Item 1: July 2003 Publications by Members of the Group:

1: Yoshinari K, Kobayashi K, Moore R, Kawamoto T, Negishi M. 
Identification of the nuclear receptor CAR:HSP90 complex in mouse liver and recruitment of protein phosphatase 2A in response to phenobarbital.
FEBS Lett. 2003 Jul 31;548(1-3):17-20. [PubMed]

2: Trievel RC, Flynn EM, Houtz RL, Hurley JH. 
Mechanism of multiple lysine methylation by the SET domain enzyme Rubisco LSMT.
Nat Struct Biol. 2003 Jul;10(7):545-52. [PubMed]

3: Cai M, Williams DC Jr, Wang G, Lee BR, Peterkofsky A, Clore GM. 
Solution structure of the phosphoryl transfer complex between the
signal-transducing protein IIAGlucose and the cytoplasmic domain of the glucose transporter IICBGlucose of the Escherichia coli glucose phosphotransferase
system.
J Biol Chem. 2003 Jul 4;278(27):25191-206. [PubMed]

Item 2: Topic Discussions

CNS TARGET FUNCTIONS: CNS has 10 target functions for crystallographic refinement.

mlf:        maximum likelihood target using amplitudes

mli:        maximum likelihood target using intensities

mlhl:      maximum likelihood target using amplitudes & phase probability distribution

residual: standard crystallographic residual

vector:    vector residual

mixed:    (1-fom)*residual + fom*vector

e2e2:     correlation coefficient using normalized E²

e1e1:     correlation coefficient using normalized E

f2f2:       correlation coefficient using F²

f1f1:       correlation coefficient using F

Please comment on their appropriate use for data sets obtained with different techniques as well as at different stages of refinement and resolution.

TIPS AND TRICKS IN CRYSTALLOGRAPHY: See previous contributions from members, click here. This section is always open for contributions.

Dr. Peter Sun (NIAID): Crystallization of protein-protein complexes. Protein-protein complexes are, in general, harder to crystallize than their non-complexed components due to their lesser solubility and instability. For example, it maybe difficult to achieve a 10 mg/ml concentration for protein-protein complexes while the solubility of their components could exceed well beyond 10mg/ml concentration. A blind application of the available crystallization screening kits often results in precipitation in majority of the conditions. Even if one can reach a desired concentration for a protein complex sample, the stability under which a complex remains often restricts its crystallization configuration space. This is mostly due to a lesser energy required to disrupt a protein complex formation than it is required to disrupt a protein structure or folding. As such, the crystallization configuration space is less restricted for tight binding protein complexes, such as antibody-antigen and cytokine-receptor complexes, and is more restricted for weakly interacting complexes, such as many transient signal transduction complexes that are at best of micromolar binding affinity. Thus, the principle concern of crystallizing a protein-protein complex is to maximize the complex solubility and limit to the conditions under which the complex remains associated. A brief survey of the crystallization conditions for 200 published protein complexes showed certain preference in their crystallization conditions compared to single proteins (1). Here is a summary of our findings: (A) The majority conditions are PEG related with ammonium sulfate represents less than 20% of the cases. As for the precipitant concentration, the average PEG used is 10-15%, which is somewhat lower than the 20-30% PEG represented in many screening kits. (B) Most of them are crystallized at or near neutral pH values with less than 10% cases having pH lower than 5 or higher than 8.5. (C) Salt concentrations are general less than 300 mM, except when ammonium sulfate is used. (D) A crystallization screen kit was assembled to represent the most favorable conditions for obtaining protein complex crystals.        

Reference: 1. Radaev S., and Sun P. Crystallization of protein-protein complexes. J. Appl. Cryst. (2002), 35:674-676.

Dr. Traci Hall (NIEHS): Crystallization of Protein-RNA complexes. Xinhua asked me to write something about how to grow crystals of protein:RNA complexes, so here are some tips from our lab.  (1) RNA source.  We order our synthetic RNA oligos from Dharmacon Research (http://www.dharmacon.com).  I’m told that TriLink is also a good source (http://www.trilinkbiotech.com). (2) Determining the minimal piece of RNA that is sufficient for binding.  A first analysis can be done with a standard band shift or filter-binding assay, but the shortest piece of RNA that binds in these assays can sometimes be longer than what is necessary for crystallization.  So, it is useful to analyze the ability to form complexes at crystallographic concentrations by gel filtration chromatography.  We use a Superdex 75 or 200 HR 10/30 column from Amersham Pharmacia for this, and it is helpful to monitor at 260 and 280 nm, if possible.  (3) Forming complexes for crystallization.  We use gel filtration chromatography to analyze complex formation at crystallographic concentrations and determine the ratio of protein:RNA to produce stoichiometric complexes.  Alternatively, we calculate a ratio and set up crystallization trials at slightly varying ratios.  It is also possible to purify protein:RNA complexes by gel filtration for crystallization.  (4) Crystallization screens.  Our favorite screens are Natrix and MembFac from Hampton Research.

Item 3: Topics Discussed

GROUP ACTIVITIES: No additional activities were suggested.

ABSORPTION CORRECTION: The effect of absorption by macromolecular crystals can only be implicitly taken into account by a purely empirical approach, the classic inter-image scaling. A significant improvement of this approach involves the use of spherical harmonics and is now programmed in some data reduction programs (eg. Scalepack and Scala), which usually leads to significantly improved intensity estimations, especially in the case of anomalous scattering. However, this approach needs to have enough redundancy of measurements and should be used with care (Full discussion).

TRENDS IN CRYSTALLOGRAPHY: Structural genomics is supposed to deliver 3-D structures for all building blocks of biological macromolecules; molecular modeling should be able to organize these building blocks into 3-D structures. However, structural genomics and molecular modeling together cannot provide extensive information on any biological process where intermolecular interactions and signaling are involved, not to mention that for any modeled structure, a real structure is the best and final validation. Modern crystallography, armed with many newly developed and advanced tools, will be doing even better mapping the reaction trajectories where dramatic conformational changes of biomolecules often occur and studying macromolecular assemblies and signaling pathways where intermolecular interactions always dictate (Full discussion).

MISSING ATOMS: In principle, including them with zero occupancy makes sense, because such atoms do not contribute to the refinement, are clearly marked as modeled rather than observed, and do not cause problems such as changes in the sequence when a lysine, for example, is replaced by an alanine (Full discussion).

NCS: NCS is part of single crystal X-ray diffraction data and a useful addition to the crystallographic symmetry. All experimental data should be used for derivation of experimental results. NCS contains "error" that should be treated properly with the use of a weighting scheme. CNS and SHELXL use different approaches in structure refinement with NCS restraints (Full discussion).