University of Groningen

Electron MicroscopyBoekema, Egbert J.; Rögner, Matthias

Published in:EPRINTS-BOOK-TITLE

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:1996

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Boekema, E. J., & Rögner, M. (1996). Electron Microscopy. In EPRINTS-BOOK-TITLE

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 27-06-2020

Chapter 20Electron Microscopy

Egbert J. Boekema* and Matthias Rögner1

Bioson Research Institute, Biophysical Chemistry, University of Groningen, Nijenborgh 4,9747 AG Groningen, The Netherlands

1Institute of Botany, University of Münster, Schlossgarten 3, D-48149 Münster, Germany

SummaryI. Principles

A.B.C.D.E.

IntroductionThe Electron MicroscopeSpecimen PreparationAveraging MethodsPossibilities of EM in Terms of Resolution and in Relation to Object Size and Specimen Preparation

II. Periodic AveragingA.B.C.D.E.

Two-Dimensional CrystallizationPeriodic Averaging by Fourier MethodsAveraging of Photosystem I CrystalsHigh-Resolution EMLight-Harvesting Complex II

325326326326327328329330330330330331331332332332332333333333335335335

III. Single Particle AveragingA. Method

1.2.3.

Alignment by Correction MethodsMultivariate Statistical AnalysisClassification Step

B.C.

Analysis of Photosystem I Trimers from CyanobacteriaAnalysis of Photosystem II Dinners from Cyanobacteria

IV. Concluding RemarksAcknowledgementsReferences

Summary

Electron microscopy (EM) in combination with image analysis is a powerful technique to study proteinstructure at low- and high resolution. Since electron micrographs of biological objects are very noisy,substantial improvement of image quality can be obtained by averaging of individual projections.Averaging procedures can be divided into crystallographic and non-crystallographic methods and bothwill be described. Crystallographic averaging, based on two-dimensional crystals of rather small proteins,has the potential of solving a structure to atomic resolution just as the more common techniques of X-ray diffraction and NMR. Single particle analysis is an alternative method for large proteins, virusesand all non-crystallizable proteins. It is a fast method to reveal the low-resolution structure with detailsin the range of maximally 10–15 Å. Results of EM on Light-harvesting complex II (LHC-II) andPhotosystem I will be presented as examples for the crystallographic averaging. Trimeric PhotosystemI complexes and dimeric Photosystem II complexes will be discussed as examples for the potential ofsingle-particle averaging.

*Correspondence: Fax: 31-50-3634800; E-mail: boekema@chem.rug.nl

325J. Amesz and A. J. Hoff (eds.), Biophysical Techniques in Photosynthesis, pp. 325–336.© 1996 Kluwer Academic Publishers. Printed in the Netherlands.

326 Egbert J. Boekema and Matthias Rögner

I. Principles

A. Introduction

Direct information about the three-dimensional(3D) structure of a protein is essential for under-standing its functional organization. At presentelectron microscopy (EM) is a widely appliedtechnique for studying the structure of proteinsand membranes; however, it is still less commonthan X-ray diffraction, where solving the 3Dstructure of proteins becomes almost routine,once suitable crystals have been obtained. On theother hand, X-ray diffraction has two disadvan-tages in comparison to EM. First, X-rays cannotbe focussed and only diffraction patterns are ob-tained, whereas EM results in direct informationin the form of images. Second, the interaction ofX-rays with material is a factor of about 10,000weaker than that of electrons. This makes EMa useful technique as images of single proteinmolecules or one-layer thick crystals can be ob-tained, whereas X-ray diffraction needs muchthicker specimens.

In this chapter on EM, the first sections willbriefly introduce: the electron microscope withsome instrumental aspects (I.B); specimen pre-paration (I.C); image analysis averaging tech-niques (I.D) and finally the possible resolutionof EM with respect to object size and specimenpreparation (I.E). Sections II.A and III.A willfocus on image analysis of periodic and non-peri-odic objects including some examples.

For more details concerning theory of the elec-tron microscope, techniques for recording the sig-nal and image analysis, we refer to the book byHawkes and Valdrè (1990), which was written forthe field of protein structure determination.

