Journal: Nat Commun / Year: 2020 Title: MetAP-like Ebp1 occupies the human ribosomal tunnel exit and recruits flexible rRNA expansion segments. Authors: Klemens Wild / Milan Aleksić / Karine Lapouge / Keven D Juaire / Dirk Flemming / Stefan Pfeffer / Irmgard Sinning / Abstract: Human Ebp1 is a member of the proliferation-associated 2G4 (PA2G4) family and plays an important role in cancer regulation. Ebp1 shares the methionine aminopeptidase (MetAP) fold and binds to mature ...Human Ebp1 is a member of the proliferation-associated 2G4 (PA2G4) family and plays an important role in cancer regulation. Ebp1 shares the methionine aminopeptidase (MetAP) fold and binds to mature 80S ribosomes for translational control. Here, we present a cryo-EM single particle analysis reconstruction of Ebp1 bound to non-translating human 80S ribosomes at a resolution range from 3.3 to ~8 Å. Ebp1 blocks the tunnel exit with major interactions to the general uL23/uL29 docking site for nascent chain-associated factors complemented by eukaryote-specific eL19 and rRNA helix H59. H59 is defined as dynamic adaptor undergoing significant remodeling upon Ebp1 binding. Ebp1 recruits rRNA expansion segment ES27L to the tunnel exit via specific interactions with rRNA consensus sequences. The Ebp1-ribosome complex serves as a template for MetAP binding and provides insights into the structural principles for spatial coordination of co-translational events and molecular triage at the ribosomal tunnel exit.
History
Deposition
Jan 14, 2020
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Header (metadata) release
Feb 19, 2020
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Map release
Feb 19, 2020
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Update
Feb 19, 2020
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Current status
Feb 19, 2020
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Download / File: emd_10608.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel size
X=Y=Z: 1.5218 Å
Density
Contour Level
By AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum
-0.395412 - 0.52741325
Average (Standard dev.)
0.001479043 (±0.008448959)
Symmetry
Space group: 1
Details
EMDB XML:
Map geometry
Axis order
X
Y
Z
Origin
0
0
0
Dimensions
360
360
360
Spacing
360
360
360
Cell
A=B=C: 547.848 Å α=β=γ: 90.0 °
CCP4 map header:
mode
Image stored as Reals
Å/pix. X/Y/Z
1.5218
1.5218
1.5218
M x/y/z
360
360
360
origin x/y/z
0.000
0.000
0.000
length x/y/z
547.848
547.848
547.848
α/β/γ
90.000
90.000
90.000
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
360
360
360
D min/max/mean
-0.395
0.527
0.001
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Supplemental data
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Sample components
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Entire : Human Ebp1-ribosome complex
Entire
Name: Human Ebp1-ribosome complex
Components
Complex: Human Ebp1-ribosome complex
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Supramolecule #1: Human Ebp1-ribosome complex
Supramolecule
Name: Human Ebp1-ribosome complex / type: complex / ID: 1 / Parent: 0 Details: Puromycin-treated ribosomes from HeLa cells in complex with recombinantly expressed Ebp1
Source (natural)
Organism: Homo sapiens (human)
Recombinant expression
Organism: Escherichia coli (E. coli)
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Buffer
pH: 7.5 Details: 20 mM HEPES KOH pH 7.5 5 mM Mg(OAc)2 175 mM KOAc 1 mM Tris(2-carboxyethyl)phosphin
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
Details
100 nM ribosome; 800 nM Ebp1
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 3370 / Average electron dose: 38.0 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) Details: Ebp1-60S density segment after 3D classification focused on ES27L and 2-body multibody refinement; filtered to local resolution Number images used: 4864
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classification
Software - Name: RELION (ver. 3.0)
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