5OYG

Structure of calcium-free mTMEM16A chloride channel at 4.06 A resolution


ELECTRON MICROSCOPY

Refinement

RMS Deviations
KeyRefinement Restraint Deviation
r_dihedral_angle_2_deg30.514
r_long_range_B_refined16.866
r_long_range_B_other16.865
r_dihedral_angle_3_deg14.099
r_dihedral_angle_4_deg13.844
r_scangle_other10.241
r_mcangle_it9.597
r_mcangle_other9.597
r_dihedral_angle_1_deg6.217
r_scbond_it5.664
RMS Deviations
KeyRefinement Restraint Deviation
r_dihedral_angle_2_deg30.514
r_long_range_B_refined16.866
r_long_range_B_other16.865
r_dihedral_angle_3_deg14.099
r_dihedral_angle_4_deg13.844
r_scangle_other10.241
r_mcangle_it9.597
r_mcangle_other9.597
r_dihedral_angle_1_deg6.217
r_scbond_it5.664
r_scbond_other5.664
r_mcbond_it5.315
r_mcbond_other5.314
r_angle_other_deg3.701
r_angle_refined_deg1.295
r_chiral_restr0.105
r_bond_refined_d0.007
r_gen_planes_refined0.005
r_gen_planes_other0.004
r_bond_other_d
r_nbd_refined
r_nbd_other
r_nbtor_refined
r_nbtor_other
r_xyhbond_nbd_refined
r_xyhbond_nbd_other
r_metal_ion_refined
r_metal_ion_other
r_symmetry_vdw_refined
r_symmetry_vdw_other
r_symmetry_hbond_refined
r_symmetry_hbond_other
r_symmetry_metal_ion_refined
r_symmetry_metal_ion_other
r_scangle_it
r_rigid_bond_restr
r_sphericity_free
r_sphericity_bonded
Sample
mTMEM16A in absence of calcium ions
Specimen Preparation
Sample Aggregation StatePARTICLE
Vitrification InstrumentFEI VITROBOT MARK IV
Cryogen NameETHANE
Sample Vitrification Details2 ul sample volume 2-4 sec blotting time
3D Reconstruction
Reconstruction MethodSINGLE PARTICLE
Number of Particles195465
Reported Resolution (Å)4.06
Resolution MethodFSC 0.143 CUT-OFF
Other Details
Refinement Type
Symmetry TypePOINT
Map-Model Fitting and Refinement
Id1
Refinement Space
Refinement ProtocolRIGID BODY FIT
Refinement Target
Overall B Value
Fitting Procedure
Details
Data Acquisition
Detector TypeGATAN K2 SUMMIT (4k x 4k)
Electron Dose (electrons/Å**2)80
Imaging Experiment1
Date of Experiment
Temperature (Kelvin)
Microscope ModelFEI TITAN KRIOS
Minimum Defocus (nm)500
Maximum Defocus (nm)3800
Minimum Tilt Angle (degrees)
Maximum Tilt Angle (degrees)
Nominal CS2.7
Imaging ModeBRIGHT FIELD
Specimen Holder ModelFEI TITAN KRIOS AUTOGRID HOLDER
Nominal Magnification36630
Calibrated Magnification36630
SourceFIELD EMISSION GUN
Acceleration Voltage (kV)300
Imaging Details
EM Software
TaskSoftware PackageVersion
PARTICLE SELECTIONRELION2.1b1
IMAGE ACQUISITIONSerialEM
CTF CORRECTIONCTFFIND4.1.8
MODEL FITTINGCoot
MODEL REFINEMENTPHENIX
MODEL REFINEMENTREFMAC5
INITIAL EULER ASSIGNMENTRELION21b1
FINAL EULER ASSIGNMENTRELION2.1b1
CLASSIFICATIONRELION2.1b1
RECONSTRUCTIONRELION2.1b1
Image Processing
CTF Correction TypeCTF Correction DetailsNumber of Particles SelectedParticle Selection Details
PHASE FLIPPING AND AMPLITUDE CORRECTIONThe contrast transfer function (CTF) parameters were estimated on the movie frames by ctffind4.11349821For the dataset collected in absence of calcium ions a total of 5063 dose-fractionated super-resolution images were recorded, 2 x 2 down-sampled by Fourier cropping (final pixel size 1.365A) and subjected to motion correction and dose-weighting of frames by MotionCor2. The contrast transfer function (CTF) parameters were estimated on the movie frames by ctffind4.1. Images showing a strong drift, higher defocus than -3.8 um or a bad CTF estimation were discarded, From a total of 4,738 images (final pixel size 1.365 A) 1,349,821 particles were extracted with a box size of 256 pixels after auto-picking and initial round of 2D classification. Initial rounds of 2D and 3D classification, resulted in a set of 467,286 particles which where subjected to particle polishing, as described above, as well as final rounds of 3D classification. The final polished and auto-refined map was calculated from 195,465 particles derived from 4726 images with a resolution of 4.86 A before masking and 4.06 A after masking and was sharpened using an isotropic b-factor of -116 A2