6T72

Structure of the RsaA N-terminal domain bound to LPS


ELECTRON MICROSCOPY
Sample
Structure of RsaA N-terminal domain bound to LPS
Specimen Preparation
Sample Aggregation StatePARTICLE
Vitrification InstrumentFEI VITROBOT MARK IV
Cryogen NameETHANE
Sample Vitrification DetailsVitrobot options: Blot time 3 seconds, Blot force -13,1, Wait time 10 seconds, Drain time 0.5 seconds,
3D Reconstruction
Reconstruction MethodSINGLE PARTICLE
Number of Particles115776
Reported Resolution (Å)3.7
Resolution MethodFSC 0.143 CUT-OFF
Other DetailsParticles 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 t ...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.
Refinement Type
Symmetry TypePOINT
Point SymmetryC1
Map-Model Fitting and Refinement
Id1
Refinement SpaceRECIPROCAL
Refinement ProtocolBACKBONE TRACE
Refinement TargetBest fit
Overall B Value85.819
Fitting Procedure
DetailsThe carbon backbone of the RsaA protein was manually traced through a single subunit of the cryo-EM density using Coot (Emsley et al., 2010). Initiall ...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.
Data Acquisition
Detector TypeGATAN K2 SUMMIT (4k x 4k)
Electron Dose (electrons/Å**2)43
Imaging Experiment1
Date of Experiment
Temperature (Kelvin)
Microscope ModelFEI TITAN KRIOS
Minimum Defocus (nm)-1000
Maximum Defocus (nm)-4000
Minimum Tilt Angle (degrees)
Maximum Tilt Angle (degrees)
Nominal CS2.7
Imaging ModeBRIGHT FIELD
Specimen Holder ModelFEI TITAN KRIOS AUTOGRID HOLDER
Nominal Magnification130000
Calibrated Magnification130000
SourceFIELD EMISSION GUN
Acceleration Voltage (kV)300
Imaging DetailsEPU software
EM Software
TaskSoftware PackageVersion
PARTICLE SELECTIONRELION3.0
IMAGE ACQUISITIONEPU
CTF CORRECTIONCTFFIND4.1.13
MODEL FITTINGCoot0.9-pre
INITIAL EULER ASSIGNMENTRELION3.0
FINAL EULER ASSIGNMENTRELION3.0
CLASSIFICATIONRELION3.0
RECONSTRUCTIONRELION3.0
MODEL REFINEMENTREFMAC5
Image Processing
CTF Correction TypeCTF Correction DetailsNumber of Particles SelectedParticle Selection Details
PHASE FLIPPING AND AMPLITUDE CORRECTIONRELION refinement with in-built CTF correction. The function is similar to a Wiener filter, so amplitude correction included.129633Particles 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.