|Entry||Database: PDB / ID: 6nyy|
|Title||human m-AAA protease AFG3L2, substrate-bound|
|Keywords||TRANSLOCASE / AAA+ / ATPase / protease / mitochondria / protein quality control / neurodegeneration / inner membrane / AFG3L2 / m/AAA protease|
|Function / homology|
Function and homology information
Processing of SMDT1 / mitochondrial protein processing / m-AAA complex / cristae formation / calcium import into the mitochondrion / mitochondrial calcium ion transmembrane transport / mitochondrial calcium ion homeostasis / righting reflex / muscle fiber development / mitochondrial fusion ...Processing of SMDT1 / mitochondrial protein processing / m-AAA complex / cristae formation / calcium import into the mitochondrion / mitochondrial calcium ion transmembrane transport / mitochondrial calcium ion homeostasis / righting reflex / muscle fiber development / mitochondrial fusion / nerve development / myelination / Hydrolases, Acting on peptide bonds (peptidases), Metalloendopeptidases / neuromuscular junction development / protein autoprocessing / protein processing / axonogenesis / regulation of multicellular organism growth / metallopeptidase activity / metalloendopeptidase activity / unfolded protein binding / mitochondrial inner membrane / proteolysis / mitochondrion / zinc ion binding / ATP binding
Peptidase M41 / AAA ATPase, AAA+ lid domain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site / Peptidase, FtsH / Peptidase M41, FtsH extracellular / AAA+ ATPase domain / Peptidase M41-like / P-loop containing nucleoside triphosphate hydrolase / ATPase family associated with various cellular activities (AAA) ...Peptidase M41 / AAA ATPase, AAA+ lid domain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site / Peptidase, FtsH / Peptidase M41, FtsH extracellular / AAA+ ATPase domain / Peptidase M41-like / P-loop containing nucleoside triphosphate hydrolase / ATPase family associated with various cellular activities (AAA) / AAA+ lid domain / Peptidase family M41 / FtsH Extracellular / AAA-protein family signature.
AFG3-like protein 2
|Biological species||Homo sapiens (human)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å|
|Authors||Lander, G.C. / Puchades, C.|
|Funding support|| United States, 1items |
|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.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
Downloads & links
A: AFG3-like protein 2
B: AFG3-like protein 2
C: AFG3-like protein 2
D: AFG3-like protein 2
E: AFG3-like protein 2
F: AFG3-like protein 2
-Protein/peptide , 2 types, 10 molecules A
B C D E F G H I J
Mass: 58331.629 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AFG3L2 / Production host: Escherichia coli (E. coli)
References: UniProt: Q9Y4W6, Hydrolases, Acting on peptide bonds (peptidases), Metalloendopeptidases
Mass: 799.871 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
-Non-polymers , 4 types, 13 molecules
Mass: 506.196 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP (energy-carrying molecule analogue) *YM
|#5: Chemical||#6: Chemical|| ChemComp-ADP / |
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: AFG3L2 / Type: ORGANELLE OR CELLULAR COMPONENT|
Details: Soluble domains of the AFG3L2, comprising the AAA+ ATPase and protease domains.
Entity ID: 1,2 / Source: RECOMBINANT
|Molecular weight||Value: .360 MDa / Experimental value: NO|
|Source (natural)||Organism: Homo sapiens (human)|
|Source (recombinant)||Organism: Escherichia coli (E. coli)|
|Buffer solution||pH: 8 |
Details: DTT and ATP were added fresh before sample preparation
|Specimen||Conc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Details: Grids were plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.).|
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
|Vitrification||Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K|
Details: Sample was blotted for 3 seconds using Whatman No. 1 filter paper immediately prio to plunge-freezing.
-Electron microscopy imaging
Model: Talos Arctica / Image courtesy: FEI Company
|Microscopy||Model: FEI TECNAI ARCTICA|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Calibrated magnification: 43478 X / Nominal defocus max: 18000 nm / Nominal defocus min: 8000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µns / Alignment procedure: COMA FREE|
|Specimen holder||Cryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 100 K / Residual tilt: 0.07 mradians|
|Image recording||Average exposure time: 7 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 5 / Num. of real images: 5707 / Details: Data collected with Leginon|
|Image scans||Sampling size: 5 µns / Width: 3710 / Height: 3838 / Movie frames/image: 30 / Used frames/image: 1-30|
|Software||Name: PHENIX / Version: 1.13_2998: / Classification: refinement|
|CTF correction||Details: CTF estimated with CTFFIND 4, local CTF estimated with|
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
|Particle selection||Num. of particles selected: 4541491 / Details: Picked with FindEM|
|Symmetry||Point symmetry: C1 (asymmetric)|
|3D reconstruction||Resolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1129437 / Algorithm: BACK PROJECTION|
Details: Multi-body refinement was used to generate 3 different reconstructions that were stitched together
Symmetry type: POINT
|Atomic model building||B value: 85 / Protocol: RIGID BODY FIT / Space: REAL|
Details: The sequence was threaded onto the PDB ID 6AZ0 and built into the EM density with Coot.
|Atomic model building||PDB-ID: 6AZ0|
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