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- EMDB-29817: ATP- and mtHsp10-bound mtHsp60 V72I -

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Basic information

Entry
Database: EMDB / ID: EMD-29817
TitleATP- and mtHsp10-bound mtHsp60 V72I
Map data
Sample
  • Complex: ATP- and mtHsp10-bound mtHsp60
    • Protein or peptide: 60 kDa heat shock protein, mitochondrial
    • Protein or peptide: 10 kDa heat shock protein, mitochondrial
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: POTASSIUM ION
Keywordschaperonin / ATPase / foldase / CHAPERONE
Function / homology
Function and homology information


coated vesicle / isotype switching to IgG isotypes / mitochondrial unfolded protein response / TFAP2A acts as a transcriptional repressor during retinoic acid induced cell differentiation / apolipoprotein A-I binding / lipopolysaccharide receptor complex / protein import into mitochondrial intermembrane space / high-density lipoprotein particle binding / migrasome / cysteine-type endopeptidase activator activity ...coated vesicle / isotype switching to IgG isotypes / mitochondrial unfolded protein response / TFAP2A acts as a transcriptional repressor during retinoic acid induced cell differentiation / apolipoprotein A-I binding / lipopolysaccharide receptor complex / protein import into mitochondrial intermembrane space / high-density lipoprotein particle binding / migrasome / cysteine-type endopeptidase activator activity / positive regulation of T cell mediated immune response to tumor cell / chaperonin ATPase / Mitochondrial protein import / positive regulation of macrophage activation / negative regulation of execution phase of apoptosis / cellular response to interleukin-7 / MyD88-dependent toll-like receptor signaling pathway / biological process involved in interaction with symbiont / 'de novo' protein folding / sperm plasma membrane / apoptotic mitochondrial changes / B cell activation / : / B cell proliferation / positive regulation of interferon-alpha production / positive regulation of interleukin-10 production / DNA replication origin binding / apolipoprotein binding / RHOG GTPase cycle / positive regulation of execution phase of apoptosis / response to unfolded protein / Mitochondrial unfolded protein response (UPRmt) / isomerase activity / chaperone-mediated protein complex assembly / sperm midpiece / clathrin-coated pit / positive regulation of interleukin-12 production / protein folding chaperone / Mitochondrial protein degradation / intrinsic apoptotic signaling pathway / response to cold / secretory granule / T cell activation / protein maturation / ATP-dependent protein folding chaperone / lipopolysaccharide binding / positive regulation of T cell activation / positive regulation of interleukin-6 production / positive regulation of type II interferon production / osteoblast differentiation / p53 binding / unfolded protein binding / protein folding / single-stranded DNA binding / double-stranded RNA binding / protein-folding chaperone binding / protein refolding / early endosome / mitochondrial inner membrane / protein stabilization / mitochondrial matrix / ubiquitin protein ligase binding / negative regulation of apoptotic process / enzyme binding / cell surface / protein-containing complex / ATP hydrolysis activity / mitochondrion / extracellular space / RNA binding / extracellular exosome / ATP binding / metal ion binding / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
Chaperonin GroES, conserved site / Chaperonins cpn10 signature. / Chaperonin 10 Kd subunit / GroES chaperonin family / GroES chaperonin superfamily / Chaperonin 10 Kd subunit / Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily ...Chaperonin GroES, conserved site / Chaperonins cpn10 signature. / Chaperonin 10 Kd subunit / GroES chaperonin family / GroES chaperonin superfamily / Chaperonin 10 Kd subunit / Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like apical domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonin Cpn60/GroEL/TCP-1 family / GroES-like superfamily
Similarity search - Domain/homology
60 kDa heat shock protein, mitochondrial / 10 kDa heat shock protein, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsBraxton JR / Shao H / Tse E / Gestwicki JE / Southworth DR
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS) United States
CitationJournal: bioRxiv / Year: 2023
Title: Asymmetric apical domain states of mitochondrial Hsp60 coordinate substrate engagement and chaperonin assembly.
Authors: Julian R Braxton / Hao Shao / Eric Tse / Jason E Gestwicki / Daniel R Southworth /
Abstract: The mitochondrial chaperonin, mtHsp60, promotes the folding of newly imported and transiently misfolded proteins in the mitochondrial matrix, assisted by its co-chaperone mtHsp10. Despite its ...The mitochondrial chaperonin, mtHsp60, promotes the folding of newly imported and transiently misfolded proteins in the mitochondrial matrix, assisted by its co-chaperone mtHsp10. Despite its essential role in mitochondrial proteostasis, structural insights into how this chaperonin binds to clients and progresses through its ATP-dependent reaction cycle are not clear. Here, we determined cryo-electron microscopy (cryo-EM) structures of a hyperstable disease-associated mtHsp60 mutant, V72I, at three stages in this cycle. Unexpectedly, client density is identified in all states, revealing interactions with mtHsp60's apical domains and C-termini that coordinate client positioning in the folding chamber. We further identify a striking asymmetric arrangement of the apical domains in the ATP state, in which an alternating up/down configuration positions interaction surfaces for simultaneous recruitment of mtHsp10 and client retention. Client is then fully encapsulated in mtHsp60/mtHsp10, revealing prominent contacts at two discrete sites that potentially support maturation. These results identify a new role for the apical domains in coordinating client capture and progression through the cycle, and suggest a conserved mechanism of group I chaperonin function.
History
DepositionFeb 16, 2023-
Header (metadata) releaseJul 12, 2023-
Map releaseJul 12, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29817.png

