+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-0552 | |||||||||
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Title | human m-AAA protease AFG3L2, substrate-bound | |||||||||
Map data | Composite map containing the stitched maps of the ATPase domains from subunits B-E, the ATPase domains from subunits A & F, and the protease domains | |||||||||
Sample |
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Keywords | AAA+ / ATPase / protease / mitochondria / protein quality control / neurodegeneration / inner membrane / AFG3L2 / m/AAA protease / translocase | |||||||||
Function / homology | Function and homology information cellular response to glutathione / m-AAA complex / mitochondrial protein processing / regulation of calcium import into the mitochondrion / mitochondrial protein quality control / Processing of SMDT1 / mitochondrial calcium ion homeostasis / membrane protein proteolysis / calcium import into the mitochondrion / cristae formation ...cellular response to glutathione / m-AAA complex / mitochondrial protein processing / regulation of calcium import into the mitochondrion / mitochondrial protein quality control / Processing of SMDT1 / mitochondrial calcium ion homeostasis / membrane protein proteolysis / calcium import into the mitochondrion / cristae formation / righting reflex / Hydrolases; Acting on acid anhydrides / nerve development / muscle cell development / Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / mitochondrial fusion / neuromuscular junction development / ATP-dependent peptidase activity / protein autoprocessing / protein maturation / regulation of multicellular organism growth / myelination / axonogenesis / Mitochondrial protein degradation / protein catabolic process / protein processing / metalloendopeptidase activity / metallopeptidase activity / unfolded protein binding / mitochondrial inner membrane / ATP hydrolysis activity / mitochondrion / proteolysis / zinc ion binding / ATP binding Similarity search - Function | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.0 Å | |||||||||
Authors | Lander GC / Puchades C | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Mol Cell / Year: 2019 Title: Unique Structural Features of the Mitochondrial AAA+ Protease AFG3L2 Reveal the Molecular Basis for Activity in Health and Disease. Authors: Cristina Puchades / Bojian Ding / Albert Song / R Luke Wiseman / Gabriel C Lander / Steven E Glynn / Abstract: Mitochondrial AAA+ quality-control proteases regulate diverse aspects of mitochondrial biology through specialized protein degradation, but the underlying mechanisms of these enzymes remain poorly ...Mitochondrial AAA+ quality-control proteases regulate diverse aspects of mitochondrial biology through specialized protein degradation, but the underlying mechanisms of these enzymes remain poorly defined. The mitochondrial AAA+ protease AFG3L2 is of particular interest, as genetic mutations localized throughout AFG3L2 are linked to diverse neurodegenerative disorders. However, a lack of structural data has limited our understanding of how mutations impact enzymatic function. Here, we used cryoelectron microscopy (cryo-EM) to determine a substrate-bound structure of the catalytic core of human AFG3L2. This structure identifies multiple specialized structural features that integrate with conserved motifs required for ATP-dependent translocation to unfold and degrade targeted proteins. Many disease-relevant mutations localize to these unique structural features of AFG3L2 and distinctly influence its activity and stability. Our results provide a molecular basis for neurological phenotypes associated with different AFG3L2 mutations and establish a structural framework to understand how different members of the AAA+ superfamily achieve specialized biological functions. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_0552.map.gz | 5.6 MB | EMDB map data format | |
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Header (meta data) | emd-0552-v30.xml emd-0552.xml | 30.8 KB 30.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_0552_fsc_1.xml emd_0552_fsc_2.xml emd_0552_fsc_3.xml | 8.6 KB 8.6 KB 8.6 KB | Display Display Display | FSC data file |
Images | emd_0552.png | 331.6 KB | ||
Filedesc metadata | emd-0552.cif.gz | 7.4 KB | ||
Others | emd_0552_additional_1.map.gz emd_0552_additional_2.map.gz emd_0552_additional_3.map.gz emd_0552_additional_4.map.gz emd_0552_half_map_1.map.gz emd_0552_half_map_2.map.gz | 7.3 MB 7.3 MB 7.4 MB 7.4 MB 7.3 MB 7.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-0552 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-0552 | HTTPS FTP |
-Validation report
Summary document | emd_0552_validation.pdf.gz | 832.9 KB | Display | EMDB validaton report |
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Full document | emd_0552_full_validation.pdf.gz | 832.4 KB | Display | |
Data in XML | emd_0552_validation.xml.gz | 8.5 KB | Display | |
Data in CIF | emd_0552_validation.cif.gz | 9.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0552 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0552 | HTTPS FTP |
-Related structure data
Related structure data | 6nyyMC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_0552.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Composite map containing the stitched maps of the ATPase domains from subunits B-E, the ATPase domains from subunits A & F, and the protease domains | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.15 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: ATPase domains from subunits B-E half map 1
