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2003 NSLS Users' Meeting
Bio-Matters: from Infrared to X-rays
Location: Bldg. 911A - Snyder Seminar Room
Date: May 21, 2003
Organizer: V. Stojanoff, Brookhaven National Lab, 631-344-8375
Co-Organizer: W. Caliebe, Brookhaven National Lab, 631-344-4744
Description: The structure determination and
the understanding of biomolecular functions constitute the main goal
in this post-genomic and proteomic era. The aim of this workshop is
to discuss the contributions of different Synchrotron based
techniques from the infrared to high-energy X-rays to the
understanding of bio-molecular structures.
Workshop participants are encouraged to present
their work in poster format. Ample time for discussions is being
provided. Poster boards (36 x 48 inches or 110 x 90 cm) will be
available at the location of the workshop to those who advise the
organizing committee before May 14th
Link to another workshop held at NSLS the Week of
May 19-23, 2003
Crystallization
Workshop May 19, 2003
Schedule:
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Chairperson: Leemor Joshua-Tor,
Cold Spring Harbor Lab |
| 8:15-8:30a.m. |
Welcome |
| 8:30-9:15a.m. |
Dr. W. Hendrickson, Howard Hughes Medical Institute
“New
Structures” |
| 9:15-9:20 a.m. |
Break |
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Chairperson: Lonny Berman, NSLS |
| 9:20-9:50 |
Dr. C. Jacobsen, S.U.N.Y at Stony
Brook
"Soft x-ray imaging and Spectromicroscopy"
Soft x-ray microscopes are able to deliver 30-50 nm
resolution images of hydrated biological specimens in
present-day zone plate microscopes, with the
potential for improvements towards 10 nm using improved zone
plates and phasing of diffraction data. When combined with near-edge absorption spectroscopy,
high resolution views of chemical contrast can be obtained and
analyzed using multivariate statistical analysis methods. These approaches are illustrated with biomedical
examples including spectromicroscopy studies of human sperm,
and imaging of several cell types. |
| 9:50-10:20 a.m. |
Dr. R. Scarrow, Haverford College
“EXAFS
studies of Metalloproteins and the Usefulness of Model
Coordination complexes”
In the past quarter century, EXAFS
(Extended X-ray Absorption Fine Structure) analysis has been
applied to a variety of metalloproteins to determine the
nature of liganding atoms (S/Cl vs. N/O), the number and
lengths of bonds, and (in proteins containing metal-oxo or
metal-sulfido clusters) metal-metal distances. Examples from
studies in my lab on the proteins lipoxygenase and
porphobilinogen synthase illustrate how EXAFS data from model
coordination complexes are used along with theoretical
calculations to develop an accurate analysis protocol. I will
also discuss how small molecule crystal structure databases
are useful in interpretation of the EXAFS results for the
metalloproteins.
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| 10:20-10:45 a.m. |
Break/Posters |
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Chairperson: Marc Allaire, NSLS |
| 10:45-11:15 a.m. |
Dr. J. Krueger, University of
North Carolina at Charlotte
“Small-angle Scattering: Solutions in Protein Structural
Analysis”
Proteins are the functional products of
gene translation and as such, structural analysis of proteins
will be a primary objective for researchers in this
genome-enabled era. An important goal of such analysis is to
discover how protein structures interact with one another to
regulate specific cellular events. Techniques capable of
providing information on the resultant large macromolecular
complexes will be a central component of any structural
biologists' toolbox. The techniques of small-angle X-ray and
neutron scattering on macromolecular complexes complement
other structural approaches, providing a low-resolution
framework from which to begin assembling molecular-level
details on individual proteins or protein subunits into their
intact, functional units. Indeed, interpretation of
small-angle scattering data is most effective when used as a
complementary tool with other structural and biochemical
information such as that obtained from selected-site
mutagenesis, circular dichroism, NMR, crystallography or
electron microscopy, providing key pieces of information that
complete a story. This seminar will present the background and
provide some current examples on the biological application of
small-angle scattering technologies. These technologies,
particularly that of neutron scattering using contrast
variation, is relatively new and not yet widely explored;
however, they show great promise in providing structural
information on protein:protein interactions that can be
difficult to obtain otherwise.
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| 11:15-11:40 p.m. |
Dr. U. Ramagopal, National Cancer
Institute and Z. Dauter, National Cancer
Institute
“SAD: Happy Phasing With Weak Anomalous Scatterers”
The recent years witnessed significant
advancements in X-ray data acquisition techniques and phasing
algorithms that made possible the successful use of a very
small anomalous diffraction signal for solving crystal
structures of macromolecules. Two examples of the
single-wavelength anomalous diffraction (SAD) approach applied
to proteins containing less sulfurs than most proteins in the
bacterial or eukariotic proteomes, with the Bijvoet ratio of
about 0.6% will be shown. In general, the simplicity of SAD as
an alternative to MAD and its applicability to all protein
crystals able to provide accurate diffraction data will be
discussed with examples.
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| 11:40-12:05 p.m. |
Dr. B. von Dreele, LANL
“Protein Crystal Structure Determination from Powder Diffraction
Data”
Although it may seem surprising, under
certain conditions there is sufficient information in a high
resolution X-ray powder diffraction pattern to permit
determination of a protein crystal structure. This includes
the use of a rigid body description for the protein molecule
and Rietveld refinement of its position and orientation as
well as use of electron density maps obtained from structure
factors extracted from the powder diffraction profile using
partial structure information. This talk will focus on some
recent examples of these methods.
