Nikolaus Grigorieff

Nikolaus Grigorieff
University of Massachusetts Medical School | UMMS · RNA Therapeutics Institute (RTI)

MSc, PhD

About

183
Publications
20,386
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18,819
Citations
Citations since 2016
71 Research Items
10924 Citations
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201620172018201920202021202205001,0001,5002,000
201620172018201920202021202205001,0001,5002,000
Additional affiliations
August 2013 - August 2021
Janelia Research Campus
Position
  • Principal Investigator
June 2000 - July 2013
Howard Hughes Medical Institute
Position
  • Investigator
January 1999 - July 2013
Brandeis University
Position
  • Professor

Publications

Publications (183)
Article
A major goal of biological imaging is localization of biomolecules inside a cell. Fluorescence microscopy can localize biomolecules inside whole cells and tissues, but its ability to count biomolecules and accuracy of the spatial coordinates is limited by the wavelength of visible light. Cryo-electron microscopy (cryo-EM) provides highly accurate p...
Article
Full-text available
Previously we showed that high-resolution template matching can localize ribosomes in two-dimensional electron cryo-microscopy (cryo-EM) images of untilted Mycoplasma pneumoniae cells with high precision (Lucas et al., 2021). Here we show that comparing the signal-to-noise ratio (SNR) observed with 2DTM using different templates relative to the sam...
Preprint
Full-text available
Electron cryo-microscopy (cryo-EM) can generate high-resolution views of cells with faithful preservation of molecular structure. In situ cryo-EM, therefore, has enormous potential to reveal the atomic details of biological processes in their native context. However, in practice, the utility of in situ cryo-EM is limited by the difficulty of reliab...
Article
Full-text available
For a more complete understanding of molecular mechanisms, it is important to study macromolecules and their assemblies in the broader context of the cell. This context can be visualized at nanometer resolution in three dimensions (3D) using electron cryo-tomography, which requires tilt series to be recorded and computationally aligned, currently l...
Preprint
Full-text available
Over the last decade, single-particle electron cryo-microscopy has become one of the main techniques contributing to the growing library of high-resolution structures of macromolecules and their assemblies. For a full understanding of molecular mechanisms, however, it is important to place them into the broader context of a cell. Traditionally, thi...
Article
Full-text available
Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frames...
Article
Full-text available
Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frames...
Article
Full-text available
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Preprint
Full-text available
Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Forster resonance energy transfer (smFRET) experiments, we studied how frames...
Article
Full-text available
Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frames...
Article
Full-text available
The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited owing to signal loss in, and misalignment of, the subtomograms. By contrast, single-particle cryo-electron microscopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We report a meth...
Article
Full-text available
The biological membranes of many cell types contain large-pore channels through which a wide variety of ions and metabolites permeate. Examples include connexin, innexin and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs), thr...
Article
Full-text available
The large (L) proteins of non-segmented, negative-strand RNA viruses are multifunctional enzymes that produce capped, methylated, and polyadenylated mRNA and replicate the viral genome. A phosphoprotein (P), required for efficient RNA-dependent RNA polymerization from the viral ribonucleoprotein (RNP) template, regulates the function and conformati...
Preprint
Full-text available
Biological membranes of many tissues and organs contain large-pore channels designed to permeate a wide variety of ions and metabolites. Examples include connexin, innexin, and pannexin, which form gap junctions and/or bona fide cell surface channels. The most recently identified large-pore channels are the calcium homeostasis modulators (CALHMs),...
Preprint
Recent advances in cryo-electron microscopy (cryo-EM) are paving the way to determining isolated three-dimensional (3D) macromolecular structures at near-atomic resolution using single-particle cryo-electron microscopy (SP-cryo-EM). However, determining the subcellular structures in intact cells and organelles using cryo-electron tomography (cryo-E...
Preprint
Full-text available
The large (L) proteins of non-segmented, negative-strand RNA viruses are multifunctional enzymes that produce capped, methylated and polyadenylated mRNAs and replicate the viral genome. A phosphoprotein (P), required for efficient RNA-dependent RNA polymerization from the viral ribonucleoprotein (RNP) template, regulates function and conformation o...
