EMDB-3245: Density map of the Sec61 channel opened by a signal sequence

Basic information

• Entry: Database: EMDB / ID: EMD-3245
• Title: Density map of the Sec61 channel opened by a signal sequence
• Map data: This is the final postprocessed, sharpened, and masked map.

Sample

• Sample: The stalled mammalian Sec61 complex opened by the pre-prolactin signal sequence.
• Complex: mammalian ribosome
• Protein or peptide: Sec61 channel

• Keywords: ribosome / Sec61 / translocation / signal sequence

Function / homology

Function:

: / Growth hormone receptor signaling / long-day photoperiodism / response to external biotic stimulus / negative regulation of nitric oxide mediated signal transduction / peptide hormone secretion / prolactin receptor binding / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / membrane docking / positive regulation of lactation / regulation of meiotic cell cycle process involved in oocyte maturation / pronephric nephron development / response to L-arginine / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein insertion into ER membrane / positive regulation of fatty acid biosynthetic process / signal transduction involved in regulation of gene expression / SRP-dependent cotranslational protein targeting to membrane, translocation / mammary gland development / blastocyst formation / signal sequence binding / post-translational protein targeting to membrane, translocation / response to food / positive regulation of endocytosis / biosynthetic process / protein transmembrane transporter activity / response to mechanical stimulus / lactation / response to nutrient levels / female pregnancy / phospholipid binding / positive regulation of receptor signaling pathway via JAK-STAT / hormone activity / positive regulation of non-canonical NF-kappaB signal transduction / positive regulation of nitric oxide biosynthetic process / ribosome binding / positive regulation of NF-kappaB transcription factor activity / endoplasmic reticulum lumen / negative regulation of gene expression / positive regulation of cell population proliferation / endoplasmic reticulum membrane / positive regulation of gene expression / negative regulation of apoptotic process / extracellular space / extracellular region / cytosol

Domain/homology:

Somatotropin/prolactin / Somatotropin hormone, conserved site / Somatotropin hormone family / Somatotropin, prolactin and related hormones signature 1. / Somatotropin, prolactin and related hormones signature 2. / Protein translocase SEC61 complex, gamma subunit / Protein translocase SecE domain superfamily / Translocon Sec61/SecY, plug domain / Plug domain of Sec61p / Protein secE/sec61-gamma signature. / 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 / Four-helical cytokine-like, core

Component:

Prolactin / Protein transport protein Sec61 subunit alpha isoform 1 / Protein transport protein Sec61 subunit gamma / Prolactin

• Biological species: Canis lupus (gray wolf)
• Method: single particle reconstruction / cryo EM / Resolution: 3.6 Å
• Authors: Voorhees RM / Hegde RS
• Citation: Journal: Science / Year: 2016; Title: Structure of the Sec61 channel opened by a signal sequence.; Authors: Rebecca M Voorhees / Ramanujan S Hegde / ; Abstract: Secreted and integral membrane proteins compose up to one-third of the biological proteome. These proteins contain hydrophobic signals that direct their translocation across or insertion into the lipid bilayer by the Sec61 protein-conducting channel. The molecular basis of how hydrophobic signals within a nascent polypeptide trigger channel opening is not understood. Here, we used cryo-electron microscopy to determine the structure of an active Sec61 channel that has been opened by a signal sequence. The signal supplants helix 2 of Sec61α, which triggers a rotation that opens the central pore both axially across the membrane and laterally toward the lipid bilayer. Comparisons with structures of Sec61 in other states suggest a pathway for how hydrophobic signals engage the channel to gain access to the lipid bilayer.

