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.
The
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
Grigorieff, N., Ceska, T. A., Downing, K. H., Baldwin,
J. M. & Henderson, R. (1996). Electron-crystallographic
refinement of the structure of bacteriorhodopsin. J.
Mol. Biol., 259, 393-421. [abstract]
Grigorieff, N. & Henderson, R. (1996). Comparison
of calculated and observed dynamical diffraction from purple
membrane: implications. Ultramicroscopy, 65,
101-107.
Grigorieff, N. (1998). Three-dimensional structure of
bovine NADH:ubiquinone oxidoreductase (complex I) at 22
Å in ice. J. Mol. Biol., 277, 1033-1046.
[abstract]
Walz, T. & Grigorieff. N (1998). Electron crystallography
of two-dimensional crystals of membrane proteins. J.
Struct. Biol., 121, 142-161. [abstract]
Smith, C. J., Grigorieff, N. & Pearse, B. M. F. (1998).
Clathrin coats at 21 Å resolution - A cellular assembly
designed to recycle multiple membrane receptors. EMBO
J., 17, 4943-4953. [abstract]
Grigorieff, N. (1999). Structure of the respiratory NADH:ubiquinone
oxidoreductase (complex I). Curr. Opinion Struct. Biol.,
9, 476-483. [abstract]
Grigorieff N. (2000). Resolution measurement in structures
derived from single particles. Acta Crystallogr D Biol
Crystallogr 56 :1270-1277 . [abstract
in PubMed]
Nogales E & Grigorieff N. (2001). Molecular Machines.
Putting the pieces together. J Cell Biol 152
:F1-F10 . [abstract
in PubMed]
Mindell, J.A., Maduke, M., Miller, C., & Grigorieff,
N. (2001). Projection structure of a ClC-type chloride channel
at 6.5 Å resolution. Nature 409:219-223.
[abstract
in PubMed]
Sokolova, O., Kolmakova-Partensky, L. & Grigorieff,
N. (2001). Three-dimensional structure of a voltage-gated
potassium channel at 2.5 nm resolution. Structure
9:215-220.
McGovern, S. L., Caselli, E., Grigorieff, N. & Shoichet,
B. K. (2002) A common mechanism underlying promiscuous inhibitors
from virtual and high-throughput screening. J. Med. Chem.
45:1712-1722. [abstract
in PubMed]
Jurica, M. S., Licklider, L. J., Gygi, S. P., Grigorieff,
N. & Moore, M. J. (2002) Purification and characterization
of native spliceosomes suitable for three-dimensional structural
studies. RNA 8: 426-439. [abstract
in PubMed]
Mindell, J. A. & Grigorieff, N. (2003). Accurate determination
of local defocus and specimen tilt in electron microscopy.
J. Struct. Biol. 142: 334-347. [abstract
in PubMed]
Furst, J., Sutton, R. B., Chen, J., Brunger, A. T. & Grigorieff,
N. (2003) Electron Cryo-Microscopy Structure of N-ethyl
Maleimide Sensitive Factor at 11A Resolution. EMBO J.,
22: 4365-4374. [abstract]
Wolf, M., Eberhart, A., Glossmann, H., Striessnig, J.
& Grigorieff, N. (2003) Visualization of the Domain Structure
of an L-Type Ca2+ Channel Using Electron Cryo-Microscopy.
J. Mol. Biol., 332: 171-182. [abstract]
Sokolova O, Accardi A, Gutierrez D, Lau A, Rigney M, Grigorieff
N. (2003) Conformational changes in the C terminus of Shaker
K+ channel bound to the rat Kvbeta2-subunit. Proc Natl
Acad Sci USA. 100(22):12607-12. [abstract]
Chen JZ, Furst J, Chapman MS, Grigorieff N. (2003) Low-resolution
structure refinement in electron microscopy. J Struct
Biol. 144(1-2):144-51. [abstract]
Jurica MS, Sousa D, Moore MJ, Grigorieff N. (2004) Three-dimensional
structure of C complex spliceosomes by electron microscopy.
Nat Struct Mol Biol. 11(3):265-9. [abstract]
Kim, L. A., Furst, J., Butler, M., Xu, S., Grigorieff,
N. & Goldstein, A. N. (2004) Subunit Composition of Kv4.2-KChiP2
Potassium Channel. J. Biol. Chem., 279: 5549-5554.
Kim, L. A., Furst, J., Gutierrez, D., Butler, M., Xu, S.,
Goldstein, A. N. & Grigorieff, N. (2004) Three-Dimensional
Structure of Kv4.2-KChiP2 Channels by Electron Microscopy
at 21 A. Neuron, 41: 513-519. [abstract]
Stewart, A. & Grigorieff, N. (2004). Noise bias in the
refinement of structures derived from single particles.
Ultramicroscopy, 102: 67-84. [abstract]
Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison,
S. C., Kirchhausen, T., & Walz, T. (2004). Molecular model
for a complete clathrin lattice from electron cryomicroscopy.
Nature, 432: 573-579. [abstract]
Fotin, A., Cheng, Y., Grigorieff, N., Walz, T., Harrison,
S. C. & Kirchhausen, T. (2004). Structure of an auxilin-bound
clathrin coat and its implications for the mechanism of
uncoating. Nature, 432: 649-653. [abstract]
Rodal AA, Sokolova O, Robins DB, Daugherty KM, Hippenmeyer
S, Riezman H, Grigorieff N, Goode BL. Conformational changes
in the Arp2/3 complex leading to actin nucleation. Nat Struct
Mol Biol. 2005 Jan;12(1):26-31. Epub 2004 Dec 12. [abstract]
View Complete Publication List on PubMed:
Nikolaus Grigorieff
Last review: January 8, 2007. E-mail comments
or questions to the webmaster.
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