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| 12:05-13:00
p.m. |
Lunch/Posters |
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Chairperson:
Wolfgang Caliebe, NSLS
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| 13:00-13:30 p.m. |
Dr. M. Chance, AECOM
“Structure and
Dynamics of Macromolecular Machines”
The study of protein and nucleic acid
dynamics with high structural resolution is of increasing
importance to the understanding of biological function. We
have developed technologies capable of analyzing protein and
nucleic acid structure, dynamics, and interactions with
millisecond time resolution and high structural resolution
using nanomoles to picomoles of material.
RNA structure and folding is essential to processes of
splicing and translation. Synchrotron footprinting of nucleic
acids has been used to follow the folding of RNA molecules
with single nucleotide resolution on millisecond timescales.
This has defined a sequential folding mechanism for the L-21
ribozyme from Tetrahymena1,2. Also, the simultaneous binding
of integration host factor to DNA with the bending of DNA was
demonstrated3.
Cofilin can regulate the assembly of
actin filaments and thus the metastatic potential of cells. It
is an important downstream component of the estrogen receptor
pathway whose activity is controlled by LIM kinase.
Synchrotron protein footprinting was used to identify the
critical residues of cofilin that bind actin monomers and
control its regulatory function4. In addition, we examined the
equilibrium and time-resolved activation of the actin binding
and severing protein gelsolin, whose function is Ca+2
sensitive5. We are currently developing further refinements of
the method so that the structure of other proteins that
regulate actin filament dynamics (such as Arp 2/3), as well as
the interactions of these proteins with filaments can be
better understood.
1-Sclavi, B., et al. Science 279:
1940-1943 (1998). 2-Ralston, C.Y. et al., Nature Structural
Biology 7: 371-374 (2000). 3-Dhavan, G.M., et al. Journal of
Molecular Biology 315: 1027-1037 (2002). 4-Guan, J-Q., et al.,
Biochemistry 41: 5765-5775 (2002). 5-Kiselar et al., Proc.
Nat. Acad. Sci. (USA), 100: 3942-3947 (2003).
|
| 13:30-14:00 p.m. |
Dr. U. Bergmann, LBNL
“Advances in
High-Resolution Hard X-ray Spectroscopy: From Vibrational
Studies to identify ligands to the Local Structure of Water”
Photon-in photon-out hard x-ray
spectroscopy involving rare events has become possible in
recent years due to powerful new synchrotron sources and
improvements in x-ray instrumentation. Examples include x-ray
fluorescence spectroscopy (XFS) of weak lines, resonant
inelastic x-ray scattering (RIXS), (non resonant) x-ray Raman
scattering (XRS) and nuclear resonant vibrational spectroscopy
(NRVS). All of these techniques are bulk probes of the local
structure, representing unique alternatives to more
traditional methods.
After introducing the instrumentation and the principle of
these techniques recent applications to biologically relevant
systems are shown. In particular we will focus on a) XRS
studies of the oxygen K-edge of water under various conditions
(including the supercritical phase) and its implications on
current structural models, b) RIXS studies of Mn and Ni
containing metalloproteins, and c) NRVS studies as a new tool
to identify ligands in Fe containing systems.
|
| 14:00-14:30
p.m. |
Break/Posters |
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Chairperson: Annie Heroux,
BNL
|
| 14:30-15:00 p.m.. |
Dr. L. Miller, NSLS
“Chemical
Imaging of Biological Tissues Using a Combination of Infrared,
UV-visible Flourescence, and X-ray Microspectroscopy”
Synchrotron infrared (IR)
microspectroscopy is a valuable technique for examining the
inherent chemical makeup of biological cells and tissues at a
spatial resolution unsurpassed by conventional IR microscopes.
As a complementary technique, x-ray fluorescence microprobe
can be used to image metal ions within biological tissue. For
both techniques, the complex composition of biological tissues
often benefits from sample visualization with fluorescence
illumination. For example, immunofluorescence, where
fluorochrome labels are attached to specific antibodies, has
become a widespread and powerful technique in cell biology and
immunology for visualizing targeted proteins.
In this work, technological improvements for combining
infrared, UV-visible fluorescence, and x-ray microspectroscopy
will be presented. In addition, biomedical applications to
Alzheimer's disease, scrapie, and bone disease will be
discussed. This work was performed at Beamlines U10B and X26A
at the National Synchrotron Light Source, Brookhaven National
Laboratory. The NSLS is supported by the United States
Department of Energy under contract DE-AC02-98CH10886. More
information on the infrared programs at the NSLS can be found
at http://infrared.nsls.bnl.gov.
|
| 15:00-15:45 p.m. |
Dr. T. Terwiliger, LANL
“Structural Genomics:
Technology for Structural Biology”
Structural genomics is a new field made
possible by advances in protein structure analysis and the
availability of genomic sequences. The overall goal of the
field is to determine the 3-dimensional structures of all
protein molecules, either by experiment or by computational
techniques. The promise of structural genomics is to provide a
broad structural foundation for understanding biology.
Achieving the high throughput required to determine thousands
of protein structures will require substantial technological
improvements in all aspects of coordination, protein
production and structure determination. Many such developments
are already well underway. One of the most important is the
automation of data collection and analysis at X-ray beamlines
worldwide. Other technologies developed for structural
genomics include work at Los Alamos on engineering of proteins
for optimal solubility, automated structure solution and phase
improvement by X-ray crystallography. Truly high-throughput
structure determination is likely to require extensive
miniaturization and parallelization of screening to identify
protein sequences, expression conditions, and crystallization
conditions that will yield material suitable for structure
determination.
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| 16:00-17:00 p.m. |
Panel Discussion on "Genomics and
Proteomics: How Can Different Methods Compliment Each Other?"
- Discussion Panel:
- W. Hendrickson, T. Terwilliger, S. Smith,
J. Frank, and N. Chayen
- .....and workshop participants
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Adjourn |
Register: Please register for the meeting and workshops (including speakers) at
http://www.nsls.bnl.gov/users/meeting/2003/registration/
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