2024-05-22 REL PDBe PDBe PDBe 2019-10-21 2019-11-06 2020-01-15 2024-05-22 Wellcome Trust 202231/Z/16/Z United Kingdom Structure of the RsaA N-terminal domain bound to LPS von Kuegelgen A Bharat TAM S-layer LPS RsaA, STRUCTURAL PROTEIN von Kugelgen A Tang H Hardy GG Kureisaite-Ciziene D Brun YV Stansfeld PJ Robinson CV Bharat TAM In Situ Structure of an Intact Lipopolysaccharide-Bound Bacterial Surface Layer. Cell 180 348 358.e15 2020 31883796 doi:10.1016/j.cell.2019.12.006 1097-4172 6t72 FULLOVERLAP Structure of RsaA N-terminal domain bound to LPS Structure of RsaA N-terminal domain bound to LPS 0 1
Structure of RsaA N-terminal domain bound to LPS
Caulobacter vibrioides CB15 YB1001 0.577 S-layer protein Caulobacter vibrioides CB15 0.025820353999999997
LPS O-antigen bound to protein
1 Caulobacter vibrioides CB15 LEVO AYTTAQLVTAYTNANLGKAPDAATTLTLDAYATQTQTGGLSDAAALTNTLKLVNSTTAVAIQTYQFFTGVAPSAAGLDFL VDSTTNTNDLNDAYYSKFAQENRFINFSINLATGAGAGATAFAAAYTGVSYAQTVATAYDKIIGNAVATAAGVDVAAAVA FLSRQANIDYLTAFVRANTPFTAAADIDLAVKAALIGTILNAATVSGIGGYATATAAMINDLSDGALSTDNAAGVNLFTA YPSSGVSGSENLYFQ P35828 CALCIUM ION 4.0078e-05 3 CA singleParticle particle 2.5 7.5 25.0 C8H18N2O4S HEPES 100.0 NaCl sodium chloride 1.0 MgCl2 magnesium chloride 1.0 CaCl2 calcium chloride
Buffer solutions were prepared fresh from sterile filtered concentrated stocksolutions. Solutions were filtered through a 0.22 um filter to avoid microbial contamination and degassed using a vacuum fold pump. The pH of the HEPES stock solution was adjusted with sodium hydroxide at 4 deg C.
 Quantifoil R2/2 COPPER/RHODIUM 200 CARBON HOLEY ARRAY GLOW DISCHARGE AIR
20 seconds, 15 mA
 ETHANE 100 283.15 FEI VITROBOT MARK IV
Vitrobot options: Blot time 3 seconds, Blot force -13,1, Wait time 10 seconds, Drain time 0.5 seconds,.
RsaA N-terminal domain with LPS
 FEI TITAN KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 50.0 2.7 -1.0 -1.0 -4.0 -4.0 130000.0 130000.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN 70.0 70.0 GIF Quantum LS 20
EPU software
GATAN K2 SUMMIT (4k x 4k) COUNTING 3838 3710 1-20 1 2422 8.0 43.0
Images were collected in movie-mode and subjected to 8 seconds of exposure where a total dose of 43 e-/A2 was applied, and 20 frames were recorded per movie.
 1
Movies were motion corrected and dose weighted with MotionCor2 (Zheng et al., 2017) implemented in Relion 3.0 (Zivanov et al., 2018). Contrast transfer functions (CTFs) of the resulting motion corrected micrographs were estimated using CTFFIND4 (Rohou and Grigorieff, 2015).
129633
Particles were automatically picked from the motion and CTF corrected micrographs using the AutoPick function in Relion 3.0 (Zivanov et al., 2018). As particle reference a 3 dimensional reconstruction from an earlier dataset with different pixelsize was used which was reconstructed using an unbiased subtomogram average structure of the same sample.
Initial model generation from cryo-ET data was performed using the Relion sub-tomogram averaging pipeline (Bharat et al., 2015; Bharat and Scheres, 2016). An unambiguous 3D reference was generated and used in the single-particle EM pipeline.
 2 C1 FOURIER SPACE 3.7 FSC 0.143 CUT-OFF RELION 3.0
Particles from two main 3D classes containing 21 or 20 RsaA subunits were combined for a focused 3D auto refinement on the central 14 subunits using the output from the 3D classification as a starting model. The final map was obtained from 115,776 particles and post-processed using a soft mask focused on the inner fourteen subunits.
115776 MAXIMUM LIKELIHOOD RELION 3.0
Angle assignment was performed within Relion 3.0.
 MAXIMUM LIKELIHOOD RELION 3.0
Angle assignment was performed within Relion 3.0.
 2 115776.0 RELION 3.0
Particles from two main 3D classes containing 21 or 20 RsaA subunits were combined for a focused 3D auto refinement on the central 14 subunits using the output from the 3D classification as a starting model. The final map was obtained from 115,776 particles and post-processed using a soft mask focused on the inner fourteen subunits.
 emd_10389.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 300 300 300 0 0 0 300 300 300 324.0 324.0 324.0 90.0 90.0 90.0 X Y Z -0.06003098 0.107363954 0.000029459738 0.0039067115 1.08 1.08 1.08 0.0195 AUTHOR cryo-EM map of the RsaA protein N-terminal domain BACKBONE TRACE
The carbon backbone of the RsaA protein was manually traced through a single subunit of the cryo-EM density using Coot (Emsley et al., 2010). Initially, side chains were assigned in regions with density corresponding to characteristic aromatic residues allowing us to deduce the register of the amino acid sequence in the map. Side chains for residues 2-243 of RsaA were thus assigned unambiguously and the structure was refined and manually rebuilt using Refmac5 (Murshudov et al., 2011) inside the CCP-EM (Burnley et al., 2017) software suite and Coot.
Best fit RECIPROCAL 85.819000000000003