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- EMDB-16509: Cryo-EM captures early ribosome assembly in action -

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Basic information

Entry
Database: EMDB / ID: EMD-16509
TitleCryo-EM captures early ribosome assembly in action
Map data
Sample
  • Complex: large ribosomal subunit precursor d1
    • RNA: 23S rRNA
    • Protein or peptide: 50S ribosomal protein L22
    • Protein or peptide: 50S ribosomal protein L24
    • Protein or peptide: 50S ribosomal protein L29
Keywordsribosome / ribosome assembly / ribosome biogenesis / total reconstitution / RNA / ribosomal protein.
Function / homology
Function and homology information


ribosome assembly / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / large ribosomal subunit / large ribosomal subunit rRNA binding / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / rRNA binding / structural constituent of ribosome ...ribosome assembly / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / large ribosomal subunit / large ribosomal subunit rRNA binding / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / rRNA binding / structural constituent of ribosome / translation / response to antibiotic / cytosol / cytoplasm
Similarity search - Function
: / Ribosomal protein L22, bacterial/chloroplast-type / : / Ribosomal proteins 50S L24/mitochondrial 39S L24 / Ribosomal protein L24 / Ribosomal protein L29, conserved site / Ribosomal protein L29 signature. / Ribosomal protein L22/L17, conserved site / Ribosomal protein L22 signature. / Ribosomal L29 protein ...: / Ribosomal protein L22, bacterial/chloroplast-type / : / Ribosomal proteins 50S L24/mitochondrial 39S L24 / Ribosomal protein L24 / Ribosomal protein L29, conserved site / Ribosomal protein L29 signature. / Ribosomal protein L22/L17, conserved site / Ribosomal protein L22 signature. / Ribosomal L29 protein / Ribosomal protein L29/L35 / Ribosomal protein L29/L35 superfamily / Ribosomal protein L24 signature. / Ribosomal protein L24/L26, conserved site / KOW (Kyprides, Ouzounis, Woese) motif. / Ribosomal protein L26/L24, KOW domain / Ribosomal protein L22/L17 / Ribosomal protein L22/L17 superfamily / Ribosomal protein L22p/L17e / Translation protein SH3-like domain superfamily / KOW motif / KOW / Ribosomal protein L2, domain 2
Similarity search - Domain/homology
Large ribosomal subunit protein uL29 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL22
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.62 Å
AuthorsNikolay R / Qin B / Lauer S
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research16GW0300 Germany
CitationJournal: Nat Commun / Year: 2023
Title: Cryo-EM captures early ribosome assembly in action.
Authors: Bo Qin / Simon M Lauer / Annika Balke / Carlos H Vieira-Vieira / Jörg Bürger / Thorsten Mielke / Matthias Selbach / Patrick Scheerer / Christian M T Spahn / Rainer Nikolay /
Abstract: Ribosome biogenesis is a fundamental multi-step cellular process in all domains of life that involves the production, processing, folding, and modification of ribosomal RNAs (rRNAs) and ribosomal ...Ribosome biogenesis is a fundamental multi-step cellular process in all domains of life that involves the production, processing, folding, and modification of ribosomal RNAs (rRNAs) and ribosomal proteins. To obtain insights into the still unexplored early assembly phase of the bacterial 50S subunit, we exploited a minimal in vitro reconstitution system using purified ribosomal components and scalable reaction conditions. Time-limited assembly assays combined with cryo-EM analysis visualizes the structurally complex assembly pathway starting with a particle consisting of ordered density for only ~500 nucleotides of 23S rRNA domain I and three ribosomal proteins. In addition, our structural analysis reveals that early 50S assembly occurs in a domain-wise fashion, while late 50S assembly proceeds incrementally. Furthermore, we find that both ribosomal proteins and folded rRNA helices, occupying surface exposed regions on pre-50S particles, induce, or stabilize rRNA folds within adjacent regions, thereby creating cooperativity.
History
DepositionJan 21, 2023-
Header (metadata) releaseApr 5, 2023-
Map releaseApr 5, 2023-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16509.png

Downloads & links

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Map

FileDownload / File: emd_16509.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.25 Å/pix.
x 300 pix.
= 375. Å		1.25 Å/pix.
x 300 pix.
= 375. Å		1.25 Å/pix.
x 300 pix.
= 375. Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.25 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-0.26748407 - 1.2925204
Average (Standard dev.)-0.00051153416 (±0.0440648)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 375.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16509_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16509_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_16509_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : large ribosomal subunit precursor d1

EntireName: large ribosomal subunit precursor d1
Components
  • Complex: large ribosomal subunit precursor d1
    • RNA: 23S rRNA
    • Protein or peptide: 50S ribosomal protein L22
    • Protein or peptide: 50S ribosomal protein L24
    • Protein or peptide: 50S ribosomal protein L29

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Supramolecule #1: large ribosomal subunit precursor d1

