  |
"Structural Characterization of Macromolecular Complexes: Modern Techniques and Strategies" Grenoble Outstation, May 3-8, 2004 |
|
| Final Course Program Organizers
|
||
Course information
The practical course we are proposing will introduce scientists to the strategies and techniques required to prepare and structurally characterize macromolecular complexes. During the course the production of individual protein, DNA and RNA components followed by complex reconstitution or alternatively purification of complexes directly from the organism will be presented. Different biochemical methods to characterize complexes (i.e. proteolysis, nucleic acids/protein band shifts, cross-linking) and to validate their composition, stoichiometry and homogeneity (i.e. mass spectroscopy, low angle X-ray/neutron scattering, ultra-centrifugation) will be introduced. Finally, the major techniques for the structure determination of macromolecular complexes: crystallization/X-ray crystallography and electron microscopy will be presented. Here we will mainly emphasize techniques specific for complexes and the combination of crystallography and EM techniques (i.e. fitting of high resolution structure into low resolution EM models, use of EM results to modify the composition of complexes to favor crystallization etc.).
The course is aimed at participants with a strong interest in the structural biology of protein-nucleic acid and protein/protein complexes. Participants will range from the molecular biologists who would like to move into structural biology to the X-ray crystallographers who have not worked before on complexes because they considered it as technically too challenging.
Content of the practical course
additional information : |
Instructors
James Conway (IBS, Grenoble), Patrick Cramer (Univ. of Munich, Munich),
Christine Ebel (IBS, Grenoble), Carola Hunte (MPI , Frankfurt), Leopold
Luna Ilag (Cambridge Univ, Cambridge), Reinhard Lührmann (MPI,
Göttingen),
Ohad Medalia (MPI, München), Kiyoshi Nagai (LMB, Cambridge),
Pär
Nordlund (Karolinska Institute, Stockholm), Carlo Petosa (EMBL
Grenoble),
Rob Russell (EMBL, Heidelberg), Guy Schoehn (IVMS, UJF Grenoble),
Bertrand
Seraphin (CGM, Gif-sur-Yvette), Holger Stark (MPI, Göttingen),
Dimitri
Svergun (EMBL, Hamburg), Song Tan (Penn State University, University
Park),
Oliver Weichenrieder (NCI, Amsterdam), Willy Wriggers (University of
Texas,
Houston), Ray Owens (Oxford), Gabriel Waksman (Birkbeck College), Chloe
Zubieta (EMBL Grenoble), Carlos Fernandz-Tornero (EMBL Grenoble).
Preliminary Schedule
In the morning seminars will introduce methods required for the
preparation,
characterization and crystallization of macromolecular complexes. In
the
afternoon practical sessions will allow participants to gain hands-on
experience
in different techniques. Evening lectures will present recent
structural
biology results.
Monday 3 May
|
Expression &
Purification of Complexes |
||
|
09:00-9.05 |
Welcome |
Christoph Müller |
|
09:05-9.45 |
High-throughput
protein production technologies for structural and functional genomics |
Pär Nordlund |
|
09:45-10.30 |
Medium-throughput
approaches to protein production in insect and mammalian cells |
Ray
Owens |
|
10:30-11.00 |
Coffee |
|
|
11:00-11.45 |
Production of protein complexes |
Song Tan |
|
11:45-12.30 |
Purification
and characterization of complexes using the TAP method |
Bertrand Seraphin |
|
12:30-14:00 |
Lunch |
|
|
14:00-18:0 |
Practicals |
|
|
19:00-20:00 |
Dinner
at Canteen |
|
|
20:00-21:30 |
Student
presentations (EMBL seminar room) |
|
Tuesday 4 May
|
Isolation
of Functional Complexes |
||
|
09:00-09:45 |
Purification
and structural analysis of RNP complexes from human cells |
Reinhard
Lührmann Göttingen |
|
09:45-10:30 |
In vitro
transcription of RNA by T7 RNA polymerase (and design of suitable
constructs) |
Oliver
Weichenrieder |
|
10:30-11:00 |
Coffee |
|
|
11:00-11:45 |
Preparation
and crystallisation of RNA/protein complexes. |
Kiyoshi
Nagai |
|
11:45-12:30 |
Preparation of Membrane Protein Complexes for Structural Studies. |
Carola
Hunte |
|
12:30-14:00 |
Lunch |
|
|
14:00-18:00 |
