The resolution dependence of optimal exposures in liquid nitrogen temperature electron cryomicroscopy of catalase crystals

Baker, Lindsay A.; Smith, Eric A.; Bueler, Stephanie A.; Rubinstein, John L.

doi:10.1016/j.jsb.2009.11.014

Cited by 76 publications

(73 citation statements)

References 39 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similarly, later frames collected during the exposure are expected to display greater radiation damage effects and can also be discarded to preserve high-resolution features, as shown in our analysis. Our finding that the use of just the first 10 e − /A 2 produces density maps with the least radiation damage is consistent with estimates of the optimal dose to obtain density maps at 3-Å resolution by using cryo-EM (42). In addition to reducing the effects of beaminduced motion and mitigating the effects of radiation damage, we show that dose fractionation can also be used to improve the accuracy of contrast transfer function (CTF) estimation, which is an important resolution-limiting factor in single-particle cryo-EM (ref.…”

Section: Significancesupporting

confidence: 82%

Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy

Bartesaghi

Matthies

Banerjee

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

202

178

View full text Add to dashboard Cite

We report the solution structure of Escherichia coli β-galactosidase (∼465 kDa), solved at ∼3.2-Å resolution by using single-particle cryo-electron microscopy (cryo-EM). Densities for most side chains, including those of residues in the active site, and a catalytic Mg 2+ ion can be discerned in the map obtained by cryo-EM. The atomic model derived from our cryo-EM analysis closely matches the 1.7-Å crystal structure with a global rmsd of ∼0.66 Å. There are significant local differences throughout the protein, with clear evidence for conformational changes resulting from contact zones in the crystal lattice. Inspection of the map reveals that although densities for residues with positively charged and neutral side chains are well resolved, systematically weaker densities are observed for residues with negatively charged side chains. We show that the weaker densities for negatively charged residues arise from their greater sensitivity to radiation damage from electron irradiation as determined by comparison of density maps obtained by using electron doses ranging from 10 to 30 e − /Å 2 . In summary, we establish that it is feasible to use cryo-EM to determine near-atomic resolution structures of protein complexes (<500 kDa) with low symmetry, and that the residue-specific radiation damage that occurs with increasing electron dose can be monitored by using dose fractionation tools available with direct electron detector technology.single-particle EM | frame alignment | CTF determination | 3D reconstruction | structure refinement R apid advances in technology for single-particle cryo-electron microscopy (cryo-EM) over the last few years have made it possible to determine high-resolution structures of large and well-ordered macromolecular assemblies such as 2D protein crystals, helical lattices, icosahedral viruses, and protein complexes with high symmetry (1-10). More recently, continuing developments in microscope hardware and image processing software have yielded near-atomic resolution information for two smallersized complexes with low symmetry, the 700-kDa proteasome (11) and a 300-kDa mammalian ion channel (12), and for larger assemblies with no symmetry such as ribosome complexes (13-15). These developments signal an important change in the way cryo-EM can now be used in structural biology. Rather than simply using cryo-EM maps, typically in the 6-to 20-Å resolution range, as an envelope in which to fit structures obtained by X-ray crystallography, there is the exciting prospect of using cryo-EM to derive de novo, high-resolution structural models of proteins in one or multiple functional conformational states.The preparation of specimens for cryo-EM involves rapid plunge freezing of a thin aqueous suspension into liquid ethane cooled by liquid nitrogen. The speed of freezing is estimated to be ∼10 7 K/s (16), enabling the rapid cooling of the aqueous medium into a glass-like state. This rapid cooling traps the macromolecular components in a near-native state, capturing the conformational distribution and spa...

show abstract

Section: Significancesupporting

confidence: 82%

Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy

Bartesaghi

Matthies

Banerjee

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

202

178

View full text Add to dashboard Cite

show abstract

“…In 2013, a new method for cryo-EM was unveiled and termed MicroED, or 3D electron crystallography of microscopic crystals (Shi et al, 2013;Nannenga et al, 2014b). With continuous rotation MicroED, which is the preferred method of data collection in MicroED, significant loss of diffraction intensity was observed at resolutions better than ~3 Å when only ~3 e -Å -2 were used, consistent with past studies (Hayward & Glaeser, 1979;Jeng & Chiu, 1984;Baker et al, 2010).…”

