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- PDB-6pdw: Msp1-substrate complex in closed conformation -

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Basic information

Entry
Database: PDB / ID: 6pdw
TitleMsp1-substrate complex in closed conformation
Components
  • Membrane-spanning ATPase-like protein
  • Unknown peptide
KeywordsPROTEIN TRANSPORT / membrane protein / tail-anchored protein / protein quality control
Function / homologyAAA+ ATPase domain / ATPase, AAA-type, core / ATPase, AAA-type, conserved site / P-loop containing nucleoside triphosphate hydrolase / AAA ATPase, AAA+ lid domain / integral component of membrane / ATP binding / Membrane-spanning ATPase-like protein
Function and homology information
Biological speciesChaetomium thermophilum (fungus)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsWang, L. / Myasnikov, A. / Pan, X. / Walter, P.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R01GM032384 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)S10OD021741 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)S10OD020054 United States
CitationJournal: Elife / Year: 2020
Title: Structure of the AAA protein Msp1 reveals mechanism of mislocalized membrane protein extraction.
Authors: Lan Wang / Alexander Myasnikov / Xingjie Pan / Peter Walter /
Abstract: The AAA protein Msp1 extracts mislocalized tail-anchored membrane proteins and targets them for degradation, thus maintaining proper cell organization. How Msp1 selects its substrates and firmly ...The AAA protein Msp1 extracts mislocalized tail-anchored membrane proteins and targets them for degradation, thus maintaining proper cell organization. How Msp1 selects its substrates and firmly engages them during the energetically unfavorable extraction process remains a mystery. To address this question, we solved cryo-EM structures of Msp1-substrate complexes at near-atomic resolution. Akin to other AAA proteins, Msp1 forms hexameric spirals that translocate substrates through a central pore. A singular hydrophobic substrate recruitment site is exposed at the spiral's seam, which we propose positions the substrate for entry into the pore. There, a tight web of aromatic amino acids grips the substrate in a sequence-promiscuous, hydrophobic milieu. Elements at the intersubunit interfaces coordinate ATP hydrolysis with the subunits' positions in the spiral. We present a comprehensive model of Msp1's mechanism, which follows general architectural principles established for other AAA proteins yet specializes Msp1 for its unique role in membrane protein extraction.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 19, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
B: Membrane-spanning ATPase-like protein
C: Membrane-spanning ATPase-like protein
D: Membrane-spanning ATPase-like protein
E: Membrane-spanning ATPase-like protein
F: Membrane-spanning ATPase-like protein
G: Unknown peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)215,86917
Polymers213,8656
Non-polymers2,00411
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Membrane-spanning ATPase-like protein


Mass: 42599.152 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (fungus) / Gene: CTHT_0034230 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G0S654
#2: Protein/peptide Unknown peptide


Mass: 869.063 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#3: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, energy-carrying molecule*YM
#4: Chemical ChemComp-BEF / BERYLLIUM TRIFLUORIDE ION


Mass: 66.007 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: BeF3
#5: Chemical
ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg

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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: Msp1-substrate complex in closed conformation / Type: COMPLEX / Entity ID: 1,2 / Source: RECOMBINANT
Molecular weightValue: 0.24 MDa / Experimental value: YES
Source (natural)Organism: Chaetomium thermophilum (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21DE3
Buffer solutionpH: 7.5
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: DIFFRACTION
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: SerialEM / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48861 / Symmetry type: POINT

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