B. The Electron Microscope

The resolution of a light microscope, which isabout 2000 Å, is mainly limited by the wavelengthof the light. Improving the resolution of a micro-scope is only possible by exploiting waves with amuch shorter wavelength. According to the wellknown formula of de Broglie, acceler-ated particles such as electrons also have a wavecharacter. At an acceleration voltage of 100,000

V the wavelength of the electron beam is 0.037 Å.This is certainly sufficient to enable microscopy atatomic resolution. Shortly after de Broglie haddescribed the wave character of particles, it wasdiscovered, and put into practice, that electronscan be focussed by electric and magnetic fieldswith axial symmetry. Based on these principles,Ernst Ruska and Max Knoll constructed the firstsimple electron microscope in 1931, a tube undervacuum with an electron source and several len-ses. Improvements in the following years en-hanced the resolution to 100 Å, already muchbetter than the resolution of a light microscope.Since the early days the electron microscope hasbeen gradually but substantially improved to aninstrument which can now routinely achieve aresolution of about 2 Å. This resolution is limitedby lens geometries and reflects compromises be-tween several optical parameters, such as minim-izing the spherical aberration, which is a kind oflens error. Overall, much further improvement inresolution cannot be expected, but 2 Å resolutionis sufficient to solve a protein structure at theatomic level.

To minimize the lens aberrations, the lenses inthe electron microscope have a small opening:the holes in lens apertures are less than 0.1 mmin diameter. This results in a large “depth offield” and “depth of focus” at the object planeand image plane, respectively. As a consequence,EM gives two-dimensional (2D) projections inwhich the upper- and lower side of a thin object(up to a few 1000 Å) are seen superimposed withthe same “sharpness”. As a result, a simple fo-cussing on selected levels in an object, as is pos-sible with a light microscope, cannot be donewith the electron microscope. To get informationabout the 3D shape of a protein, the specimenmust be tilted in the microscope and the variousprojections must be compared or combined intoa 3D reconstruction.

Imaging by EM of, for example, a thin metalfoil or a gold cluster will easily provide projec-tions with atomic resolution, but obtaining struc-tures of proteins at high resolution is much harderwork. Why? The contrast in the electron micro-scope is caused by scattering. Electrons are de-flected at atomic nuclei through large angles andby other electrons through small angles. Since the

Electron microscopy 327

scattering by nuclei is proportional to the atomicnumber, biological material, containing only thelighter elements, will give images with very lowcontrast. Besides, radiation damage by an elec-tron beam easily destroys biological samples. Ra-diation damage cannot be avoided, but only mini-mized by cooling the specimen to liquid nitrogenor liquid helium temperature and by minimizingthe electron dose. As a result, electron microg-raphs are noisy and objects are hardly visible.Therefore, image analysis techniques have beendeveloped to improve the signal recorded in thenoisy EM pictures.

C. Specimen Preparation

Since modern electron microscopes have enoughresolving power for structural studies of macrom-olecules, other factors than instrumental ones areof importance. The specimen preparation methodis one of these factors and it strongly determinesthe ultimate resolution that can be achieved. Inthe negative staining technique the contrast isenhanced by embedding proteins or protein crys-tals in a heavy metal salt solution. Upon drying,the metal salts fill spacings and cavities aroundthe molecules, but do not penetrate the proteininterior. As a result, negatively stained specimensshow protein envelopes with good contrast, butwith a resolution that usually does not exceed 15Å, due to the graininess of the contrasting agent.Because of its simplicity, negative staining hasbeen widely applied. Single particles as well ascrystals of photosynthetic membrane proteins,such as Photosystem I and II, have been success-fully prepared by negative staining (Boekema,1991; Boekema et al., 1994).

As an alternative for negative staining, Unwinand Henderson (1975) pioneered the embeddingof proteins in other media, such as glucose. Itwas demonstrated for crystals that images with aresolution of better than 10 Å could be achieved.This opened the way to high-resolution EM forbiological macromolecules. A second importantdiscovery in this context was the reduction ofradiation damage, leading to a better signal.Specimen holders were developed that could becooled down to liquid nitrogen-or liquid heliumtemperature. It was found for organic- and pro-

tein crystals that at the temperature of liquid ni-trogen radiation damage is roughly reduced by afactor of 3–5 and by a factor of 10–20 at liquidhelium temperature (Zemlin, 1992). Presently,the instrumental difficulties of practical cryo-EMhave been mostly solved and the performanceof the newest microscopes at low temperature isalmost as good as at room temperature.

