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- EMDB-8267: CryoEM Reconstruction of Hsp104 Hexamer -

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Basic information

Entry
Database: EMDB / ID: EMD-8267
TitleCryoEM Reconstruction of Hsp104 Hexamer
Map dataHexamer Reconstruction
Sample
  • Complex: Hexamer Complex of Hsp104
    • Protein or peptide: Heat shock protein 104Heat shock response
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
KeywordsHsp104 / AAA+ protein / CHAPERONE
Function / homology
Function and homology information


trehalose metabolism in response to heat stress / TRC complex / cellular heat acclimation / protein folding in endoplasmic reticulum / post-translational protein targeting to endoplasmic reticulum membrane / stress granule disassembly / chaperone cofactor-dependent protein refolding / protein unfolding / nuclear periphery / ADP binding ...trehalose metabolism in response to heat stress / TRC complex / cellular heat acclimation / protein folding in endoplasmic reticulum / post-translational protein targeting to endoplasmic reticulum membrane / stress granule disassembly / chaperone cofactor-dependent protein refolding / protein unfolding / nuclear periphery / ADP binding / unfolded protein binding / protein-folding chaperone binding / protein refolding / ATP hydrolysis activity / ATP binding / nucleus / identical protein binding / cytosol / cytoplasm
Similarity search - Function
ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp amino terminal domain, pathogenicity island component / Clp repeat (R) domain profile. / Clp, repeat (R) domain / Clp, N-terminal domain superfamily ...ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp amino terminal domain, pathogenicity island component / Clp repeat (R) domain profile. / Clp, repeat (R) domain / Clp, N-terminal domain superfamily / ClpA/B family / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Heat shock protein 104
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.64 Å
AuthorsYokom AL / Gates SN
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM099836 United States
American Heart Association15PRE25090089 United States
National Science Foundation (NSF, United States)DGE-1321851 United States
Muscular Dystrophy AssociationMDA277268 United States
CitationJournal: Nat Struct Mol Biol / Year: 2016
Title: Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation.
Authors: Adam L Yokom / Stephanie N Gates / Meredith E Jackrel / Korrie L Mack / Min Su / James Shorter / Daniel R Southworth /
Abstract: Hsp104, a conserved AAA+ protein disaggregase, promotes survival during cellular stress. Hsp104 remodels amyloids, thereby supporting prion propagation, and disassembles toxic oligomers associated ...Hsp104, a conserved AAA+ protein disaggregase, promotes survival during cellular stress. Hsp104 remodels amyloids, thereby supporting prion propagation, and disassembles toxic oligomers associated with neurodegenerative diseases. However, a definitive structural mechanism for its disaggregase activity has remained elusive. We determined the cryo-EM structure of wild-type Saccharomyces cerevisiae Hsp104 in the ATP state, revealing a near-helical hexamer architecture that coordinates the mechanical power of the 12 AAA+ domains for disaggregation. An unprecedented heteromeric AAA+ interaction defines an asymmetric seam in an apparent catalytic arrangement that aligns the domains in a two-turn spiral. N-terminal domains form a broad channel entrance for substrate engagement and Hsp70 interaction. Middle-domain helices bridge adjacent protomers across the nucleotide pocket, thus explaining roles in ATP hydrolysis and protein disaggregation. Remarkably, substrate-binding pore loops line the channel in a spiral arrangement optimized for substrate transfer across the AAA+ domains, thereby establishing a continuous path for polypeptide translocation.
History
DepositionJul 1, 2016-
Header (metadata) releaseJul 27, 2016-
Map releaseJul 27, 2016-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5kne
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8267.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHexamer Reconstruction
Voxel sizeX=Y=Z: 2 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.115307875 - 0.27497694
Average (Standard dev.)-0.000066139706 (±0.014375578)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions144144144
Spacing144144144
CellA=B=C: 288.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z288.000288.000288.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-0.1150.275-0.000

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Supplemental data

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Sample components

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Entire : Hexamer Complex of Hsp104

EntireName: Hexamer Complex of Hsp104
Components
  • Complex: Hexamer Complex of Hsp104
    • Protein or peptide: Heat shock protein 104Heat shock response
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: Hexamer Complex of Hsp104

SupramoleculeName: Hexamer Complex of Hsp104 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Heat shock protein 104

MacromoleculeName: Heat shock protein 104 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 95.967398 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: QFTERALTIL TLAQKLASDH QHPQLQPIHI LAAFIETPED GSVPYLQNLI EKGRYDYDLF KKVVNRNLVR IPQQQPAPAE ITPSYALGK VLQDAAKIQK QQKDSFIAQD HILFALFNDS SIQQIFKEAQ VDIEAIKQQA LELRGNTRID SRGADTNTPL E YLSKYAID ...String:
QFTERALTIL TLAQKLASDH QHPQLQPIHI LAAFIETPED GSVPYLQNLI EKGRYDYDLF KKVVNRNLVR IPQQQPAPAE ITPSYALGK VLQDAAKIQK QQKDSFIAQD HILFALFNDS SIQQIFKEAQ VDIEAIKQQA LELRGNTRID SRGADTNTPL E YLSKYAID MTEQARQGKL DPVIGREEEI RSTIRVLARR IKSNPCLIGE PGIGKTAIIE GVAQRIIDDD VPTILQGAKL FS LDLAALT AGAKYKGDFE ERFKGVLKEI EESKTLIVLF IDEIHMLMGN GKDDAANILK PALSRGQLKV IGATTNNEYR SIV EKDGAF ERRFQKIEVA EPSVRQTVAI LRGLQPKYEI HHGVRILDSA LVTAAQLAKR YLPYRRLPDS ALDLVDISCA GVAV ARDSK PEELDSKERQ LQLIQVEIKA LERDEDADST TKDRLKLARQ KEASLQEELE PLRQRYNEEK HGHEELTQAK KKLDE LENK ALDAERRYDT ATAADLRYFA IPDIKKQIEK LEDQVAEEER RAGANSMIQN VVDSDTISET AARLTGIPVK KLSESE NEK LIHMERDLSS EVVGQMDAIK AVSNAVRLSR SGLANPRQPA SFLFLGLSGS GKTELAKKVA GFLFNDEDMM IRVDCSE LS EKYAVSKLLG TTAGYVGYDE GGFLTNQLQY KPYSVLLFDE VEKAHPDVLT VMLQMLDDGR ITSGQGKTID CSNCIVIM T SNLGAEFINS QQGSKIQEST KNLVMGAVRQ HFRPEFLNRI SSIVIFNKLS RKAIHKIVDI RLKEIEERFE QNDKHYKLN LTQEAKDFLA KYGYSDDMGA RPLNRLIQNE ILNKLALRIL KNEIKDKETV NV

UniProtKB: Heat shock protein 104

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Macromolecule #2: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 2 / Number of copies: 11 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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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

Concentration0.7 mg/mL
BufferpH: 7.5
GridModel: C-flat / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
Details: Plunged into liquid ethane (FEI VITROBOT MARK IV).
DetailsClean protein sample incubated with AMP-PNP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-40 / Number grids imaged: 2 / Number real images: 3661 / Average exposure time: 0.2 sec. / Average electron dose: 1.6 e/Å2
Details: Movies contained 38 of 40 frames from an 8 sec exposure (200 milliseconds per frame).
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 240005
Details: Particles were selected using automated picking within the Appion package.
Startup modelType of model: EMDB MAP
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 5.64 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 172043
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsBackbone atoms fit into reconstruction density.
Output model

PDB-5kne:
CryoEM Reconstruction of Hsp104 Hexamer

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