A national facility for biological cryo-electron microscopy

Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic
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  research papers Acta Cryst.  (2015). D 71 , 127–135 doi:10.1107/S1399004714025280  127 Acta Crystallographica Section D BiologicalCrystallography ISSN 1399-0047 A national facility for biological cryo-electronmicroscopy Helen R. Saibil, a * KayGru¨newald b and David I.Stuart b,c a Crystallography, Institute for Structural andMolecular Biology, Birkbeck College,Malet Street, London WC1E 7HX, England, b Division of Structural Biology, WellcomeTrust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, England, and c Diamond Light Source, Didcot OX11 0DE,EnglandCorrespondence e-mail:h.saibil@mail.cryst.bbk.ac.uk Three-dimensional electron microscopy is an enormouslypowerful tool for structural biologists. It is now able to providean understanding of the molecular machinery of cells, diseaseprocesses and the actions of pathogenic organisms fromatomic detail through to the cellular context. However,cutting-edge research in this field requires very substantialresources for equipment, infrastructure and expertise. Here,a brief overview is provided of the plans for a UK nationalthree-dimensional electron-microscopy facility for integratedstructural biology to enable internationally leading researchon the machinery of life. State-of-the-art equipment operatedwith expert support will be provided, optimized for bothatomic-level single-particle analysis of purified macromole-cules and complexes and for tomography of cell sections. Theaccess to and organization of the facility will be modelled onthe highly successful macromolecular crystallography (MX)synchrotron beamlines, and will be embedded at the DiamondLight Source, facilitating the development of user-friendlyworkflows providing near-real-time experimental feedback. Received 9 June 2014Accepted 18 November 2014 1. Introduction From the srcins of electron microscopy (EM) the method hasmade major contributions to biology, but the recent technicaland methodological developments outlined below haveexpanded both the scope and the precision of the method, sothat cryo-transmission EM is now a central pillar of structuralbiology (Henderson, 2004; Frank, 2006; Cheng & Walz, 2009;Luc ˇ ic ˇ   et al. , 2013). It covers a wide range of sample size andresolution from tomographic analysis of sections of whole cellsat 20–40 A˚resolution down to single-particle analysis of lessthan 200 kDa molecular mass at around 3 A˚resolution (seeFig. 1 for an overview). Furthermore, it is a powerful toolnot only for detailed structure determination but also forthe characterization and quality control of assemblies beingexpressed and purified for structural analysis. In this context,we envisage the national facility fitting in by providing accessat the top end of an increasingly broad use of the method.The national facility will work to encourage growth in the EMcommunity by providing training programmes to generate anexpanding, more confident and more demanding user base. Inparticular, we expect a broadening of the user community toencompass structural and cell biologists who are not EMspecialists but are attracted by the potential of the method.This will markedly increase the demand for top-end equip-ment, just as high-end MX beamlines have enabled progresson some of the most biologically significant but challengingcrystallographic problems, empowering a user base that nowhas very high expectations of facilities.  Transmission electron micro-scopy of vitrified samples wasdeveloped over 30 years ago(Dubochet  et al. , 1988). In recentyears, the capabilities of single-particle EM and electron tomo-graphy (ET) have expandeddramatically, benefitting fromhardware and software advances,notably stable and coherent elec-tron sources (field emission guns),stable specimen stages (includingmulti-sample cartridges), auto-mation and high-speed directelectron detectors, along withpowerful statistical methods forreconstruction and sorting outheterogeneity in data sets(Jensen, 2010 a , b , c ; Suloway  et al. ,2005; Ruskin  et al. , 2013; Li  et al. ,2013; McMullan  et al. , 2014;Scheres, 2012; Orlova & Saibil,2011). Further developments, e.g.  phase plates and aberrationcorrection, are ongoing (Dai etal. ,2013; Wang  etal. , 2011). However,the continuing hardware devel-opments are increasingly puttingstate-of-the-art facilities for thistype of work out of the reach of most laboratories. It is becomingprohibitively expensive to acquireand run high-end cryo-EMs andelectron detectors, and thedemands on engineering exper-tise, high-tech infrastructure andbuilding services strain thecapabilities of most universitiesand small institutes. Furthermore,the logistical problems of providing a safe, functioning 24/7facility are more easily solved ina central facility operating withsupport staff available day andnight. For this reason, the UKstructural biology community hasprioritized the establishment of acentral facility for high-end EM. 2. Methods Work on the structural biologyof macromolecular machinery isnow moving beyond the opera-tion of individual proteins toencompass whole systems. Illus-trations of the power of this research papers 128  Saibil  et al.   