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Electron cryo-‐microscopy at Yale
A discussion with the Basic Science Strategic Planning Commi<ee of the Yale Medical School D. M. Engelman, with help from colleagues
02/03/15
Some highlights of EM history
• EM yields images vastly superior to opIcal microscopy (1930’s-‐’60s) • First 3D reconstrucIon of a virus parIcle (Derosier and Klug, 1969) • Transmembrane alpha helices imaged in 3D (Unwin and Henderson, 1975) • Frozen hydrated samples successfully prepared for EM (Dubochet, 1983) • First 3D reconstrucIon of an isolated biomolecule: the ribosome
(Frank, 1984) • It is predicted that EM can solve biomolecular structures to atomic resoluIon
(Henderson, 1995) • PracIcal methods for cryo-‐electron tomography developed
(Baumeister, mid-‐90’s) • Maximum likelihood technique introduced for EM image analysis
(Sigworth, 1998) • The first atomic chain traces from EM images are generated,
from filamentous biological assemblies (Namba, Unwin; 2003)
InformaIon content goes as the inverse cube of the resoluIon, so a 3 A structure has more than 10x the informaIon content of a 7A structure,
125x that of a 15A structure from negaIve stain.
Technical improvements enable chemical resoluIon in very recent work (“near-‐atomic”)
New detectors are advancing the art: Given that radiaIon damage strictly limits the number of electrons that can be used, cryo-‐EM images are intrinsically noisy and it is important to detect the available electrons as efficiently as possible. Consider ribosomes:
Perfect image
Perfect Detector, 20 e-‐/A2
New detectors
Old CCD
One of the great leaps is the realizaIon that specimen moIon is a major limit on resoluIon, new camera takes short exposures to “freeze” the moIon
In favorable cases, can separate classes of parIcles— different conformaIons, for example
Some examples of recent work using new advances: First de novo atomic models by cryo-‐EM (very recent)! Examples:
Using the K2 detector, the Cheng group at UCSF solved the structure of the transient receptor potenIal caIon channel subfamily V member 1 (TRPV1) channel; using the Falcon detector, the Vonck group at Max Planck solved the structure of the F420-‐reducing [NiFe] hydrogenase (FRH); and at the MRC, the structure of the large subunit of the mitochondrial ribosome from yeast was solved. In all three cases, resoluIons beyond 3.5 Å were obtained, at which most of the amino acid side-‐chains are clearly visible, and near-‐complete atomic models could be built de novo
No need for crystals! (but, sIll other challenges of homogeneity, purity, etc.)
Access to larger complexes and uses of tomography move
structural biology toward cell biology
By broadening the scope of structures we are finding unsuspected homologies, and hence new generalizaIons
Determining the structural variaIons that correspond to geneIc diversity
could be a key tool in personalized medicine.
Views of complexes are proving useful in drug development
What are the benefits? Seeing the chemistry is informaIve and enabling: ligand interacIons
The other main applicaIon: electron tomography
The morphome has been suggested as a term for the distribuIon of ma<er in a 3D object, Such as an organelle, cell, or organism. Morphomics methods characterize or quanIfy 3D data, each with a characterisIc window of informaIon: high resoluIon light mic., cryo-‐em, x-‐ray, and electron tomography. UnificaIon of these views will give the best understanding of life in different states and levels of organizaIon, basically using stereo views and tying the data sets together. An important bridge from the chemistry levels (x-‐ray, cryo em) to the light microscopy is the use of electron tomography. Tomography can be done with the same equipment as cryo-‐em: use thin secIons (either fixed, embedded or frozen), Ilt series to get 3D via reconstrucIon, combinaIon of secIons.
Examples of tomography: one of the first studies (Baumeister)
Upper row: first electron tomographic invesIgaIon of a eukaryoIc cell; the slime mould Dictyostelium discoideum embedded in vitrified ice. Cyan: Ribosomes, orange-‐red: the acIn filament network, and blue: the cell membrane. (Medalia et al. Science 298, 2002).
Arrows: side views of nuclear pore complexes Arrowheads: Ribosomes connected to the outer nuclear membrane Right: Structure of the Dictyostelium nuclear pore complex aqer classificaIon and averaging of subtomograms (Beck et al.; Science, 306, 2004; Nature 449, 2007).
Combining data—tomography plus single parIcle averaging
Cryo-‐electron tomography is invading the realm of single-‐parIcle cryo-‐EM
ApplicaIon of cryo-‐electron tomography and sub-‐tomogram averaging methods resolves the structure of the capsid larce within intact immature HIV-‐1 parIcles at 8.8A resoluIon, allowing unambiguous posiIoning of all α-‐helices. The resulIng model reveals terIary and quaternary structural interacIons that mediate HIV-‐1 assembly. (Briggs, 2015)
Concept of integrated approach: organizaIon of macromolecules in a cell
ComputaIon is deeply integrated as a part of the science
• ComputaIonal image processing, for example, analysis takes longer than data collecIon in cryo, tomography of cells, crystallography
• Macromolecular modeling at different scales—how to incorporate chemistry in structural images?
• Data integraIon—for the morphome, combining informaIon at different scales and of different kinds
• Database management and visualizaIon—handling very large databases, for example in em images
• Macromolecular simulaIons at long and large scales
Example: computaIon as a component of genomics—expect similariIes for cryo, tomo, morphome ( image from M. Gerstein)
Electrons interact with molecules ~106 Imes more strongly than X-‐rays do, and so can produce diffracIon data from extremely small crystals (up to 6 orders of magnitude smaller in volume than those typically used for X-‐ray crystallography).
And, another use: electron crystallography from microcrystals
Three-‐dimensional electron crystallography of protein microcrystals. Shi et al. eLife 2013;2:e01345. Nannenga et al. eLife 2014;3:e03600.
Circles: crystals used for X-‐ray diffracIon. Arrows: Crystals used for electron diffracIon.
What are others doing? As the home of one of the founders of the electron microscopy of the cell, George Palade, And as a leader in structural biology, and where a key enabling technical advance in cryoEM was made, Yale needs to remain at the technological curng edge of electron microscopy
Among other efforts, new faciliIes are commi<ed, and most are operaIonal at: MRC-‐LMB MPI Biochemistry Munich, MPI-‐CBG Dresden Monash University (Melbourne) MPI Frankfurt, Görngen NaIonal University Singapore U. Stockholm Univ. Munich Netherlands Centre for Electron Microscopy (2x), Str. Biol. Centre Strasbourg ETH Zürich Univ Zürich And, we must realize, others, for example in China.
Florida State University McGill University Purdue Rockefeller U. Sanford-‐Burnham (La Jolla) UC Berkeley UCSF U. Virginia U. Michigan UCLA, NY consorIum (Manha<an) Janelia Farm HHMI (2x)
What is the need at Yale?
• New camera on exisIng scope is a good step, but not state of the art
• New Krios would be the choice, both for opIcs and for sample changer
• A suitable locaIon now exists in Bass.
• Cost is steep-‐-‐~$10MM purchase plus ~~$ 0.5-‐1.0MM/year to staff and run.
• Users now go elsewhere— advantages are help, limited cost, lack of commitment long term • But, limited scheduling availability, uncertain future, need to screen condiIons, not much capacity for new problems, missed opportuniIes • What do we see as potenIal demand at Yale? Survey now under way.
• And, we need to think clearly about computaIon