Cryo-EM was further stimulated by the discov-ery of vitrification of protein solutions (Adrian etal., 1984). By rapidly cooling a protein solution,the formation of ice crystals can be avoided andproteins embedded in a thin layer of amorphousice are obtained. Contrast is caused by the differ-ence in density between amorphous ice (0.93

and protein and is ratherlow in comparison to negative staining. However,there are several advantages of cryo-EM of vitr-ified specimens: Specimen flattening and otherdrying artifacts are circumvented. Moreover,cryo-images better reflect the true density of aprotein, because the contrast directly originatesfrom scattering by the protein rather than fromthe surrounding stain (Fig. 1). Also, negativestain interaction with the protein is often quitecomplex. In thinner stain layers, the upper partof the protein could easily be less well embeddedin the stain layer, as pointed out in Fig. 1. Thismeans that the contribution of the upper- andlower half of a protein in the final recorded imagedo not have the same weighting.

Other techniques are less important for highresolution structural work. Freeze-fracture tech-niques have been widely applied in research onphotosynthesis. Cell or membranes are rapidlyfrozen, cleaved and replicated. The replicas giveuseful information about the overall size and dis-tribution of the complexes embedded in thesemembranes (Staehelin, 1988). But the resolutionis rather limited. Only particle diameters and theoverall shape of membrane protein can be ob-tained. The main value of these techniques liesin the imaging of the in vivo situation of themembranes. It can reveal crystalline packing ofphotosystems and sometimes the multimeric stateof large protein complexes. Evidence for the exis-tence of a dimeric organization of PS II in vivowas obtained by freeze-fracturing (Mörschel andSchatz, 1987).

328 Egbert J. Boekema and Matthias Rögner

D. Averaging Methods

In EM image analysis, improving the signal of anobject recorded in a noisy electron micrograph isperformed by averaging. By adding hundreds or,if possible, thousands of projections the signalimproves substantially and trustworthy electrondensity maps are obtained. There are two generalmethods for averaging of 2D projections, de-pending on the object. One method is based on

filtering images of periodic objects, which are usu-ally 2D crystals. The other one deals with single-particle projections.

Periodic averaging takes advantage of the factthat in crystals, protein molecules are arrangedin a regular packing. This means that neighbor-to-neighbor distances have a fixed value. In otherwords: the precise position of the molecules canbe easily determined, even if the crystal is re-corded with a low electron dose to prevent radi-ation damage and the molecules can be barelyseen. As a result, higher resolution can be ob-tained. If 2D crystals with a diameter of at leastseveral can be grown, EM can be performedunder cryo-EM conditions at high resolution. Forsome small membrane proteins with a mass of20–40 kDa, averaging over very large areas re-sulted in projection structures with a resolutionbetter than 5 Å. For bacteriorhodopsin, thethree-dimensional structure could be determinedentirely from EM data by fitting the amino acidchain into the electron density map (Hendersonet al., 1990). Another example is in the field ofphotosynthesis, where a second high resolutionstructure determination, that of the light-har-vesting complex II (LHC-II) from pea was re-cently completed (Kühlbrandt et al., 1994). Thecrystallographic method, which is based on Four-ier methods, is further described in section II,where results on LHC-II will be discussed in moredetail.

Projections of single particles can be averagedafter they have been brought into equivalent posi-tions by shifting them rotationally and transla-tionally. This a-periodic averaging technique orsingle particle analysis is able to reveal the pre-dominant projections of protein molecules (Franket al., 1988). The fact that crystallization of theprotein is not required is an advantage of thismethod. A disadvantage is that the maximal pos-sible resolution by single-particle analysis is re-stricted to about 10–25 Å. This limit is set by thesignal-to-noise ratio, which is related to the sizeof the object and has a relative low value forsmall objects. The resolution is also limited bythe fact that the particles are not fixed in a definiteposition, as in a crystal. Small tilts from a com-mon predominant position cause slight differ-ences between similar-looking projections, result-ing in an ensemble of projections that are all

Electron microscopy 329

slightly different. Averaging all of them wouldgive a sum in which the finest details would beblurred out.

