III: Single particle cryoem - practical approaches

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1 III: Single particle cryoem - practical approaches Single particle EM analysis can be performed at both 2D and 3D. Single particle EM (both negative stain and cryo) is to extract structural information (both 2D and 3D) of macromolecules by averaging a large number of molecules without crystals. Good single particle cryoem: * Good sample: biochemically well behaved samples; * Good images: image quality matches resolution; * Good results: claimed resolution matches structural features; A better structure has less ambiguity. Single particle v.s crystallography Crystallography is also an averaging technique: * all molecules are aligned by forming a crystal; * crystallinity guaranteed homogeneity, i.e. crystal is formed by both compositional and conformational identical molecules; * large crystals contain more coherently packed molecules; * diffraction patterns can assess the quality of a crystal; Analogous to X-ray crystallography: * averaging by computation instead of by crystallization; * accuracy of alignment is similar as order of packing molecules in crystals; * number of particle images is similar as the size of the crystals; Single particle: * image of every individual molecule are aligned computationally; * homogeneity is not guaranteed; * R. Henderson pointed out in 1995 that atomic resolution can be reached as far as one can align image accurate enough. He also predicted how many images are needed to achieve this level of resolution.

2 What limits resolution of single particle cryo-em reconstructions? * Modern microscope has ~2Å point resolution and ~1Å information limit; * Homogeneity is not guaranteed by biochemical process. * Classification and image alignment are not independent processes. * Influence of CTF. Atomic resolution imaging by TEM graphene, Nature Mat, 2011, 10, 165 Electron optic system of a modern electron microscope is of sufficient quality to image radiation resistant material (typically inorganic) at atomic resolution (~2Å or better).

3 Influence of CTF on image contrast graphene, Nature Mat, 2011, 10, 165 Particle picking: manual v.s. automation * Manual picking is more reliable, but tedious; * Automated picking is model based and prone to model induced bias; * A good practice is to start with manual picking followed by automated picking. * Sufficient image contrast is required for either manual or automated particle picking. Model induced bias in Single particle cryoem Considering that low-does images of frozen hydrated sample are always extremely noisy, reference model induced bias is very strong. It is therefore possible to produce a wrong structure. Model induced bias: all iterative refinement procedures are prone to model induced bias. Image quality, i.e. image contrast and resolution, is critical in preventing model induced bias.

4 Demonstration of reference induced bias Note: The averaged image after reference based alignment is strongly biased towards the reference. 100 images 1000 images reference From Niko Gorigorieff Difficulties of apply high-resolution single particle cryoem to study small proteins * Images of individual small molecule do not contain sufficient structural information for accurate image alignment; * 3D reconstruction calculated from images of small molecules are strongly influenced by the initial model and is difficult to be validated. * Iterative procedure is highly model biased; * Low SNR makes it very hard to validate the result; Nature Structural & Molecular Biology (2012) Difficulties of apply high-resolution single particle cryoem to study small proteins * Images of individual small molecule do not contain sufficient structural information for accurate image alignment; * 3D reconstruction calculated from images of small molecules are strongly influenced by the initial model and is difficult to be validated. * Iterative procedure is highly model biased; * Low SNR makes it very hard to validate the result; Nature Structural & Molecular Biology (2012)

5 Difficulties of apply high-resolution single particle cryoem to study small proteins * Images of individual small molecule do not contain sufficient structural information for accurate image alignment; * 3D reconstruction calculated from images of small molecules are strongly influenced by the initial model and is difficult to be validated. * Iterative procedure is highly model biased; * Low SNR makes it very hard to validate the result; Nature Structural & Molecular Biology (2012) Signs of model induced bias Richard Henderson: Avoiding the pitfalls of single particle cryo-electron microscopy: Einstein from noise, PNAS, 2013, Marin van Heel: Finding trimeric HIV-1 envelop glycoproteins in random noise, PNAS, 2013, Sriram Subramaniam: Structure of trimeric HIV-1 envelop glycoproteins, PNAS, 2013, Negative stain EM of 20S complex 20S particles on a thin layer of carbon film are in side view orientations.

6 cryo-em of 20S complex in ice!"#$%&'()*+, -.&**$%&'()*+, 20S particles have a preferred orientation when suspended in ice. cryo-em of 20S complex on a carbon film /&'012 3"4'+15,$")+ 20S particles on a thin layer of carbon film are in side view orientations. cryo-em of 20S complex on a carbon film 3"4'+15,$")+ /&'012 We assumed that there are particles on carbon film, and they are mostly side views.

7 cryo-em of 20S complex Using detergent Nonidet P-40 change the preferred orientations. But very few particles are in ice. Zhao, Wu, et al. (2015) Nature Alarming example: Structure of IP3 receptor Structure of the type 1 onsoitol 1,4,5- trisphosphate receptor revealed by electron cryomicroscopy. JBC 2003, 278, Insoitol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped Ligand-binding domains. JMB 2004, 336, Alarming example: Structure of IP3 receptor da Fonseca P C A et al. PNAS 2003; 100:

8 Alarming example: Structure of IP3 receptor IP3 receptor (Ludtke, et. al. 2011, Structure) Tilt pair validation For both cryoem and X-ray crystallography, biochemistry is similarly important Sample requirement for structural analysis (X-ray crystallography): * high purity; - clean SDS PAGE gel (better with silver stain) * homogeneous conformation; - nice size exclusion chromatography profile * stable complex; - does not fall apart during crystallization Same are required for high-resolution single particle cryoem structural studies.

