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- EMDB-44675: Structure of the human mitochondrial Hsp70 (mortalin; R126W mutan... -

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Basic information

Entry
Database: EMDB / ID: EMD-44675
TitleStructure of the human mitochondrial Hsp70 (mortalin; R126W mutant) bound to nucleotide exchange factor GrpEL1 (WT)
Map dataUnsharpened map
Sample
  • Complex: Mortalin(R126W) in complex with GrpEL1(WT)
    • Protein or peptide: Stress-70 protein, mitochondrial
    • Protein or peptide: GrpE protein homolog 1, mitochondrial
    • Protein or peptide: Substrate peptide
KeywordsHsp70 / chaperone / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.96 Å
AuthorsMorizono MA / McGuire KL / Birouty NI / Herzik Jr MA
Funding support United States, 2 items
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, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateJan 15, 2025-
Current statusJan 15, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44675.png

Downloads & links

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Map

FileDownload / File: emd_44675.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 384 pix.
= 359.04 Å		0.94 Å/pix.
x 384 pix.
= 359.04 Å		0.94 Å/pix.
x 384 pix.
= 359.04 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.935 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0286
Minimum - Maximum-0.44928378 - 0.75636536
Average (Standard dev.)0.00002484845 (±0.008587063)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 359.04 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44675_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Unsharpened map

Fileemd_44675_additional_1.map
AnnotationUnsharpened map
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Additional map: Unsharpened map

Fileemd_44675_additional_2.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

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Half map: Unsharpened map

Fileemd_44675_half_map_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unsharpened map

Fileemd_44675_half_map_2.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Mortalin(R126W) in complex with GrpEL1(WT)

EntireName: Mortalin(R126W) in complex with GrpEL1(WT)
Components
  • Complex: Mortalin(R126W) in complex with GrpEL1(WT)
    • Protein or peptide: Stress-70 protein, mitochondrial
    • Protein or peptide: GrpE protein homolog 1, mitochondrial
    • Protein or peptide: Substrate peptide

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Supramolecule #1: Mortalin(R126W) in complex with GrpEL1(WT)

SupramoleculeName: Mortalin(R126W) in complex with GrpEL1(WT) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 152.5 kDa/nm

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Macromolecule #1: Stress-70 protein, mitochondrial

MacromoleculeName: Stress-70 protein, mitochondrial / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 64.524137 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASEAIKGAV VGIDLGTTNS CVAVMEGKQA KVLENAEGAR TTPSVVAFTA DGERLVGMPA KRQAVTNPNN TFYATKRLIG WRYDDPEVQ KDIKNVPFKI VRASNGDAWV EAHGKLYSPS QIGAFVLMKM KETAENYLGH TAKNAVITVP AYFNDSQRQA T KDAGQISG ...String:
MASEAIKGAV VGIDLGTTNS CVAVMEGKQA KVLENAEGAR TTPSVVAFTA DGERLVGMPA KRQAVTNPNN TFYATKRLIG WRYDDPEVQ KDIKNVPFKI VRASNGDAWV EAHGKLYSPS QIGAFVLMKM KETAENYLGH TAKNAVITVP AYFNDSQRQA T KDAGQISG LNVLRVINEP TAAALAYGLD KSEDKVIAVY DLGGGTFDIS ILEIQKGVFE VKSTNGDTFL GGEDFDQALL RH IVKEFKR ETGVDLTKDN MALQRVREAA EKAKCELSSS VQTDINLPYL TMDSSGPKHL NMKLTRAQFE GIVTDLIRRT IAP CQKAMQ DAEVSKSDIG EVILVGGMTR MPKVQQTVQD LFGRAPSKAV NPDEAVAIGA AIQGGVLAGD VTDVLLLDVT PLSL GIETL GGVFTKLINR NTTIPTKKSQ VFSTAADGQT QVEIKVCQGE REMAGDNKLL GQFTLIGIPP APRGVPQIEV TFDID ANGI VHVSAKDKGT GREQQIVIQS SGGLSKDDIE NMVKNAEKYA EEDRRKKERV EAVNMAEGII HDTETKMEEF KDQLPA DEC NKLKEEISKM RELLARKDSE TGENIRQAAS S

UniProtKB: Stress-70 protein, mitochondrial

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Macromolecule #2: GrpE protein homolog 1, mitochondrial

MacromoleculeName: GrpE protein homolog 1, mitochondrial / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.178029 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
TLLEEKVKLE EQLKETVEKY KRALADTENL RQRSQKLVEE AKLYGIQAFC KDLLEVADVL EKATQCVPKE EIKDDNPHLK NLYEGLVMT EVQIQKVFTK HGLLKLNPVG AKFDPYEHEA LFHTPVEGKE PGTVALVSKV GYKLHGRTLR PALVGVVKEA S A

UniProtKB: GrpE protein homolog 1, mitochondrial

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Macromolecule #3: Substrate peptide

MacromoleculeName: Substrate peptide / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 771.942 Da
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
VLLLDVT

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.298 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
100.0 mMNaClSodum chloride
0.5 mMC9H15O6PTris(2-carboxyethyl)phosphine

Details: 20mM Tris pH 8, 100mM NaCl, 0.5mM TCEP
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER
Details: Custom manual plunger. Greater than 95% humidity..

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 93.0 K / Max: 123.0 K
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 4 / Number real images: 4570 / Average exposure time: 6.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3304588
Startup modelType of model: NONE / Details: ab initio reconstruction, cryoSPARC
Final reconstructionNumber classes used: 44 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 138296
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
Final 3D classificationNumber classes: 50 / Avg.num./class: 3000 / Software - Name: cryoSPARC (ver. 4.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

1dkg

chain_id: D, residue_range: 3-383, source_name: PDB, initial_model_type: experimental model

4po2

chain_id: A, residue_range: 386-613, source_name: PDB, initial_model_type: experimental model

1dkg

chain_id: A, residue_range: 34-197, source_name: PDB, initial_model_type: experimental model

1dkg

chain_id: B, residue_range: 38-195, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 97.9
Output model

PDB-9bls:
Structure of the human mitochondrial Hsp70 (mortalin; R126W mutant) bound to nucleotide exchange factor GrpEL1 (WT)

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