History

Deposition	Nov 14, 2015	-
Header (metadata) release	Nov 25, 2015	-
Map release	Jan 13, 2016	-
Update	Jan 13, 2016	-
Current status	Jan 13, 2016	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_3245.map.gz / Format: CCP4 / Size: 276 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: This is the final postprocessed, sharpened, and masked map.
• Voxel size: X=Y=Z: 1.34 Å

Density

Contour Level	By AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum	-0.40719661 - 0.66735524
Average (Standard dev.)	0.00116352 (±0.01930853)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 420 420 420 Spacing 420 420 420
Cell	A=B=C: 562.8 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.34	1.34	1.34
M x/y/z	420	420	420
origin x/y/z	0.000	0.000	0.000
length x/y/z	562.800	562.800	562.800
α/β/γ	90.000	90.000	90.000
start NX/NY/NZ	-190	-190	-189
NX/NY/NZ	380	380	380
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	420	420	420
D min/max/mean	-0.407	0.667	0.001

Supplemental data

Supplemental map: run1 half1 class001 unfil 1.map

• File: run1_half1_class001_unfil_1.map

Supplemental map: run1 half2 class001 unfil.map

• File: run1_half2_class001_unfil.map

Sample components

Entire : The stalled mammalian Sec61 complex opened by the pre-prolactin s...

• Entire: Name: The stalled mammalian Sec61 complex opened by the pre-prolactin signal sequence.

Components

• Sample: The stalled mammalian Sec61 complex opened by the pre-prolactin signal sequence.
• Complex: mammalian ribosome
• Protein or peptide: Sec61 channel

Supramolecule #1000: The stalled mammalian Sec61 complex opened by the pre-prolactin s...

• Supramolecule: Name: The stalled mammalian Sec61 complex opened by the pre-prolactin signal sequence.; type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: monomer / Number unique components: 2

Supramolecule #1: mammalian ribosome

• Supramolecule: Name: mammalian ribosome / type: complex / ID: 1 / Name.synonym: 80S ribosome / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
• Source (natural): Organism: Canis lupus (gray wolf) / synonym: Dog / Tissue: pancreas / Organelle: Endoplasmic Reticulum / Location in cell: Endoplasmic Reticulum

Macromolecule #1: Sec61 channel

• Macromolecule: Name: Sec61 channel / type: protein_or_peptide / ID: 1 / Name.synonym: Sec61 translocon / Details: The heterotrimeric mammalian Sec61 channel / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
• Source (natural): Organism: Canis lupus (gray wolf) / synonym: Dog / Tissue: pancreas / Organelle: Endoplasmic reticulum / Location in cell: Endoplasmic reticulum

Experimental details

Structure determination

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

Sample preparation

• Buffer: pH: 7.5 ; Details: 50 mM HEPES pH 7.5, 200 mM KOAc, 15 mM MgOAc2, 1 mM DTT, 0.25% digitonin
• Grid: Details: 400 mesh holey carbon grid coated with a continuous layer of amorphous carbon
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK II; Method: Incubate for 30 seconds at 4 C and blot for 9 seconds before plunging

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 59000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
• Alignment procedure: Legacy - Astigmatism: Objective astigmatism was corrected at 60,000 times magnification
• Date: Mar 9, 2015
• Image recording: Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1489 / Average electron dose: 27 e/Ų; Details: Every image is the average of 17 frames recorded by the direct electron detector.
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• CTF correction: Details: Each particle
• Final two d classification: Number classes: 5
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 101339
• Details: The particles were selected using the automated particle selection in Relion.

Atomic model buiding 1

Initial model

PDB ID:

3jaj

• Software: Name: Chimera and Coot
• Refinement: Space: REAL / Protocol: RIGID BODY FIT

Output model

PDB-3jc2:
The structure of the mammalian Sec61 channel opened by a signal sequence

Atomic model buiding 2

Initial model

PDB ID:

1rh5

• Software: Name: Chimera, coot, and Refmac
• Refinement: Space: RECIPROCAL / Protocol: FLEXIBLE FIT

Output model

PDB-3jc2:
The structure of the mammalian Sec61 channel opened by a signal sequence

Atomic model buiding 3

Initial model

PDB ID:

3j7q

• Software: Name: Chimera, coot, and Refmac
• Refinement: Space: RECIPROCAL / Protocol: FLEXIBLE FIT

Output model

PDB-3jc2:
The structure of the mammalian Sec61 channel opened by a signal sequence