NikoNikolaus Grigorieff, Ph.D.
Visiting Professor of Biochemistry
Group Leader, Janelia Research Center, HHMI

High-resolution electron cryo-microscopy of macromolecular complexes
Ph.D., University of Bristol

Lab Webpage

Nikolaus Grigorieff is a Visiting Professor of Biochemistry at Brandeis University. He does not have a lab at Brandeis and will not accept application from students to work in his lab. His primary affiliation is as a Group Leader at the Janelia Research Campus (Howard Hughes Medical Institute).

Our laboratory is studying the three-dimensional (3D) structure of proteins and protein complexes using high-resolution electron microscopy (EM). We generally focus on proteins that are difficult to study by more traditional techniques such as X-ray crystallography and NMR. For example, membrane proteins are usually too large for NMR analysis, or are difficult to crystallize for X-ray crystallography. Large protein assemblies, such as the spliceosome, pose additional problems because they undergo constant changes in composition and conformation.

Using EM, individual protein molecules and complexes can be visualized if their total molecular weight is larger than about 200 kDa, thus avoiding the need for crystals. This "single particle" approach requires extremely small amounts of material, typically only a few tens of picomols. Single particle EM is therefore ideally suited for the structural analysis of larger proteins and protein assemblies. Images from single protein particles are usually dominated by noise, because the sample exposure to a high-energy beam must be kept small in order to limit protein damage. To obtain a well-defined structure of the particle, many thousands of images have to be aligned with each other and averaged using computer image processing. Depending on the protein particle, sample preparation technique and instrumentation, a resolution better than 6 Angstroms can be obtained. We are developing new image processing methods to push the current limit to higher resolution.

imageThe Spliceosome

An important goal in my laboratory is to understand the structural underpinnings of gene splicing. This work is carried out in collaboration with the Moore laboratory at Brandeis University to obtain purified, homogeneous splicing complexes that are suitable for single particle EM. The spliceosome removes introns from nascent transcripts, an essential step in eukaryotic gene expression. Most introns interrupt precursors to messenger RNAs (pre-mRNAs), and their precise excision is required to create readable mRNAs. Spliceosomes are ribosome-sized (50 - 60 S) complexes composed of pre-mRNA, four small nuclear ribonucleoprotein (snRNP) particles, and a host of associated protein factors. The snRNPs (U1, U2, U4/6, and U5) are, in turn, multicomponent complexes, each containing at least one small stable RNA molecule (snRNA) and five or more tightly bound polypeptides. In all, it has been estimated that nuclear pre-mRNA splicing requires the action of over 100 different gene products. We have recently obtained images of purified spliceosomes (C complex) that have been used to determine an initial 3D structure of the spliceosome (Figure 1). Our goal is now to improve this structure using cryo-electron microscopy of unstained specimens. The 3D structure of one or more of the spliceosomal complexes, at a resolution of about 20 Angstroms or higher, will be invaluable for a better understanding of the inner workings of this large molecular machine.

The catalytically competent C complex stands at the end of an ordered pathway by which the snRNPs assemble to form spliceosomes. To better understand this assembly, and how splice sites are recognized, we are also working on earlier splicing complexes. Finally, together with the Moore laboratory, we study the exon junction complex (EJC), a post-splicing complex that remains on the spliced mRNA substrate. The EJC targets the spliced mRNA for nuclear export and is involved in determining its fate in subsequent processing, such as translation by the ribosome.