E. Possibilities of EM in Terms ofResolution and in Relation to Object Sizeand Specimen Preparation

With the present state of the art in EM, as de-scribed in the previous sections, we can give anoverview of the possibilities of EM in the field ofproteins. Fig. 2 describes the potential of 5 typesof EM approaches in the field of proteins. Thethickness of the lines in Fig. 2 indicates the suit-ability and the attainable resolution in relation tothe molecular mass of a protein.

Approach 1. Single-particle averaging of nega-tively stained specimens is able to resolve thestructure up to 15 Å in favorable cases. This resol-ution is sufficient for the localization of subunitpositions in projections. Examples will be givenfor Photosystem I and II.

Approach 2. If the negative staining method isreplaced by cryo-EM of vitrified solutions, singleparticle averaging can be applied to objects with amass of at least a few hundred kDa. From smallerproteins, especially those with a mass of 100 kDaor lower, the projections from single moleculesas recorded by EM are too noisy for accurateaveraging. Approach 2 works better than ap-

proach 1 for larger objects, for reasons mentionedbefore, such as removing the flattening upon dry-ing. Another reason is the lower contrast of cryo-EM, which becomes relatively disadvantageousfor small molecules, as we will briefly explain.The contrast in biological material is largelycaused by the scattering of the electron cloud ofthe C, N and O elements. This contrast is calledphase contrast. The contrast originating from in-teraction with atomic nuclei, the scattering con-trast, is relatively unimportant. Enhancement ofphase contrast is possible by a stronger defo-cussing of the objective lens. But this coincideswith a degradation and loss of fine details in theimage. The smaller the object, the larger the de-focussing needed to see the object at all.

Approach 3. Periodic averaging of stained ob-jects. The advantage of a periodic object is itsregular packing. This enables an accurate deter-mination of the positions of the molecules andthus an accurate averaging of projections. Forcrystals of about in size, a resolution ofabout 15–25 Å is usual. Small proteins (10–50 kDa) are advantageous, because they allowaveraging over a larger number of molecules perarea.

Approach 4. Cryo-EM of unstained, periodicobjects. As for approach 2, the contrast comeslargely from phase contrast. But crystals can beconsidered as a “big object” and can be recordedwith small defocus values. Although the signal ofone individual molecule is almost buried in thenoise, it will still be interpretable, because it isaveraged periodically. Small crystals of organicmolecules and small proteins of a few in sizecan be analyzed to high resolution. For such smallmacromolecules, EM provides the same qualityas X-ray diffraction, although the image analysisis not as straightforward.

Approach 5. Based on perfect, large 2D crys-tals atomic resolution is possible for proteins upto at least 50 kDa. For larger proteins, the resol-ution will gradually decrease. For proteins with amass between 300 and 600 kDa, like PhotosystemI and II and ATP synthase, solving the structureat high-resolution by EM would be difficult. Thepreferential size of 2D crystals of such objects isat least Such large crystals are difficultto grow and have not yet been obtained.

330 Egbert J. Boekema and Matthias Rögner

II. Periodic Averaging

A. Two-Dimensional Crystallization

As indicated in Fig. 2, small proteins should becrystallized into 2D crystals, to obtain the beststructural information. The best 2D crystals pro-duced in vitro have been grown from detergent-solubilized, purified material. Starting fromhighly purified protein, crystallization conditionscan be controlled more easily and results aremore reproducible (Kühlbrandt, 1992; Jap et al.,1992; Engel et al., 1992). Reconstitution of mem-brane proteins into lipid bilayers by detergentremoval is currently the most universal methodof 2D crystallization. A suspension of a lipid-detergent mixture is usually added to a detergent-solubilized protein preparation. Crystallization ofthe protein into sheets or vesicle crystals is theninduced by removal of detergent by means ofdialysis or absorption.

B. Periodic Averaging by Fourier Methods

Usually, images of 2D crystals are recorded onelectron micrographs. Fourier analysis has beenproven to be very valuable in the processing ofmicrographs. The Fourier transform is a fre-quency decomposition in reciprocal space. A 2DFourier transform calculated from an image givesa 2D diffraction pattern. If the image shows agood 2D crystal, its transform will show a patternwith many spots laying on a regular pattern, asin X-ray diffraction patterns. These spots repre-sent the structure factor amplitudes and have anamplitude (peak height) and a phase. But thenoise in the images will also show up in the dif-fraction pattern. An illustration of the Fouriertechniques will be given in the next section; itshows an effective way to get rid of the noise.

elongatus (Böttcher et al., 1992) by removal ofdetergent (Fig. 3A). In a 2D Fourier transformof the PS I crystal (Fig. 3B), we see spots atregular distances. They tell us about the latticeparameters and about resolution. In this case, thelattice of the crystal is rectangular.