A national facility for biological cryo-electron microscopy  Acta Cryst.  (2015). D 71 , 127–135 Figure 1 Overview of current methods in biological cryo-EM. ( a ) Single-particle reconstruction of the TrpV channel(Liao  et al. , 2013; EMD-5778). Left, projected views representing idealized single-particle data. Right,three-dimensional reconstruction of the mainly   -helical tetramer, coloured by subunit, with the fittedsecondary structure. ( b ) Zernicke phase-contrast cryo-ET of a virus-infected cyanobacterial cell. Left,section through the tomogram; right, segmented view of the cell with the viruses in pink. Reproduced bypermission from Macmillan Publishers Ltd, Dai  et al.  (2013), copyright (2013). ( c ) Electron diffraction of lysozyme microcrystals. Left, optical micrograph of small crystals with microcrystals indicated by arrows.Right, representation of the three-dimensional electron diffraction data. Figures reproduced or modifiedfrom Shi  et al.  (2013) under a Creative Commons Attribution license.  complex approach come, for example, from virus, proteasome,ribosome and membrane-inserted structures (Li  et al. , 2013;Liao  et al. , 2013; Zhang  et al. , 2010; Amunts  et al. , 2014; Fig. 1).Until now, such high-resolution studies have been performedon highly purified samples, but it is now clear that the nextmajor challenge is to obtain the same level of understandingin the context of cells and tissues (Al-Amoudi  et al. , 2007;Luc ˇ ic ˇ   et al. , 2013). To achieve this integration of molecular andcellular information will require significant further develop-ment of the methods of correlative microscopy (Faas  et al. ,2013) and multiscale imaging (Russel  et al. , 2012). We willbriefly summarize the major methods of electron imaging,which use transmission microscopy applied to biologicalspecimens cryo-preserved in vitreous ice. 2.1. Single-particle analysis This method is suitable for purified complexes that are largeenough to give sufficient signal for detection and alignment(Frank, 2002; van Heel  et al. , 2000). In practice, the lower limithas until recently been about 200 kDa, although in theory100 kDa should be achievable and we expect further progresstowards this (Henderson, 2004). In contrast there is no uppermass limit for single particles, but the sample thickness mustbe well under 1  m m for TEM to avoid limitations caused bymultiple scattering of electrons passing through the specimenand other electron optical problems. The principle is simple:a single projection image is obtained from each of a largenumber of similar particles. If the particles are in randomorientations then combining the information from thedifferent particles will enable a full three-dimensional recon-struction of the average structure. This process is verycomputationally intensive compared with crystallographysince the process of crystallization precisely defines the rela-tive position and orientation of all of the particles in thecrystal, whereas in cryo-EM single-particle analysis theseparameters must be determined for each particle using aniterative computational procedure. With a state-of-the-artsystem, the resolution obtainable from suitable single-particlesamples is often limited by the order and homogeneity of the sample, with atomic detail resolvable in the best cases. Inpractice, the criteria for good samples are usually similar forEM and X-ray crystallography and so the availability of ‘crystallization grade’ material is an excellent starting point fora single-particle analysis (although the concentration requiredfor EM analysis is usually considerably less than that requiredfor crystallization). It is important to note that, aside fromsample considerations, biological structure determination bycryo-EM is strictly limited by the inherently low signal-to-noise ratio and by radiation damage to the specimen. Thiscontributes to the requirement of at least tens of thousands of asymmetric units for a high-resolution reconstruction. There-fore, improvements in signal contrast in cutting-edge systems( e.g.  phase plates), increased stability in the microscope and,most notably, increased speed and detective quantum effi-ciency of direct electron detectors provide major advantages.Movie-mode acquisition with direct detectors enablescorrection for beam-induced motion by realignment of sub-frames, leading to a marked increase in the efficiency of themethod at higher resolution (Li  et al. , 2013). With sufficientlylarge and rigid biological assemblies (Zhang  et al. , 2010) anddirect detectors it is becoming routine to determine a single-particle structure in atomic detail (at 3–4 A˚resolution) usingstate-of-the-art equipment (Amunts  et al. , 2014; Li  et al. , 2013;Liao  et al. , 2013; Fig. 1 a ). With a growing number of analysesresulting in resolutions better than 4 A˚, there is increasingcrossover in computational methods for structure analysisand refinement between X-ray crystallography and electronmicroscopy. Thus, the leading X-ray refinement programs, PHENIX   and  REFMAC   (Moriarty  et al. , 2014; Murshudov  et al. , 2011), are both now capable of performing model refine-ment against electron-microscopy maps. There is still scope forimprovement in the methods, with relatively few published‘fully refined’ EM structures so far (Amunts  et al. , 2014).Moreover, there are still problems to solve in methods forvalidation and resolution determination (Chen  et al. , 2013). 