9 Difference samples resulted different 3D reconstructions Difference samples resulted different 3D reconstructions 6.9Å 12Å Difference samples resulted different 3D reconstructions 6.9Å claimed 7.5Å in 2004

10 In principle, common line approach should give us an answer. But in real world, it may not be. A problem that we have to deal with all the time: images of two particles are different, are they from different type of particles or from different views of the same particle? * Classification of different conformations/composition and determining 3D orientations of particle images is intertwined together, and cannot be separated completely. IN project Early stage of retroviral infection: integration into host chromatin - Shenping Wu, (Angela Lai), Akram Alian, Sarah Griner and Bob Stroud Suzuki, Y. and Craigie, R Nature Reviews Microbiology. Crystal structure of integrase dimer Cheng, J.C.H., et al PNAS. Wang, J.Y., et al EMBO J.

11 Superposition of the two structures generates the model of the full length integrase dimer Wang, J.Y., et al EMBO J. Our goal To obtain a structure of tetramer full length integrase, in complex with DNA, by single particle EM. HIV-IN DNA Purification Chromatograph, Sarah Negatively stained HIV-IN DNA complex Angela Lai

12 Class averages of integrase particle Angela Lai 3D reconstruction of Integrase To obtain this structure, we have to make a assumption that all particles we selected are integrase tetramers. Angela Lai Placing atomic structure into 3D reconstruction By placing atomic models of integrase into 3D reconstruction, we generated a model. * Problem: the size exclusion column cannot really separate tetramer and dimer, and we are not sure if the particles contains DNA or not. Angela Lai

13 Difficulties of apply high-resolution single particle cryoem to study small proteins * Images of individual small molecule do not contain sufficient structural information for accurate image alignment; * 3D reconstruction calculated from images of small molecules are strongly influenced by the initial model and is difficult to be validated. A milestone work of single particle cryoem: 3.3Å reconstruction of icosahedral virus from: Hong Zhou laboratory - Zhang et al. (2010) Cell, 141, Image of icosahedral virus particles from: Hong Zhou laboratory - Zhang et al. (2010) Cell, 141,

14 Comparison with Image of smaller proteins * Difficult to visualize; * Difficult to align; * Difficult to validate; Tfr-Tf: ~300kDa Vglut: ~50kDa Strategy to deal with small proteins Solution: Below 100,000 molecular weight, some kind of crystal or other geometrically ordered aggregate is necessary to provide a sufficiently high combined molecular weight to allow for the alignment. - Richard Henderson, Quarterly Reviews of Biophysics, 28 (1995) Previously tested methods: formation of monolayer crystal, fuse the target protein into an icosahedral virus, etc. Our strategy: use one or more monoclonal Fab to form a stable and rigid complex with a target protein. Benefit for single particle cryoem of small proteins: - Enlarge the target proteins for better visualization; - providing fiducial markers for image alignment; - providing internal control for 3D reconstruction validation; Negative stain EM image of Fab - Fabs have a well-defined characteristic shape that is easy to be recognized in negative stain EM. Wu et al., Structure, 2012

15 Negative stain EM image of Fab - Fabs have a well-defined characteristic shape that is easy to be recognized in negative stain EM. Wu et al., Structure, 2012 Negative stain EM image of Fab - Fabs have a well-defined characteristic shape that is easy to be recognized in negative stain EM. Wu et al., Structure, 2012 Fab assisted structural analysis Zhou et al, Nature 2001 Fab Matriptase Hibbs et al, Nature 2011 Matriptase Fab Schneider et al, JMB 2012 Wu et al, Structure 2012

16 Test: small soluble proteins Example 1: human PSCK9-J16 complex (Pfizer) * PCSK9 has a molecular weight of ~70kDa; * Fab J16 was generated by using hybridoma technology; Fab generated by using hydridoma EM image of negatively stained Fab. technology (Pfizer Inc.) Wu et al., Structure, 2012 Test: small soluble proteins Dimeric HIV integrase: * dimer has a molecular weight of ~65kDa; * 4 Fabs identified from phage displayed library panning experiment; Wu et al., Structure, 2012 Test: small soluble proteins CryoEM of IN-Fab complex Sample: IN-Fab complex: ~165kDa; Quantifoil grid, Vitrobot; Microscope: TF20 operated at 200kV; TVIPS 8K CMOS camera; Image condition: 70um objective aperture; Defocus 2 ~ 5um;~25e/A2 dose; Image processing: Negative stain reconstruction as initial model; CTFFIND; Frealign on GPU (GeFrealign); Wu et al., Structure, 2012

17 CryoEM of ABC-AH5 complex - K2 Summit camera Shenping Wu CryoEM of ABC-AH5 complex - K2 Summit camera Class averages of particle images recorded with the K2 Summit. Shenping Wu 3D reconstruction of TmrAB-AH5 complex at 8.3 Å resolution 3D reconstruction of TrmAB transporter-ah5 complex at ~8.3 Å resolution. Shenping Wu