ELECTRON MICROSCOPY

Sample

Native chicken ZPD homopolymeric filament

Specimen Preperation
Sample Aggregation State FILAMENT
Vitrification Instrument ?
Cryogen Name NITROGEN
Sample Vitrification Details ?
3D Reconstruction
Reconstruction Method SINGLE PARTICLE
Number of Particles 498339
Reported Resolution (Å) 4.60
Resolution Method FSC 0.143 CUT-OFF
Other Details ?
Refinement Type
Symmetry Type POINT
Map-Model Fitting and Refinement
ID 1
Refinement Space REAL
Refinement Protocol FLEXIBLE FIT
Refinement Target ?
Overall B Value ?
Fitting Procedure ?
Details Model building was initiated using a local installation of AlphaFold 3 to predict a minimal filament fragment comprising one full-length subunit (chain A) and two partial subunits (chains B and C). The top-ranked prediction was rigid-body fitted into an initial 8.6 A-resolution map (postprocessed with EMReady2) using UCSF Chimera, followed by flexible fitting with Namdinator. Non-resolved terminal regions were trimmed, and well-defined N-glycan densities were manually built in Coot. The model was refined by real-space refinement in Phenix using NCS constraints and increased non-bonded interaction weights, followed by ADP refinement against the unsharpened map. This model served as a starting point for extension with an additional EGF and ZP-N domain from a fourth subunit (chain D). The extended model was docked into the present 4.6 A-resolution map, manually adjusted, and subjected to flexible fitting using the cryo-EM minimizer from cg2all; subsequently, it was refined using Refmac Servalcat task of CCP-EM Doppio, applying global NCS restraints, ProSMART-derived self-restraints, and increased non-bonded interaction weights. Following additional rounds of manual model rebuilding in Coot and real-space refinement in PHENIX (as described above), with positional refinement performed against a LocScale2-postprocessed map and ADP refinement against the unsharpened map, the model was validated using MolProbity and PHENIX. Note that the EGF domain of chain A (and, to a lesser extent, portions of its ZP-N domain near the postprocessed map boundary and the distal regions of the EGF domains in chains C and D) are weakly defined in the density, consistent with their elevated B-factors. These regions were retained in the model to preserve biological completeness, with their conformations constrained by NCS during refinement.
Data Acquisition
Detector Type FEI FALCON IV (4k x 4k)
Electron Dose (electrons/Å2) 53
Imaging Experiment
Date of Experiment ?
Temprature (Kelvin)
Microscope Model TFS KRIOS
Minimum Defocus (nm) 700
Maximum Defocus (nm) 2800
Minimum Tilt Angle (degrees) ?
Maximum Tilt Angle (degrees) ?
Nominal CS 2.7
Imaging Mode BRIGHT FIELD
Specimen Holder Model FEI TITAN KRIOS AUTOGRID HOLDER
Nominal Magnification 165000
Calibrated Magnification ?
Source FIELD EMISSION GUN
Acceleration Voltage (kV) 300
Imaging Details ?
Imaging Experiment
Task Software Package Version
PARTICLE SELECTION cryoSPARC ?
IMAGE ACQUISITION EPU ?
MODEL FITTING UCSF Chimera ?
MODEL FITTING ISOLDE ?
MODEL FITTING Coot ?
RECONSTRUCTION cryoSPARC ?
RECONSTRUCTION EMReady ?
MODEL REFINEMENT REFMAC ?
MODEL REFINEMENT Servalcat ?
MODEL REFINEMENT PHENIX 2.0_5936
Image Processing
CTF Correction Type CTF Correction Details Number of Particles Selected Particle Selection Details
PHASE FLIPPING AND AMPLITUDE CORRECTION ?
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