Practicals
|
|
|
19:00-20:00 |
Dinner
at Canteen |
|
|
20:00-21:00 |
Student
presentations (EMBL seminar room) |
|
Wednesday 5 May
|
Characterization
of macromolecular complexes |
||
|
09:00-09:45 |
Small
angle scattering: nothing is too (macromolecular) complex |
Dimitri
Svergun |
|
09:45-10:30 |
Analytical
ultracentrifugation for the characterization of macromolecular
assemblies and interactions |
Christine
Ebel |
|
10:30-11:00 |
Coffee |
|
|
11:00-12:00 |
Mass
spectrometry of cellular machineries |
Leopold
Luna Ilag Cambridge |
|
12:00-12:45 |
Calorimetric
studies of the interaction of the SH2 domains with their cognate
tyrosine-phosphorylated targets |
Gabriel
Waksman |
|
13:00-14:00 |
Lunch |
|
|
14:00-18:00 |
Free Afternoon |
|
|
20:00 |
Workshop
Banquet |
|
Thursday 6 May
|
Electron
microscopy |
||
|
09:00-09:45 |
Combining
cryo-electron microscopy and crystallography for studying
macromolecular assemblies |
James
Conaway |
|
09:45-10:30 |
3D
structure determination of macromolecular complexes by single particle
electron cryo-microscopy |
Holger
Stark Göttingen |
|
10:30-11:00 |
Coffee |
|
|
11:00-11:45 |
Structural
analysis of intact cells and organelles using cryo-electron tomography |
Ohad
Medalia |
|
11:45-12:30 |
Strategies
for rigid body and flexible fitting of biophysical data |
Willy
Wriggers |
|
12:45-13:45 |
Lunch |
|
|
14:00-18:00 |
Practicals
|
|
|
19:00-20:00 |
Dinner
at Canteen |
|
|
20:00-21:00 |
Electron-microscopy
of the signal recognition particle (tentative title, EMBL seminar
room): |
Roland
Beckmann |
Friday 7 May
|
Crystallization/crystallography
of macromolecular complexes |
||
|
09:00-09:30 |
High-throughput
crystallization |
Jochen
Müller-Dieckmann |
|
09:30-10:15 |
Strategies
for crystallizing protein and protein/DNA complexes |
S. Tan |
|
10:15-10:45 |
Coffee |
|
|
10:45-11:30 |
RNA polymerase II structure: from core to
transient functional complexes |
Patrick
Cramer |
|
11:30-12:00 |
Adenovirus
penton structure |
Chloe
Zubieta |
|
12:00-12:30 |
Structural studies on
yeast RNA polymerase III |
Carlos
Fernadez-Tornero |
|
12:30-13:30 |
Lunch |
|
|
|
ESRF
safety training for Saturday Practicals |
|
|
14:00-18:00 |
Practicals
|
|
|
19:00-20:00 |
Dinner
at Canteen |
|
|
20:00 |
Structure-based assembly of
protein complexes in yeast (tentative title, EMBL seminar room) |
Rob Russell |
Saturday 8 May
|
X-Ray
structure analysis (Chair: Carlo Petosa,
Chadwick Amphitheatre,
ILL) |
||
|
09:00-09:45 |
Solving structures by X-ray crystallography |
Gordon
Leonard |
|
09:45-10:30 |
Structure of a photosynthetic membrane protein complex |
Janet
Smith |
|
10:30-11:00 |
Coffee |
|
|
|
|
|
|
|
Lunch |
|
Students
We accept 16-20 students for the course, based on the need of their
host laboratory to get updated in current state of the art methodology
while having projects that can benefit from it. Late Ph.D. students and
early post-docs will be the desired audience and ideally all
participants
will be on that level. Applicants should preferentially come from an
EMBO
member country, an associated country or eastern countries selected for
participation in the EMBO course program. Participants will bring
posters
and present their own work in a 5 min presentation at the evening of
the
first two days. Participants can also bring their own material in
coordination
with the organizers.
Course Fee
The course will be free of charge. Housing and meals will be provided.
Participants have to arrange for their own travel. Some support for
travel
can be provided upon request.
Sponsors
The course is supported by EMBO, SPINE, MAX-INF and the ESRF.
Application
The practical course is open for PhD students, postdocs and more senior
participants (preferentially) coming from an EMBO member country, an
associated
country or eastern countries selected for participation in the EMBO
course
program.
Subscriptions for this course are now closed
DEADLINE FOR APPLICATION: March 1, 2004
In case of questions contact the course secretary Mary-Jane Villot (villot@embl-grenoble.fr) or Christoph W. Müller (mueller@embl-grenoble.fr).
38042 Grenoble Cedex 9, France
Tel: +33 476 20 71 23