Single-Particle Cryo-EM of Arabinosyltransferase EmbB from Mycobacterium smegmatis, Collected using both Falcon III and K2 Detectors

REL 2020-05-14 2020-07-30 2020-07-30 Single-Particle Cryo-EM of Arabinosyltransferase EmbB from Mycobacterium smegmatis, Collected using both Falcon III and K2 Detectors 0000-0001-6656-6320 Yong Zi Tan Department of Physiology and Cellular Biophysics, Columbia University 1150 Saint Nicolas Ave New York New York United States 10032 0000-0003-3293-2200 Filippo Mancia Department of Physiology and Cellular Biophysics, Columbia University 1150 Saint Nicolas Ave New York New York United States 10032 0000-0002-0624-5020 Bridget Carragher New York Structural Biology Center/Columbia University 89 Convent Ave New York New York United States 10027 0000-0003-2394-0831 Clinton S Potter New York Structural Biology Center/Columbia University 89 Convent Ave New York New York United States 10027 Tan YZ Rodrigues J Keener JE Zheng RB Brunton R Kloss B Giacometti SI Rosário AL Zhang L Niederweis M Clarke OB Lowary TL Marty MT Archer M Potter CS Carragher B Mancia F 4.5 10.6019/EMPIAR-10420 EMDB EMD-21983 Tan YZ Rodrigues J Keener JE Zheng RB Brunton R Kloss B Giacometti SI Rosário AL Zhang L Niederweis M Clarke OB Lowary TL Marty MT Archer M Potter CS Carragher B Mancia F Tan YZ Rodrigues J Keener JE Zheng RB Brunton R Kloss B Giacometti SI Rosário AL Zhang L Niederweis M Clarke OB Lowary TL Marty MT Archer M Potter CS Carragher B Mancia F Cryo-EM structure of arabinosyltransferase EmbB from Mycobacterium smegmatis Nature communications Nat Commun 1 11 3396 3396 2020 English 10.1038/s41467-020-17202-8 32636380 Unaligned and compressed multiframe movies from Falcon III Detector /data/01_Raw_Frames/18nov21d micrographs - multiframe MRCS MRCS 2159 80 1 78 SIGNED 16 BIT INTEGER 4096 0.665 4096 0.655

Images were recorded in two sessions. This first session was on a Titan Krios electron microscope (FEI) equipped with a Falcon III direct detector operating at 0.665 Å per pixel in electron counting mode using the Leginon software package. Pixel size was calibrated after obtaining a preliminary map by docking with a crystal structure of a homolog of the soluble part of EmbB (PDB ID: 3PTY). Data collection was performed using a dose of ~78.02 e-/Å2 across 80 frames (1080 msec per frame) at a dose rate of ~0.40 e–/pix/sec, using a set defocus range of -0.5 μm to -2.5 μm. 100 micron objective aperture was used. The movies were recorded over three days using an image beam shift data collection strategy. These files have been CTF corrected using the parameters in: data/02_Aligned_Micrographs/micrographs_ctf_18nov21d_FalconIII.star

Unaligned and compressed multiframe movies from K2 Summit Detector /data/01_Raw_Frames/19may24c micrographs - multiframe TIFF TIFF 7943 80 1 80 UNSIGNED 16 BIT INTEGER 3710 0.667089 3838 0.667089

Images were recorded in two sessions. This second session was on a Titan Krios electron microscope (FEI) equipped with a K2 summit direct detector operating at 0.667 Å per pixel in counting mode using the Leginon software package. Pixel size was calibrated in-house using a proteasome test sample. Data collection was performed using a dose of ~77.53 e-/Å2 across 80 frames (100 msec per frame) at a dose rate of ~4.3 e–/pix/sec, using a set defocus range of -0.3 μm to -2.9 μm. 100 micron objective aperture was used. The movies were recorded over three days using an image beam shift data collection strategy. These files have been CTF corrected using the parameters in: data/02_Aligned_Micrographs/micrographs_ctf_19may24c_K2.star

Aligned and dose-weighted micrographs from Falcon III Detector /data/02_Aligned_Micrographs/18nov21d micrographs - single frame MRC MRC 1955 1 32 BIT FLOAT 4096 0.665 4096 0.665

For this first Falcon III dataset, movie frames were aligned using MotionCor2 with 5 by 5 patches and B-factor of 500 for global alignment and 100 for local alignment through the Relion package.

Aligned and dose-weighted micrographs from K2 Summit Detector /data/02_Aligned_Micrographs/19may24c micrographs - single frame MRC MRC 6025 1 32 BIT FLOAT 3710 0.667089 3838 0.667089

For the second K2 summit dataset, movie frames were aligned using MotionCor2 with 5 by 5 patches and B-factor of 500 for global alignment and 100 for local alignment through the Relion package.

Final Particle Stack with Refined Euler Angles and Shifts /data/03_Final_Stack picked particles - single frame - processed MRCS MRCS 1 57970 32 BIT FLOAT 256 1.0 256 1.0

Combined particle stack from both datasets.

data/02_Aligned_Micrographs/*/*.mrc data/03_Final_Stack/EmbB-Combined18nov21d-19may24c.star data/03_Final_Stack/