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EMD-5127 – PDB 3IYDThree-dimensional EM structure of
an intact activator-dependent transcription initiation complex
EMD-5127 – PDB 3IYDThree-dimensional EM structure of
an intact activator-dependent transcription initiation complex
Hudson, B.P., Quispe, J., Lara-Gonzalez, S., Berman, H.M., Arnold, E., Ebright, R.H.,
Lawson, C.L. (2009)
Proc. National Acad. Sci. USA 106, 19830-19835
Funding:NIH GM021589
Catherine Lawson, PIHelen Berman, co-PI
Brian BenoffJens BirktoftBrian Hudson **Samuel Lara-GonzalezAndrew NapoliRamya RaoKaitlyn Turo
http://roma.rutgers.edu
Collaborators
RutgersRichard EbrightEddy Arnold
ScrippsJoel QuispeBridget CarragherClint Potter
Acknowledgements Acknowledgements Acknowledgements Acknowledgements
3 components:Activator (50 kDa)
RNA polymerase (300 kDa)Promoter DNA (~100 base pairs)
Total: ~ 500 kDa
Bacterial Transcription ActivationBacterial Transcription Activation
Sample ReconstitutionSample ReconstitutionCAP (-61.5)
+1
CGCAATAAATGTGATCTAGATCACATTTTAGGCAAAAAAGGCTTTACACTTTATGCTTCCGGCTCGT TGTGAGCGGATAACAAGGCGTTATTTACACTAGATCTAGTGTAAAATCCGTTTTTTCCGAAATGTGAAATACGAAGGCCGAGCA ACACTCGCCTATTGTTC
-35 -10 ATAATCGCACCTTA
ATAATCACACCTTA
EM SpecimenEM Specimen
• Carbon sandwich stain technique
Sample
Uranyl Formate Stain
Carbon Film
Carbon Film
Grid
Ohi et al, Biol. Proced. Online (2004), 6, 23-34.
EM ReconstructionEM Reconstruction
• EMAN projection matching
• thermophilic RNAP starting model yielded density for E. coli-specific insertions at expected positions
First refinement cycleFirst refinement cycleprojections
averages
projections
averages
Particles (~17000 total)Particles (~17000 total)
Final refinement cycleFinal refinement cycle
Resolution EstimationResolution Estimation
“Class I” Map + Interpretation“Class I” Map + Interpretation
EMDB IDEMD-5127
PDB ID3iyd
T. thermophilus RNAP holoenzyme from Mukhopadhyay et al., 2008 (PDBid 3DXJ).
CAP + CTD + DNAfrom Benoff et al., 2002 (PDBid 1LB2),
CTD + 70 region 4 + DNA from Lara et al, manuscript in preparation
DNA positions -23 to +20 from Lawson et al., 2004.
E. coli subunit N-terminal domain dimer from Zhang & Darst, 1998 (PDBid 1BDF).
E. coli 70 regions 1.2 to 2 from Malhotra et al., 1996 (PDBid 1SIG).
E. coli ’GNCD from Chlenov et al., 2005 (PDBid 2AUK).
Initial Model FittingInitial Model Fitting
Coordinate Model GenerationCoordinate Model Generation• Rigid body fitting, multiple models (Chimera)
• DNA helical path adjusted around -35 element (3DNA)
• Unified RNAP homology model created (Modeller)
• Domain-wise fitting (Yup.scx, improved overall map-model correlation coefficient from 0.77 to 0.85)
• Geometry regularization (phenix.pdbtools)
Coordinate Model QualityCoordinate Model Quality
Phenix.pdbtools geometry regularization:
• No close contacts• RMSD bonds 0.005 Å• RMSD angles 0.8 degrees• Unrealistic phi-psi angles:
500/4000 residues (12%)
“the value of such a reconstruction is … in having a model for E. coli RNAP (which contains significant differences from the thermophilic enzymes) and in providing a framework for testing positions/interactions involving parts of the complex that have not been available previously. “
Reviewer CommentReviewer Comment
component Avg. map value at fitted atoms
EOTEST mapsShift (Å)
EOTEST mapsRotation(°)
CAP dimer 3.3 1.4 2.9
Tth RNAP 6.0 0.1 0.3
RNAP α2NTD dimer
7.0 0.2 2.8
RNAP σ70 region 1.2-2
5.8 0.9 6.7
RNAP β’ GNCD 3.3 n.a. n.a.
Fitting StatisticsFitting Statistics
Euler Frequency Distribution Euler Frequency Distribution