The 36th Mid-Atlantic Macromolecular Crystallography Meeting

Item 1: December 2005 Publications by Members:

1:  Townsend DM,  Findlay VJ,  Fazilev F, Ogle M, Fraser J, Saavedra J, Ji X, Keefer L, Tew KD.
 A Glutathione S-Transferase {pi} Activated Pro-drug Causes Kinase Activation Concurrent with S-glutathionylation of Proteins.
Mol Pharmacol. 2005 Dec 1; PMID: 16288082

2:  Hosseinpour F, Moore R, Negishi M, Sueyoshi T.
 Serine-202 regulates the nuclear translocation of constitutive
active/androstane receptor CAR.
Mol Pharmacol. 2005 Dec 23; PMID: 16377764

3:  Dauter Z.
 Current state and prospects of macromolecular crystallography.
Acta Crystallogr D Biol Crystallogr. 2006 Jan;62(Pt 1):1-11. Epub 2005 Dec 14. PMID: 16369088

4:  Li M, Laco GS, Jaskolski M, Rozycki J, Alexandratos J, Wlodawer A, Gustchina A.
 Crystal structure of human T cell leukemia virus protease, a novel target for anticancer drug design.
Proc Natl Acad Sci U S A. 2005;102(51):18332-7. PMID: 16352712

5:  Clore GM, Schwieters CD.
 Concordance of Residual Dipolar Couplings, Backbone Order Parameters and Crystallographic B-factors for a Small alpha/beta Protein: A Unified Picture of High Probability, Fast Atomic Motions in Proteins.
J Mol Biol. 2005 Dec 9; PMID: 16343537

6:  Cho S, Swaminathan CP, Yang J, Kerzic MC, Guan R, Kieke MC, Kranz DM, Mariuzza RA, Sundberg EJ.
 Structural basis of affinity maturation and intramolecular cooperativity in a
protein-protein interaction.
Structure (Camb). 2005 Dec;13(12):1775-87. PMID: 16338406

7:  Saxena AK, Singh K, Su HP, Klein MM, Stowers AW, Saul AJ, Long CA, Garboczi DN.
 The essential mosquito-stage P25 and P28 proteins from Plasmodium form tile-like triangular prisms.
Nat Struct Mol Biol. 2005 Dec 4; PMID: 16327807

8:  Nallamsetty S, Austin BP, Penrose KJ, Waugh DS.
 Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli.
Protein Sci. 2005 Dec;14(12):2964-71. PMID: 16322578

9:  Buchanan SK.
 Bacterial metal detectors.
Mol Microbiol. 2005 Dec;58(5):1205-9. PMID: 16313609

10:  Koike C, Moore R, Negishi M.
 Localization of the nuclear receptor CAR at the cell membrane of mouse liver.
FEBS Lett. 2005 Dec 19;579(30):6733-6. PMID: 16310787

11:  Laronde-Leblanc N, Wlodawer A.
 The RIO kinases: An atypical protein kinase family required for ribosome
biogenesis and cell cycle progression.
Biochim Biophys Acta. 2005 Dec 30;1754(1-2):14-24. PMID: 16182620

Item 2: Tips and Tricks

This section is always open for contributions. Click for Introduction and tips and tricks in Crystallization, Post-crystallization treatments for improving diffraction quality of protein crystals, Derivatization, Diffraction, Symmetry, Structure Solution, Structure Refinement, and Structure Analysis.

Item 3: Topic Discussion

Click for previous discussions on: PHASER, HKL2000, Parallel Protein Expression, Structural Genomics, NCS, Missing Atoms, Trends in Crystallography, and Absorption Correction.

PHASER: MR with Maximum Likelihood

Drs. Mariusz Jaskolski (UAM) & Alexander Wlodawer (NCI):

Although numerous crystal structures of several retroviral proteases (PR) have been solved in the past (e.g. from HIV, RSV, FIV, EIAV) and all these enzymes share significant sequence and structure homology, the new structure of human T-cell leukemia virus (HTLV) PR could not be solved (in our hands) by any of the standard MR programs (AMoRe, EPMR, MolRep). However, the structure was solved in a straightforward manner using the PHASER program, as implemented in ccp4i.

Some explanation of the failure of the "older" MR programs is provided by the comparatively low levels of sequence identity/similarity between the models and the target (ranging in simple CLUSTAL comparisons from 23.9/59.3% for FIV PR to 32.3/63.6% for HIV PR) and by the complicated architecture of the unknown structure (three homodimeric molecules forming a highly pseudosymmetric trimer), combined with high degree of uncertainty about the asymmetric unit contents.

In automatic runs, which included the full available resolution of 2.6 A, PHASER correctly solved the trimeric model, but failed (correctly again) to located any additional copies of the molecule due to packing considerations. The solutions could be unambiguously identified by their high Z-score parameter (> 10) for the more similar models (HIV PR, EIAV PR). The less similar models had Z-score values just below 8, and corresponded to a correct (7.96, RSV PR) or incorrect (7.80, FIV PR) solution. The quality of the solutions is also recognizable from the final LL-gain parameter, which at a value of 110 could distinguish between incorrect and correct solutions, increased to about 180 for "strong", unambiguous solutions (Z-score about 11), and rose sharply to 270 for the absolutely best case (Z-score 14.54).

In post factum analyses, the levels of sequence identity/similarity are reflected in the r.m.s. deviations between the Ca atoms of the corresponding models and the final HTLV PR structure, which are high even for the best models: HIV PR 1.6 A, EIAV PR 1.7 A, FIV PR 1.8 A, RSV PR 1.9 A.

In practical terms, the structure was cracked using the best available model of HIV PR determined at atomic resolution (Z-score 11.16). However, in a posteriori calculations the clearest solution was obtained with a medium-resolution HIV PR model, which in Ca comparison with the final target did not show any obvious superiority. This observation reinforces the notion that the performance of even the most robust MR algorithms may critically depend on the initial conditions and that, whenever available, different models should be tried.

Item 4: Lectures

Dr. Zbigniew Dauter's Lectures at the NIH (03/29-31)