EMDB-1858: Structure of the ribosome-SecYE complex in the membrane environment

Basic information

• Entry: Database: EMDB / ID: EMD-1858
• Title: Structure of the ribosome-SecYE complex in the membrane environment
• Map data: This map represents an E.coli 70S ribosome carrying an elongation arrested nascent chain of 118 amino acid residues, with the first 102 residues representing the N-terminus of the membrane protein FtsQ with the signal anchor, a tRNA in the P-site and the E.coli SecYEG complex embedded in a E.coli lipid bilayer (Nanodisc).

Sample

• Sample: An active E.coli SecYEG complex embedded in a lipid bilayer (Nanodisc), bound to a translating E.coli ribosome
• Complex: 70S ribosome nascent chain complex
• Ligand: Nd-SecYEG

Function / homology

Function:

cell septum assembly / high-density lipoprotein particle receptor binding / Defective ABCA1 causes TGD / Scavenging by Class B Receptors / HDL clearance / spherical high-density lipoprotein particle / positive regulation of hydrolase activity / regulation of intestinal cholesterol absorption / negative regulation of response to cytokine stimulus / protein oxidation / vitamin transport / phosphatidylcholine-sterol O-acyltransferase activator activity / Chylomicron remodeling / positive regulation of phospholipid efflux / high-density lipoprotein particle binding / cholesterol import / Chylomicron assembly / positive regulation of cholesterol metabolic process / protein transport by the Sec complex / intracellular protein transmembrane transport / negative regulation of heterotypic cell-cell adhesion / high-density lipoprotein particle remodeling / blood vessel endothelial cell migration / ABC transporters in lipid homeostasis / phospholipid efflux / apolipoprotein receptor binding / high-density lipoprotein particle clearance / negative regulation of cell adhesion molecule production / negative regulation of cytokine production involved in immune response / apolipoprotein A-I receptor binding / HDL assembly / peptidyl-methionine modification / negative regulation of very-low-density lipoprotein particle remodeling / cholesterol transfer activity / reverse cholesterol transport / phosphatidylcholine biosynthetic process / high-density lipoprotein particle assembly / very-low-density lipoprotein particle / protein-transporting ATPase activity / lipoprotein biosynthetic process / glucocorticoid metabolic process / positive regulation of CoA-transferase activity / phosphatidylcholine metabolic process / lipid storage / phospholipid homeostasis / high-density lipoprotein particle / triglyceride homeostasis / regulation of Cdc42 protein signal transduction / cholesterol transport / chemorepellent activity / HDL remodeling / FtsZ-dependent cytokinesis / cholesterol efflux / Scavenging by Class A Receptors / endothelial cell proliferation / cholesterol binding / negative regulation of interleukin-1 beta production / positive regulation of Rho protein signal transduction / negative chemotaxis / adrenal gland development / cell division site / stringent response / cholesterol biosynthetic process / mRNA base-pairing translational repressor activity / plasma membrane => GO:0005886 / ornithine decarboxylase inhibitor activity / misfolded RNA binding / transcription antitermination factor activity, RNA binding / Group I intron splicing / positive regulation of cholesterol efflux / RNA folding / endocytic vesicle / protein secretion / protein targeting / transcriptional attenuation / negative regulation of tumor necrosis factor-mediated signaling pathway / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / Scavenging of heme from plasma / Retinoid metabolism and transport / positive regulation of phagocytosis / translational termination / DnaA-L2 complex / positive regulation of substrate adhesion-dependent cell spreading / four-way junction DNA binding / negative regulation of translational initiation / translation repressor activity / endocytic vesicle lumen / positive regulation of stress fiber assembly / negative regulation of DNA-templated DNA replication initiation / cholesterol metabolic process / regulation of mRNA stability / heat shock protein binding / ribosome assembly / mRNA regulatory element binding translation repressor activity / response to reactive oxygen species / assembly of large subunit precursor of preribosome / positive regulation of RNA splicing

Domain/homology:

Cell division protein FtsQ / Cell division protein FtsQ/DivIB, C-terminal / POTRA domain, FtsQ-type / Cell division protein FtsQ/DivIB, C-terminal / POTRA domain, FtsQ-type / SecE subunit of protein translocation complex, bacterial-like / SecE superfamily / Protein translocase subunit SecY / Apolipoprotein A/E / Apolipoprotein A1/A4/E domain / Protein secE/sec61-gamma signature. / POTRA domain / POTRA domain profile. / Protein secY signature 1. / Protein secY signature 2. / SecE/Sec61-gamma subunits of protein translocation complex / Protein translocase complex, SecE/Sec61-gamma subunit / SecY/SEC61-alpha family / SecY domain superfamily / SecY conserved site / SecY / Ribosomal protein L1, bacterial-type / Ribosomal protein L1, conserved site / Ribosomal protein L1 / Ribosomal protein L1 signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L25, short-form / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L1p/L10e family / Ribosomal protein L11, bacterial-type / Ribosomal protein S21 superfamily / Ribosomal protein S21 / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / Ribosomal protein L11, conserved site / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L11 signature. / Ribosomal protein L16 signature 1. / : / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / Ribosomal protein L16, conserved site / Ribosomal protein L16 signature 2. / Ribosomal protein L11, N-terminal / Ribosomal protein L17 signature. / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11, RNA binding domain / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L28/L24 superfamily / Ribosomal protein L36 signature. / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L32p, bacterial type / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L28 / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L5, bacterial-type / Ribosomal protein L6, bacterial-type / Ribosomal protein L18, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein L20 signature. / Ribosomal protein S3, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein S19, bacterial-type / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily

Component:

