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- PDB-7pxd: Substrate-engaged mycobacterial Proteasome-associated ATPase in c... -

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Basic information

Entry
Database: PDB / ID: 7pxd
TitleSubstrate-engaged mycobacterial Proteasome-associated ATPase in complex with open-gate 20S CP - composite map (state B)
Components
  • (Proteasome subunit ...) x 2
  • AAA ATPase forming ring-shaped complexes
  • Prokaryotic ubiquitin-like protein Pup
KeywordsCYTOSOLIC PROTEIN / AAA motor / ATPAse / mycobacterium / proteasome activator / 20S CP
Function / homology
Function and homology information


protein pupylation / proteasome binding / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / proteasomal protein catabolic process / proteasome complex / modification-dependent protein catabolic process / protein tag activity ...protein pupylation / proteasome binding / proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / proteasomal protein catabolic process / proteasome complex / modification-dependent protein catabolic process / protein tag activity / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
Prokaryotic ubiquitin-like protein Pup / Pup-like protein / Proteasome ATPase / Proteasomal ATPase, N-terminal OB domain / Proteasomal ATPase OB N-terminal domain / Proteasome, alpha subunit, bacterial / Proteasome subunit beta, actinobacteria / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal domain / : ...Prokaryotic ubiquitin-like protein Pup / Pup-like protein / Proteasome ATPase / Proteasomal ATPase, N-terminal OB domain / Proteasomal ATPase OB N-terminal domain / Proteasome, alpha subunit, bacterial / Proteasome subunit beta, actinobacteria / Proteasomal ATPase OB C-terminal domain / Proteasomal ATPase OB C-terminal domain / : / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / ATPase, AAA-type, conserved site / AAA-protein family signature. / Nucleophile aminohydrolases, N-terminal / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Prokaryotic ubiquitin-like protein Pup / Proteasome subunit beta / AAA ATPase forming ring-shaped complexes / Proteasome subunit alpha
Similarity search - Component
Biological speciesMycobacterium tuberculosis (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsJomaa, A. / Kavalchuk, M. / Weber-Ban, E.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis of prokaryotic ubiquitin-like protein engagement and translocation by the mycobacterial Mpa-proteasome complex.
Authors: Mikhail Kavalchuk / Ahmad Jomaa / Andreas U Müller / Eilika Weber-Ban /
Abstract: Proteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. In ...Proteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. In mycobacteria and other members of Actinobacteria, prokaryotic ubiquitin-like protein (Pup) serves as a degradation tag post-translationally conjugated to target proteins for their recruitment to the mycobacterial proteasome ATPase (Mpa). Here, we use single-particle cryo-electron microscopy to determine the structure of Mpa in complex with the 20S core particle at an early stage of pupylated substrate recruitment, shedding light on the mechanism of substrate translocation. Two conformational states of Mpa show how substrate is translocated stepwise towards the degradation chamber of the proteasome core particle. We also demonstrate, in vitro and in vivo, the importance of a structural feature in Mpa that allows formation of alternating charge-complementary interactions with the proteasome resulting in radial, rail-guided movements during the ATPase conformational cycle.
History
DepositionOct 8, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 19, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 26, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Jul 17, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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 / _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
0: Proteasome subunit alpha
1: AAA ATPase forming ring-shaped complexes
2: Proteasome subunit alpha
4: Proteasome subunit alpha
6: Proteasome subunit alpha
8: Proteasome subunit alpha
A: AAA ATPase forming ring-shaped complexes
B: AAA ATPase forming ring-shaped complexes
C: AAA ATPase forming ring-shaped complexes
D: AAA ATPase forming ring-shaped complexes
E: AAA ATPase forming ring-shaped complexes
F: AAA ATPase forming ring-shaped complexes
G: Prokaryotic ubiquitin-like protein Pup
H: Proteasome subunit beta
I: Proteasome subunit alpha
J: Proteasome subunit beta
K: Proteasome subunit alpha
L: Proteasome subunit beta
M: Proteasome subunit beta
N: Proteasome subunit beta
O: Proteasome subunit alpha
P: Proteasome subunit beta
Q: Proteasome subunit alpha
R: Proteasome subunit beta
S: Proteasome subunit beta
T: Proteasome subunit alpha
U: Proteasome subunit beta
V: Proteasome subunit beta
W: Proteasome subunit beta
X: Proteasome subunit alpha
Y: Proteasome subunit beta
Z: Proteasome subunit alpha
a: Proteasome subunit beta
b: Proteasome subunit beta
d: Proteasome subunit alpha
f: Proteasome subunit alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,284,10248
Polymers1,280,91436
Non-polymers3,18912
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Proteasome subunit ... , 2 types, 28 molecules 02468IKOQTXZdfHJLMNPRSUVWYab

