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Micron 96 (2017) 57–64

Contents lists available at ScienceDirect

Micron

j ourna l ho me page: www.elsev ier .com/ locate /micron

eview

ffects of radiation damage in studies of protein-DNA complexes byryo-EM

. Mishyna a,∗, O. Volokh a, Ya. Danilova a, N. Gerasimova a, E. Pechnikova b,.S. Sokolova a,∗

Lomonosov Moscow State University, 119234, Moscow, RussiaThermo Fisher Scientific, Materials & Structural Analysis, 5651 GG Eindhoven, Netherlands

r t i c l e i n f o

rticle history:eceived 7 December 2016eceived in revised form 18 February 2017ccepted 18 February 2017vailable online 21 February 2017

a b s t r a c t

Nucleic acids are responsible for the storage, transfer and realization of genetic information in the cell,which provides correct development and functioning of organisms. DNA interaction with ligands ensuresthe safety of this information. Over the past 10 years, advances in electron microscopy and image process-ing allowed to obtain the structures of key DNA-protein complexes with resolution below 4 Å. However,radiation damage is a limiting factor to the potentially attainable resolution in cryo-EM. The prospect andlimitations of studying protein-DNA complex interactions using cryo-electron microscopy are discussed

eywords:NAroteinsryo-electron microscopy (cryo-EM)rotein-DNA complexesadiation damage

here. We reviewed the ways to minimize radiation damage in biological specimens and the possibilitiesof using radiation damage (so-called ‘bubblegrams’) to obtain additional structural information.

© 2017 Elsevier Ltd. All rights reserved.

ubblegrams

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572. Structural basis of protein-DNA interactions studied by cryo-EM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

2.1. Large protein-DNA complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582.2. Nucleoid in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592.3. Viral protein-DNA complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

3. Radiation damage in cryo-EM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614. ‘Bubblegrams’ reveal the disguised protein structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .625. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

. Introduction

DNA organization into chromatin protects its primary structurerom damage, provides vital functions of the cell, and mediates theransfer of genetic material between cells. Ligands (ions, antibiotics,

Low molecular weight substances, such as metal ions, the onlytraces of which can be found in cells, strongly affect the structuralcharacteristics of DNA by introducing single- and double-strandbreaks which lead to inhibition of the protein synthesis (Sissi et al.,2005). Copper, manganese and nickel ions (Desoize, 2002) bear

igh-molecular weight compounds) actively participate in the for-ation of the chromatin structure (Alberts et al., 2002; Boyle et al.,

008 Travers and Muskhelishvili, 2015).

∗ Corresponding authors.E-mail addresses: maryia.mishyna@gmail.com (M. Mishyna),

okolova@mail.bio.msu.ru (O.S. Sokolova).

ttp://dx.doi.org/10.1016/j.micron.2017.02.004968-4328/© 2017 Elsevier Ltd. All rights reserved.

strong carcinogenic and mutagenic effects on DNA. Simultaneously,metals such as platinum are used in various drugs, for instance cis-platin (Bury et al., 1987; Desoize and Madoulet, 2002; Mohamedand Soliman, 2010).

The second type of DNA-binding ligands includes small organic

molecules, such as dyes, antibiotics and anti-cancer drugs. Theseligands can be divided into two classes, depending on their inter-actions with the DNA: (i) ligands with a chain structure (Fig. 1A)

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58 M. Mishyna et al. / Micro

Fig. 1. The variety of DNA-ligand interactions. (A) small molecules – chain structure.Left, netropsin. Right, interaction of netropsin with DNA; (B) small molecules – inter-calators. Left, actinomycin D. Right, interaction of actinomycin D with DNA; (C,D)macromolecular protein-DNA complexes. (C) crystal structure of the nucleosome.PDB ID – 1KX5 (Davey et al., 2002); (D) cryo-EM structure of chromatin remodelingfe

(i(ddcDlwu

apntgtW1

was resolved.

actor ISW1a, bound to a nucleosome. n – nucleosome; EMDB ID – 1878 (Yamadat al., 2011).