Protein translocase subunit SecY / Protein translocase subunit SecE / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Apolipoprotein A-I / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein uL1 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS9 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Small ribosomal subunit protein bS21 / Large ribosomal subunit protein bL25 / Protein translocase subunit SecE / Protein translocase subunit SecY / Cell division protein FtsQ

• Biological species: Escherichia coli (E. coli)
• Method: single particle reconstruction / cryo EM / Resolution: 7.1 Å
• Authors: Frauenfeld J / Gumbart J / Sluis EO / Funes S / Gartmann M / Beatrix B / Mielke T / Berninghausen O / Becker T / Schulten K / Beckmann R
• Citation: Journal: Nat Struct Mol Biol / Year: 2011; Title: Cryo-EM structure of the ribosome-SecYE complex in the membrane environment.; Authors: Jens Frauenfeld / James Gumbart / Eli O van der Sluis / Soledad Funes / Marco Gartmann / Birgitta Beatrix / Thorsten Mielke / Otto Berninghausen / Thomas Becker / Klaus Schulten / Roland Beckmann / ; Abstract: The ubiquitous SecY-Sec61 complex translocates nascent secretory proteins across cellular membranes and integrates membrane proteins into lipid bilayers. Several structures of mostly detergent-solubilized Sec complexes have been reported. Here we present a single-particle cryo-EM structure of the SecYEG complex in a membrane environment, bound to a translating ribosome, at subnanometer resolution. Using the SecYEG complex reconstituted in a so-called Nanodisc, we could trace the nascent polypeptide chain from the peptidyltransferase center into the membrane. The reconstruction allowed for the identification of ribosome-lipid interactions. The rRNA helix 59 (H59) directly contacts the lipid surface and appears to modulate the membrane in immediate vicinity to the proposed lateral gate of the protein-conducting channel (PCC). On the basis of our map and molecular dynamics simulations, we present a model of a signal anchor-gated PCC in the membrane.

History

Deposition	Jan 17, 2011	-
Header (metadata) release	Apr 21, 2011	-
Map release	Apr 21, 2011	-
Update	Dec 11, 2013	-
Current status	Dec 11, 2013	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_1858.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: This map represents an E.coli 70S ribosome carrying an elongation arrested nascent chain of 118 amino acid residues, with the first 102 residues representing the N-terminus of the membrane protein FtsQ with the signal anchor, a tRNA in the P-site and the E.coli SecYEG complex embedded in a E.coli lipid bilayer (Nanodisc).
• Voxel size: X=Y=Z: 1.2375 Å

Density

Contour Level	By AUTHOR: 0.5 / Movie #1: 0.7
Minimum - Maximum	-3.53351 - 6.82334
Average (Standard dev.)	0.0507365 (±0.456442)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order Y X Z Origin -160 -160 -159 Dimensions 320 320 320 Spacing 320 320 320
Cell	A=B=C: 396 Å α=β=γ: 90 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.2375	1.2375	1.2375
M x/y/z	320	320	320
origin x/y/z	0.000	0.000	0.000
length x/y/z	396.000	396.000	396.000
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-160	-160	-159
NX/NY/NZ	320	320	320
MAP C/R/S	2	1	3
start NC/NR/NS	-160	-160	-159
NC/NR/NS	320	320	320
D min/max/mean	-3.534	6.823	0.051

Supplemental data

Sample components

Entire : An active E.coli SecYEG complex embedded in a lipid bilayer (Nano...

• Entire: Name: An active E.coli SecYEG complex embedded in a lipid bilayer (Nanodisc), bound to a translating E.coli ribosome

Components

• Sample: An active E.coli SecYEG complex embedded in a lipid bilayer (Nanodisc), bound to a translating E.coli ribosome
• Complex: 70S ribosome nascent chain complex
• Ligand: Nd-SecYEG

Supramolecule #1000: An active E.coli SecYEG complex embedded in a lipid bilayer (Nano...

• Supramolecule: Name: An active E.coli SecYEG complex embedded in a lipid bilayer (Nanodisc), bound to a translating E.coli ribosome; type: sample / ID: 1000 ; Details: The heterotrimeric SecYEG complex was embedded in a lipid bilayer (nascent HDL, Nanodisc); Oligomeric state: 70S ribosome bound to one copy of the E.coli SecYEG complex in a lipid bilayer; Number unique components: 2
• Molecular weight: Theoretical: 2.7 MDa

Supramolecule #1: 70S ribosome nascent chain complex

• Supramolecule: Name: 70S ribosome nascent chain complex / type: complex / ID: 1 / Name.synonym: 70S RNC / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
• Source (natural): Organism: Escherichia coli (E. coli)
• Molecular weight: Theoretical: 2.7 MDa

Macromolecule #1: Nd-SecYEG

• Macromolecule: Name: Nd-SecYEG / type: ligand / ID: 1 / Name.synonym: SecYEG embedded in a Nanodisc / Number of copies: 1 / Oligomeric state: Heterotrimeric / Recombinant expression: Yes
• Source (natural): Organism: Escherichia coli (E. coli) / Location in cell: Plasma membrane
• Recombinant expression: Organism: Escherichia coli (E. coli)

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Buffer: pH: 7.2 ; Details: 20 mM Hepes (pH 7.2), 100 mM KOAc, 10 mM Mg(OAc)2, 1 mM DTT, 250 microg/ml chloramphenicol
• Vitrification: Cryogen name: ETHANE / Instrument: OTHER

Electron microscopy

• Microscope: FEI POLARA 300
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Calibrated magnification: 38000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 39000
• Sample stage: Specimen holder: FEI Polara cartridge system / Specimen holder model: OTHER
• Image recording: Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 4.76 µm / Average electron dose: 22 e/Ų / Details: Scanned at 5334 dpi
• Experimental equipment: Model: Tecnai Polara / Image courtesy: FEI Company

Image processing

• CTF correction: Details: Defocus group volumes
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.1 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 85664