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

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Basic information

Entry
Database: PDB / ID: 6nyy
Titlehuman m-AAA protease AFG3L2, substrate-bound
Components
  • AFG3-like protein 2
  • Substrate
KeywordsTRANSLOCASE / AAA+ / ATPase / protease / mitochondria / protein quality control / neurodegeneration / inner membrane / AFG3L2 / m/AAA protease
Function / homology
Function and homology information


cellular response to glutathione / m-AAA complex / mitochondrial protein processing / mitochondrial protein quality control / regulation of calcium import into the mitochondrion / 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 / mitochondrial protein quality control / regulation of calcium import into the mitochondrion / 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 / Mitochondrial protein degradation / myelination / axonogenesis / 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
: / Peptidase M41, FtsH extracellular / FtsH Extracellular / Peptidase, FtsH / Peptidase M41 / Peptidase M41-like / Peptidase family M41 / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site ...: / Peptidase M41, FtsH extracellular / FtsH Extracellular / Peptidase, FtsH / Peptidase M41 / 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
ADENOSINE-5'-DIPHOSPHATE / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Mitochondrial inner membrane m-AAA protease component AFG3L2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsLander, G.C. / Puchades, C.
Funding support United States, 1items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 22, 2019Provider: repository / Type: Initial release
Revision 1.1Aug 7, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Sep 18, 2019Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.3Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.5Mar 20, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • EMDB-0552
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
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
G: Substrate
H: Substrate
I: Substrate
J: Substrate
hetero molecules


Theoretical massNumber of molelcules
Total (without water)355,60023
Polymers353,18910
Non-polymers2,41113
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein / Protein/peptide , 2 types, 10 molecules ABCDEFGHIJ

#1: Protein
AFG3-like protein 2 / Paraplegin-like protein


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
#2: Protein/peptide
Substrate


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)

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Non-polymers , 4 types, 13 molecules

#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 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 weightValue: .360 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Details: DTT and ATP were added fresh before sample preparation
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPES1
2100 mMPotassium ChlorideKCl1
35 mMMagnesium ChlorideMgCl1
41 mMAdenosine TriPhosphateATP1
51 mMDithiothreitolDTT1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 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
VitrificationInstrument: 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.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / 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 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 100 K / Residual tilt: 0.07 mradians
Image recordingAverage 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 scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 30 / Used frames/image: 1-30

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1FindEMparticle selection
2Leginon3.2image acquisition
4CTFFIND4CTF correction
5RELION3CTF correctionlocal CTF estimation and CTF correction of particles
8UCSF Chimera1.11.2model fitting
10RELION3initial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
14PHENIX1.11.1_2580model refinement
CTF correctionDetails: CTF estimated with CTFFIND 4, local CTF estimated with
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4541491 / Details: Picked with FindEM
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 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 buildingB 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 buildingPDB-ID: 6AZ0

6az0


Accession code: 6AZ0 / Source name: PDB / Type: experimental model

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