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- EMDB-30352: Split conformation 1 of CtHsp104 (Hsp104 from Chaetomium Thermophilum) -

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Basic information

Entry
Database: EMDB / ID: EMD-30352
TitleSplit conformation 1 of CtHsp104 (Hsp104 from Chaetomium Thermophilum)
Map data
Sample
  • Complex: Split conformation 1 of CtHsp104 (Hsp104 from Chaetomium Thermophilum)
    • Other: Heat shock protein
Function / homology
Function and homology information


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 speciesChaetomium thermophilum (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsInoue Y / Hanazono Y / Noi K / Kawamoto A / Kimatsuka M / Harada R / Takeda K / Iwamasa N / Shibata K / Noguchi K ...Inoue Y / Hanazono Y / Noi K / Kawamoto A / Kimatsuka M / Harada R / Takeda K / Iwamasa N / Shibata K / Noguchi K / Shigeta Y / Namba K / Ogura T / Miki K / Shinohara K / Yohda M
Funding support Japan, 4 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)P15J08261 Japan
Japan Society for the Promotion of Science (JSPS)JP16H04572 Japan
Japan Society for the Promotion of Science (JSPS)JP16H00753 Japan
Japan Society for the Promotion of Science (JSPS)18H04690 Japan
CitationJournal: Structure / Year: 2021
Title: Split conformation of Chaetomium thermophilum Hsp104 disaggregase.
Authors: Yosuke Inoue / Yuya Hanazono / Kentaro Noi / Akihiro Kawamoto / Masato Kimatsuka / Ryuhei Harada / Kazuki Takeda / Ryoichi Kita / Natsuki Iwamasa / Kyoka Shibata / Keiichi Noguchi / Yasuteru ...Authors: Yosuke Inoue / Yuya Hanazono / Kentaro Noi / Akihiro Kawamoto / Masato Kimatsuka / Ryuhei Harada / Kazuki Takeda / Ryoichi Kita / Natsuki Iwamasa / Kyoka Shibata / Keiichi Noguchi / Yasuteru Shigeta / Keiichi Namba / Teru Ogura / Kunio Miki / Kyosuke Shinohara / Masafumi Yohda /
Abstract: Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. ...Hsp104 and its bacterial homolog ClpB form hexameric ring structures and mediate protein disaggregation. The disaggregated polypeptide is thought to thread through the central channel of the ring. However, the dynamic behavior of Hsp104 during disaggregation remains unclear. Here, we reported the stochastic conformational dynamics and a split conformation of Hsp104 disaggregase from Chaetomium thermophilum (CtHsp104) in the presence of ADP by X-ray crystallography, cryo-electron microscopy (EM), and high-speed atomic force microscopy (AFM). ADP-bound CtHsp104 assembles into a 6 left-handed spiral filament in the crystal structure at a resolution of 2.7 Å. The unit of the filament is a hexamer of the split spiral structure. In the cryo-EM images, staggered and split hexameric rings were observed. Further, high-speed AFM observations showed that a substrate addition enhanced the conformational change and increased the split structure's frequency. Our data suggest that split conformation is an off-pathway state of CtHsp104 during disaggregation.
History
DepositionJul 1, 2020-
Header (metadata) releaseMay 5, 2021-
Map releaseMay 5, 2021-
UpdateJul 14, 2021-
Current statusJul 14, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0084
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0084
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30352.png

Downloads & links

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Map

FileDownload / File: emd_30352.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.88 Å
Density
Contour LevelBy AUTHOR: 0.0084 / Movie #1: 0.0084
Minimum - Maximum-0.024000276 - 0.05420004
Average (Standard dev.)0.00038396646 (±0.0023178826)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 253.44 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.880.880.88
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z253.440253.440253.440
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.0240.0540.000

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Supplemental data

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Sample components

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Entire : Split conformation 1 of CtHsp104 (Hsp104 from Chaetomium Thermophilum)

EntireName: Split conformation 1 of CtHsp104 (Hsp104 from Chaetomium Thermophilum)
Components
  • Complex: Split conformation 1 of CtHsp104 (Hsp104 from Chaetomium Thermophilum)
    • Other: Heat shock protein

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Supramolecule #1: Split conformation 1 of CtHsp104 (Hsp104 from Chaetomium Thermophilum)

SupramoleculeName: Split conformation 1 of CtHsp104 (Hsp104 from Chaetomium Thermophilum)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: Heat shock protein

MacromoleculeName: Heat shock protein / type: other / ID: 1 / Classification: other
Source (natural)Organism: Chaetomium thermophilum (fungus)
SequenceString: MVDSRNADTE QENENLSKFC IDMTAMARE GKIDPVIGRE E EIRRVIRI LSRRTKNNPV LI GEPGVGK TTIVEGLAQR IVN ADVPDN LAACKLLSLD VGAL VAGSK YRGEFEERMK GVLKE IQES KETIILFVDE IHLLMG AGS SGEGGMDAAN LLKPMLA RG ...String:
MVDSRNADTE QENENLSKFC IDMTAMARE GKIDPVIGRE E EIRRVIRI LSRRTKNNPV LI GEPGVGK TTIVEGLAQR IVN ADVPDN LAACKLLSLD VGAL VAGSK YRGEFEERMK GVLKE IQES KETIILFVDE IHLLMG AGS SGEGGMDAAN LLKPMLA RG QLHCIGATTL AEYRKYIE K DAAFERRFQQ VLVKEPSIT ETISILRGLK EKYEVHHGVN IADAAIVAA ANLAARYLTS R RLPDSAVD LIDEAAAAVR VA RESQPEI IDSLERRLRQ LKI EIHALS REKDEASKAR LAQA KQDAQ NVEEELRPLR EKYER ERQR GKAIQEAKMK LEALRV KAE DASRMGDHSR AADLQYY AI PEQEAIIKRL EAEKAAAD A ALNANGADVG GSMITDVVG PDQINEIVAR WTGIPVTRLK TSEKEKLLH MEQALSKIVV G QKEAVQSV SNAIRLQRSG LS NPNQPPS FLFCGPSGTG KTL LTKALA EFLFDDPKSM IRFD MSEYQ ERHSLSRMIG APPGY VGHD AGGQLTEALR RRPFSI LLF DEVEKAAKEV LTVLLQL MD DGRITDGQGR VVDAKNCI V VMTSNLGAEY LSRANNGKD GKIDPTTREL VMNTLRNYFL PEFLNRISS IVIFNRLTRR E IRKIVDLR IAEIQKRLTD ND RNVTIKV SDEAKDKLGA QGY SPVYGA RPLQRLLEKE VLNR LAILI LRGQIREGEV ACVEL VDGK VQVLPNHPDS EPEDVD VDM ESDDAVDEVA PDSMDED IY ND
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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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

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: MOLYBDENUM / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Specialist opticsSpherical aberration corrector: Microscope was modified with a Cs corrector
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number real images: 4198 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1738736
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER
Details: making the initial model of CtHsp104 using cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationSoftware - Name: RELION (ver. 2.1)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 111567

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Atomic model buiding 1

Initial modelPDB ID:

5zui

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 208

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