EMDB-1738: Pyruvate carboxylase from S. aureus after addition of ...

Entry
Summary
Database / ID	EM DATA BANK (EMDB) / 1738
Title	Pyruvate carboxylase from S. aureus after addition of acetyl-CoA and AMP-PNP
Map	Pyruvate carboxylase from S. aureus after addition of acetyl-CoA and AMP-PNP
Sample	Pyruvate carboxylase from S. aureus after addition of acetyl-Coa and AMP-PNP
Keywords	Pyruvate carboxylase, Biotin-dependent, Acetyl-CoA, Multifunctional, Pyruvate, Oxaloacetate, AMP-PNP, EC 6.4.1.1, Ligase
Authors	Lasso G, Yu LPC, Gil D, Xiang S, Tong L, Valle M
Date	Deposition: 2010-06-01, Header release: 2010-06-11, Map release: 2011-05-19, Last update: 2011-05-19
EMDB Sites	EMDB @PDBe (EU), EMDB @RCSB (USA)
Structure Visualization
Movies	Movie Page #1: Surface view with section colored by density value, Surface level: 3.44, Made by UCSF CHIMERA #2: Surface view colored by cylindrical radius, Surface level: 3.44, Made by UCSF CHIMERA
Supplemental images	EMD1738.jpg
Structure viewers	Yorodumi, Launch PeppeR (About PeppeR), Volume viewer (RCSB, PDBe)
Related Structure Data
Related Entries	EMDB-1736 Cite EMDB-1737 Cite EMDB-1741 Cite EMDB-1742 Cite EMDB-1743 Cite EMDB-1744 Cite Cite: data citing same article
Similar strucutres (beta)	Diagram EMDB-1737 EMDB-1743 EMDB-1744 EMDB-1736 EMDB-1742 EMDB-1741 List of similar structure data about Omokage system

Article
Citation - Primary
Article	Structure, Vol. 18, Issue 10, Page 1300-10, Year 2010
Title	Cryo-EM analysis reveals new insights into the mechanism of action of pyruvate carboxylase.
Authors	Gorka Lasso, Linda P C Yu, David Gil, Song Xiang, Liang Tong, Mikel Valle Structural Biology Unit, Center for Cooperative Research in Biosciences bioGUNE, 48160 Derio, Spain.
Keywords	Bacterial Proteins (chemistry), Binding Sites, Carbon-Nitrogen Ligases (chemistry, 6.3.-), Carboxyl and Carbamoyl Transferases (chemistry, 2.1.3.-), Catalytic Domain, Cryoelectron Microscopy, Crystallography, X-Ray, Models, Molecular, Protein Binding, Protein Folding, Protein Multimerization, Protein Structure, Tertiary, Pyruvate Carboxylase (chemistry, 6.4.1.1), Recombinant Proteins (chemistry), Staphylococcus aureus (enzymology), Substrate Specificity, biotin carboxylase (6.3.4.14)
Links	PII: S0969-2126(10)00297-2, DOI: 10.1016/j.str.2010.07.008, PubMed: 20947019, PMC: PMC2956116

File

Projections & Slices

Size of images:

Images are generated by Spider package.

Density

Data Type

Image stored as Reals

Space Group Number

Map Geometry

Unit Cell

A = 332.76 A , B = 332.76 A , C = 332.76 A ,
alpha = 90 degrees , beta = 90 degrees , gamma = 90 degrees

Pixel Spacing

X = 2.82 A , Y = 2.82 A , Z = 2.82 A

CCP4 map header info

Annotation Details

Pyruvate carboxylase from S. aureus after addition of acetyl-CoA and AMP-PNP

Sample
Name	Pyruvate carboxylase from S. aureus after addition of acetyl-Coa and AMP-PNP
Number of Components	1
Oligomeric State	Homotetramer
Theoretical Mass	0.52 MDa
Component #1: protein - PC
Scientific name	Pyruvate carboxylase
Common Name	PC
Theoretical Mass	0.52 MDa
Oligomeric Details	Homotetramer
Scientific Name of Species	Staphylococcus aureus (NCBI Taxonomy: 1280)
Recombinant expression	Yes
Engineered Source	Vector: pET28a Exp System: Escherichia coli BL21 Star

Experiment
Sample Preparation
Staining	Cryo-EM
Specimen Conc	0.1 mg/ml
Specimen Support Details	Quantifoil R2/2, 100 holey carbon films, cu 200 mesh
Specimen State	particle
Buffer	Details: 20mM Tris-HCl, 2mM NaCl, 2mM DTT, 2mM acetyl-CoA, 2mM AMP-PNP pH: 7.5
Vitrification
Method	Blot for 1.5 seconds
Cryogen Name	ETHANE
Time Resolved State	45 sec
Details	Vitrification instrument: Vitrobot (FEI)
Humidity	100
Instrument	FEI VITROBOT
Temperature	277 Kelvin
Imaging
Microscope	JEOL 2200FS
Electron Gun
Electron Source	FIELD EMISSION GUN
Accelerating Voltage	200 kV
Electron Dose	11 e/A**2
Illumination Mode	FLOOD BEAM
Lens
Magnification	Nominal: 50000 X,
Nominal Cs	2.0 mm
Imaging Mode	BRIGHT FIELD
Defocus	1254 nm - 6349 nm
Energy Filter	Type: Omega , Window:
Specimen Holder
Holder	Single tilt cryoholder ( GATAN LIQUID NITROGEN )
Temperature	99 Kelvin
Camera
Detector	Kodak film SO-163
Image Acquisition
Scanner	ZEISS SCAI
Number of Digital Images	49
Sampling Size	2.82 microns

Processing
Method	single particle reconstruction
3 D reconstruction
Algorithm	Single particle
Euler Angles Details	Spider
Software	Spider
CTF Correction	By defocus groups (Wiener filter)
Resolution By Author	12.3
Resolution Method	FSC at 0.15 cut-off
Single Particle
Number of Projections	15268
Atomic Model Fitting
Model #0
Details	Protocol: Initial rigid body followed by flexible fitting. The initial model was constructed based on 3BG5. Particular domains were modelled by homology modelling or manually fitted into the electron density. The model was finally refined using flex-em
Software	flex-em
Refinement Protocol	flexible
Refinement Space	REAL

Data from EMDB

Header (meta data in XML format)

Map data

Images

FTP directory

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, 26.7 KB

movie #2

	.mp4 (H.264/MPEG-4 AVC format), 3.1 MB .webm (WebM/VP8 format), 4.7 MB Session file for UCSF-Chimera, 26.3 KB