Davey et al., 2002) that fit into grooves on the DNA surface andnteract with phosphate groups and bases; (ii) intercalating agentsFig. 1B) which lodge between DNA base pairs, push them apart andeform the structure of the helix (examples: transcription blockersistamycin-A and actinomycin-D). Interactions with small ligandshange the physical, chemical and mechanical properties of theNA helix, its flexibility and the distance between bases. Small

igands can disrupt the interactions of DNA with large enzymes,hich, in turn, lead to transcription blocking, and are also often

sed for therapeutic purposes (Sheng et al., 2013).Large proteins form the third type of DNA-ligand interactions

nd are generally found in chromatin (Fig. 1C) and its multi-le remodeling factors (Fig. 1D). The structural-functional role ofuclear chromatin in eukaryotes is substantial: it exposes the DNAo cellular enzyme systems, executing the natural mechanism ofene activity regulation. DNA fragments that are associated with

he nucleosomes become inaccessible to nucleases (Noll, 1974;

igler and Axel, 1976) and small intercalating ligands (Hayes et al.,990).

n 96 (2017) 57–64

Since the conformational state of the macromolecular complexgenerally reflects its functional activity (Alberts et al., 2002), knowl-edge of the spatial structure enables the researcher to interpretand predict the changes in the functioning molecule. Interactions ofDNA with ions, low molecular weight substances and small proteinsare investigated by X-ray crystallography, computational dockingand molecular dynamic simulations (Lengauer and Rarey, 1996;Rossmann, 2000; Volokh et al., 2015). Complexes of DNA with largerproteins are investigated using cryo-electron microscopy (cryo-EM).

The cryo-EM approach is set in motion by spreading solublehomogenous protein-DNA complexes across a hole in a carbonfilm, creating a thin layer from a few hundred to a few thousandÅngstroms (Milne et al., 2013; Thompson et al., 2016). Then, thefilm is promptly frozen by plunging it into cryogen (McDonald andAuer, 2006; Thompson et al., 2016; Tivol et al., 2008). Imaging ofvitrified specimens, choice of electron source, electron detector andhardware have been previously discussed in detail (Cheng, 2015;Milne et al., 2013; Thompson et al., 2016). Image processing of2D projections to a 3D reconstruction and its refined structure isachieved using specialized computer programs, such as the: EMAN(Ludtke et al., 1999), RELION (Scheres, 2012), SPRING (Desfosseset al., 2014), Bsoft (Heymann et al., 2008), Frealign (Lyumkis et al.,2013), etc.

One of the main limitations related to DNA-ligand structuralevaluation is lack of pure and stable complexes with no or lit-tle flexibility. To improve and facilitate applications of electronmicroscopy to those objects both sample preparations, as well asdifferent microscopy techniques, are being developed. During thelast decade, the resolution, achieved for 3D reconstructions of DNA-protein complexes went down from about 30 Å to 3.9 Å (Table 1).

The latest developments include stain-free sample prepara-tion using a super-hydrophobic surface with further imagingusing high-resolution transmission electron microscopy (HRTEM)(Gentile et al., 2012). Using this approach we can detect the DNAsdouble helix and measure its helical characteristics without stain-agent bias, which can lead to new opportunities for studying DNAin different complexes with ligands, including small organic andinorganic molecules with a potential resolution of 1.5 Å (Mariniet al., 2016). Along with the development of classical TEM methods,a DNA damage response imaging technique with use of positronemission tomography (PET) and single-photon emission computedtomography (SPECT) was recently developed (Knight et al., 2017).These promising approaches use DNA ligands radiotracers to visu-alize DNA damage response, which can lead to new opportunitiesin the oncology field.

2. Structural basis of protein-DNA interactions studied bycryo-EM

2.1. Large protein-DNA complexes

3D structures of large protein complexes with nucleic acid(RNA), obtained from single particle reconstruction, have beenstudied since the 1960s. In fact, due to their large size, relative sta-bility and the availability of established purification protocols, the70S ribosome and plant RNA-containing viruses were the first tobe analyzed by single particle electron microscopy (Frank, 2002).In contrast, with the exception of the DNA itself, DNA-containingprotein complexes have avoided cryo-EM studies for a long time. Inparticular, using the cryo-approach, the hexagonal packing of DNAin the head of the giant bacteriophage (Fokine et al., 2005, 2004)

Structural studies of DNA-protein complexes began with theinterpretation of chromatin on raw cryo-images (Athey et al., 1990).This approach did not provide much resolution, but demonstrated

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M. Mishyna et al. / Micron 96 (2017) 57–64 59

Table 1The protein-DNA complexes, solved by cryo-EM and image processing.