Based on the Fourier transform of the image,a filtering is performed. Computationally, a maskis constructed that is superimposed on the Fouriertransform (Fig. 3C). The mask has holes thatneatly surround the peaks, which contain thecrystalline information. The mask shields thespace between the peaks, which represents noisein the crystal image. With a reverse Fourier-trans-formation (Fig. 3D) a real image is generatedagain, in which much of the noise is removed.This procedure is called Fourier-peak filteringand the comparison between Figs. 3E and F illus-

C. Averaging of Photosystem I Crystals

At medium resolution no complicated strategy isnecessary since correcting for image aberrations isonly necessary for high-resolution EM (see II.D).We will show by a simple example the basics ofperiodic averaging. Monomeric Photosystem I(PS I) has been crystallized into two-dimensionalarrays from the cyanobacterium Synechococcus

Electron microscopy 331

trates the considerable improvement in signal-to-noise ratio.

Further improvement in filtering is also pos-sible. As crystals never have a perfect lattice, asmall bending in the plane results in moleculesbeing slightly displaced from their ideal position.Corrections can be made by “cutting” the crystalinto pieces containing one or several molecularprojections and shifting them into their ideal posi-tion. This is done by correlation methods, whichhave also been used in single particle averagingmethods (Frank, 1982). An application of thismethod is the analysis of PS I crystals (Böttcheret al., 1992).

D. High-Resolution EM

While a low-resolution structure by single particleaveraging can be obtained within weeks, obtain-ing a high-resolution structure from EM data maytake years. One of the limits of a structure deter-mination at high resolution is merely the produc-tion of well-ordered, large crystals. Once this pre-requisite is fulfilled, a resolution better than 10Å is possible. However, recording the highestquality images and extensive processing are alsotime consuming. The signal recorded by EM suf-fers from aberrations, which increase in severityas the required resolution becomes higher (Hend-erson et al., 1986). Images providing structuralinformation beyond a resolution of about 5 Åneed extensive processing, which is carried out inFourier space on the raw image amplitudes andphases. The phases are corrected for the effectsof the contrast transfer function, beam tilt andphase origin (Henderson et al., 1986). A furthertreatment and discussion of these image distor-tions and their analysis and correction is beyondthe scope of this contribution. For the interestedreader we refer again to the book of Hawkesand Valdrè (1990), as a useful source of furtherinformation, and to the paper of Henderson etal. (1990).

The microscope can be configured in two dif-ferent ways: a) image mode and b) diffractionmode, i.e. recording the image formed in theback-focal plane of the objective lens. Electrondiffraction (ED) patterns of crystals contain manyspots, as in X-ray diffraction patterns and alsorepresent the structure factors without the phase

information that is present in calculated Fouriertransforms. ED patterns usually have muchstronger spots than those from calculated trans-forms. The reason possibly lies in the specimenmovement during image recording. Fourier trans-formation and diffraction in an optical systemhave the property that they are “translation in-variant”. This means that movements during therecording of an ED pattern are less dramatic thanduring the recording of a real image, because theydo not result in blurring the ED. Therefore spotsin ED are much stronger and this makes re-cording of only ED for high resolution worktempting. But then a “phase problem”, as in X-ray diffraction, is created and phases need to begenerated. Isomorphous replacement, as used inX-ray diffraction, is not a useful method for phas-ing the ED data from 2D protein crystals becausethe scattering contrast of heavy atoms is low forelectrons and the noise level in the patterns isrelatively high. To compute a protein map athigh-resolution by Fourier methods, images, arerecorded as well. They are necessary to extractthe phase from each of the spots. The phases(from the images) and amplitudes (from ED) arefinally combined and corrected for some imageerrors, briefly mentioned in section I.D. This iscalled a “Fourier synthesis”. An example of theFourier techniques for high-resolution structuredetermination will be given in the next section.

