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- EMDB-0308: Rea1 Wild type ADP state (tail part) -

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Basic information

Entry
Database: EMDB / ID: EMD-0308
TitleRea1 Wild type ADP state (tail part)
Map data
Sample
  • Complex: Rea1 (MIDASIN) tail with ADP
    • Protein or peptide: Midasin,Midasin,Midasin
Function / homology
Function and homology information


protein-RNA complex remodeling / regulation of ribosomal subunit export from nucleus / preribosome, large subunit precursor / ribosomal large subunit export from nucleus / rRNA processing / ribosomal large subunit assembly / nucleolus / ATP hydrolysis activity / mitochondrion / nucleoplasm ...protein-RNA complex remodeling / regulation of ribosomal subunit export from nucleus / preribosome, large subunit precursor / ribosomal large subunit export from nucleus / rRNA processing / ribosomal large subunit assembly / nucleolus / ATP hydrolysis activity / mitochondrion / nucleoplasm / ATP binding / nucleus
Similarity search - Function
Midasin / Midasin, AAA lid domain 7 / Midasin AAA lid domain 5 / : / Midasin AAA lid domain / Midasin AAA lid domain / Midasin AAA+ ATPase lid domain / ATPase, dynein-related, AAA domain / AAA domain (dynein-related subfamily) / Sigma-54 interaction domain, ATP-binding site 1 ...Midasin / Midasin, AAA lid domain 7 / Midasin AAA lid domain 5 / : / Midasin AAA lid domain / Midasin AAA lid domain / Midasin AAA+ ATPase lid domain / ATPase, dynein-related, AAA domain / AAA domain (dynein-related subfamily) / Sigma-54 interaction domain, ATP-binding site 1 / VWFA domain profile. / von Willebrand factor, type A / von Willebrand factor A-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) / Saccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSosnowski P / Urnavicius L / Boland A / Fagiewicz R / Busselez J / Papai G / Schmidt H
Funding support France, 1 items
OrganizationGrant numberCountry
French National Research AgencyATIP-Avenir France
CitationJournal: Elife / Year: 2018
Title: The CryoEM structure of the ribosome maturation factor Rea1.
Authors: Piotr Sosnowski / Linas Urnavicius / Andreas Boland / Robert Fagiewicz / Johan Busselez / Gabor Papai / Helgo Schmidt /
Abstract: The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly ...The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualise the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide-induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA +ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.
History
DepositionOct 19, 2018-
Header (metadata) releaseDec 12, 2018-
Map releaseDec 12, 2018-
UpdateSep 30, 2020-
Current statusSep 30, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0444
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0444
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6hyd
  • Surface level: 0.0444
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0308.png

Downloads & links

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Map

FileDownload / File: emd_0308.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.0444 / Movie #1: 0.0444
Minimum - Maximum-0.28497687 - 0.38296542
Average (Standard dev.)0.00012719262 (±0.0043909685)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.2850.3830.000

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Supplemental data

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Sample components

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Entire : Rea1 (MIDASIN) tail with ADP

EntireName: Rea1 (MIDASIN) tail with ADP
Components
  • Complex: Rea1 (MIDASIN) tail with ADP
    • Protein or peptide: Midasin,Midasin,Midasin

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Supramolecule #1: Rea1 (MIDASIN) tail with ADP

SupramoleculeName: Rea1 (MIDASIN) tail with ADP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Midasin,Midasin,Midasin