SupramoleculeName: large ribosomal subunit precursor d1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli) / Strain: Can20-12E

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Macromolecule #1: 23S rRNA

MacromoleculeName: 23S rRNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli) / Strain: Can20-12E
Molecular weightTheoretical: 941.612375 KDa
SequenceString: GGUUAAGCGA CUAAGCGUAC ACGGUGGAUG CCCUGGCAGU CAGAGGCGAU GAAGGACGUG CUAAUCUGCG AUAAGCGUCG GUAAGGUGA UAUGAACCGU UAUAACCGGC GAUUUCCGAA UGGGGAAACC CAGUGUGUUU CGACACACUA UCAUUAACUG A AUCCAUAG ...String:
GGUUAAGCGA CUAAGCGUAC ACGGUGGAUG CCCUGGCAGU CAGAGGCGAU GAAGGACGUG CUAAUCUGCG AUAAGCGUCG GUAAGGUGA UAUGAACCGU UAUAACCGGC GAUUUCCGAA UGGGGAAACC CAGUGUGUUU CGACACACUA UCAUUAACUG A AUCCAUAG GUUAAUGAGG CGAACCGGGG GAACUGAAAC AUCUAAGUAC CCCGAGGAAA AGAAAUCAAC CGAGAUUCCC CC AGUAGCG GCGAGCGAAC GGGGAGCAGC CCAGAGCCUG AAUCAGUGUG UGUGUUAGUG GAAGCGUCUG GAAAGGCGCG CGA UACAGG GUGACAGCCC CGUACACAAA AAUGCACAUG CUGUGAGCUC GAUGAGUAGG GCGGGACACG UGGUAUCCUG UCUG AAUAU GGGGGGACCA UCCUCCAAGG CUAAAUACUC CUGACUGACC GAUAGUGAAC CAGUACCGUG AGGGAAAGGC GAAAA GAAC CCCGGCGAGG GGAGUGAAAA AGAACCUGAA ACCGUGUACG UACAAGCAGU GGGAGCACGC UUAGGCGUGU GACUGC GUA CCUUUUGUAU AAUGGGUCAG CGACUUAUAU UCUGUAGCAA GGUUAACCGA AUAGGGGAGC CGAAGGGAAA CCGAGUC UU AACUGGGCGU UAAGUUGCAG GGUAUAGACC CGAAACCCGG UGAUCUAGCC AUGGGCAGGU UGAAGGUUGG GUAACACU A ACUGGAGGAC CGAACCGACU AAUGUUGAAA AAUUAGCGGA UGACUUGUGG CUGGGGGUGA AAGGCCAAUC AAACCGGGA GAUAGCUGGU UCUCCCCGAA AGCUAUUUAG GUAGCGCCUC GUGAAUUCAU CUCCGGGGGU AGAGCACUGU UUCGGCAAGG GGGUCAUCC CGACUUACCA ACCCGAUGCA AACUGCGAAU ACCGGAGAAU GUUAUCACGG GAGACACACG GCGGGUGCUA A CGUCCGUC GUGAAGAGGG AAACAACCCA GACCGCCAGC UAAGGUCCCA AAGUCAUGGU UAAGUGGGAA ACGAUGUGGG AA GGCCCAG ACAGCCAGGA UGUUGGCUUA GAAGCAGCCA UCAUUUAAAG AAAGCGUAAU AGCUCACUGG UCGAGUCGGC CUG CGCGGA AGAUGUAACG GGGCUAAACC AUGCACCGAA GCUGCGGCAG CGACGCUUAU GCGUUGUUGG GUAGGGGAGC GUUC UGUAA GCCUGCGAAG GUGUGCUGUG AGGCAUGCUG GAGGUAUCAG AAGUGCGAAU GCUGACAUAA GUAACGAUAA AGCGG GUGA AAAGCCCGCU CGCCGGAAGA CCAAGGGUUC CUGUCCAACG UUAAUCGGGG CAGGGUGAGU CGACCCCUAA GGCGAG GCC GAAAGGCGUA GUCGAUGGGA AACAGGUUAA UAUUCCUGUA CUUGGUGUUA CUGCGAAGGG GGGACGGAGA AGGCUAU GU UGGCCGGGCG ACGGUUGUCC CGGUUUAAGC GUGUAGGCUG GUUUUCCAGG CAAAUCCGGA AAAUCAAGGC UGAGGCGU G AUGACGAGGC ACUACGGUGC UGAAGCAACA AAUGCCCUGC UUCCAGGAAA AGCCUCUAAG CAUCAGGUAA CAUCAAAUC GUACCCCAAA CCGACACAGG UGGUCAGGUA GAGAAUACCA AGGCGCUUGA GAGAACUCGG GUGAAGGAAC UAGGCAAAAU GGUGCCGUA ACUUCGGGAG AAGGCACGCU GAUAUGUAGG UGAGGUCCCU CGCGGAUGGA GCUGAAAUCA GUCGAAGAUA C CAGCUGGC UGCAACUGUU UAUUAAAAAC ACAGCACUGU GCAAACACGA AAGUGGACGU AUACGGUGUG ACGCCUGCCC GG UGCCGGA AGGUUAAUUG AUGGGGUUAG CGCAAGCGAA GCUCUUGAUC GAAGCCCCGG UAAACGGCGG CCGUAACUAU AAC GGUCCU AAGGUAGCGA AAUUCCUUGU CGGGUAAGUU CCGACCUGCA CGAAUGGCGU AAUGAUGGCC AGGCUGUCUC CACC CGAGA CUCAGUGAAA UUGAACUCGC UGUGAAGAUG CAGUGUACCC GCGGCAAGAC GGAAAGACCC CGUGAACCUU UACUA UAGC UUGACACUGA ACAUUGAGCC UUGAUGUGUA GGAUAGGUGG GAGGCUUUGA AGUGUGGACG CCAGUCUGCA UGGAGC CGA CCUUGAAAUA CCACCCUUUA AUGUUUGAUG UUCUAACGUU GACCCGUAAU CCGGGUUGCG GACAGUGUCU GGUGGGU AG UUUGACUGGG GCGGUCUCCU CCUAAAGAGU AACGGAGGAG CACGAAGGUU GGCUAAUCCU GGUCGGACAU CAGGAGGU U AGUGCAAUGG CAUAAGCCAG CUUGACUGCG AGCGUGACGG CGCGAGCAGG UGCGAAAGCA GGUCAUAGUG AUCCGGUGG UUCUGAAUGG AAGGGCCAUC GCUCAACGGA UAAAAGGUAC UCCGGGGAUA ACAGGCUGAU ACCGCCCAAG AGUUCAUAUC GACGGCGGU GUUUGGCACC UCGAUGUCGG CUCAUCACAU CCUGGGGCUG AAGUAGGUCC CAAGGGUAUG GCUGUUCGCC A UUUAAAGU GGUACGCGAG CUGGGUUUAG AACGUCGUGA GACAGUUCGG UCCCUAUCUG CCGUGGGCGC UGGAGAACUG AG GGGGGCU GCUCCUAGUA CGAGAGGACC GGAGUGGACG CAUCACUGGU GUUCGGGUUG UCAUGCCAAU GGCACUGCCC GGU AGCUAA AUGCGGAAGA GAUAAGUGCU GAAAGCAUCU AAGCACGAAA CUUGCCCCGA GAUGAGUUCU CCCUGACCCU UUAA GGGUC CUGAAGGAAC GUUGAAGACG ACGACGUUGA UAGGCCGGGU GUGUAAGCGC AGCGAUGCGU UGAGCUAACC GGUAC UAAU GAACCGUGAG GCUUAACCUU