E. Light-Harvesting Complex II

The light-harvesting chlorophyll a/b protein com-plex associated with photosystem II (LHC-II)harvests light energy and is capable of passing itto photosystem II. Detergent-solubilized, purifiedLHC-II forms large, highly ordered 2D crystals,which are ideal objects for recording high-resol-ution EM. Based on the averaging methods de-veloped by Henderson et al. (1990), Kühlbrandtet al. (1994) were able to calculate a 3D structureat 3.4 Å resolution. Images were recorded withan electron microscope capable of resolving 2 Åobject features at a specimen temperature of 4.2K. Tilted and untilted images were collected fora 3D reconstruction. Since it is difficult to recordimages at tilt angles above 60°, there was a regionin the Fourier space where amplitudes and phasescould not be measured. This caused a reduction

332 Egbert J. Boekema and Matthias Rögner

in the resolution in the direction perpendicular tothe membrane plane to between 4.6 and 4.7 Å.But due to the high quality of the phases it waspossible to trace the polypeptide chain in the 3Delectron density map. About 80% of the aminoacids could be fitted, as well as the tetrapyrrolerings of 12 chlorophylls and two carotenoids (Fig.4).

LHC-II is the second protein solved by EM toatomic resolution. No doubt, the work on LHC-II is a major step forward both for EM and forphotosynthesis. To stress the similarities with X-ray crystallography, the term electron crystal-lography has been introduced for high-resolutionprotein determination by EM.

III. Single-Particle Averaging

A. Method

Isolated proteins prepared on a carbon supportfilm exhibit a full range of rotational and transla-tional orientations in the plane of the supportfilm. As a consequence, the projections of theproteins have a random orientation within thisplane and computer averaging of such projectionscan only be achieved after an alignment proce-dure. Also, proteins often are attached in variousways to the support film (Fig. 1) and this resultsin a further variety in the obtained projections.To separate the various predominant projections,multivariate statistical analysis together withautomated classification was introduced (VanHeel and Frank, 1981). The alignment procedure,multivariate statistical analysis and classificationform the main steps in the single particle averag-ing procedure (Frank et al, 1988). These stepswill be briefly discussed.

1. Alignment by Correlation Methods

The first step in comparing images of projectionsof biomolecules is to bring them into register inthe plane: the alignment process. A referenceimage is taken and each image in the data setis compared with this reference; rotational andtranslational cross-correlation functions are com-puted to determine the best angular and transla-tional shifts to bring the image into a positionmost similar to the reference image. The align-ment procedure is an iterative process becausesums of projections align the data set better thandoes a noisy projection of just one molecule. Inpractice, several references are used and resultsare combined to circumvent a possibly wrongchoice of the first reference.

2. Multivariate Statistical Analysis

Once a data set of typically hundreds of projec-tions has been aligned, they can be compared insome numerical way. In particular, correspon-dence analysis, a special form of principal compo-nent analysis, is used to extract relevant featureinformation from the data set (Van Heel andHarauz, 1988). Each image of × pixels can be

Electron microscopy 333

represented as a point in an × dimensional “hyp-er”space, and the entire set of images forms acloud in this space. Correspondence analysis de-termines a new, rotated-coordinate system inwhich the first axis represents the direction ofthe greatest inter-image variance, the second axisrepresents the direction of the largest remaininginter-image variance, and so on. The cloud ofimages can now be described with respect to thisnew coordinate system. By describing each imagewith respect to only the first few components, theimages can be considered as points in a muchsmaller (than n × n) dimensional space. In thisway a very large reduction in the amount of datato be analyzed is achieved.

3. Classification Step

After the data compression by correspondenceanalysis, the grouping together of those imagesthat are most similar is achieved by automaticclassification schemes. Output of the classificationare “classes” of groups of projections that aremost similar. In the classification process, projec-tions are shifted between the classes to optimizethe variance between the classes and to minimizethe variance of the members belonging to theclasses. The number of classes to be chosen issomewhat arbitrary, but usually a number of 6–12 are chosen. The differences between classesmay represent real structural features (as will beillustrated by two examples) or are merely noise-related when there is not more than one type ofprojection present in the data set.

