Improvement of cryo-EM maps by density modification.

A density-modification procedure for improving maps from single-particle electron cryogenic microscopy (cryo-EM) is presented. The theoretical basis of the method is identical to that of maximum-likelihood density modification, previously used to improve maps from macromolecular X-ray crystallography. Key differences from applications in crystallography are that the errors in Fourier coefficients are largely in the phases in crystallography but in both phases and amplitudes in cryo-EM, and that half-maps with independent errors are available in cryo-EM. These differences lead to a distinct approach for combination of information from starting maps with information obtained in the density-modification process. The density-modification procedure was applied to a set of 104 datasets and improved map-model correlation and increased the visibility of details in many of the maps. The procedure requires two unmasked half-maps and a sequence file or other source of information on the volume of the macromolecule that has been imaged

An Expanded Conformation of Single-Ring GroEL-GroES Complex Encapsulates an 86 kDa Substrate

SummaryElectron cryomicroscopy reveals an unprecedented conformation of the single-ring mutant of GroEL (SR398) bound to GroES in the presence of Mg-ATP. This conformation exhibits a considerable expansion of the folding cavity, with ∼80% more volume than the X-ray structure of the equivalent cis cavity in the GroEL-GroES-(ADP)7 complex. This expanded conformation can encapsulate an 86 kDa heterodimeric (αβ) assembly intermediate of mitochondrial branched-chain α-ketoacid dehydrogenase, the largest substrate ever observed to be cis encapsulated. The SR398-GroES-Mg-ATP complex is found to exist as a mixture of standard and expanded conformations, regardless of the absence or presence of the substrate. However, the presence of even a small substrate causes a pronounced bias toward the expanded conformation. Encapsulation of the large assembly intermediate is supported by a series of electron cryomicroscopy studies as well as the protection of both α and β subunits of the substrate from tryptic digestion

Details of ssDNA annealing revealed by an HSV-1 ICP8–ssDNA binary complex

Infected cell protein 8 (ICP8) from herpes simplex virus 1 was first identified as a single-strand (ss) DNA-binding protein. It is essential for, and abundant during, viral replication. Studies in vitro have shown that ICP8 stimulates model replication reactions, catalyzes annealing of complementary ssDNAs and, in combination with UL12 exonuclease, will catalyze ssDNA annealing homologous recombination. DNA annealing and strand transfer occurs within large oligomeric filaments of ssDNA-bound ICP8. We present the first 3D reconstruction of a novel ICP8–ssDNA complex, which seems to be the basic unit of the DNA annealing machine. The reconstructed volume consists of two nonameric rings containing ssDNA stacked on top of each other, corresponding to a molecular weight of 2.3 MDa. Fitting of the ICP8 crystal structure suggests a mechanism for the annealing reaction catalyzed by ICP8, which is most likely a general mechanism for protein-driven DNA annealing

Structure of the SecY channel during initiation of protein translocation

Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY- or eukaryotic Sec61-complexes, and are translocated across the membrane during their synthesis1,2. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase3-5. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome–nascent chain complex binds to the SecY/Sec61 complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here, we have addressed this question by determining structures of inactive and active ribosome–channel complexes with cryo-electron microscopy. Non-translating ribosome–SecY channel complexes derived from Methanococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome–channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the N- and C-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than directly entering the channel

MRC2014: Extensions to the MRC format header for electron cryo-microscopy and tomography

Open Access funded by Medical Research CouncilThe MRC binary file format is widely used in the three-dimensional electron microscopy field for storing image and volume data. Files contain a header which describes the kind of data held, together with other important metadata. In response to advances in electron microscopy techniques, a number of variants to the file format have emerged which contain useful additional data, but which limit interoperability between different software packages. Following extensive discussions, the authors, who represent leading software packages in the field, propose a set of extensions to the MRC format standard designed to accommodate these variants, while restoring interoperability. The MRC format is equivalent to the map format used in the CCP4 suite for macromolecular crystallography, and the proposal also maintains interoperability with crystallography software. This Technical Note describes the proposed extensions, and serves as a reference for the standard.We thank Chris Booth and Steffen Meyer from Gatan Inc. for clarifying the format definition used by Digital Micrograph. Acknowledgement for support from National Institute of Health, USA includes: NIGMS grant P41GM103310 (AC and SD), NIBIB grant 5R01-EB005027 (DM), and R01GM080139 (SJL). RH and MW would like to thank the UK Medical Research Council for the award of Partnership Grant MR/J000825/1 to support the establishment of CCP-EM. RH and JS are also supported by MRC grant U105184322

In situ structure and assembly of the multidrug efflux pump AcrAB-TolC

Abstract: Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors

Ribosome Binding of a Single Copy of the SecY Complex: Implications for Protein Translocation

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain

Ribosome Binding of a Single Copy of the SecY Complex: Implications for Protein Translocation

The SecY complex associates with the ribosome to form a protein translocation channel in the bacterial plasma membrane. We have used cryo-electron microscopy and quantitative mass spectrometry to show that a nontranslating E. coli ribosome binds to a single SecY complex. The crystal structure of an archaeal SecY complex was then docked into the electron density maps. In the resulting model, two cytoplasmic loops of SecY extend into the exit tunnel near proteins L23, L29, and L24. The loop between transmembrane helices 8 and 9 interacts with helices H59 and H50 in the large subunit RNA, while the 6/7 loop interacts with H7. We also show that point mutations of basic residues within either loop abolish ribosome binding. We suggest that SecY binds to this primary site on the ribosome and subsequently captures and translocates the nascent chain