File | emd_0552_additional_1.map | ||||||||||||
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Annotation | ATPase domains from subunits B-E half map 1 | ||||||||||||
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Density Histograms |
-Additional map: ATPase domains from subunits B-E half map 2
File | emd_0552_additional_2.map | ||||||||||||
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Annotation | ATPase domains from subunits B-E half map 2 | ||||||||||||
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Density Histograms |
-Additional map: ATPase domains from subunits A & F half map 2
File | emd_0552_additional_3.map | ||||||||||||
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Annotation | ATPase domains from subunits A & F half map 2 | ||||||||||||
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Density Histograms |
-Additional map: ATPase domains from subunits A & F half map 2
File | emd_0552_additional_4.map | ||||||||||||
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Annotation | ATPase domains from subunits A & F half map 2 | ||||||||||||
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Density Histograms |
-Half map: Protease domains half map 1
File | emd_0552_half_map_1.map | ||||||||||||
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Annotation | Protease domains half map 1 | ||||||||||||
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Density Histograms |
-Half map: Protease domains half map 2
File | emd_0552_half_map_2.map | ||||||||||||
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Annotation | Protease domains half map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : AFG3L2
Entire | Name: AFG3L2 |
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Components |
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-Supramolecule #1: AFG3L2
Supramolecule | Name: AFG3L2 / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 Details: Soluble domains of the AFG3L2, comprising the AAA+ ATPase and protease domains. |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 360 KDa |
-Macromolecule #1: AFG3-like protein 2
Macromolecule | Name: AFG3-like protein 2 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO EC number: Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 58.331629 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: SNARRGPAGI GRTGRGMGGL FSVGETTAKV LKDEIDVKFK DVAGCEEAKL EIMEFVNFLK NPKQYQDLGA KIPKGAILTG PPGTGKTLL AKATAGEANV PFITVSGSEF LEMFVGVGPA RVRDLFALAR KNAPCILFID QIDAVGRKRG RGNFGGQSEQ E NTLNQLLV ...String: SNARRGPAGI GRTGRGMGGL FSVGETTAKV LKDEIDVKFK DVAGCEEAKL EIMEFVNFLK NPKQYQDLGA KIPKGAILTG PPGTGKTLL AKATAGEANV PFITVSGSEF LEMFVGVGPA RVRDLFALAR KNAPCILFID QIDAVGRKRG RGNFGGQSEQ E NTLNQLLV EMDGFNTTTN VVILAGTNRP DILDPALLRP GRFDRQIFIG PPDIKGRASI FKVHLRPLKL DSTLEKDKLA RK LASLTPG FSGADVANVC NEAALIAARH LSDSINQKHF EQAIERVIGG LEKKTQVLQP EEKKTVAYHQ AGHAVAGWYL EHA DPLLKV SIIPRGKGLG YAQYLPKEQY LYTKEQLLDR MCMTLGGRVS EEIFFGRITT GAQDDLRKVT QSAYAQIVQF GMNE KVGQI SFDLPRQGDM VLEKPYSEAT ARLIDDEVRI LINDAYKRTV ALLTEKKADV EKVALLLLEK EVLDKNDMVE LLGPR PFAE KSTYEEFVEG TGSLDEDTSL PEGLKDWNKE REKEKEEPPG EKVAN UniProtKB: Mitochondrial inner membrane m-AAA protease component AFG3L2 |
-Macromolecule #2: Substrate
Macromolecule | Name: Substrate / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 799.871 Da |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: AAAAAAAAAA A |
-Macromolecule #3: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 3 / Number of copies: 6 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 3 / Formula: ANP |
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Molecular weight | Theoretical: 506.196 Da |
Chemical component information | ChemComp-ANP: |
-Macromolecule #5: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ChemComp-ADP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 2 mg/mL | ||||||||||||||||||
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Buffer | pH: 8 Component:
Details: DTT and ATP were added fresh before sample preparation | ||||||||||||||||||
Grid | Model: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 6 sec. / Pretreatment - Atmosphere: OTHER Details: Grids were plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.). | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK III Details: Sample was blotted for 3 seconds using Whatman No. 1 filter paper immediately prio to plunge-freezing.. |
-Electron microscopy
Microscope | FEI TECNAI ARCTICA |
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Temperature | Min: 100.0 K / Max: 100.0 K |
Alignment procedure | Coma free - Residual tilt: 0.07 mrad |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 1-30 / Number grids imaged: 5 / Number real images: 5707 / Average exposure time: 7.0 sec. / Average electron dose: 40.0 e/Å2 / Details: Data collected with Leginon |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Calibrated magnification: 43478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 18.0 µm / Nominal defocus min: 8.0 µm / Nominal magnification: 36000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Details | The sequence was threaded onto the PDB ID 6AZ0 and built into the EM density with Coot. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Overall B value: 85 |
Output model | PDB-6nyy: |