ca-dependent cysteine protease abundant in skeletal muscle. Loss-of-function mutations in its single-copy gene cause a dystrophy of the limb-girdle muscles. These ...Calpain-3 is an intracellular Ca-dependent cysteine protease abundant in skeletal muscle. Loss-of-function mutations in its single-copy gene cause a dystrophy of the limb-girdle muscles. These mutations, of which there are over 500 in humans, are spread all along this 94-kDa multidomain protein that includes three 40+-residue sequences (NS, IS1, and IS2). The latter sequences are unique to this calpain isoform and are hypersensitive to proteolysis. To investigate the whole enzyme structure and how mutations might affect its activity, we produced the proteolytically more stable 85-kDa calpain-3 ΔNS ΔIS1 form with a C129A inactivating mutation as a recombinant protein in Escherichia coli. During size-exclusion chromatography, this calpain-3 was consistently eluted as a much larger 0.5-MDa complex rather than the expected 170-kDa dimer. Its size, which was confirmed by SEC-MALS, Blue Native PAGE, and AUC, made the complex amenable to single-particle cryo-EM analysis. From two data sets, we obtained a 3.85-Å reconstruction map that shows the complex is a trimer of calpain-3 dimers with six penta-EF-hand domains at its core. Calpain-3 has been reported to bind the N2A region of the giant muscle protein titin. When this 37-kDa region of titin was co-expressed with calpain-3, the multimer was reduced to a 320-kDa particle, which appears to be the calpain dimer bound to several copies of the titin fragment. We suggest that newly synthesized calpain-3 is kept as an inactive hexamer until it binds the N2A region of titin in the sarcomere, whereupon it dissociates into functional dimers.
History
DepositionFeb 7, 2024-
Header (metadata) releaseJan 8, 2025-
Map releaseJan 8, 2025-
UpdateFeb 5, 2025-
Current statusFeb 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43646.png

Downloads & links

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Map

FileDownload / File: emd_43646.map.gz / Format: CCP4 / Size: 229.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 392 pix.
= 323.792 Å		0.83 Å/pix.
x 392 pix.
= 323.792 Å		0.83 Å/pix.
x 392 pix.
= 323.792 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.826 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.095
Minimum - Maximum-1.8404273 - 2.4458504
Average (Standard dev.)0.00028960686 (±0.033306006)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions392392392
Spacing392392392
CellA=B=C: 323.792 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43646_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43646_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Calpain-3 hexamer particle

EntireName: Calpain-3 hexamer particle
Components
  • Complex: Calpain-3 hexamer particle
    • Protein or peptide: Human calpain-3

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Supramolecule #1: Calpain-3 hexamer particle

SupramoleculeName: Calpain-3 hexamer particle / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 514 KDa

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Macromolecule #1: Human calpain-3

MacromoleculeName: Human calpain-3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: calpain-3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MISRNFPIIG VKEKTFEQLH KKCLEKKVLY VDPEFPPDET SLFYSQKFPI QFVWKRPPEI CENPRFIIDG ANRTDICQGE LGDAWFLAAI ACLTLNQHLL FRVIPHDQSF IENYAGIFHF QFWRYGEWVD VVIDDCLPTY NNQLVFTKSN HRNEFWSALL EKAYAKLHGS ...String:
MISRNFPIIG VKEKTFEQLH KKCLEKKVLY VDPEFPPDET SLFYSQKFPI QFVWKRPPEI CENPRFIIDG ANRTDICQGE LGDAWFLAAI ACLTLNQHLL FRVIPHDQSF IENYAGIFHF QFWRYGEWVD VVIDDCLPTY NNQLVFTKSN HRNEFWSALL EKAYAKLHGS YEALKGGNTT EAMEDFTGGV AEFFEIRDAP SDMYKIMKKA IERGSLMGCS IDTIIPVQYE TRMACGLVRG HAYSVTGLDE VPFKGEKVKL VRLRNPWGQV EWNGSWSDRW KDWSFVDKDE KARLQHQVTE DGEFWMSYED FIYHFTKLEI CNLTADALQS DKLQTWTVSV NEGRWVRGCS AGGCRNFPDT FWTNPQYRLK LLEEDDDPDD SEVICSFLVA LMQKNRRKDR KLGASLFTIG FAIYEVPKEM HGNKQHLQKD FFLYNASKAR SKTYINMREV SQRFRLPPSE YVIVPSTYEP HQEGEFILRV FSEKRNLSEE VENTISVDRP VKKKKTKPII FVSDRANSNK ELGVDQESEE GKGKTSPDKQ KQSPQPQPGS SDQESEEQQQ FRNIFKQIAG DDMEICADEL KKVLNTVVNK HKDLKTHGFT LESCRSMIAL MDTDGSGKLN LQEFHHLWNK IKAWQKIFKH YDTDQSGTIN SYEMRNAVND AGFHLNNQLY DIITMRYADK HMNIDFDSFI CCFVRLEGMF RAFHAFDKDG DGIIKLNVLE WLQLTMYALE HHHHHH

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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

Concentration2.76 mg/mL
BufferpH: 7.6
Component:
ConcentrationFormulaName
100.0 mMNaClsodium chloride
20.0 mMTris-HClTris hydrochloride
2.0 mMEDTAEthylenediaminetetraacetic acid
10.0 mM2-ME2-mercaptoethanol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Average exposure time: 2.5 sec. / Average electron dose: 60.0 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: 3.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
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

Particle selectionNumber selected: 334733
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.85 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 334733
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.2)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 4.2)
FSC plot (resolution estimation)

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