EMD-3541 Experiments and Validation

Structure of a spliceosome remodeled for exon ligation

Single particle reconstruction
Overview of EMD-3541
Sample name: Saccharomyces cerevisiae spliceosome. Complex C just after Prp16-mediated remodeling
Organisms: Saccharomyces cerevisiae, Baker's yeast
Fitted atomic model: 5mq0
Related EM entry by publication: EMD-3539, EMD-3542

Map parameters

Recommended contour level: 0.03 (author)
Number of grid points: 412 × 412 × 412
Voxel size: 1.43 × 1.43 × 1.43 Å
Minimum density: -0.033
Maximum density: 0.094
Average density: 0.00
Standard deviation: 0.003

Sample information

Sample name: Saccharomyces cerevisiae spliceosome. Complex C just after Prp16-mediated remodeling
Ligands: MAGNESIUM ION, POTASSIUM ION, INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE, GUANOSINE-5'-TRIPHOSPHATE, ZINC ION
Nucleic acids: Yeast UBC4 gene for ubiquitin-conjugating enzyme, 5'-EXON OF UBC4 PRE-MRNA, S.cerevisiae chromosome II reading frame ORF YBR230c, Saccharomyces cerevisiae strain T.52_2H chromosome XII sequence, Saccharomyces cerevisiae strain WI_C_MBSP_4 chromosome VII sequence, 3'-EXON OF UBC4 PRE-MRNA, BOUND BY PRP22 HELICASE
Proteins: Saccharomyces cerevisiae spliceosome. Complex C just after Prp16-mediated remodeling, Pre-mRNA-splicing factor 8, Pre-mRNA-splicing factor SNU114, Pre-mRNA-splicing factor CWC22, Pre-mRNA-splicing factor PRP46, Pre-mRNA-processing protein 45, Pre-mRNA-splicing factor BUD31, Pre-mRNA-splicing factor CWC2, Pre-mRNA-splicing factor SLT11, Pre-mRNA-splicing factor CEF1, Pre-mRNA-splicing factor CWC15, Pre-mRNA-splicing factor CWC21, Pre-mRNA-splicing factor CLF1, Pre-mRNA-splicing factor SYF1,PRE-MRNA-SPLICING FACTOR SYF1, Pre-mRNA-splicing factor 18, Pre-mRNA-splicing factor SLU7, Pre-mRNA-processing factor 17, UNKNOWN PROTEIN, Pre-mRNA-splicing factor SYF2, Small nuclear ribonucleoprotein-associated protein B, Small nuclear ribonucleoprotein Sm D3, Small nuclear ribonucleoprotein E, Small nuclear ribonucleoprotein F, Small nuclear ribonucleoprotein G, Small nuclear ribonucleoprotein Sm D1, Small nuclear ribonucleoprotein Sm D2, Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP22, U2 small nuclear ribonucleoprotein A', U2 small nuclear ribonucleoprotein B'', Pre-mRNA-splicing factor SNT309, Pre-mRNA-processing factor 19

Validation information

Experimental information

 

Image processing

Applied symmetry: C1
Software: RELION
Number of particles: 29527
Details: A 3D reconstruction was obtained after refinement of a subset of particles obtained by classification with a mask around Prp22.

Fitting

Fitting protocol: flexible
Target criteria: Fourier Shell Correlation
Overall B value: 330.0
Refinement space: RECIPROCAL
Details: Used secondary structure restraints generated in ProSMART and LibG.

Imaging

Session
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 2.0
Energy filter: GIF Quantum
Nominal magnification: 81000.0
Defocus max (nm): 4,500.00
Defocus min (nm): 500.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Details: GIF Quantum energy filter, 20 eV slit width
Acquisition
Number of images: 3596
Details: Total dose: 40 electrons/Angstrom^2 over 16 seconds. 20 movie frames collected at 1.25 frames per second.

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.30
Buffer: NP-40 is also called IGEPAL CA-630
Vitrification
Apparatus: FEI VITROBOT MARK III
Cryogen: ETHANE
Humidity: 100
Details: 3.5 microlitres sample were applied to the grid, left for 25 seconds and then blotted for 3.0-3.5 seconds before plunging..