Article
Full-text available
Noroviruses are a leading cause of foodborne illnesses worldwide. Although GII.4 strains have been responsible for most norovirus outbreaks, the assembled virus shell structures have been available in detail for only a single strain (GI.1). We present high-resolution (2.6- to 4.1-Å) cryoelectron microscopy (cryo-EM) structures of GII.4, GII.2, GI.7...
Article
Full-text available
Rotaviruses, like other non-enveloped, double-strand RNA viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the "double-layer particle," or DLP). The RdRp, designated VP1...
Preprint
Rotaviruses, like other non-enveloped, double-strand RNA (dsRNA) viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the "double-layer particle" or DLP). The RdRp, designa...
Preprint
Full-text available
Noroviruses are a leading cause of food-borne illnesses worldwide. Although GII.4 strains have been responsible for most norovirus outbreaks, the assembled virus shell structures have been available in detail for only a single strain (GI.1). We present high-resolution (2.6-4.1 Å) cryo-electron microscopy (cryo-EM) structures of GII.4, GII.2, GI.7 a...
Article
Full-text available
Systemic AA amyloidosis is a worldwide occurring protein misfolding disease of humans and animals. It arises from the formation of amyloid fibrils from the acute phase protein serum amyloid A. Here, we report the purification and electron cryo-microscopy analysis of amyloid fibrils from a mouse and a human patient with systemic AA amyloidosis. The...
Article
Single-particle electron cryo-microscopy and computational image classification can be used to analyze structural variability in macromolecules and their assemblies. In some cases, a particle may contain different regions that each display a range of distinct conformations. We have developed strategies, implemented within the Frealign and cisTEM im...
Article
Severing to build microtubules Microtubules are essential intracellular polymers, built from tubulin subunits, that establish cell shape, move organelles, and segregate chromosomes during cell division. Vemu et al. show that microtubule-severing enzymes extract tubulin subunits along the microtubule shaft. This nanoscale damage is repaired by the i...
Preprint
Full-text available
Single-particle electron cryo-microscopy and computational image classification can be used to analyze structural variability in macromolecules and their assemblies. In some cases, a particle may contain different regions that each display a range of distinct conformations. We have developed strategies, implemented within the Frealign and cis TEM i...
Preprint
The resolution of subtomogram averages calculated from cryo-electron tomograms (cryo-ET) of crowded cellular environments is often limited due to signal loss in, and misalignment of the subtomograms. In contrast, single-particle cryo-electron microcopy (SP-cryo-EM) routinely reaches near-atomic resolution of isolated complexes. We developed a novel...
Preprint
Systemic AA amyloidosis is a worldwide occurring disease of humans and animals that arises from the misfolding of serum amyloid A protein. To provide insights into the molecular basis of this disease we used electron cryo-microscopy and determined the structure of an ex vivo amyloid fibril purified from AA amyloidotic mice at 3.0 A resolution. The...
Article
Full-text available
The advent of direct electron detectors has enabled the routine use of single-particle cryo-electron microscopy (EM) approaches to determine structures of a variety of protein complexes at near-atomic resolution. Here, we report the development of methods to account for local variations in defocus and beam-induced drift, and the implementation of a...
Preprint
Full-text available
A bacteriophage-encoded tubulin homologue, PhuZ, harnesses dynamic instability to position genomes of ϕKZ-like bacteriophage at the midline of their Pseudomonas hosts, facilitating phage infectivity. While much has been learned about molecular origins of microtubule dynamics, how GTP binding and hydrolysis control dynamics in the divergent 3-strand...
Article
Alternative gene splicing gives rise to N-methyl-D-aspartate (NMDA) receptor ion channels with defined functional properties and unique contributions to calcium signaling in a given chemical environment in the mammalian brain. Splice variants possessing the exon-5-encoded motif at the amino-terminal domain (ATD) of the GluN1 subunit are known to di...
Article
Full-text available
We have developed new open-source software called cisTEM (computational imaging system for transmission electron microscopy) for the processing of data for high-resolution electron cryo-microscopy and single-particle averaging. cisTEM features a graphical user interface that is used to submit jobs, monitor their progress, and display results. It im...
Data
Source data for the curves shown in Figure 7C.
Data
Source code for cisTEM 1.0 beta.
Preprint
Full-text available
