With the addition of Cr radiation to the crystallographer's toolkit, in-house X-ray sources can routinely provide this softer wavelength as an option. This report discusses the results of SAD and anomalous scattering aided molecular replacement experiments with Cr radiation. We also report the results of data collection with a new imaging plate detector (R-AXIS HR) that allows the collection of data with Cr radiation from a single crystal in one diffraction experiment suitable for both phasing and refinement.
Many examples of S-SAD and Se-SAD phasing have been reported for data collected with Cu radiation (λ = 1.54 Å), at the K absorption edge of selenium and the K edge and L edge of many other elements. To the left is a plot of depositions of MAD vs SAD structures since the inception of MAD. It is a landmark that the rate of of deposition of SAD structures in the RSCB PDB1 now exceeds that of MAD structures.
Furthermore, a number of successful SAD experiments using Cr radiation (λ = 2.29 Å), which can double the anomalous signal compared to Cu radiation, have been published by several groups. To the left is a collage of deposited and released structures. Several more have more been deposited but not released and yet others are proprietary and will not be released for some time. Please keep and eye on this page for new releases as they become available.
The in-house phasing approach we describe is called "know before you go". It provides a method to improve the efficiency of the solution of macromolecular crystal structures and usage of the synchrotron beam time by enabling the collection of phasing data in the home laboratory.
This chart shows the strength of the anomalous signal for many elements with Cu and Cr radiation. Note that the signal for Cr radiation is twice that of Cu for many elements of interest particularly for S and Se. This means that with careful data collection there will always be twice the anomalous scattering signal from a given protein—greatly enhancing the possibility of structure solution.
This slides reinforces the concept displayes in the previous slide. Here we compare the AD signal from the same crystal collected with Cu then Cr radiation. Note the increased density for the sulfur sites in the Cr data. Contours are at 4σ. Yang et al. Acta Cryst., D59, 1943-1957 (2003).
Efforts to uses Cr radiation in the past were hampered by the existing technology. BC Wang and others tried but failed. It was the confluence of modern rotating anode generators, multilayer optics, sensitive detectors, cryocrystallography and superb software that has allowed macromolecular crystallography to flourish that has also allowed CrSAD to be an effective tool.
odCPO was one of our first great successes. Our collaborators generated a proteolytic fragment of 267 AA and sent crystals to Rigaku. Using CrSAD data they were able to solve the structure of the fragment which then provided a modelk for molecular replacement for the larger, complete protein. The paper was submitted within two weeks of data collection.
This is a interesting case. The iron signal dominated the solutions from the Cu data sets and caused difficulty with phase extension. With Cr data, the iron and sulfur have the same signal which allowed easy resolution of the latter atoms. Phase extension was trivial with the correct sulfur positions.
This example shows the strength of the signal. Under most circumstances one might look for better cryoconditions on such a sample. However, that was not an option and this particualr crystal was the only one available. Data collection and processing were undertaken with careful attention to the rejection of ice ring reflections. The results are quite amazing for such a poor quality freeze.
Experimental details: Se-Met protein crystal from Dr. Su's lab in PKU. The protein has 277 aa with 9 Se and 1 S atom. A single scan (320°) of data was collected on a single sm 430 crystal.
An example of using phased molecular replacement. A human natural product could not derivitized and because it was too sensitive to soaks. Our colleagues use a very low redundancy CrSAD to locate sulfur sites with AutoSHARP. Those sites were then used with FFEAR and homology model to locate the first of seven monomers. AMORE was used to find the remaining six - 1694 AA in all.
Another example of using phased molecular replacement. In the second case, the anomalous signal was used to validate to the correct the molecular replacement solution in P1 from twinned data. A very impressive feat.
To extract the more information from the CrSAD experiment we developed a Cr anode for our flagship generator, the FR-E+ SuperBright. With an Osmic™ optic and helium beam path we can generate more usable Cr Kα photons than many beamlines.
In order to address the issue of adequate resolution we have developed curved, dual IP detector to collect data of sufficient resolution to perform both SAD phasing and structural refinement with Cr radiation.
We chose the cancer targer HSP90 as our test case. We collected data on a MicroMax-007 HF generator with VariMax Cr optic and R-AXIS HR. Data were processed with HKL2000 and refinement statistics are shown on the left.
The experimental electron denisty maps and the Ramachandran plot on this slide show that a good quality structure is possible with these tools.
Know Before You Go!
