EMDB-1667: Cryo-EM structure of the active yeast Ssh1 complex bou...

Entry
Summary
Database / ID	EM DATA BANK (EMDB) / 1667
Title	Cryo-EM structure of the active yeast Ssh1 complex bound to the programmed yeast 80S ribosome bearing a P-site tRNA
Map	This map represents a yeast 80S ribosome bearing a nascent chain of the first 120 amino acid of the type I signal anchor membrane protein DPAP-B, a tRNA in the P-site and the yeast Ssh complex bound at the exit tunnel.
Sample	An active yeast Ssh1 complex bound to a translating yeast ribosome
Keywords	Ribosome, protein exit tunnel, cotranslational protein translocation, protein conducting channel, signal sequence
Authors	Becker T, Mandon E, Bhushan S, Jarasch A, Armache JP, Funes S, Jossinet F, Gumbart J, Mielke T, Berninghausen O, Schulten K, Westhof E, Gilmore R, Beckmann R
Date	Deposition: 2009-12-03, Header release: 2009-12-16, Map release: 2010-12-22, Last update: 2011-07-15
EMDB Sites	EMDB @PDBe (EU), EMDB @RCSB (USA)
Structure Visualization
Movies	Movie Page #1: Surface view with section colored by density value, Surface level: 1.5, Made by UCSF CHIMERA #2: Surface view colored by height, Surface level: 1.5, Made by UCSF CHIMERA #3: Surface view with fitted model, atomic models: PDB-2ww9, Surface level: 1.5, Made by UCSF CHIMERA #4: Simplified surface model with fitted atomic model: PDB-2ww9, Made by Jmol
Supplemental images	1667.gif 1667_EMD_166...
Structure viewers	Yorodumi, Launch PeppeR (About PeppeR), Volume viewer (RCSB, PDBe)
Related Structure Data
Related Entries	PDB-2ww9 CiteFit EMDB-1651 Cite EMDB-1652 Cite EMDB-1668 Cite EMDB-1669 Cite PDB-2wwa Cite PDB-2wwb Cite PDB-3izb PDB-3izd PDB-3ize PDB-3izf PDB-3izs Cite: data citing same article Fit: output model of fitting
Similar strucutres (beta)	Diagram EMDB-1668 EMDB-1669 EMDB-1666 EMDB-1076 EMDB-1125 EMDB-1217 List of similar structure data about Omokage system

Article
Citation - Primary
Article	Science, Vol. 326, Issue 5958, Page 1369-73, Year 2009
Title	Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome.
Authors	Thomas Becker, Shashi Bhushan, Alexander Jarasch, Jean-Paul Armache, Soledad Funes, Fabrice Jossinet, James Gumbart, Thorsten Mielke, Otto Berninghausen, Klaus Schulten, Eric Westhof, Reid Gilmore, Elisabet C Mandon, Roland Beckmann Gene Center Munich and Center for Integrated Protein Science, Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
Keywords	Animals, Binding Sites, Cryoelectron Microscopy, Dogs, Image Processing, Computer-Assisted, Membrane Proteins (chemistry), Models, Molecular, Protein Biosynthesis, Protein Conformation, Protein Multimerization, Protein Structure, Secondary, Protein Transport, Proteins (chemistry), Ribosomes (metabolism), SSH1 protein, S cerevisiae, Saccharomyces cerevisiae Proteins (chemistry)
Links	DOI: 10.1126/science.1178535, PubMed: 19933108, PMC: PMC2920595

Map

File

EMD-1667.map ( map file in CCP4 format, 199346 KB )

Projections & Slices

Size of images:

Axes	Z (Sec.)	X (Row.)	Y (Col.)
Surface
Projections
Slices (1/3)
Slices (1/2)
Slices (2/3)

Images are generated by Spider package.

Density

Histogram

Histogram (log scale)

Contour Level:	1.5 (by author), 1.5 (movie #1):
Minimum - Maximum:	-4.08212 - 9.04266
Average (Standard dev.):	0.0505784 (0.670568)

Data Type

Image stored as Reals

Space Group Number

Map Geometry

Axis order	Y	X	Z
Dimensions	368	368	368
Origin	-184	-184	-183
Limit	183	183	184
Spacing	368	368	368

Unit Cell

A= B= C: 455.4 A
Alpha=beta=gamma: 90 degrees

Pixel Spacing

X= Y= Z: 1.2375 A

CCP4 map header info

mode	Image stored as Reals
A/pix X/Y/Z	1.2375	1.2375	1.2375
M x/y/z	368	368	368
origin x/y/z	0.000	0.000	0.000
length x/y/z	455.400	455.400	455.400
alpha/beta/gamma	90.000	90.000	90.000
start NX/NY/NZ	-184	-184	-183
NX/NY/NZ	368	368	368
MAP C/R/S	2	1	3
start NC/NR/NS	-184	-184	-183
NC/NR/NS	368	368	368
start NC,NX/NR,NY/NS,NZ
NC,NX/NR,NY/NS,NZ
D min/max/mean	-4.082	9.043	0.051

Annotation Details

This map represents a yeast 80S ribosome bearing a nascent chain of the first 120 amino acid of the type I signal anchor membrane protein DPAP-B, a tRNA in the P-site and the yeast Ssh complex bound at the exit tunnel.