B. Analysis of Photosystem I Trimers fromCyanobacteria

A first practical example of single particle analysisconcerns Photosystem I. In cyanobacteria, suchas the thermophilic Synechococcus elongatus andthe mesophilic Synechocystis PCC 6803, PS I isarranged as a trimeric complex with a diameterof about 200 Å in the membrane (Boekema etal., 1989; Kruip et al., 1993). From electron mic-rographs of PS I from Synechocystis PCC 6803(Kruip et al., 1993), top view projections (Fig. 5)were extracted, aligned, treated by correspon-dence analysis and classified. The analysis re-sulted in the separation of the top view projec-

tions into two types, which are mirror-related(Fig. 6). These two types are generated becauseparticles are asymmetric and can be attached tothe carbon support film in two ways (upside-upand upside-down or “flip” and “flop”). It shouldbe noted that it is not easy to judge by eyewhether the PS I trimer projections (Fig. 5) be-long to the flip or flop-type, but after a 3–foldaveraging most of them (but not all) can be classi-fied by eye.

C. Analysis of Photosystem II Dimers fromCyanobacteria

Photosystem II is another membrane protein witha size large enough to enable single particle analy-sis. Dimeric particles were purified from the Syne-chococcus elongatus (Rögner et al., 1987; Dekker

334 Egbert J. Boekema and Matthias Rögner

et al., 1988). We visualized these particles in thepresence of the detergent dodecyl maltoside bynegative staining with uranyl acetate (Boekemaet al., 1994). Averaged top views of dimeric PSII show that the dimers are built up from twomonomers which are arranged in an anti-parallelway (Fig. 7A). In contrast to the example ofPS I top view projections, there was only onepredominant top view present in the data set.The dimer has dimensions of 120 × 155 Å in themembrane (corrected for detergent). The sideviews, however, show more variations. Originalimages clearly show protrusions in the side-viewposition (see Fig. 9 in Boekema et al., 1994).Often two protrusions can be seen on one dimer,but sometimes only one is visible. The protrusions

are thought to represent the 33 kDa oxygen-evolving subunit resulting in a maximal height of90 Å for the particles. Image analysis of sideviews gives another example of the usefulness ofsingle particle analysis. By correspondence analy-sis and classification, a discrimination betweendifferent views was possible, which we interpretas overlap- and non-overlap views. In the non-overlap views two protrusions can be seen (Fig.7D, E), whereas the overlap view shows one pro-trusion (Fig. 7F). Note that the non-overlap viewshows a longer projection than the overlap views,indicating a different position of the respectivedimers on the carbon support.

Electron microscopy 335

IV. Concluding Remarks

In conclusion, EM techniques are available tostudy isolated (membrane) protein complexesfrom very small to very large size. In combinationwith techniques used in cell biology for studyingwhole cells or cell fragments, EM has the possibil-ity and the potential to supply the field of photo-synthesis with a detailed picture of the photosyn-thetic membrane and its interacting proteins. Forstructure determination, crystals are more usefulthan single particles. But not in all cases will itbe possible to grow good crystals in a short termfor such complicated structures as PS I, PS II andthe synthase complex including theirinteracting donor-, acceptor- and regulatory mol-ecules and proteins. In the meantime a combi-nation of low-resolution EM reconstructions ofthese photosynthetic membrane complexes withatomic structures of their individual components,determined by EM, NMR or X-ray diffraction,will be useful for understanding the structure andfunction of these proteins.

Acknowledgements

We are grateful to Dr. W. Keegstra for his helpwith computer image analysis, Dr. G. Perkins fordiscussion and Mr. K. Gilissen for photography.Work has been supported by the NetherlandsFoundation for Chemical Research (SON) withfinancial aid from the Netherlands Organizationfor Scientific Research (NWO), by a grant fromthe European Union BIO2CT-930078 (EJB), theDeutsche Forschungsgemeinschaft (MR) and agrant from NEDO/RITE, Japan (MR).