#1: Protein
Proteasome subunit alpha / 20S proteasome alpha subunit / Proteasome core protein PrcA


Mass: 26911.039 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Details: The first 7 residues were removed (open gate proteasome)
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: prcA_1, prcA, prcA_2, C0094_11490, ERS007657_01774, ERS007661_00268, ERS007663_02522, ERS007665_00481, ERS007720_00613, ERS007722_01881
Production host: Escherichia coli (E. coli) / References: UniProt: A0A655IUE1
#4: Protein
Proteasome subunit beta / 20S proteasome beta subunit / Proteasome core protein PrcB


Mass: 30332.006 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Details: Propetide (the first N-terminal 57 residues) is removed
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: prcB, C0094_11495, DSI38_15610, E5M05_20980, E5M52_20895, E5M78_20920, ERS007665_00482, ERS007670_00434, ERS007679_01035, ERS007720_00612, ERS007722_01880, ERS007741_02624, ERS013471_03478, ...Gene: prcB, C0094_11495, DSI38_15610, E5M05_20980, E5M52_20895, E5M78_20920, ERS007665_00482, ERS007670_00434, ERS007679_01035, ERS007720_00612, ERS007722_01880, ERS007741_02624, ERS013471_03478, ERS023446_02554, ERS024276_00650, ERS094182_01868, F6W99_00699, GCL30_10845, SAMEA2683035_00452
Production host: Escherichia coli (E. coli)
References: UniProt: A0A045HFG5, proteasome endopeptidase complex

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Protein , 2 types, 8 molecules 1ABCDEFG

#2: Protein
AAA ATPase forming ring-shaped complexes / ARC


Mass: 67487.930 Da / Num. of mol.: 7
Source method: isolated from a genetically manipulated source
Details: C-terminal extension of Mpa containing GQYL motif which interacts with the proteasome
Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: arc, mpa, C0094_11520, DSI38_15585, E5M05_19030, E5M52_17490, E5M78_17520, ERS007661_01151, ERS007665_00732, ERS007679_01634, ERS007681_02311, ERS007703_01049, ERS007720_01453, ERS007722_00998, ...Gene: arc, mpa, C0094_11520, DSI38_15585, E5M05_19030, E5M52_17490, E5M78_17520, ERS007661_01151, ERS007665_00732, ERS007679_01634, ERS007681_02311, ERS007703_01049, ERS007720_01453, ERS007722_00998, ERS007741_01455, ERS023446_02559, ERS024276_00114, ERS027646_02035, ERS027659_02128, ERS027661_00811, ERS094182_01863, F6W99_00704, FRD82_11355, GCL30_10870, SAMEA2683035_00457
Production host: Escherichia coli (E. coli) / References: UniProt: A0A045JPX7
#3: Protein Prokaryotic ubiquitin-like protein Pup / Bacterial ubiquitin-like modifier


Mass: 7095.416 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: GS residues are left from the TEV cleavage site / Source: (gene. exp.) Mycobacterium tuberculosis (bacteria)
Gene: pup, C0094_11500, DSI38_15605, E5M05_20975, E5M52_20890, E5M78_20915, ERS007661_02567, ERS007665_00483, ERS007670_00435, ERS007679_01034, ERS007681_03228, ERS007688_03984, ERS007720_00611, ...Gene: pup, C0094_11500, DSI38_15605, E5M05_20975, E5M52_20890, E5M78_20915, ERS007661_02567, ERS007665_00483, ERS007670_00435, ERS007679_01034, ERS007681_03228, ERS007688_03984, ERS007720_00611, ERS007722_01879, ERS007741_02623, ERS013471_03479, ERS023446_02555, ERS024276_00651, ERS027646_03180, ERS027661_04264, ERS094182_01867, F6W99_00700, FRD82_16980, GCL30_10850, SAMEA2683035_00453
Production host: Escherichia coli (E. coli) / References: UniProt: A0A045GWT8

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Non-polymers , 2 types, 12 molecules

#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: Mycobacterial Proteasome-associated ATPase in complex with substrate and open-gate 20SCP
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 1.1 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
2EPUimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 860718
3D reconstructionResolution: 4 Å / Resolution method: OTHER / Num. of particles: 50814 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
15KWA

5kwa

15KWA1PDBexperimental model
25LZP

5lzp

15LZP2PDBexperimental model

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