N-ethyl Maleimide Sensitive Factor (NSF)

NSF belongs to the family of AAA ATPases and is an essential component of the protein machinery that regulates vesicle fusion with target membranes, for example at synaptic terminals. NSF associates with a-SNAP (Soluble NSF Attachment Protein) to disassemble SNARE (Soluble NSF Attachment Protein REceptor) complexes. SNAREs, together with other proteins, facilitate docking and fusion of vesicles, and they are recycled and reactivated through disassembly by NSF. NSF functions as a homo-hexamer and each protomer contains three domains. The N-terminal domain of NSF is essential for the binding of a-SNAP and is followed by ATPase domains D1 and D2. Binding and hydrolysis of ATP by the D1 domain induces conformational changes in NSF leading to disassembly of the SNARE complex. The Brunger laboratory determined the crystal structures of the N and D2 domains, and of a-SNAP and a SNARE complex. Together with the Brunger laboratory, we recently obtained a structure at 11 Å resolution of NSF bound to a-SNAP and a SNARE that revealed the arrangement of the D1 and D2 domains within the NSF hexamer. Other parts of the structure, including the N domain and a-SNAP/SNARE complex, appeared to be disordered and were not resolved at the same level of detail. Our goal is to visualize these parts of the structure at higher resolution using improved preparations of the complex, and novel image processing techniques that can accommodate sample heterogeneity.

Chloride Ion Channels

Cl- channels play a multitude of roles in biological membranes. In contrast to cation-conducting channels, which service ions with fixed, defined gradients, Cl- channels handle a biologically ambidextrous ion whose cytoplasmic concentration, and hence equilibrium potential, varies greatly with cellular context. The ClC family of Cl- channels is the only family identified so far. ClC channels come in different functional flavors (voltage-gated, osmosensitive, and inwardly or outwardly rectifying), but all eukaryotic ClCs are built from polypeptides of ~100 kD with a characteristic transmembrane topology signature. ClC channels are homodimers with one independent pore per monomer. In addition, they have a slow common gate.

Together with the Miller laboratory at Brandeis University, we obtained 2D crystals of a ClC homologue from E. coli called EriC. Using cryo-EM, these crystals diffract to 6 Angstrom resolution when embedded in glucose, which is sufficient to resolve a-helices. A projection structure was calculated from images of these 2D crystals, revealing a dimer with at least two water-filled pores. Subsequently, Rod MacKinnon and co-workers used X-ray crystallography to solve the 3D structure of EriC, providing a much more detailed picture of the channel. Recently, however, the Miller laboratory found that EriC is not a channel but a chloride-proton antiporter. Despite the high-resolution X-ray structure, it remains unclear how this antiporter transports ions and protons. We are conducting experiments to detect conformational changes in the channel by subjecting our 2D crystals to varying environmental conditions, such as chloride concentration and pH. (This work is supported by a grant from the National Institutes of Health.)

Amyloid Fibrils

Amyloid fibrils are peptide or protein aggregates that form under certain conditions in vitro or in vivo. For example, the amyloid fibril plaques found in brain tissue of Alzheimer patients are formed from the peptide Ab and are associated with neurodegeneration. Amyloid formation is also observed with other diseases, such as type II Diabetes and Creutzfeldt-Jakob. Amyloid structures represent an alternative to the native folding pattern of many peptides and proteins. A characteristic motif of this folding pattern is the cross-b structure in which the peptides or proteins associate by b-sheet formation within protofilaments making up a fibril. In collaboration with Marcus Fändrich (Leibniz-Institute for age research, Jena, Germany) we study the molecular architecture of amyloid fibrils associated with human disease. Our goal is to identify fundamental principles of amyloid formation, and potential targets for disease treatment.

Selected Publications

Structural polymorphism of Alzheimer Abeta and other amyloid fibrils. Fändrich M, Meinhardt J, Grigorieff N. Prion. 2009 Apr 24;3(2). [abstract]

Molecular interactions in rotavirus assembly and uncoating seen by high-resolution cryo-EM. Chen JZ, Settembre EC, Aoki ST, Zhang X, Bellamy AR, Dormitzer PR, Harrison SC, Grigorieff N. Proc Natl Acad Sci U S A. 2009 Jun 30;106(26):10644-8. [abstract]

Pentameric assembly of potassium channel tetramerization domain-containing protein 5. Dementieva IS, Tereshko V, McCrossan ZA, Solomaha E, Araki D, Xu C, Grigorieff N, Goldstein SA. J Mol Biol. 2009 Mar 20;387(1):175-91. [abstract]