References

Adrian M, Dubochet J, Lepault J, McDowall AW (1984)Cryoelectron microscopy of viruses. Nature 308: 32–36

Boekema EJ (1991) Negative staining of integral membraneproteins. Micron and Microsc Acta 22: 361–369

Boekema EJ, Dekker JP, Rögner M, Witt I, Witt HT andvan Heel, MG (1989) Refined analysis of the trimeric struc-ture of the isolated Photosystem I complex from the therm-ophilic cyanobacterium Synechococcus sp. Biochim Bi-ophys Acta 974: 81–87

Boekema EJ, Boonstra AF, Dekker JP and Rögner (1994)Electron microscopic structural analysis of Photosystem I,Photosystem II, and the cytochrome b6/f complex from

green plants and cyanobacteria. J Bioenerg Biomembr 26:17–29

Böttcher B, Gräber P and Boekema EJ (1992) The structureof Photosystem I from the thermophilic cyanobacteriumSynechococcus sp. determined by electron microscopy oftwo-dimensional crystals. Biochim Biophys Acta 1100:125–136

Dekker JP, Boekema EJ, Witt HT Rögner M (1988) Refinedpurification and further characterization of oxygen-evolv-ing and Tris-treated Photosystem II particles from the ther-mophilic cyanobacterium Synechococcus sp. Biochim Bi-ophys Acta 936: 307–318

Engel A, Hoenger A, Hefti A, Henn C, Ford RC, Kistler Jand Zulauf M (1992) Assembly of 2–D membrane proteincrystals: dynamics, crystal order, and fidelity of structureanalysis by electron microscopy. J Struct Biol 109: 219–234

Frank J (1982) New methods for averaging non-periodic ob-jects and distorted crystals in biologic electric microscopy.Optik 63: 67–89

Frank J, Radermacher M, Wagenknecht T and Verschoor A(1988) Studying ribosome structure by electron microscopyand computer-image processing. Methods in Enzymology164: 3–35

Hawkes PW and Valdrè U (1990) Biophysical Electron Micro-scopy. Basic concepts and modern techniques. AcademicPress, London

Henderson R, Baldwin JM, Downing KH, Lepault J andZemlin F (1986) Structure of purple membrane from Halo-bacterium halobium: recording, measurement and evalu-ation of electron micrographs at 3.5 Å resolution. Ultram-icroscopy 19: 147–178

Henderson R, Baldwin JM, Ceska TA, Zemlin F, BeckmannE and Downing KH (1990) Model for the structure ofbacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol 213: 899–920.

Jap BK, Zulauf M, Scheybani T, Hefti A, Baumeister W,Aebi U and Engel A (1992) 2D crystallization: from art toscience. Ultramicroscopy 46: 45–84

Kruip J, Boekema EJ, Bald D, Boonstra AF and Rögner M(1993) Isolation and structural characterization of monom-eric and trimeric Photosystem I complexesand from the cyanobacterium Synechocystis PCC6803. J Biol Chem 268: 23353–23360

Kühlbrandt W (1992) Two-dimensional crystallization ofmembrane proteins. Quaterly Rev of Biophys 25: 1–49

Kühlbrandt W, Wang DN and Fujiyoshi Y (1994) Atomicmodel of plant light-harvesting complex by electron crystal-lography. Nature 367: 614–621

Mörschel E and Schatz GH (1987) Correlation of photosyst-em-II complexes with exoplasmic freeze-fracture particlesof thylakoids of the cyanobacterium Synechococcus sp.Planta 172: 145–154

Rögner M, Dekker JP, Boekema EJ and Witt HT (1987) Size,shape and mass of the oxygen-evolving photosystem IIcomplex from the thermophilic cyanobacterium Synechoc-occus sp. FEBS Lett 219: 207–211

Staehelin LA (1988) Chloroplast structure and supramolecularorganization of photosynthetic membranes. In: StaehelinLA and Arntzen CJ (eds.) Photosynthesis III, pp. 1–84,Springer-Verlag, Berlin

336 Egbert J. Boekema and Matthias Rögner

Unwin PNT and Henderson R (1975) Molecular structuredetermination by electron microscopy of unstained crystal-line specimens. J Mol Biol 94: 425–440

Van Heel M and Frank J (1981) Use of multivariate statisticsin analysing the images of biological macromolecules. Ul-tramicroscopy 6: 187–194

Van Heel H and Harauz G (1988) Biological macromoleculesexplored by pattern recognition. Scanning Microscopy,Supplement 2: 295–301

Zemlin F (1992) Desired features of a cryoelectron micro-scope for the electron crystallography of biological ma-terial. Ultramicroscopy 46: 25–32