MacromoleculeName: Midasin,Midasin,Midasin / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 192.045828 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: PIEESLAAVI PISHLGEVGK WANNVLNCTE YSEKKIAERL YVFITFLTDM GVLEKINNLY KPANLKFQKA LGLHDKQLTE ETVSLTLNE YVLPTVSKYS DKIKSPESLY LLSSLRLLLN SLNALKLINE KSTHGKIDEL TYIELSAAAF NGRHLKNIPR I PIFCILYN ...String:
PIEESLAAVI PISHLGEVGK WANNVLNCTE YSEKKIAERL YVFITFLTDM GVLEKINNLY KPANLKFQKA LGLHDKQLTE ETVSLTLNE YVLPTVSKYS DKIKSPESLY LLSSLRLLLN SLNALKLINE KSTHGKIDEL TYIELSAAAF NGRHLKNIPR I PIFCILYN ILTVMSENLK TESLFCGSNQ YQYYWDLLVI VIAALETAVT KDEARLRVYK ELIDSWIASV KSKSDIEITP FL NINLEFT DVLQLSRGHS ITLLWDIFRK NYPTTSNSWL AFEKLINLSE KFDKVRLLQF SESYNSIKDL MDVFRLLNDD VLN NKLSEF NLLLSKLEDG INELELISNK FLNKRKHYFA DEFDNLIRYT FSVDTAELIK ELAPASSLAT QKLTKLITNK YNYP PIFDV LWTEKNAKLT SFTSTIFSSQ FLEDVVRKSN NLKSFSGNQI KQSISDAELL LSSTIKCSPN LLKSQMEYYK NMLLS WLRK VIDIHVGGDC LKLTLKELCS LIEEKTASET RVTFAEYIFP ALDLAESSKS LEELGEAWIT FGTGLLLLFV PDSPYD PAI HDYVLYDLFL KTKTFSQNLM KSWRNVRKVI SGDEEIFTEK LINTISDDDA PQSPRVYRTG MSIDSLFDEW MAFLSST MS SRQIKELVSS YKCNSDQSDR RLEMLQQNSA HFLNRLESGY SKFADLNDIL AGYIYSINFG FDLLKLQKSK DRASFQIS P LWSMDPINIS CAENVLSAYH ELSRFFKKGD MEDTSIEKVL MYFLTLFKFH KRDTNLLEIF EAALYTLYSR WSVRRFRQE QEENEKSNMF KFNDNSDDYE ADFRKLFPDY EDTALVTNEK DISSPENLDD IYFKLADTYI SVFDKDHDAN FSSELKSGAI ITTILSEDL KNTRIEELKS GSLSAVINTL DAETQSFKNT EVFGNIDFYH DFSIPEFQKA GDIIETVLKS VLKLLKQWPE H ATLKELYR VSQEFLNYPI KTPLARQLQK IEQIYTYLAE WEKYASSEVS LNNTVKLITD LIVSWRKLEL RTWKGLFNSE DA KTRKSIG KWWFYLYESI VISNFVSEKK ETAPNATLLV SSLNLFFSKS TLGEFNARLD LVKAFYKHIQ LIGLRSSKIA GLL HNTIKF YYQFKPLIDE RITNGKKSLE KEIDDIILLA SWKDVNVDAL KQSSRKSHNN LYKIVRKYRD LLNGDAKTII EAGL LY(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) RNIDTVASNM DSYLEKISSQ EFPNFADLAS DFYAEAERLR KETPNVYTKE NKKRLAYLKT QKSKLLGDAL KELRRIGLKV NFREDIQKV QSSTTTILAN IAPFNNEYLN SSDAFFFKIL DLLPKLRSAA SNPSDDIPVA AIERGMALAQ SLMFSLITVR H PLSEFTND YCKINGMMLD LEHFTCLKGD IVHSSLKANV DNVRLFEKWL PSLLDYAAQT LSVISKYSAT SEQQKILLDA KS TLSSFFV HFNSSRIFDS SFIESYSRFE LFINELLKKL ENAKETGNAF VFDIIIEWIK ANKGGPIKKE QKRGPSVEDV EQA FRRTFT SIILSFQKVI GDGIESISET DDNWLSASFK KVMVNVKLLR SSVVSKNIET ALSLLKDFDF TTTESIYVKS VISF TLPVI TRYYNAMTVV LERSRIYYTN TSRGMYILST ILHSLAKN

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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

Concentration1 mg/mL
BufferpH: 7.2
Component:
ConcentrationFormulaName
150.0 mMC8H18N2O4SHEPES
10.0 mMMg(CH3COO)2Magnesium Acetate
5.0 mMC14H24N2O10EGTA
5.0 mMC4H10O2S2DTT
3.0 mMC10H15N5O10P2ADPAdenosine diphosphate

Details: ADP was added 5 minute before the plunging
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 3.4 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 105000
Specialist opticsSpherical aberration corrector: Titan Krios Cs Corrector / Energy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
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 - Dimensions - Width: 3712 pixel / Digitization - Dimensions - Height: 3840 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 2-35 / Number grids imaged: 14 / Number real images: 23230 / Average exposure time: 0.2 sec. / Average electron dose: 46.2 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware: (Name: Gctf, RELION)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 432556
FSC plot (resolution estimation)

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