6B5B

Cryo-EM structure of the NAIP5-NLRC4-flagellin inflammasome

ELECTRON MICROSCOPY

Refinement

RMS Deviations
Key	Refinement Restraint Deviation
f_dihedral_angle_d	10.157
f_angle_d	1.013
f_chiral_restr	0.051
f_plane_restr	0.006
f_bond_d	0.004

Sample
NAIP5-NLRC4-flagellin inflammasome
Sample Components
NAIP5
NLRC4
flagellin

Specimen Preparation
Sample Aggregation State	PARTICLE
Vitrification Instrument	FEI VITROBOT MARK II
Cryogen Name	ETHANE
Sample Vitrification Details

3D Reconstruction
Reconstruction Method	SINGLE PARTICLE
Number of Particles	252214
Reported Resolution (Å)	5.2
Resolution Method	FSC 0.143 CUT-OFF
Other Details
Refinement Type
Symmetry Type	POINT
Point Symmetry	C1

Map-Model Fitting and Refinement
Id	1
Refinement Space	REAL
Refinement Protocol	FLEXIBLE FIT
Refinement Target	Correlation coefficient
Overall B Value	167
Fitting Procedure
Details	Homology models predicted by I-TASSER server were used as initial model sources. The main structural template identified by I-TASSER for NAIP5 was the ...	Homology models predicted by I-TASSER server were used as initial model sources. The main structural template identified by I-TASSER for NAIP5 was the crystal structure of NLRC4 in the inactive conformation (PDB ID: 4KXF) that covered all the domains except the N-terminal BIR region, where homology models from several BIR domains were recognized (PDB IDs: 1SE0, 2VM5, and 1OXQ for BIR1, BIR2, and BIR3, respectively). Predictions were first rigid body docked into the density map using Chimera and/or ADP_EM. Then, the docked models were flexibly fitted with iMODFIT, if necessary. Finally, all models were refined in Phenix.

Data Acquisition
Detector Type	GATAN K2 SUMMIT (4k x 4k)
Electron Dose (electrons/Å**2)	45.8

Imaging Experiment	1
Date of Experiment
Temperature (Kelvin)
Microscope Model	FEI TITAN KRIOS
Minimum Defocus (nm)	1800
Maximum Defocus (nm)	4000
Minimum Tilt Angle (degrees)
Maximum Tilt Angle (degrees)
Nominal CS
Imaging Mode	BRIGHT FIELD
Specimen Holder Model
Nominal Magnification	22500
Calibrated Magnification
Source	FIELD EMISSION GUN
Acceleration Voltage (kV)	300
Imaging Details

EM Software
Task	Software Package	Version
PARTICLE SELECTION	RELION	1.4
IMAGE ACQUISITION	SerialEM
CTF CORRECTION	Gctf	0.5
MODEL FITTING	UCSF Chimera	1.10.1
MODEL FITTING	ADP_EM	1.21
MODEL FITTING	iMODFIT	1.45
INITIAL EULER ASSIGNMENT	EMAN2	2.05
FINAL EULER ASSIGNMENT	RELION	1.4
CLASSIFICATION	RELION	1.4
RECONSTRUCTION	RELION	1.4
MODEL REFINEMENT	PHENIX	1.11.1-2575
MODEL REFINEMENT	I-TASSER	5.1

Image Processing
CTF Correction Type	CTF Correction Details	Number of Particles Selected	Particle Selection Details
PHASE FLIPPING AND AMPLITUDE CORRECTION		856358

RCSB PDB Core Operations are funded by the U.S. National Science Foundation (DBI-2321666), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198.