Section: Introductionsupporting

confidence: 54%

“…Past studies of radiation damage in cryo-EM described the exposure-dependent decay of diffraction intensities up to and including 3 Å resolution Hayward & Glaeser, 1979;Jeng & Chiu, 1984;Baker et al, 2010). No data was analyzed at resolutions better than 3 Å likely because such data was not recorded at the time.…”

Section: Discussionmentioning

confidence: 99%

Analysis of global and site-specific radiation damage in cryo-EM

Hattne

Shi

Glynn

et al. 2018

Preprint

View full text Add to dashboard Cite

SummaryMicro-crystal electron diffraction (MicroED) is an emerging method in cryo-EM for structure determination using nanocrystals. It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.

show abstract

“…Images were acquired under low-dose conditions at defoci between 3 and 7 μm. For untilted images, electron exposures of 25-30 e − /Å 2 were used to optimize signal-to-noise ratios for spatial frequencies corresponding to information between 20 and 50 Å (43). For pairs of tilted images, this exposure was divided evenly between the first image (with a goniometer setting of +15°) and the second image (with a goniometer setting of −15°).…”

Section: Methodsmentioning

confidence: 99%

Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Baker

Watt

Runswick

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

115

109

View full text Add to dashboard Cite

Mitochondrial ATP synthase is responsible for the synthesis of ATP, a universal energy currency in cells. Whereas X-ray crystallography has revealed the structure of the soluble region of the complex and the membrane-intrinsic c-subunits, little is known about the structure of the six other proteins (a, b, f, A6L, e, and g) that comprise the membrane-bound region of the complex in animal mitochondria. Here, we present the structure of intact bovine mitochondrial ATP synthase at ∼18 Å resolution by electron cryomicroscopy of single particles in amorphous ice. The map reveals that the a-subunit and c 8 -ring of the complex interact with a small contact area and that the b-subunit spans the membrane without contacting the c 8 -ring. The e-and g-subunits extend from the a-subunit density distal to the c 8 -ring. The map was calculated from images of a preparation of the enzyme solubilized with the detergent dodecyl maltoside, which is visible in electron cryomicroscopy maps. The structure shows that the micelle surrounding the complex is curved. The observed bend in the micelle of the detergent-solubilized complex is consistent with previous electron tomography experiments and suggests that monomers of ATP synthase are sufficient to produce curvature in lipid bilayers.A TP synthases are responsible for the synthesis of ATP from ADP and inorganic phosphate. In mammalian mitochondria, the enzyme is an ∼600-kDa membrane protein complex comprised of a catalytic F 1 region and a membrane-bound F O region. The F 1 region consists of subunits α 3 β 3 γδε (1, 2) and the F O region consists of subunits abc 8 defg(A6L)F 6 (3). The F 1 and F O regions are connected by a central stalk, comprised of the γ-, δ-, and ε-subunits from the F 1 region, and a peripheral stalk, comprised of the oligomycin sensitivity conferral protein (OSCP) and subunits b, d, and F 6 from the F O region (4, 5). Previously, electron cryomicroscopy (cryo-EM) of intact detergent-solubilized bovine ATP synthase particles embedded in a thin layer of amorphous ice revealed the overall shape of the complex at 32 Å resolution (6), and subsequent analysis of the Saccharomyces cerevisiae enzyme produced a similar map at 24 Å resolution (7). X-ray crystallography of subcomplexes of the bovine enzyme has defined the arrangement of subunits in the F 1 region (8) and the peripheral stalk (9, 10) and showed the presence of a ring of eight c-subunits in the F O region (11). The structure and arrangement of the remaining subunits in the membrane-bound region of the enzyme are not known.The ATP synthase functions by a rotary catalytic mechanism. Proton translocation through the F O region requires the a-, b-, and c-subunits (12-14) and induces rotation of the membranebound c 8 -ring (15). The structure of the c 8 -ring is thought to be stabilized by binding of cardiolipin to a lysine residue conserved throughout animalia that has been shown to be trimethylated at the ε-amino group in all animal ATP synthases tested (11,16,17). The c 8 -ring is attached to the central st...

show abstract

The resolution dependence of optimal exposures in liquid nitrogen temperature electron cryomicroscopy of catalase crystals

Cited by 76 publications

References 39 publications

Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy

Structure of β-galactosidase at 3.2-Å resolution obtained by cryo-electron microscopy

Analysis of global and site-specific radiation damage in cryo-EM

Arrangement of subunits in intact mammalian mitochondrial ATP synthase determined by cryo-EM

Contact Info

Product

Resources

About