Experimental
Assignment Strategies
There are many different NMR experiments that a
spectroscopist can collect. However, certain combinations of
experiments are better than others for determining protein NMR
resonance assignments. The better strategies typically use more
experiments. However, more experiments means more time spent
collection, processing, and peak picking these experiments.
However, certain datasets are harder than others and require a more
rigorous approach. Below are listed the major types of experimental
strategies that AutoAssign can handle. They are broken down into
categories based on the number of resonances or rungs used to linked
nearest neighbor spin systems together. Typically a 5-rung
strategy is better than a 4-rung strategy from an assignment robustness
perspective. However, some experiments are simply better than
others depending on their implementation and on the sample
analyzed. For example, an HNcaCO experiment is typically
horrible for any decent-sized protein, unless the sample is
significantly deuterated. Collecting an HNcaCO will probably
not improve the assignment results for a 30kDa protein that is not
deuterated.
Also, the experiment names below refer to a family
of related
experiments that provide the given type of resonance information.
For
example, "HNCACB" refers to the family of straight-through and
out-and-back experiments that provide amide H, amide N, and
intra/sequential
CA/CB resonances.
AutoAssign also needs a "ROOT" spectrum for creating
spin systems. This is typically an NH-HSQC, but it could also be
an HNCO or HNcoCA peak list. These 3 experiments are usable
because only one peak exists for each spin system. An "alternate root" spectrum
can be specified as well. The HNCO is the alternate root by
default, but the HNcoCA could be the alternate root as well. An
alternate root spectrum is used to help detect HN-overlapped spin
systems.
5-Rung Matching
- [NH-HSQC], HNCO, HNcaCO, {HNCA, HNCACB OR
HNCACB-phased},
{HNcoCA, HNcoCACB OR HNcoCACB-phased}, {HNHA, HNHAHB OR HNHAHB-phased},
{HNcoHA, HNcoHAHB OR HNcoHAHB-phased}
4-Rung Matching
- {NH-HSQC AND/OR HNCO}, {HNCA, HNCACB
OR
HNCACB-phased},
{HNcoCA, HNcoCACB OR HNcoCACB-phased}, {HNHA, HNHAHB OR HNHAHB-phased},
{HNcoHA, HNcoHAHB OR HNcoHAHB-phased}
- [NH-HSQC], HNCO, HNcaCO, {HNCA,
HNCACB OR HNCACB-phased},
{HNcoCA, HNcoCACB OR HNcoCACB-phased}, HNHA, HNcoHA, [HNcoHAHB]
- [NH-HSQC], HNCO, HNcaCO, {HNCA,
HNCACB OR HNCACB-phased},
{HNcoCA, HNcoCACB OR HNcoCACB-phased}, HNHA, HNcoHAHB-phased
3-Rung Matching
- [NH-HSQC], HNCO, HNcaCO, {HNCA,
HNCACB OR HNCACB-phased},
{HNcoCA,
HNcoCACB OR HNcoCACB-phased}, [HNcoHA], [{HNcoHA, HNcoHAHB OR
HNcoHAHB-phased}]
- {NH-HSQC AND/OR HNCO}, {HNCA, HNCACB OR
HNCACB-phased},
{HNcoCA,
HNcoCACB OR HNcoCACB-phased}, HNHA, HNcoHA, [HNcoHAHB]
- {NH-HSQC AND/OR HNCO}, {HNCA, HNCACB OR
HNCACB-phased},
{HNcoCA,
HNcoCACB OR HNcoCACB-phased}, HNHA, HNcoHAHB-phased
2-Rung Matching
- {NH-HSQC AND/OR HNCO}, {HNCA, HNCACB OR
HNCACB-phased},
{HNcoCA,
HNcoCACB OR HNcoCACB-phased}, {HNcoHA,
HNcoHAHB OR HNcoHAHB-phased}
Experiments in "[]" are optional to the strategy.
Experiments in "{}" represent equivalent sets of experiments that
provide the same information for the strategy.
Experiments with a "-phased" suffix refers to a type of experiment
where the resonances in its 3rd dimension are distinguishable by the
sign of their intensity.
Resonances in lower case are not detected.