Shared Facilities: NMR Suite
James Graham Brown Cancer Center, 529 South Jackson Street.
Introduction
The JGBCC NMR Shared Facility occupies a suite in the Molecular Imaging Research
Center (MIRC), located in the rear of the Cancer Center. The NMR Shared
Facility is operated as part of the Structural Biology Program, under the direction
of Dr. Andrew Lane. The new purpose-built NMR facility was formally opened
in June, 2002. This facility currently houses two Varian Inova NMR spectrometers,
which are available for collaborative studies.
- 4-channel Varian Inova 14.1 T system (600 MHz for protons), equipped with
a 5 mm HCN cold probe, 5 and 8 mm inverse probes, a 4 mm flow probe and flow
system, and an automatic sample changer. This configuration enables a wide
range of 3D experiments to be carried out on isotopically labeled proteins
including deuterium decoupling. The cold probe gives a welcome increase in
sensitivity which is especially valuable for less soluble proteins or complexes,
dilute metabolite samples, larger systems, and for metastable systems.
- The second system operating at 18.8 T (800 MHz for protons) was funded
through an $2M EPScor grant (P.I. Prof. R. Wittebort, Dept. Chemistry) and
was installed and commissioned early in 2003. The 18.8 T system greatly
aids the study of larger proteins and complexes by the agency of the TROSY
technique. It is also equipped with accessories for solid state (CP-MAS)
NMR including a 3.2 mm HCN MAS probe.
Contacts
For information about NMR time scheduling, inquiries about collaborative
projects, or general information about NMR please contact Dr. Arumugam, Dr.
R. Wittebort (solid state NMR) Dr. T. Fan (Metabolomics) or Dr. Lane.
Andrew Lane, Director - anlane01@louisville.edu or 502-852-3067
Dr. S. Arumugam, Manager - 502 562-6832 or 502 562-6073
NMR Center provides maintenance, expertise and advice. Experiments
are modified or installed as needed. We undertake development work
for new and existing projects. Probe temperatures have been calibrated and an online manual for
offsets is available. Additional spectrometer-specific information is kept in folders on
the consoles for users to consult.
As isotopically labeled source molecules are best bought in
bulk, we purchase commonly used compounds in bulk, and pass on the savings
to users. We will also keep good quality NMR tubes, including Shigemis
for recharge.
Access to the NMR Suite is restricted, and entry is by Prox card.
Training is mandatory.[the detection system is both fragile and expensive-enormous
damage can be done by an unwary user].
Users have their own areas on the spectrometer
data station. User groups will have a principal person who will be responsible
for any trained members of that group. Alternatively, NMR can be done in
collaboration with trained user groups, such as with Drs. Arumugam, Fan,
Wittebort or Lane.
As most projects require 2D and 3D spectra, the time is allocated
in units of 24 hours.
NMR Center Advisory Committee:
- S. Arumugam (Manager) (ex officio)
- A.N. Lane (Director) (ex officio)
- D. M. Miller (Director, JG Brown Cancer Center)
- J.W. Eaton (Deputy Director, JG Brown Cancer Center)
- M.C. Maurer (Assoc. Professor of Chemistry)
- G. R. Pack (Chair, Dept. Chemistry)
- J. O. Trent (Assoc. Professor Medicine)
- R.J. Wittebort (Professor Chemistry)
Facilities
NMR Suite
The NMR suite comprises a large magnet room, a separate utility room, and a control room where the consoles are sited.
The control room also contains the wet laboratory area which is equipped
with a digital pH meter with long solid state probe, 4°C refrigerator, 2-compartment –25°C
freezer, small chemical fume hood, balance, bench space, deionised water and
general laboratory equipment. Limited supplies of NMR tubes, solvents and supplies
are available at cost on request.
The off-line computing area houses 2 Sun workstations (1 Sun
Sparc Ultra 5, 1 Sun Blade 10000 with 512 MB RAM); 2 SGI Octanes, one with
18” flat screen, the other with 19” monitor and Xtallise stereo
view system. Large (180 GB) disks are available on these. 1 Dell workstation
running Linux and a second running Windows with ACD software installed.
Backup systems include CD-RW and DVD-R. All systems are connect by
internal Ethernet. Console computers are attached directly to HP Laser printers. An
A3 inkjet printer is also available. Software: InsightII and Biopolymer on the SGIs; VNMR on the Suns;
nmrpipe on the Suns and Linux workstation. ACD software suite on the Windows PC
Cyana. The small library contains NMR and reference books and some journals.
The NMR suite in the BCC currently houses two four-channel Varian
Inova spectrometers, operating at 14.1 T (600 MHz for protons) and 18.8 T
(800 MHz for protons). The 14.1 Tesla instrument was commissioned
in July 2002, and is fully operational; the 18.1 Tesla spectrometer was commissioned
in February 2003 and is fully operational.
The 14.1 T instrument is a JGBrown Cancer Center instrument and is used
primarily for cancer-related projects.
The 18.8 T instrument was funded by NSF (P.I. Prof. R. Wittebort,
Dept. Chemistry), and is shared between the research groups in BCC, Department
of Chemistry and researchers at the University of Kentucky, Lexington.
NMR Facilities
14.1 T spectrometer
14.1 T Oxford shielded magnet- (4.2 K)
Varian Inova 4-channel console
Can pulse simultaneously on H,H X and X including 2H decoupling
There are six probes available for this instrument. All are equipped
with a z-axis coil for pulsed field gradients that enable investigators to
look at the structure, kinetics and dynamics of molecules of a wide range
of sizes.
- 5-mm 1H{13C/15N} triple resonance probe
- 5 mm 1H{13C/31P} triple resonance probe
- 5 mm-1H-19F/15N-31P switchable probe
- 4-mm 1H{13C/15N} triple resonance flow probe (120 mL). The flow probe has its own flow control system (VAST) with which an array of dilute samples e.g. from a 96 well plate can be looked at in quick succession.
- 8 mm 1H-X inverse probe for large samples and tissue work.
- 5-mm 1H{13C/15N} triple resonance cold probe. This is now the default probe.
The spectrometer is also equipped with a Sample Management System
(SMS) Controller and a Robotic arm that can automatically change the samples
in a rack and is controlled by the spectrometer host computer. This can be
programmed to continually acquire different sets of data on different samples
without manual intervention, and is ideal for high throughput applications.
The automation programs have been modified to enhance the flexibility. Further
work is in progress.
A VAST flow/sampling system is available for flow NMR.
Sample Spectra:
18.8 T spectrometer
18.8 T Oxford non-shielded magnet 63 mm bore (2.2 K)
Varian Inova 4-channel console with triple axis gradients
The 800 MHz instrument has the capability to investigate materials both in
the solid and solution phases. The probes for this instrument include:
- 5-mm 1H{13C/15N} triple resonance, triaxis pfg probe for studies directed towards macromolecules
- 3.2-mm CP- MAS probe for studying solid samples. The 18.8 T system will greatly aid in the study of larger proteins and complexes by the agency of the TROSY technique.
- 5 mm- XH pfg probe for X observe with 1H decoupling
- 5 mm HCP pfg probe
- 5 mm inverse HX probe optimized for low g