Protein-DNA complex Resolution, Å(FSC)

Number ofparticles used

Microscope/Image Detector

Software package usedfor image processing

Reference

N4 virus 30 (0.5) 2096–2506 CM200 (Philips)/Zeiss scanner EMAN (Choi et al., 2008)SWR1 28 (0.5) 32,000 Tecnai F20(FEI)/CCD camera

(Gatan)EMANCTFTILTIMAGICSPIDERRELION

(Nguyen et al., 2013)

RSC–nucleosome 25 (0.5) ∼37,000 CM200 (Philips)/Zeiss scannerTecnai F20 (FEI)/Zeiss scanner

SPIDER (Chaban et al., 2008)

SWI/SNF-Nucleosome 23 (0.5) no information Tecnai 12 (FEI)/CCD camera(Gatan)

EMAN (Dechassa et al., 2008)

Elongation complex(RNAP–nucleosome)

22 (0.5) 8500 JEM-2100 (JEOL)/UltraScan4000 CCD (Gatan)

EMAN2IMAGIC5

(Gaykalova et al., 2015)

OCCM 14 ∼90,000 JEM-2010F (JEOL)/UltraScan4000 CCD (Gatan)

EMAN2 (Sun et al., 2013)

PolIII-clamp-exonuclease-�c

8 (0.143) 63,215 Titan Krios (FEI)/K2 summit(Gatan)

MOTIONCORRCTFFIND3RELION 1.3EMAN2

(Fernandez-Leiro et al.,2015)

Nucleosome-NuA4 7.9 (0.143) 168,802–390,201 Titan Krios/Falcon II (FEI) CTFFIND3RELION 1.3

(Xu et al., 2016)

MCM2–7 helicase 3.8 85,366 Titian Krios (FEI)/K2 summit(Gatan)

RELION 1.3 (Li et al., 2015)

CMG helicase 3.7–4.8 (0.143) 687,794 Titian Krios (FEI)/K2 Summit(Gatan)

CTFFIND4RELION 1.3RELION 1.4

(Yuan et al., 2016)

i ArctiKrios

n)

tacaEdrps

w

r2(scrosscstttiecnhotrFoe

Nucleosome corereconstituted withDNA

3.9 (0.143) 26,060 TecnaTitan

(Gata

he variable thickness of 30 nm chromatin fibers in vitrified icend allowed the authors to suggest a solid-solenoid model of thehromatin structure. This model was later supported by Robinsonnd co-authors (Robinson et al., 2006). Another raw-imaging cryo-M study held ten years later (Bednar et al., 1999) suggested that,uring transcription through the nucleosome, the histone octameremains structured and does not lose single histones. Thus, as aart of chromatin, nucleosomes are stable upon changes in the DNAtructure (Odell et al., 2013).

Large DNA-containing complexes are well suited to be studiedith the single particle cryo-EM approach (Fig. 1B,C).

Using cryo-EM, several reconstructions of large chromatin-emodeling factors were obtained: SWR1 (∼1 MDa) (Nguyen et al.,013), SWI/SNF (∼1.2 MDa) (Dechassa et al., 2008), RSC (∼1.2 MDa)Chaban et al., 2008), ISW1a-�ATPase in complex with nucleo-ome (Fig. 1D) (∼500 kDa) (Yamada et al., 2011). All complexes areomposed of protein and at least a small fragment of DNA. Theesolution of all reconstructions was below 2 nm, mostly becausef their flexibility. There is a common practice of using crystaltructures of known domains and/or subcomplex parts for the con-truction and interpretation of a molecular model of the wholeomplex (Wriggers and He, 2015). In the absence of the crystaltructures in databases, homology modeling is a good alternativehat may be used. Homology modeling is based on the observationhat the spatial structure of proteins is much more conservativehan their amino acid sequence (Pils et al., 2005). 30% sequencedentity is considered to be sufficient for the construction of mod-ls. To interpret the cryo-EM structure of the ISW1a-(�ATPase)omplex with nucleosome, crystal structures of HSS-Ioc3 anducleosome were docked into the EM density (Davey et al., 2002). Itas been demonstrated that ISW1a may interact with two portionsf the DNA linker simultaneously. First, the ISW1a complex binds tohe linker, which will later be translocated. Once the linker section