Downloads & links

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Map

FileDownload / File: emd_29817.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 450 pix.
= 417.15 Å		0.93 Å/pix.
x 450 pix.
= 417.15 Å		0.93 Å/pix.
x 450 pix.
= 417.15 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.927 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-3.5503817 - 8.196474
Average (Standard dev.)0.00054472376 (±0.16912472)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 417.15 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map

Fileemd_29817_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

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Slices (1/2)
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Half map: #2

Fileemd_29817_half_map_1.map
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Histogram (log scale)

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Half map: #1

Fileemd_29817_half_map_2.map
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Sample components

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Entire : ATP- and mtHsp10-bound mtHsp60

EntireName: ATP- and mtHsp10-bound mtHsp60
Components
  • Complex: ATP- and mtHsp10-bound mtHsp60
    • Protein or peptide: 60 kDa heat shock protein, mitochondrial
    • Protein or peptide: 10 kDa heat shock protein, mitochondrial
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: POTASSIUM ION

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Supramolecule #1: ATP- and mtHsp10-bound mtHsp60

SupramoleculeName: ATP- and mtHsp10-bound mtHsp60 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: 60 kDa heat shock protein, mitochondrial

MacromoleculeName: 60 kDa heat shock protein, mitochondrial / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO / EC number: chaperonin ATPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 58.321 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SNAAKDVKFG ADARALMLQG VDLLADAVAV TMGPKGRTVI IEQSWGSPKV TKDGVTVAKS IDLKDKYKNI GAKLIQDVAN NTNEEAGDG TTTATVLARS IAKEGFEKIS KGANPVEIRR GVMLAVDAVI AELKKQSKPV TTPEEIAQVA TISANGDKEI G NIISDAMK ...String:
SNAAKDVKFG ADARALMLQG VDLLADAVAV TMGPKGRTVI IEQSWGSPKV TKDGVTVAKS IDLKDKYKNI GAKLIQDVAN NTNEEAGDG TTTATVLARS IAKEGFEKIS KGANPVEIRR GVMLAVDAVI AELKKQSKPV TTPEEIAQVA TISANGDKEI G NIISDAMK KVGRKGVITV KDGKTLNDEL EIIEGMKFDR GYISPYFINT SKGQKCEFQD AYVLLSEKKI SSIQSIVPAL EI ANAHRKP LVIIAEDVDG EALSTLVLNR LKVGLQVVAV KAPGFGDNRK NQLKDMAIAT GGAVFGEEGL TLNLEDVQPH DLG KVGEVI VTKDDAMLLK GKGDKAQIEK RIQEIIEQLD VTTSEYEKEK LNERLAKLSD GVAVLKVGGT SDVEVNEKKD RVTD ALNAT RAAVEEGIVL GGGCALLRCI PALDSLTPAN EDQKIGIEII KRTLKIPAMT IAKNAGVEGS LIVEKIMQSS SEVGY DAMA GDFVNMVEKG IIDPTKVVRT ALLDAAGVAS LLTTAEVVVT EIPKEEKDPG MGAMGGMGGG MGGGMF

UniProtKB: 60 kDa heat shock protein, mitochondrial

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Macromolecule #2: 10 kDa heat shock protein, mitochondrial

MacromoleculeName: 10 kDa heat shock protein, mitochondrial / type: protein_or_peptide / ID: 2 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.218934 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SNAMAGQAFR KFLPLFDRVL VERSAAETVT KGGIMLPEKS QGKVLQATVV AVGSGSKGKG GEIQPVSVKV GDKVLLPEYG GTKVVLDDK DYFLFRDGDI LGKYVD

UniProtKB: 10 kDa heat shock protein, mitochondrial

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 14 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 14 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 5 / Number of copies: 14 / Formula: K
Molecular weightTheoretical: 39.098 Da

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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

Concentration4.8 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa / Details: Pelco easiGlow, 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: Wait time 10 sec, blot time 3 sec, blot force 0.

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Number real images: 7460 / Average exposure time: 10.0 sec. / Average electron dose: 66.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 53937 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: OTHER / Details: Ab initio reconstruction from cryoSPARC
Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 81840
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: FLEXIBLE FIT
Output model

PDB-8g7n:
ATP- and mtHsp10-bound mtHsp60 V72I

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