Supplement
Images
Images	1667.gif 1667_EMD_166...

Sample
Name	An active yeast Ssh1 complex bound to a translating yeast ribosome
Number of Components	2
Oligomeric State	80S Ribosome bound to one copy of the heterotrimeric Ssh1 complex
Theoretical Mass	4.2MDa
Details	80S ribosomes and the detergent solubilized Ssh1 complex were reconstituted in vitro by adding 1 pmol of ribosome and Ssh1 complex in 5 fold molar excess
Mass-estimation Method	Known for 80S ribosomes
Experimental Mass	4.2MDa
Component #1: ribosome-eukaryote - Yeast 80S ribosome bound to the yeast Ssh1 complex
Scientific name	Yeast 80S ribosome bound to the yeast Ssh1 complex
Theoretical Mass	4.2 MDa
Experimental Mass	4.2 MDa
Eukaryote	ALL
Component #2: ligand - Ssh1 complex
Scientific name	Ssh1 complex
Theoretical Mass	0.0715 MDa
Details	His FLAG-tagged
Oligomeric Details	Heterotrimer
Number of Copies	1
Scientific Name of Species	Saccharomyces cerevisiae
Common Name of Species	Baker's yeast
NCBI taxonomy	4932
Strain	RGY1592
Recombinant expression	Yes
Natural Source	Cell: Rough microsomes Cell Location: Endoplasmic reticulum Organelle: Endoplasmic reticulum membrane
Engineered Source	NCBI taxonomy: 4932 Expression system: Saccharomyces cerevisiae

Experiment
Sample Preparation
Staining	cryo-EM
Specimen Support Details	Quantifoil grids (3/3) with 2 nm carbon on top
Specimen State	particle
Buffer	Details: 20 mM HEPES/KOH, pH 7.5 100 mM KOAc, 10 mM Mg(OAc)2, 1.5 mM DTT, 0.1 % (w/v) digitonin pH: 7.5
Vitrification
Method	Blot for 10 seconds before plunging, use 2 layer of filter paper
Cryogen Name	ETHANE
Details	Vitrification instrument: Vitrobot
Humidity	95
Instrument	FEI VITROBOT
Imaging
Microscope	FEI POLARA 300
Electron Gun
Electron Source	FIELD EMISSION GUN
Accelerating Voltage	300 kV
Electron Dose	25 e/A**2
Illumination Mode	FLOOD BEAM
Lens
Magnification	Nominal: 39000, Calibrated: 38000
Astigmatism	Objective lens astigmatism was corrected at 100000 times magnification
Nominal Cs	2.26 mm
Imaging Mode	BRIGHT FIELD
Defocus	1200 nm - 4500 nm
Specimen Holder
Holder	FEI Polara cartridge system
Model	OTHER
Tilt Angle	0 degrees - 0 degrees
Temperature	84 K
Camera
Detector	Kodak SO163
Image Acquisition
Od Range	1.2
Scanner	PRIMESCAN
Number of Digital Images	185
Sampling Size	4.76
Quant Bit Number	16
Details	Scanned at 5334 dpi

Processing
Method	single particle reconstruction
3D reconstruction
Algorithm	Projection matching
Euler Angles Details	SPIDER
Software	SPIDER
CTF Correction	defocus group volumes
Details	Map was filtered between 8.3 and 10.3 Angstrom to better visualize the Ssh1 complex
Resolution By Author	8.6 A
Resolution Method	FSC at 0.5 cut-off
Single Particle
Number of Projections	35800
Details	Particles were selected using the program SIGNATURE and visually inspected. This map resulted from sorting against the ES27 exit position and subsequent sorting for tRNA and the Ssh1 complex.
Atomic Model Fitting
Fitted Coordinate
PDB entry ID	PDB-2ww9

Download

Data from EMDB

Header (meta data in XML format)

emd-1667.xml (9.7 KB)

Map data

emd_1667.map.gz (18.9 MB)

Images

1667.gif (97.1 KB)

1667_EMD_1667_active_yeast.jpg (232.8 KB)

FTP directory

ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1667

Movie files

movie #1

	.mp4 (H.264/MPEG-4 AVC format), 3.5 MB .webm (WebM/VP8 format), 5.4 MB Session file for UCSF-Chimera, 26.4 KB

movie #2

	.mp4 (H.264/MPEG-4 AVC format), 3.2 MB .webm (WebM/VP8 format), 4.9 MB Session file for UCSF-Chimera, 26.5 KB

movie #3

	.mp4 (H.264/MPEG-4 AVC format), 3.7 MB .webm (WebM/VP8 format), 5.3 MB Session file for UCSF-Chimera, 2.2 MB

movie #4

	.mp4 (H.264/MPEG-4 AVC format), 3.6 MB .webm (WebM/VP8 format), 5.1 MB