eaches the desired length, the ISW1a blocks further translocation.or the interpretation of the SWR1 structure, the crystal structuref the RuvBL1 hexamer (Matias et al., 2006) was docked into thelectron density (Nguyen et al., 2013).

ca/Falcon2 (FEI)(FEI)/K2 Summit

EMAN2RELION 1.3

(Chua et al., 2016)

Recently, a breakthrough in cryo-EM methodology (Glaeser,2016a), due to the invention of direct electron detectors andsubsequent correction of beam-induced specimen movement(Henderson, 2015), allowed to receive high-resolution structuresof DNA-protein complexes: the nucleosome (Chua et al., 2016) andthe transcription initiation complex, which included the RNA poly-merase and DNA in open and closed conformations (Plaschka et al.,2016). At high resolution, the side chains of the amino acids aredetectable. But, in this case, inevitable electron damage should betaken into account. Experience shows that most sensitive to radi-ation are the cysteine, aspartate and glutamate residues (Fig. 2D)(Allegretti et al., 2014). Some disulfide bonds in a protein moleculebecame distorted at an electron dose as little as 5 e/Å2, and anaccelerating voltage of 1 MeV (Baker and Rubinstein, 2010).

2.2. Nucleoid in E. coli

In prokaryotes, the circular DNA does not form a pronounced‘chromatin’, but has been shown spatially ordered (Ryan andShapiro, 2003; Shapiro and Losick, 2000). A number of DNA-bindingproteins in bacteria that control the dynamic reorganization ofbacterial nucleoids form a dense net in the cytoplasm and are capa-ble to produce an even more complex structure, which resemblesthe mitotic apparatus in eukaryotes (Niki et al., 2000; Niki andHiraga, 1998). Interestingly, DNA-binding proteins in bacteria arecapable of crystallization under stress conditions (Frenkiel-Krispinet al., 2004). A range of scientific papers published recently (Gallatet al., 2014; Koopmann et al., 2012) suggest in vivo crystallizationas a new line of research in the field of structural biology. Theadvantages of in vivo crystallization are: the production of post-translationally modified proteins, easiness of isolation by spinningdown the crystals after cell lysis, and the possibility of analyzingcrystals by TEM.

The structure of in vivo crystals can be investigated using elec-tron tomography. This approach deals with large cellular organelles(Bharat and Scheres, 2016) and whole prokaryotic cells (Kishimoto-Okada et al., 2010; Murata et al., 2016). Moreover, recently it

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60 M. Mishyna et al. / Micron 96 (2017) 57–64

Fig. 2. The source and results of electron damage. (A) The schematic for progress of radiation damage in the TEM; (B) Influence of radiation damage onto resolution of the3D reconstruction of protein molecule. Reconstruction of amino acids 124–144 of F420 hydrogenase calculated from images obtained (left) with low electron dose ≈10 e/Å2

(resolution 3.36 Å), (middle) an increased dose of ≈24 e/Å2 (resolution 3.94 Å), and (right) high-dose ≈49 e/Å2 (resolution 4.16 Å). The side chains of Asp125 and Glu132u om (AB fter irg

hBit9m0mgbr

2

p

nderwent damage already after the first irradiation. Reprinted with permission fracteriophage EL, frozen in amorphous ice, no damage (left). Similar bacteriophage aas (arrows). Reprinted with permission from RAS publishing agency.

as demonstrated good results with single molecules (Wan andriggs, 2016). The electron tomography approach consists of tak-

ng a series of images of the object at different angular orientationso obtain a better angular resolution; the sample may be turned by0◦ to collect a new series of images. Currently, images of macro-olecular complexes may be obtained at a resolution greater than

.85 nm (Beck and Baumeister, 2016) by combining and processingany subtomograms. The main obstacle of working with the tomo-

raphic reconstruction − is radiation damage of samples obtainedy prolonged exposure to the electron beam. Therefore, the totaladiation dose in electron tomography should not exceed 50e/Å2.

