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- PDB-9blt: Structure of the human mitochondrial Hsp70 (mortalin; R126W mutan... -

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Basic information

Entry
Database: PDB / ID: 9blt
TitleStructure of the human mitochondrial Hsp70 (mortalin; R126W mutant) bound to nucleotide exchange factor GrpEL1 (Y173A mutant)
Components
  • GrpE protein homolog 1, mitochondrial
  • Stress-70 protein, mitochondrial
  • Substrate peptide
KeywordsCHAPERONE / Hsp70 / nucleotide exchange factor / mitochondria / cryoEM
Function / homology
Function and homology information


PAM complex, Tim23 associated import motor / negative regulation of hemopoiesis / MIB complex / negative regulation of hematopoietic stem cell differentiation / SAM complex / negative regulation of erythrocyte differentiation / TIM23 mitochondrial import inner membrane translocase complex / inner mitochondrial membrane organization / Cristae formation / Complex III assembly ...PAM complex, Tim23 associated import motor / negative regulation of hemopoiesis / MIB complex / negative regulation of hematopoietic stem cell differentiation / SAM complex / negative regulation of erythrocyte differentiation / TIM23 mitochondrial import inner membrane translocase complex / inner mitochondrial membrane organization / Cristae formation / Complex III assembly / adenyl-nucleotide exchange factor activity / Complex I biogenesis / calcium import into the mitochondrion / protein import into mitochondrial matrix / Mitochondrial protein import / iron-sulfur cluster assembly / non-chaperonin molecular chaperone ATPase / mitochondrial nucleoid / : / Regulation of HSF1-mediated heat shock response / Mitochondrial unfolded protein response (UPRmt) / heat shock protein binding / Mitochondrial protein degradation / protein folding chaperone / Gene and protein expression by JAK-STAT signaling after Interleukin-12 stimulation / protein export from nucleus / erythrocyte differentiation / intracellular protein transport / ATP-dependent protein folding chaperone / regulation of erythrocyte differentiation / unfolded protein binding / protein folding / protein-folding chaperone binding / protein refolding / mitochondrial inner membrane / mitochondrial matrix / positive regulation of apoptotic process / focal adhesion / ubiquitin protein ligase binding / negative regulation of apoptotic process / nucleolus / protein homodimerization activity / ATP hydrolysis activity / mitochondrion / RNA binding / extracellular exosome / nucleoplasm / ATP binding / identical protein binding / cytoplasm
Similarity search - Function
GrpE nucleotide exchange factor / GrpE nucleotide exchange factor, head / GrpE nucleotide exchange factor, coiled-coil / GrpE / grpE protein signature. / Chaperone DnaK / Heat shock hsp70 proteins family signature 2. / Heat shock hsp70 proteins family signature 1. / Heat shock hsp70 proteins family signature 3. / Heat shock protein 70, conserved site ...GrpE nucleotide exchange factor / GrpE nucleotide exchange factor, head / GrpE nucleotide exchange factor, coiled-coil / GrpE / grpE protein signature. / Chaperone DnaK / Heat shock hsp70 proteins family signature 2. / Heat shock hsp70 proteins family signature 1. / Heat shock hsp70 proteins family signature 3. / Heat shock protein 70, conserved site / Heat shock protein 70kD, peptide-binding domain superfamily / Heat shock protein 70 family / Hsp70 protein / Heat shock protein 70kD, C-terminal domain superfamily / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Stress-70 protein, mitochondrial / GrpE protein homolog 1, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsMorizono, M.A. / McGuire, K.L. / Birouty, N.I. / Herzik Jr., M.A.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R35GM138206-04 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5T32GM139795-03 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural insights into GrpEL1-mediated nucleotide and substrate release of human mitochondrial Hsp70.
Authors: Marc A Morizono / Kelly L McGuire / Natalie I Birouty / Mark A Herzik /
Abstract: Maintenance of protein homeostasis is necessary for cell viability and depends on a complex network of chaperones and co-chaperones, including the heat-shock protein 70 (Hsp70) system. In human ...Maintenance of protein homeostasis is necessary for cell viability and depends on a complex network of chaperones and co-chaperones, including the heat-shock protein 70 (Hsp70) system. In human mitochondria, mitochondrial Hsp70 (mortalin) and the nucleotide exchange factor (GrpEL1) work synergistically to stabilize proteins, assemble protein complexes, and facilitate protein import. However, our understanding of the molecular mechanisms guiding these processes is hampered by limited structural information. To elucidate these mechanistic details, we used cryoEM to determine structures of full-length human mortalin-GrpEL1 complexes in previously unobserved states. Our structures and molecular dynamics simulations allow us to delineate specific roles for mortalin-GrpEL1 interfaces and to identify steps in GrpEL1-mediated nucleotide and substrate release by mortalin. Subsequent analyses reveal conserved mechanisms across bacteria and mammals and facilitate a complete understanding of sequential nucleotide and substrate release for the Hsp70 chaperone system.
History
DepositionMay 1, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 15, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Stress-70 protein, mitochondrial
B: GrpE protein homolog 1, mitochondrial
C: GrpE protein homolog 1, mitochondrial
D: Substrate peptide


Theoretical massNumber of molelcules
Total (without water)101,4684
Polymers101,4684
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Stress-70 protein, mitochondrial / 75 kDa glucose-regulated protein / GRP-75 / Heat shock 70 kDa protein 9 / Mortalin / MOT / Peptide- ...75 kDa glucose-regulated protein / GRP-75 / Heat shock 70 kDa protein 9 / Mortalin / MOT / Peptide-binding protein 74 / PBP74


Mass: 64524.137 Da / Num. of mol.: 1 / Mutation: R126W
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HSPA9, GRP75, HSPA9B, mt-HSP70 / Production host: Escherichia coli (E. coli) / References: UniProt: P38646
#2: Protein GrpE protein homolog 1, mitochondrial / HMGE / Mt-GrpE#1


Mass: 18085.934 Da / Num. of mol.: 2 / Mutation: Y173A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GRPEL1, GREPEL1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9HAV7
#3: Protein/peptide Substrate peptide


Mass: 771.942 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
Has protein modificationN

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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: Mortalin(R126W)-GrpEL1(Y173A) with SBD-a lid / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8 / Details: 20mM Tris pH 8, 100mM NaCl, 0.5mM TCEP
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris BufferC4H11NO31
2100 mMSodium chlorideNaCl1
30.5 mMTris(2-carboxyethyl)phosphineC9H15O6P1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE-PROPANE / Humidity: 95 % / Chamber temperature: 277 K / Details: Custom manual plunger. Greater than 95% humidity

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 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): 123 K / Temperature (min): 93 K
Image recordingAverage exposure time: 6 sec. / Electron dose: 60 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 3669
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 20 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.3.0particle selection
2EPU4.4.1image acquisition
4cryoSPARC4.3.1CTF correction
7PHENIX1.21model fitting
9cryoSPARC4.3.1initial Euler assignment
10cryoSPARC4.3.1final Euler assignment
11cryoSPARC4.3.1classification
12cryoSPARC4.3.13D reconstruction
13PHENIX1.21model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2091743
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 51324 / Symmetry type: POINT
Atomic model buildingB value: 83.2 / Protocol: OTHER / Space: REAL
Atomic model buildingDetails: Model from mortalin-GrpEL1(WT) / Source name: Other / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037197
ELECTRON MICROSCOPYf_angle_d0.4789710
ELECTRON MICROSCOPYf_dihedral_angle_d4.347986
ELECTRON MICROSCOPYf_chiral_restr0.041132
ELECTRON MICROSCOPYf_plane_restr0.0031266

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