We have developed new open-source software called cis TEM (computational imaging system for transmission electron microscopy) for the processing of data for high-resolution electron cryo-microscopy and single-particle averaging. cis TEM features a graphical user interface that is used to submit jobs, monitor their progress, and display results. It...
Article
Full-text available
In bacteria, mRNA transcription and translation are coupled to coordinate optimal gene expression and maintain genome stability. Coupling is thought to involve direct interactions between RNA polymerase (RNAP) and the translational machinery. We present cryo-EM structures of E. coli RNAP core bound to the small ribosomal 30S subunit. The complex is...
Article
Full-text available
Single-particle electron cryo-microscopy (cryo-EM) has become a popular method for high-resolution study of the structural and functional properties of proteins. However, sufficient expression and purification of membrane proteins holds many challenges. We describe methods to overcome these obstacles using ClC-rm1, a prokaryotic chloride channel (C...
Data
ClC-ec1 was over-expressed in BL21(DE3) E. coli cells and purified. The sample was 99% pure after eluting at pH 8 from the Co2+ resin. The purified sample was run on SEC and the protein was eluted as a single peak (left upper panel), then run as a single band on SDS-PAGE (right upper panel). Purified protein was confirmed by mass spectrometry. The...
Article
Full-text available
Gene translation depends on accurate decoding of mRNA, the structural mechanism of which remains poorly understood. Ribosomes decode mRNA codons by selecting cognate aminoacyl-tRNAs delivered by elongation factor Tu (EF-Tu). Here we present high-resolution structural ensembles of ribosomes with cognate or near-cognate aminoacyl-tRNAs delivered by E...
Data
3D reconstruction. Stack of sections from a reconstruction of the rotavirus RNA polymerase VP1 bound near a fivefold vertex in a double-layered particle (DLP; odd frames), interleaved with sections through a simulated DLP based on the model used for VP1 template generation (even frames). Voxel size is 0.1023 nm. DOI: http://dx.doi.org/10.7554/eLife...
Data
List of files included in Supplementary file 4. Columns Δf1, Δf2, and αast provide defocus parameters (Rohou and Grigorieff, 2015) assumed in template generation. DOI: http://dx.doi.org/10.7554/eLife.25648.018
Data
Sample input files for TEM-simulator v.1.3 (Rullgard et al., J. Microscopy 243(3):234–256, 2011) to calculate expected intensity distributions at the detector, and expected output files. Images generated by TEM-simulator and used here were downsampled twofold, to a pixel size of 0.0965 nm, by Fourier cropping before substituting each pixel intensit...
Data
CCG values from the constrained VP1 search. Indices for the five-dimensional matrix (2 × 3296 × 12 × 5 × 5) correspond to: target or control template (1 or 2); DLP image number (1–3296); vertex number (1–12); position within the vertex (1–5); pixel neighborhood (1–5), with #3 the center pixel and 1, 2, 4, and 5 the adjacent pixels. DOI: http://dx.d...
Article
Structure-based vaccine design depends on extensive structural analyses of antigen–antibody complexes. Single-particle electron cryomicroscopy (cryoEM) can circumvent some of the problems of x-ray crystallography as a pipeline for obtaining the required structures. We have examined the potential of single-particle cryoEM for determining the structu...
Article
Full-text available
ArfA rescues ribosomes stalled on truncated mRNAs by recruiting release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2 Å resolution cryo-EM structures - determined from a single sample - of the 70S ribosome with ArfA•RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstre...
Article
Full-text available
The HIV-1 envelope spike [Env; trimeric (gp160)3 cleaved to (gp120/gp41)3] induces membrane fusion, leading to viral entry. It is also the viral component targeted by neutralizing antibodies. Vaccine development requires production, in quantities suitable for clinical studies, of a recombinant form that resembles functional Env. HIV-1 gp140 trimers...
Data
Resolution of the final cryo-EM reconstruction.FSC values from Frealign between unfiltered reconstructions of two independently refined half data sets indicating the resolution of the reconstruction (FSC).DOI: http://dx.doi.org/10.7554/eLife.21829.004
Data
Validation of the structure model.FSC values calculated between the structure model and the half map used for refinement, the other half map, and the full map.DOI: http://dx.doi.org/10.7554/eLife.21829.005
Preprint
ArfA rescues ribosomes stalled on truncated mRNAs by recruiting the release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2-Å resolution cryo-EM structures – determined from a single sample – of the 70S ribosome with ArfA∙RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel down...
Chapter
Frealign is a software tool designed to process electron microscope images of single molecules and complexes to obtain reconstructions at the highest possible resolution. It provides a number of refinement parameters and options that allow users to tune their refinement to achieve specific goals, such as masking to classify selected regions within...