GENBANK: GENBANK: CP011113.2

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Macromolecule #2: 50S ribosomal protein L22

MacromoleculeName: 50S ribosomal protein L22 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: Can20-12E
Molecular weightTheoretical: 12.253359 KDa
SequenceString:
METIAKHRHA RSSAQKVRLV ADLIRGKKVS QALDILTYTN KKAAVLVKKV LESAIANAEH NDGADIDDLK VTKIFVDEGP SMKRIMPRA KGRADRILKR TSHITVVVSD R

UniProtKB: Large ribosomal subunit protein uL22

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Macromolecule #3: 50S ribosomal protein L24

MacromoleculeName: 50S ribosomal protein L24 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: Can20-12E
Molecular weightTheoretical: 11.078874 KDa
SequenceString:
AAKIRRDDEV IVLTGKDKGK RGKVKNVLSS GKVIVEGINL VKKHQKPVPA LNQPGGIVEK EAAIQVSNVA IFNAATGKAD RVGFRFEDG KKVRFFKSNS ETI

UniProtKB: Large ribosomal subunit protein uL24

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Macromolecule #4: 50S ribosomal protein L29

MacromoleculeName: 50S ribosomal protein L29 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: Can20-12E
Molecular weightTheoretical: 7.286464 KDa
SequenceString:
MKAKELREKS VEELNTELLN LLREQFNLRM QAASGQLQQS HLLKQVRRDV ARVKTLLNEK AGA

UniProtKB: Large ribosomal subunit protein uL29

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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: 7.6
GridModel: Quantifoil / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 82.0 K / Max: 83.0 K
SoftwareName: Leginon
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average exposure time: 10.0 sec. / Average electron dose: 62.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Cs: 2.0 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 31000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 653029
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.62 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 9076
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.3)
Final 3D classificationSoftware: (Name: cryoSPARC (ver. 3.3), RELION (ver. 3.1))
FSC plot (resolution estimation)

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Atomic model buiding 1

SoftwareName: Coot (ver. 0.9.6.2)
RefinementSpace: REAL
Output model

PDB-8c9c:
Cryo-EM captures early ribosome assembly in action

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