2.1.1. Computational sorting to resolve dynamics frommultiple snapshots . Software developments now make itpossible to extract multiple high-quality structures from datasets of heterogeneous complexes in different functional states(Clare  et al. , 2009, 2012; Fischer  et al. , 2010; Fig. 2), but reso-lution is often limited by the throughput of data collectionand processing. Depending on how many conformations arepresent and how much they differ, various statistical methodscan be used either in two dimensions or three dimensions todetect and separate the different conformations. If an accurateseparation is achieved, then the resolution of each structurewill be determined by the number of asymmetric units and theconformational homogeneity/rigidity of that subset. For moredifficult and labile complexes, biochemical approaches suchas Grafix, which involves fractionation of complexes in thepresence of dilute glutaraldehyde (Kastner  et al. , 2008), canbe used to stabilize transient assemblies. These approachesprovide a series of snapshots that can provide fundamentalinsight into the dynamics of macromolecular machinescarrying out key activities such as protein synthesis or folding,and can map major conformational changes (Clare  et al. , 2012;Fischer  et al. , 2010; Zhang  et al. , 2008; Scheres, 2012). 2.2. Electron cryo-tomography (cryo-ET) Tomography provides lower resolution information butallows a full three-dimensional reconstruction of a singleobject by recording a series of views (Mastronarde, 2005).The method provides mechanistic details of nanoscale cellularprocesses such as cargo transport, virus uncoating, membranemodulation and membrane trafficking (Luc ˇ ic ˇ   et al. , 2013; Dai et al. , 2013). It reveals a new landscape of previously unknowndetails of cell structure, enabling the analysis of macro-molecular complexes functioning in their native environment(Al-Amoudi  et al. , 2007; Fig. 1 b ). In addition, the methodprovides ideal tools to reveal new information on host–pathogen interactions at the supramolecular level (Zeev-Ben-Mordehai  et al. , 2014). research papers Acta Cryst.  (2015). D 71 , 127–135 Saibil  et al.   A national facility for biological cryo-electron microscopy  129  A major limitation is that only the thinnest (<<1  m m)regions of cells can be studied intact. However, specimenthinning by cryo sectioning or ion beam milling relieves thisrestriction, giving access to all parts of the cell and enablingthe study of cellular processes that take place, for example, inand around the nucleus. Vitreous sectioning using a diamondknife is relatively cheap and gives access to any cell or tissueregions that can be prepared by high-pressure freezing, whichis suitable for specimens around 100  m m thick and up to a fewmillimetres in diameter (see, for example, Al-Amoudi  et al. ,2007). The main limitation of this approach is mechanicaldamage during sectioning, which causes variable mechanicalcompression and crevassing, limiting the useful sectionthickness to   50 nm. This is an insufficient depth of viewfor large assemblies such as nuclear pores. In the alternativetechnique of ion beam milling, the cryo-sample is mounted in ascanning EM and a beam of heavy ions is used to ablate thesurface to produce a lamella. This method has fundamentaladvantages, since it reduces the problems of mechanicaldamage and can produce 200–400 nm thick lamellae of thesample (Marko  et al. , 2006; Rigort  et al. , 2012). However, ionbeam milling is not yet routine and requires expensivespecialist equipment, and issuesof locating objects of interestwithin the specimen andcapturing them in a lamellaharbouring these objects have notyet been solved.For cryo-ET the conflictingrequirements of low electron doseto minimize radiation damage andhigher dose for sufficient signalto noise provide the majorlimitations for three-dimensionalreconstruction. A full tilt seriesmust be collected of each object,with repeated electron exposuresdelivering a high cumulative dose,and each tilt exposure must havesufficient signal for alignment.Although these requirementslimit cryo-ET to nanometre reso-lution, it is playing an increasinglyimportant role in defining cellularcomplexes and in extendingstructural biology from the mole-cular level to the cellular level.Furthermore, recent advances inelectron detection, and likelyfurther improvements, are havinga major impact on the effective-ness of electron tomography byproviding more recorded signalfor a given electron dose. 2.3. Sub-tomogram averaging It is possible to extract sub-regions from tomograms foralignment, classification andaveraging, establishing a metho-dological continuum betweentomography and single-particleanalyses (Gru ¨ newald  et al. , 2003;Bartesaghi  et al. , 2012) andproviding intermediate resolu-tion. Structures that cannot beisolated intact but that are present research papers 130  Saibil  et al.   