Initial Model Initial Model Generation Generation Workshop on - PowerPoint PPT Presentation

Initial Model Initial Model Generation Generation Workshop on Advanced Topics Workshop on Advanced Topics in EM Structure Determination in EM Structure Determination The Scripps Research Institute The Scripps Research Institute La Jolla, November 2007 La Jolla, November 2007

The issue: Structures of the IP3 receptor as determined by single particle EM Jiang et al., Serysheva et al., Jiang et al., Sato et al., 2002 2003 2003 2004

Structure determination by Structure determination by single particle EM single particle EM Sample Sample – Negative staining Negative staining – – Cryo Cryo- -negative staining negative staining Specimen preparation – Specimen preparation – Vitrification Vitrification – EM specimen EM specimen – Images of Images of untilted untilted samples samples – – Tilt pairs Tilt pairs Data collection – Data collection – Tilt series – Tilt series Set of 2D images Set of 2D images – Random conical tilt Random conical tilt – – Ab initio Ab initio angle assignment angle assignment 3D reconstruction – 3D reconstruction – Tomographic – Tomographic reconstruction reconstruction Initial 3D model Initial 3D model Refinement of orientation parameters, Refinement of orientation parameters, Refinement Refinement magnification, and CTF parameters , and CTF parameters magnification Refined 3D model Refined 3D model Angular distribution Angular distribution Comparison with 2D data set Comparison with 2D data set Verification Verification Comparison with known structures known structures Comparison with

Sample Sample Images that are used for a 3D reconstruction Images that are used for a 3D reconstruction have to be of identical molecules ! have to be of identical molecules ! Sample can be heterogeneous Sample can be heterogeneous even if it is it is biochemically biochemically homogeneous homogeneous even if – conformational heterogeneity conformational heterogeneity – – unstable complexes unstable complexes – Before attempting any 3D reconstruction: Before attempting any 3D reconstruction: Understand your sample ! Understand your sample !

Sample Sample We always prepare negatively stained specimens first We always prepare negatively stained specimens first good contrast & often preferred orientations good contrast & often preferred orientations (depends somewhat on preparation method) (depends somewhat on preparation method) We always calculate class averages (0 (0° ° images) images) We always calculate class averages different averages = different conformations different averages = different conformations (but can also be different orientations) (but can also be different orientations) We usually calculate 3D reconstructions (RCT) We usually calculate 3D reconstructions (RCT) different 3D maps = different conformations different 3D maps = different conformations (but can also be deformations) (but can also be deformations)

Sample - - Negative staining Negative staining Sample Many preparation artifacts (incomplete stain embedding, Many preparation artifacts (incomplete stain embedding, adsorption deformations, specimen flattening upon drying) adsorption deformations, specimen flattening upon drying) Limitation of the resolution to about 20 Å Å Limitation of the resolution to about 20 Particles adopt preferred orientations Particles adopt preferred orientations on the continuous carbon film ! on the continuous carbon film ! Need to record images BUT: : Very useful for Need to record images BUT Very useful for of tilted specimens heterogeneous samples of tilted specimens heterogeneous samples 3D reconstruction by 3D reconstruction by RCT RCT is very reliable is very reliable

Random conical tilt reconstruction Random conical tilt reconstruction Tiltaxis Tiltaxis Radermacher et al . (1987) Radermacher et al . (1987)

Random conical tilt reconstruction Random conical tilt reconstruction 3D reconstruction of specimen specimen at different tilt angles IMAGING BACKPROJECTION Different projection views ASSIGN ORIENTATIONAL PARAMETERS x, y and Φ

RCT – – AMPA receptor AMPA receptor RCT Heterogeneity due to different conformations Heterogeneity due to different conformations Fractions from Superdex-200 Crude M AMPA 13 14 15 16 17 18 19 20 21 200 116 95 66 45 31 21.5

RCT – – AMPA receptor AMPA receptor RCT Heterogeneity due to different conformations Heterogeneity due to different conformations

RCT – – AMPA receptor AMPA receptor RCT Heterogeneity due to different conformations Heterogeneity due to different conformations

RCT - - Sec23/Sec24 complex Sec23/Sec24 complex RCT Heterogeneity due to different orientations Heterogeneity due to different orientations Negligible deformations Negligible deformations 0° ° tilt tilt 60° ° tilt tilt 0 60

RCT - - Sec23/Sec24 complex Sec23/Sec24 complex RCT Heterogeneity due to different orientations Heterogeneity due to different orientations Lederkremer et al. , 2001 Bi et al. , 2002 Lederkremer et al. , 2001 Bi et al. , 2002

RCT - - Tf Tf- -TfR TfR complex complex RCT Heterogeneity due to different orientations Heterogeneity due to different orientations Severe deformations Severe deformations Face view Top view Face view Top view

RCT - - Tf Tf- -TfR TfR complex complex RCT Heterogeneity due to different orientations Heterogeneity due to different orientations Severe deformations Severe deformations Face view Top view Conventional negative staining Conventional negative staining

Cryo- -negative staining negative staining Cryo Addition of glycerol Addition of glycerol minimizes adsorption artifacts minimizes adsorption artifacts prevents specimen flattening prevents specimen flattening serves as cryo cryo- -protectant protectant serves as Carbon sandwich Carbon sandwich reduces incomplete stain embedding reduces incomplete stain embedding Freezing Freezing prevents specimen flattening prevents specimen flattening

RCT - - Tf Tf- -TfR TfR complex complex RCT Heterogeneity due to different orientations Heterogeneity due to different orientations Severe deformations Severe deformations Conventional negative staining Cryo- -negative staining negative staining Conventional negative staining Cryo 0° ° tilt tilt 0° ° tilt tilt 0 0 60° ° tilt tilt 60° ° tilt tilt 60 60

RCT - - Tf Tf- -TfR TfR complex complex RCT Heterogeneity due to different orientations Heterogeneity due to different orientations Severe deformations Severe deformations Face view Top view Cryo- Cryo -negative staining negative staining

Single particles in ice Single particles in ice y β γ 5 parameters 5 parameters to determine to determine x α

Angular reconstitution (Imagic Imagic) ) Angular reconstitution ( van Heel, 1987 van Heel, 1987 1. chose 3 projection images that are 1. chose 3 projection images that are perpendicular views of the particle perpendicular views of the particle (anchor set) (anchor set) 2. add in further projections and keep 2. add in further projections and keep refining refining Serysheva et al. , 1995 Serysheva et al. , 1995

Angular reconstitution - - Ryanodine Ryanodine receptor receptor Angular reconstitution Serysheva et al. , 1995 Serysheva et al. , 1995

Angular reconstitution - - Tf Tf- -TfR TfR complex complex Angular reconstitution Vitrified ice Vitrified ice

Angular reconstitution - - Tf Tf- -TfR TfR complex complex Angular reconstitution Angular reconstitution Atomic model

Angular reconstitution (IMAGIC) Angular reconstitution (IMAGIC) Structure depends critically on the anchor set Structure depends critically on the anchor set (these should ideally be 3 perpendicular views, (these should ideally be 3 perpendicular views, which is hard to know with an unknown molecule) ) which is hard to know with an unknown molecule Structure also depends on the order in which Structure also depends on the order in which additional projections are included additional projections are included Angular reconstitution is best for: Angular reconstitution is best for: large specimens with symmetry, large specimens with symmetry, and available structural information structural information and available (can be obtained from (can be obtained from random conical tilt) random conical tilt)