2019-03-20 REL RCSB RCSB RCSB 2018-12-06 2019-02-13 2019-02-13 2019-03-20 Single-particle cryo-EM reconstruction of the catalytic subunit of protein kinase A bound to ATP and IP20 using 200 keV Herzik Jr MA Wu M Lander GC Herzik Jr MA Wu M Lander GC High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM. Nat Commun UK 10 1032 1032 2019 30833564 doi:10.1038/s41467-019-08991-8 2041-1723 EMD-0409 associated EM volume catalytic subunit of mouse protein kinase A bound to ATP and IP20 catalytic subunit of mouse protein kinase A bound to ATP and IP20 0 1 Mus musculus Escherichia coli 0.043 catalytic subunit of protein kinase A Mus musculus Escherichia coli LEVO GNAAAAKKGSEQESVKEFLAKAKEDFLKKWETPSQNTAQLDQFDRIKTLGTGSFGRVMLVKHKESGNHYAMKILDKQKVV KLKQIEHTLNEKRILQAVNFPFLVKLEFSFKDNSNLYMVMEYVAGGEMFSHLRRIGRFSEPHARFYAAQIVLTFEYLHSL DLIYRDLKPENLLIDQQGYIQVTDFGFAKRVKGRTWTLCGTPEYLAPEIILSKGYNKAVDWWALGVLIYEMAAGYPPFFA DQPIQIYEKIVSGKVRFPSHFSSDLKDLLRNLLQVDLTKRFGNLKNGVNDIKNHKWFATTDWIAIYQRKVEAPFIPKFKG PGDTSNFDDYEEEEIRVSINEKCGKEFTEF 2.7.11.1 singleParticle particle 5 7.0 10.0 MOPS 50.0 NaCl Sodium chloride 3.0 MnCl2 Manganese chloride 0.2 ATP 2.0 DTT 10.0 Inhibitor peptide IP20 Quantifoil, UltrAuFoil, R1.2/1.3 GOLD 300 PLASMA CLEANING OTHER
Grids were plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.)
 ETHANE 90 277.15 HOMEMADE PLUNGER
Sample was manually blotted for 4-5 seconds using Whatman No. 1 filter paper immediately prior to plunge-freezing..
FEI TALOS ARCTICA FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 200 70.0 2.7 5.0 16.0 73000. FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN GATAN K2 SUMMIT (4k x 4k) COUNTING 2 3176 11.0 69.0 GATAN K2 SUMMIT (4k x 4k) COUNTING 3710 3838 5.0 1-44 2 2402 11.0 69.0
Specimen was tilted at 30 degrees
 1
Counting mode, 250 ms frames, exposure rate of ~1.95 e- pixel-1 s-1, total exposure of ~69 e- angstrom-2 (1.57 e- angstrom-2 frame-1).
788060
Template-picked
 CTFFIND 4
An ab initio model was generated from 314,001 template-picked particles using cryoSPARC and low-pass filtered to 20 A for use as an initial model for 3D refinement.
 C1 BACK PROJECTION 6.0 FSC 0.143 CUT-OFF RELION 3.0 74413 MAXIMUM LIKELIHOOD RELION 3.0 MAXIMUM LIKELIHOOD RELION 3.0 emd_0409.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 128 128 128 0 0 0 128 128 128 143.0016 143.0016 143.0016 90.0 90.0 90.0 X Y Z -0.054195397 0.06922188 0.00016517921 0.0033557545 1.1172 1.1172 1.1172 0.012 AUTHOR Single-particle cryo-EM reconstruction of the catalytic subunit of protein kinase A bound to ATP and IP20 (unsharpened) emd_0409_additional.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 128 128 128 0 0 0 128 128 128 143.0016 143.0016 143.0016 90.0 90.0 90.0 X Y Z -0.10072195 0.11183412 0.00016660415 0.004566603 1.1172 1.1172 1.1172 AUTHOR Single-particle cryo-EM reconstruction of the catalytic subunit of protein kinase A bound to ATP and IP20 (sharpened) emd_0409_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 128 128 128 0 0 0 128 128 128 143.0016 143.0016 143.0016 90.0 90.0 90.0 X Y Z -0.055288184 0.06959547 0.00016851285 0.0034565239 1.1172 1.1172 1.1172 AUTHOR Even half map emd_0409_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 128 128 128 0 0 0 128 128 128 143.0016 143.0016 143.0016 90.0 90.0 90.0 X Y Z -0.05643959 0.0708219 0.00014687848 0.0034593656 1.1172 1.1172 1.1172 AUTHOR Odd half map