A national facility for biological cryo-electron microscopy  Acta Cryst.  (2015). D 71 , 127–135 Figure 2 Dynamics of GroEL–ATP (Clare  et al. , 2012). Cryo-EM maps (transparent surfaces) and flexible fittingshow some of the main structural states determined by multivariate statistical analysis of a 60 000-particledata set. Helices H and I, which denote the hydrophobic binding sites for non-native proteins, are shown inorange/red and the GroES lid is shown in green. ( a ) Apo GroEL, ( b ) GroEL–ATP 7 , Rs1 state, ( c ) GroEL–ATP 7 , Rs-open state, ( d ) GroEL–GroES–ATP.  as multiple copies in intact systems are computationallyextracted from tomograms and treated as three-dimensionalsingle particles. Alignment, classification and averaging of such tomogram subregions increases their signal-to-noise leveland combines different orientations, leading to significantimprovements in resolution (Schur  et al. , 2013). This canreveal molecular-level three-dimensional structures of previously inaccessible cellular assemblies, and has reachedsubnanometre resolution in favourable situations. Thismethod is still at an early stage of development and is likely tobe applicable to many important biological questions. 2.4. Correlative microscopy In order to determine the molecular structures of cellularassemblies, a useful approach is to image the same structureswith both cryo-ET and fluorescence microscopy (Fig. 3). Therapidly expanding power of fluorescence microscopy is usedto identify areas and events of interest within cells, whichare then examined in molecular detail by electron imaging.Correlative microscopy is well established for plastic-embedded EM samples, but fluorescence cryo-imaging is stillin its infancy, with limited commercial equipment and nocommercial cryo-immersion objectives, thus rendering it hardto fully exploit the developing panoply of super-resolutionlight microscopy methods. Nevertheless, various correlativestudies, including the first super-resolution imaging of flash-frozen live cells, have been reported (van Driel  et al. , 2009;Hagen  et al. , 2012; Chang  et al. , 2014; Perkovic  et al. , 2014;Kaufmann  et al. , 2014). Furthermore, the complete correlativechain requires live cell imaging, rapid cryo-preservation at aselected time, sample thinning and cryo-fluorescent imagingfollowed by EM. To obtain time and space correlationthroughout this chain remains a matter of active development.The same methodology is also required for the full exploita-tion of soft X-ray microscopy, which will be offered atbeamline B24 of Diamond. This emerging imaging area buildson sample-preparation procedures srcinally established forcryo-EM and therefore fits well into the correlative pipeline(Hagen  et al. , 2012). 2.5. Electron crystallography Electron microscopes configured for single-particle analysisand tomography can also be used for recording electrondiffraction. An important early development in three-dimen-sional EM was electron crystallography of two-dimensionalcrystals, particularly of membrane proteins (Henderson &Unwin, 1975). Two-dimensional crystals provide extendedrods of diffraction perpendicular to the plane of the crystals.To obtain the three-dimensional structure, data must becombined from many such crystals recorded at different tiltangles, thereby sampling different positions along thediffraction rods. In addition to electron diffraction, images of the crystals are recorded to allow correction of lattice disorderby using correlation methods to search for the actual locationsof unit cells. In practice, this approach has had limited appli-cation, primarily because of the difficulty of producing suffi-ciently large and well ordered two-dimensional crystals.However, a common byproduct of attempts to grow three-dimensional crystals for MX are microcrystals. If they are lessthan   500 nm thick, and can be vitrified in a thin layer on anEM grid, they can be analysed by electron crystallography. Ina recent application of this approach (microED), a series of electron diffraction patterns, each at very low electron dose, isrecorded during continuous tilt of the sample stage (Nannenga et al. , 2014). Combining diffraction data from a few crystalswith different orientations on the EM grid provided acomplete data set enabling structure determination at 2.9 A˚resolution (Shi  et al. , 2013; Fig. 1 c ). Currently, phasing of suchdata is performed by molecular replacement. This is poten-tially a significant development for challenging crystallo-graphic problems. research papers Acta Cryst.  (2015). D 71 , 127–135 Saibil  et al.   A national facility for biological cryo-electron microscopy  131 Figure 3 Integrated structural biology approach combining correlative light microscopy, soft X-ray cryo-microscopy and electron cryo-microscopy with high-resolution structure information, thus enabling the dissection of dynamic processes at different levels of resolution and complexity. The biologicalprocess is visualized by light microscopy (LM), transmission X-ray cryo-microscopy (cryo-TXM), electron cryo-tomography (cryo-ET), single-particlecryo-EM and/or macromolecular crystallography (MX). Adapted from Zeev-Ben-Mordehai  et al.  (2014).
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