CO CRYSTALLIZATION OF MULTIPROTEIN-DNA COMPLEXES FOR STRUCTURAL ANALYSIS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。MATalpha2/MCM1/DNA复合体的结构已被解析为2.25埃(Tan S.，Richmond T.，Natural(1998)391，p660-666)。初步结构揭示了MATalpha2的臂域的一种不寻常的构象(残基112-130，它将MATalpha2的同源结构域连接到MCM1结合口袋)。在一个拷贝(称为MATalpha2-cis)中，该区域形成两个反平行的β片状构象，而在另一个(MATalpha2-tras)拷贝中，该区域形成(来自121-128残基的)α螺旋。这是造成不同构象的两个原因：(1)MCM1结合口袋和MATalpha2同源结构域之间的间距不同；(2)MATalpha2-反式与MCM1的对称相关拷贝接触，其中弯曲角度改变90度。我们已经用新的底物共晶MATalpha2和MCM1，这些底物在MATalpha2同源结构域和MCM1结合口袋之间的间距发生了变化。我们的晶体在我们自己的X射线源上的衍射很弱，而且通常在一维上很薄(通常是-0.2×15×02)。我们正在寻找衍射的改进，以便可以在同步加速器源上解决结构问题。我们的计划是以更高的分辨率收集衍射数据。如果获得足够高的分辨率，我们应该能够利用这些数据通过分子置换技术来求解结构。我们还想测试MATALPHA1/MCM1/DNA和Dot1/核小体的晶体，这些晶体以前在我们自己的X射线源上没有产生衍射。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The structure of the MATalpha2/MCM1/DNA complex has been solved to 2.25 angstroms previously (Tan S., Richmond T., Nature (1998) 391, p660-666). The preliminary structure has revealed an unusual conformation of the arm domain of MATalpha2 (residues 112-130 which links the MATalpha2 homeodomain to the MCM1 binding pocket). In one copy (called MATalpha2-cis)this region forms two anti-paralell beta sheets conformation while in the other (MATalpha2-trans) copy, the region forms a alpha helix (from residues 121-128). Their are two reasons for the different conformation which are (1) the spaciing between the MCM1-binding pocket and the MATalpha2 homeodomain are different and (2) the MATalpha2-trans makes a contact with a symmetry related copy of MCM1 in which the bending angle is altered by 90 degrees. We have cocrystallized MATalpha2 and MCM1 with new substrates that have an altered spacing between the MATalpha2 homeodomain and the MCM1 binding pocket. Our crystals diffract weakly at our home source xrays and are often thin in one dimension (typically-.2x.15x.02). We are looking for diffraction improvement so that the structure could be solved at a synchotron source. Our plan is to collect diffraction data at higher resolution. If high enough resolution is obtained, we should be able to use the data to solve the structure by molecular replacement techniques. We would also like to test crystals of MATalpha1/MCM1/DNA and Dot1/nucleosomes which previously do not yeild diffraction on our home source xrays.