.3. Viral protein-DNA complexes

Likewise, pro- and eukaryotic DNA, like viral DNA, forms com-lexes with proteins more often than previously thought. A number

llegretti et al., 2014). (C) Electron damage in cryo-EM leads to a bubble formation.radiation with the high dose of electrons (right). Noticeable are bubbles of hydrogen

of internal phage proteins were discovered by proteomics analyses(Thomas et al., 2016, 2012). They can serve as scaffolds, proteases,RNA polymerases, or perform transport functions. Often, they canbe found attached to the bacteriophage portal, such as the ver-tex ‘core’ of the T7 phage, via which DNA enters the capsid duringassembly and exits during infection. It has a cylindrical form (Stevenet al., 1983) and consists of three proteins that possess differentsymmetries (Agirrezabala et al., 2005; Cerritelli et al., 2003; Guoet al., 2013).

In the nineteen eighties, the much larger internal protein struc-ture which spanned the capsids of giant phiKZ-like bacteriophages,encased within genomic DNA, was discovered. The authors called

it the ‘inner body’ (Krylov et al., 1984, 1978; Krylov and Zhazykov,1978). It forms an elongated cylindrical structure (Sokolova et al.,2014; Wu et al., 2012) and is currently thought to serve as a struc-tural device for arranging genomic DNA inside a giant phage head

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Micron 96 (2017) 57–64 61

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3

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Fig. 3. Bubblegrams reveal protein encased with DNA. (A) Phage Lin68 at low elec-tron dose conditions (≈20 e/Å2s, exposition 0.5 s); (B) the same field of view witha high electron dose (≈20 e/Å2s, exposition 1.0 s). White arrows are pointing to thebubblegrams that reveal the inner bodies. Bar – 200 nm; (C) phage Luz24 at lowelectron dose conditions. Black arrows are pointing to the phage tails. White arrow-

M. Mishyna et al. /

Krylov et al., 1984; Thomas et al., 2012). It may also participate inNA injection into bacteria (Krylov et al., 1984). The nature of inter-ctions between the DNA and the proteins that form the inner bodys unknown. This structure has recently been extensively studiedy cryo-EM (Cheng et al., 2014; Sokolova et al., 2014). In such cases,adiation damage effects can provide useful structural informationsee below).

. Radiation damage in cryo-EM

In an electron microscope, electrons are the source of imageormation. Energy transmitted from the electrons to the samplean ionize atoms and thereby cause X-ray emission and otherorms of radiation damage and a consequent rearrangement ofhemical bonds. As a result, the fine details are lost and the rawata may not adequately reflect the original structure (Glaeser,008; Karuppasamy et al., 2011). The precise effect of radiationamage depends on the chemical composition of the moleculeFromm et al., 2015). Thus, radiation damage is a limiting factor tohe achievable resolution in biological specimens (Glaeser, 2008;aruppasamy et al., 2011).

The energy of electrons is significantly higher (more than hun-reds of electron volts) than the energy of covalent bonds (fewlectron volts). The greatest damage to the sample occurs at high-st exposure, when the incident electrons lose from ≈5 to ≈100 eVf their energy (on average ≈20 eV) (Glaeser et al., 1971; Grant andrigorieff, 2015; Langmore and Smith, 1992). The study of the effectf radiation damage on the tobacco mosaic virus structure revealed

ts first visible effects as the dose increased beyond 14.3 e−/Å2.nitially, radiation affects carboxylate residues (mainly, negativelyharged), followed by other intermediate and large size side chains,hich leads to resolution deterioration of the main chain (Fromm

t al., 2015). Radiation damage effects are detectable at high spa-ial frequencies, but can be observed at low resolution too (Conwayt al., 1993).

