2016-07-13 REL 2016-02-18 PDBe PDBe 2016-03-09 2016-06-29 Atomic cryoEM structure of Hsp90/Cdc37/Cdk4 complex Verba KA, Wang RYR, Arakawa A, Liu Y, Shirouzu M, Yokoyama S, Agard DA Hsp90, Cdc37, Cdk4, chaperone, kinase, unfolding Verba KA, Wang RYR, Arakawa A, Liu Y, Shirouzu M, Yokoyama S, Agard DA Atomic structure of Hsp90-Cdc37-Cdk4 reveals that Hsp90 traps and stabilizes an unfolded kinase SCIENCE 352 1542 1547 2016 27339980 doi:10.1126/science.aaf5023 emd_3344.map.gz Image stored as Reals 128 128 128 0 0 0 127 127 127 128 128 128 312.32 312.32 312.32 90.0 90.0 90.0 X Y Z -0.01775946 0.11031735 0.00048062 0.00577961 1
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2.44 2.44 2.44 0.013 Reconstruction of Hsp90:Cdc37:Cdk4 complex. Cdk4 is tagged at the C terminus with T4 lysozyme. Part of series of maps, the highest resolution map being EMD-3337, others being EMD-3338, EMD-3339, EMD-3340, EMD-3341, EMD-3342, EMD-3343.
emd_3344.tif
emd_3344_fsc.xml 3 Complex of Human Hsp90 beta, human Cdc37 and human Cdk4-T4 lysozyme One Hsp90 homodimer binds to one Cdc37 and one Cdk4-T4 Lysozyme 0.26
All three proteins were co-expressed in Saccharomyces cerevisiae cells.
As cloned, verified by SDS-PAGE 0.26 protein Heat Shock Protein HSP 90 beta Hsp90 0.083 Homo sapiens Human 2 true Dimer P08238 GO:0000052 IPR001404 cytoplasm 83nu Saccharomyces cerevisiae protein Hsp90 co-chaperone Cdc37 Cdc37 0.0445 true Homo sapiens 1 Human GO:0000002 Q16543 throughout Saccharomyces cerevisiae 83nu protein Cyclin-dependent kinase 4 Cdk4 0.0337
T4 lysozyme was fused recombinantly at the C terminus of Cdk4, with a GS linker in between. This was done to verify placement of proteins in the complex. Therefore, the total mass of Cdk4-T4Lys is 51kDa rather that 33.7kDa for pure Cdk4.
Homo sapiens 1 Human true P11802 IPR000719 GO:0000038 throughout 83nu Saccharomyces cerevisiae Single blot from 4 to 6 seconds, at 20C ETHANE 90 FEI VITROBOT MARK III 95 At high mag via FT. FIELD EMISSION GUN 40 BRIGHT FIELD 1000 5000 FLOOD BEAM Polara 6 cartridge loader GATAN K2 SUMMIT (4k x 4k) 2.0 FEI TECNAI F30 25-SEP-2015 OTHER 300 31000 1082 8
30 frames, 6 seconds total exposure
 rigid body REAL Chimera 5fwk A B E K REAL rigid body Chimera 2LZM A particle 0.27 Glow discharged for 30 sec, C-flat 400 mesh 1.2/1.3 thick carbon grids (Protochips)
20mM Tris pH 7.5, 150mM NaCl, 10mM KCl, 20mM NaMoO4, 1mM DTT, 0.085mM DDM
7.5 singleParticle Relion 10 FSC 0.143, gold-standard 1 C1 29146
Image stacks were dose weighted, drift corrected, binned to 1.22A/pix and summed using new UCSF DriftCorr program. The particles were picked and the CTF was estimated the same way as other maps in the series. Rounds of 2D classification (300 classes, 50 iterations) followed by 3D classification (2 classes, 50 iterations) in Relion 1.4 were used to eliminate low quality particles. Using the final set of particles, 3D Auto-refine feature in Relion 1.4 was used to generate the final maps.