Actin filament labels for localizing protein components in large complexes viewed by electron microscopy. Stroupe ME, Xu C, Goode BL, Grigorieff N. RNA. 2009;15(2):244-8. [abstract]

Increased sulfate uptake by E. coli overexpressing the SLC26-related SulP protein Rv1739c from Mycobacterium tuberculosis. Zolotarev AS, Unnikrishnan M, Shmukler BE, Clark JS, Vandorpe DH, Grigorieff N, et al. Comp Biochem Physiol A Mol Integr Physiol. 2008;149(3):255-66. [abstract]

Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction. Zhang X, Settembre E, Xu C, Dormitzer PR, Bellamy R, Harrison SC, et al. Proc Natl Acad Sci U S A. 2008;105(6):1867-72. [abstract]

Paired beta-sheet structure of an Abeta(1-40) amyloid fibril revealed by electron microscopy. Sachse C, Fandrich M, Grigorieff N. Proc Natl Acad Sci U S A. 2008;105(21):7462-6. [abstract]

Abeta(1-40) Fibril Polymorphism Implies Diverse Interaction Patterns in Amyloid Fibrils. Meinhardt J, Sachse C, Hortschansky P, Grigorieff N, Fandrich M. J Mol Biol. 2008. [abstract]

A dose-rate effect in single-particle electron microscopy. Chen JZ, Sachse C, Xu C, Mielke T, Spahn CM, Grigorieff N. J Struct Biol. 2008;161(1):92-100. [abstract]

A maximum likelihood approach to two-dimensional crystals. Zeng X, Stahlberg H, Grigorieff N. J Struct Biol. 2007;160(3):362-74. [full text in PubMed Central] [abstract]

Ab initio resolution measurement for single particle structures. Sousa D, Grigorieff N. J Struct Biol. 2007;157(1):201-10. [abstract]

Conformational changes in actin-binding proteins, revealed by single particle electron microscopy. Sokolova O, Maiti S, Grigorieff N, Lappalainen P, Goode BL. Febs Journal. 2007;274:107.

High-resolution Electron Microscopy of Helical Specimens: A Fresh Look at Tobacco Mosaic Virus. Sachse C, Chen JZ, Coureux PD, Stroupe ME, Fandrich M, Grigorieff N. J. Mol Biol. 2007;371(3):812-35. [abstract]

FREALIGN: high-resolution refinement of single particle structures. Grigorieff N. J Struct Biol. 2007; 157(1):117-25. [abstract]

SIGNATURE: a single-particle selection system for molecular electron microscopy. Chen JZ, Grigorieff N. J Struct Biol. 2007;157(1):168-73. [abstract]

Ewald sphere correction for single-particle electron microscopy. Wolf M, Derosier DJ, Grigorieff N. Ultramicroscopy. 2006;106(4-5):376-82. [abstract]

The three-dimensional architecture of the EJC core. Stroupe ME, Tange TO, Thomas DR, Moore MJ, Grigorieff N. J Mol Biol. 2006;360(4):743-9. [abstract]

Quaternary structure of a mature amyloid fibril from Alzheimer's abeta(1-40) Peptide.Sachse C, Xu C, Wieligmann K, Diekmann S, Grigorieff N, Fandrich M. J Mol Biol. 2006;362(2):347-54. [abstract]

Structure determination of clathrin coats to subnanometer resolution by single particle cryo-electron microscopy. Fotin A, Kirchhausen T, Grigorieff N, Harrison SC, Walz T, Cheng Y. J Struct Biol. 2006;156(3):453-60. [abstract]

Conformational changes in the Arp2/3 complex leading to actin nucleation. Rodal AA, Sokolova O, Robins DB, Daugherty KM, Hippenmeyer S, Riezman H, et al. Nat Struct Mol Biol. 2005;12(1):26-31. [abstract]

View Complete Publication List on PubMed: Nikolaus Grigorieff


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