Electron losses are responsible for the valence electron excita-ion (which forms covalent bonds), breaking ties, the emission ofecondary electrons, and the appearance of free radicals in bio-ogical objects. This process is called the primary damage. Freeadicals, in turn, trigger a cascade of chemical reactions referredo as the secondary damage (Fig. 2A). Breaking of chemical bondsccurs at all temperatures; therefore, cooling does not affect therimary damage of samples. However, liquid nitrogen temperaturelows down the motion of the molecules, i.e. inhibits the secondaryamage (Grant and Grigorieff, 2015; Henderson et al., 1990).

The Cryo-EM technique developed several approaches to min-mize damage caused by electron radiation: (i) the reduction ofhe exposure time. Typical electron doses for biological objectsange from 1 to 20 e/Å2. Although some biological samplesan withstand 100–500 e/Å2 (depending on the chemical com-osition and temperature), the structural details required forigh-resolution reconstruction undergo irreversible changes atxposures of ∼10 e/Å2 or less. Thus, radiation damage determinesxperimental conditions and limits the resolution of 3D structuresGrant and Grigorieff, 2015; Orlova and Saibil, 2010). (ii) The reduc-ion of the electron dose. Low-dose systems are widely used toeduce electron damage in search, alignment and focus modes,ntil, finally, to block the beam before the final step, capturing an

mage. However, short exposures lead to noisy images and, as aonsequence, to the lack of high-resolution data. This, in turn, com-licates subsequent image processing. The ability of the detector

o count each electron becomes critical for such short exposuresBaker and Rubinstein, 2010; Karuppasamy et al., 2011; McMullant al., 2014) (iii) The introduction of direct electronic detectors toryo-EM not only allowed to omit the intermediate step of signal-

head is pointing to the empty capsid. Bar – 100 nm; (D) Class-sum image of alignedhigh-dose Luz24 images with gas bubble, located close to the portal. Black arrow ispointing to the tail; white arrow – to the portal.

to light-conversion of electrons (Faruqi and Henderson, 2007), butalso to have fast readout rates of up to 400 frames per second in‘movie mode’ (Shrum et al., 2012). Through a special procedurecalled ‘motion correction’ (Henderson, 2015) all images of parti-cles obtained from the selected frames are aligned and summed(Fig. 3B). Thus, the beam-induced movement of particles in ice dur-ing the exposure is compensated, and a higher resolution of the3D reconstruction may be obtained. Moreover, this multiple frametechnique allows adjusting the effects of radiation damage. For thisaim, only a fraction of images with minimal damage is chosen forfurther processing. The combination of these approaches signifi-cantly improved the resolution of the 3D reconstructions in the pasttwo or three years − up to 3–4 Å (Allegretti et al., 2014; Campbellet al., 2015; Liu et al., 2016).

Tertiary protein damage, due to the formation of gas bub-

bles, occurs when samples are subjected to high energy electronirradiation (more than 40 e/Å2) (Fig. 2C). Hydrogen gas bubblesappear in the irradiated sample during a process described below(Dubochet et al., 1988; Leapman and Sun, 1995). First, radiolysis

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6 Micro

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f water occurs under the influence of an electron beam: H2O ⇒+ + OH−. In ice, free radicals are combined back into H2O, but in

lose proximity to the protein molecule they react with the hydro-en atoms of organic compounds: OH• + R−H ⇒ RO• + H2 (Leapmannd Sun, 1995). Bubbles are generally formed near that surfacef the molecule that is in contact with the ice, and is not insidehe protein (Hankamer et al., 2007). The gas is released in suchmounts that the pressure inside the bubbles may reach thousandsf atmospheres (Leapman and Sun, 1995).

. ‘Bubblegrams’ reveal the disguised protein structure

Interestingly, in some rare cases, electron damaging effectsay provide useful structural information. This especially con-

erns complexes formed by protein and DNA. Upon irradiationf such heterogeneous systems by an increased electron dose≈40–50 e/Å2), gas bubbles formed faster and at a lower dose thanpon irradiation of the protein–protein complexes. It is thoughthat tightly packed DNA prevents the diffusion of hydrogen gasway from the protein, which leads to a rapid local accumulationf radiolysis products and to earlier bubbling of proteins (Black andhomas, 2012; Sokolova et al., 2014; Wu et al., 2012). As a result,as bubbles may form a pattern that precisely outlines the protein-NA complex. Interestingly, the same electron dose does not induceubbles in free DNA embedded in ice (Chen et al., 2008). Theattern of radiation damage is clearly visible and may help in situa-ions where the protein is indistinguishable against the backgroundf the surrounding nucleic acid, providing information about thexistence and size of the protein structures. Some authors have sug-ested the term «bubblegram» for this phenomenon (Cheng et al.,014; Wu et al., 2012).

