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- EMDB-0552: human m-AAA protease AFG3L2, substrate-bound -

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Basic information

Entry
Database: EMDB / ID: EMD-0552
Titlehuman m-AAA protease AFG3L2, substrate-bound
Map dataComposite 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
  • Organelle or cellular component: AFG3L2
    • Protein or peptide: AFG3-like protein 2
    • Protein or peptide: Substrate
  • Ligand: ZINC ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
KeywordsAAA+ / ATPase / protease / mitochondria / protein quality control / neurodegeneration / inner membrane / AFG3L2 / m/AAA protease / translocase
Function / homology
Function and homology information


m-AAA complex / mitochondrial protein processing / Processing of SMDT1 / calcium import into the mitochondrion / mitochondrial calcium ion homeostasis / membrane protein proteolysis / cristae formation / righting reflex / nerve development / muscle cell development ...m-AAA complex / mitochondrial protein processing / Processing of SMDT1 / calcium import into the mitochondrion / mitochondrial calcium ion homeostasis / membrane protein proteolysis / cristae formation / righting reflex / nerve development / muscle cell development / Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / mitochondrial fusion / ATP-dependent peptidase activity / neuromuscular junction development / protein autoprocessing / regulation of multicellular organism growth / myelination / axonogenesis / metalloendopeptidase activity / protein processing / metallopeptidase activity / unfolded protein binding / mitochondrial inner membrane / ATP hydrolysis activity / mitochondrion / proteolysis / zinc ion binding / ATP binding
Similarity search - Function
Peptidase M41, FtsH extracellular / FtsH Extracellular / Peptidase M41 / Peptidase, FtsH / Peptidase M41-like / Peptidase family M41 / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. ...Peptidase M41, FtsH extracellular / FtsH Extracellular / Peptidase M41 / Peptidase, FtsH / Peptidase M41-like / Peptidase family M41 / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsLander GC / Puchades C
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R21 AG061697 01 United States
CitationJournal: 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
DepositionFeb 12, 2019-
Header (metadata) releaseMay 22, 2019-
Map releaseMay 22, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nyy
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0552.png

Downloads & links

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Map

FileDownload / File: emd_0552.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite 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 sizeX=Y=Z: 1.15 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.028558984 - 0.46374786
Average (Standard dev.)0.009907013 (±0.023940364)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 147.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z147.200147.200147.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0290.4640.010

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Supplemental data

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Additional map: ATPase domains from subunits B-E half map 1

Fileemd_0552_additional_1.map
AnnotationATPase domains from subunits B-E half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram (log scale)

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Additional map: ATPase domains from subunits B-E half map 2

Fileemd_0552_additional_2.map
AnnotationATPase domains from subunits B-E half map 2
Projections & Slices
AxesZYX

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Histogram

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Additional map: ATPase domains from subunits A & F half map 2

Fileemd_0552_additional_3.map
AnnotationATPase domains from subunits A & F half map 2
Projections & Slices
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Additional map: ATPase domains from subunits A & F half map 2

Fileemd_0552_additional_4.map
AnnotationATPase domains from subunits A & F half map 2
Projections & Slices
AxesZYX

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Half map: Protease domains half map 1

Fileemd_0552_half_map_1.map
AnnotationProtease domains half map 1
Projections & Slices
AxesZYX

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Half map: Protease domains half map 2

Fileemd_0552_half_map_2.map
AnnotationProtease domains half map 2
Projections & Slices
AxesZYX

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Sample components

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Entire : AFG3L2

EntireName: AFG3L2
Components
  • Organelle or cellular component: AFG3L2
    • Protein or peptide: AFG3-like protein 2
    • Protein or peptide: Substrate
  • Ligand: ZINC ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: AFG3L2

SupramoleculeName: 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.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 360 KDa

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Macromolecule #1: AFG3-like protein 2

MacromoleculeName: 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
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 58.331629 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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: AFG3-like protein 2

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Macromolecule #2: Substrate

MacromoleculeName: Substrate / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 799.871 Da
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
AAAAAAAAAA A

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 6 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 3 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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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 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
25.0 mMHEPES
100.0 mMPotassium ChlorideKCl
5.0 mMMagnesium ChlorideMgCl
1.0 mMAdenosine TriPhosphateATPAdenosine triphosphate
1.0 mMDithiothreitolDTT

Details: DTT and ATP were added fresh before sample preparation
GridModel: 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.).
VitrificationCryogen 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..

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 43478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 18.0 µm / Nominal defocus min: 8.0 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 100.0 K / Max: 100.0 K
Alignment procedureComa free - Residual tilt: 0.07 mrad
Image recordingFilm 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
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4541491 / Details: Picked with FindEM
Startup modelType of model: EMDB MAP
EMDB ID:

7023


Details: YME1 EMDB-7023 map, low pass filtered to 30A
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Details: Multi-body reconstruction
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0)
Details: Multi-body refinement was used to generate 3 different reconstructions that were stitched together
Number images used: 1129437
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6az0


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsThe sequence was threaded onto the PDB ID 6AZ0 and built into the EM density with Coot.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 85
Output model

PDB-6nyy:
human m-AAA protease AFG3L2, substrate-bound

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