Bubblegrams were initially observed in structural studies of bac-eriophage capsids containing DNA (Sokolova et al., 2014; Thomast al., 2008). When taking a pair of images of the same phage particlet a lower (≈10–20 e/Å2) and higher electron dose (≈30–40 e/Å2),t was noticed that, while particles in the first image (Fig. 3A) wereot damaged and the ‘inner body’ was invisible in the backgroundf the surrounding DNA, in the second high dose image, bubblesf hydrogen were formed, outlining a specific elongated shape ofhe internal protein structure (Fig. 3B). The linear dimensions ofubblegrams allowed to predict the approximate size and posi-ion of this ‘inner body’. Both high-dose and low-dose images havedentical orientations. Thus, they made possible to determine theuler angles of the intact inner body within the non-radiated capsid.hese angles were used to calculate the three-dimensional recon-truction of the inner body (Wu et al., 2012).

Another example reveals the location of the portal vertex in theuz24-like bacteriophage (Fig. 3C). This phage has an icosahedralapsid and a short conical tail. Its inner protein core is located nearhe phage vertex. When the phage particles were subjected to highlectron dose radiation, the hydrogen gas produced the bubble athe protein core end, which is normally concealed by the surround-ng DNA. After aligning the particles and producing the class-summages, the orientation of the vertex can be revealed (Fig. 3D).

Recently, the birth of the gas bubble process was studied in detailsing the T7 bacteriophage (Cheng et al., 2014). It turned out thathe emission of gas begins only after the accumulation of a criticaloncentration of radiolysis products. Power electron dose causeshe occurrence of bubbles and must be evaluated beforehand to

inimize the damage. Another option to be considered − temper-

ture. Diffusion of radiolysis products from their place of origin ishat much slower, the lower the temperature. Therefore, the accu-

ulation of radiolysis products at helium temperatures can leado earlier electron-beam damage than at the temperature of liq-

n 96 (2017) 57–64

uid nitrogen. This type of radiation damage has been observed inelectron tomographic experiments (Bammes et al., 2010).

5. Conclusions

Latest achievements in cryo-EM that we have started usingin past two to three years, and particularly the direct detec-tors, enabled to collect movies and use only several frames thataccumulated the low electron dose for the final reconstruction.Additionally, the phase plates provide improved contrast andsignal-to-noise ratio. This approach enabled the three-dimensionalreconstructions of macromolecules with resolutions close to 2 Å(Bartesaghi et al., 2015; Banerjee et al., 2016; Kimanius et al., 2016)or even below (Merk et al., 2016). Still, obtaining a reconstructionwith a resolution below 3 Å remains challenging. A large numberof DNA-protein complexes remain in low resolution or have evenavoided structural investigations altogether.

Yet, electron beam damage of macromolecules doesn’t seem tobe a resolution limiting factor anymore. Now other factors came tothe fore: beam-induced motion, the detective quantum efficiency(DQE) of detectors, the flexibility of macromolecules (Bartesaghiet al., 2015; Glaeser, 2016b). Moreover, even the electron-beamdamage could provide additional structural information. Newapproaches in image collection have recently made it possible touse radiation damage to localize objects concealed in vitrified ice,determine their orientation and, subsequently, determine theirthree-dimensional structure.

Acknowledgements

Authors would like to thank Dr. Konstantin Miroshnikov andAnton Sedov for generous donation of phage Luz24, and Ms LisaTrifonova for proofreading the manuscript. Cryo-EM has beenperformed at user facility “Center for structural diagnostics of mate-rialsfrom FRC RAS “Crystallography and photonics”. This work wassupported by grants from RFBR (16-34-50185 to OSS and 16-34-00658 mol ɑ to EP) and from Ministry of Science and Education ofRussian Federation (RFMEFI61616X0070).

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