SBGrid will host Quo Vadis 2016: Advanced Crystallography, a 2-day course focused on indexing, integration, and scaling of challenging datasets and initial model building in corresponding experimental maps. The workshop will be held May 23-24 at Harvard Medical School in Boston. Agenda below. For information on lodging and location, please see the event website

This course will cover diverse strategies that support macromolecular structure determination. Computational phasing of structures with Phenix and CCP4 are not covered in this course.

Prerequisites:

• Knowledge of Linux/OS X command line tools or SBGrid101.
• Knowledge of processing of well-behaved datasets (i.e. ~2 Å resolution, no twinning or anisotropy)
• Experience with model building in 2-3 Å electron density maps.

6-8pm: Join us for pizza and drinks to learn your way around a Linux machine.

Monday, May 23:
SBGrid 251 - Advanced Data Collection and Processing Techniques

• 8:30am:   Breakfast.
• 9-10am:  Fundamental concepts and parameters of X-ray data processing (Kay Diederichs).
• 10-11am:  Tutorial: Indexing, integration, and scaling with XIA2 (includes XDS, DIALS, and other extensions) with a demonstration of how to process some of the most challenging datasets in the SBGrid Reference Collection (Graeme Winter).
• 11-12:30pm:  Data Processing Workshop: students will start with one of the datasets reviewed by Graeme and, for these selected datasets, we will provide instructions with a bit more detail. Students will then form 4-person groups and each group will process two or more reference datasets.
• 12:30-1:30pm:  Lunch and Meet with the Experts: students will continue working on datasets.
• 1:30-3:00pm:  Data collection strategies (James Holton).
• 3:00-3:30pm:  Data Processing Workshop, continued: student groups will meet again and prepare for presentations.
• 3:30-4:00pm:  Structural Biology Data Grid: an integrated platform for data publication and analysis (Piotr Sliz)..
• 4:00-5pm:  Student Presentations: each group will present their Table I and compare their results to published statistics. Instructors will provide feedback
• 5-7pm:  Posters, Meet with the Experts, and Dinner Buffet.
• 7-8pm:  Keynote Lecture: Experimental Phasing: theory and practice with Wayne Hendrickson.

Tuesday, May 24:
SBGrid 253 - Advanced Model Building Techniques

• 8:30am:   Breakfast.
• 9-11am:   Challenges of model building: building in experimental maps, low-resolution maps, and MR-maps computed with partial models. Tools and techniques to track correctness of model building in Coot (Paul Emsley and Jason Key).
• 11-12pm:  Model Building Workshop: students will work with reference structures and will be provided with experimental maps.
• 12-12:30pm:   Structure Modeling with Data-guided Molecular Dynamics Simulations: The Flexible Fitting Paradigm (Abhishek Singha Roy from the Theoretical and Computational Biophysics Group at U Illinois).
• 12:30-1:30pm:  Lunch and Meet with the Experts. Students continue working on datasets.
• 1:30-2:30pm:  Special Keynote: Using MicroED to Decipher Amyloid Structures with Atomic Resolution with Jose Rodriguez.
• 2:30-3:30pm:   Students will meet and prepare for presentations.
• 3:30-4:30pm:  Student Presentations.
Reference Datasets
Datasets can be downloaded from the Structural Biology Data Grid

Reference Case 1:   MR/Multi-crystal averaging.
Titus Boggon's Laboratory -- Dataset 5
Datasets from 5 crystals of SNX17 FERM domain in complex with a peptide corresponding to KRIT1’s NPxY2 motif. Separate integration of the datasets and scaling together allows a complete 3.0 Å dataset for molecular replacement solution (original paper used 4GXB as a search model) and structure refinement.

Reference Case 2:   MR/Low-resolution, twinned with rotational pseudosymmetry.
Richard Baxter's Laboratory -- Dataset 117
3.70 Å dataset collected on a crystal of thioester-containing protein 1 *S1 allele (TEP1*S1). Initial data processing suggested P43212, but one of the two molecules (~1300 aa. each) in the ASU overlapped with its symmetry-mate. Comparison of alternative scenarios in refinement identified the true space group as P43 with twinning and rotational pseudosymmetry. Refinement was completed with TLS, NCS (local) and external restraints derived by ProSMART using TEP1*R1 (PDB 4D94) as reference.

Reference Case 3:   U SAD/Low-resolution.
Yorgo Modis's Laboratory -- Dataset 62
4.5 Å dataset of a uranyl acetate derivative used for a challenging structure determination by SAD. Certain images had streaky features and were excluded from data reprocessing. The height and definition of peaks in anomalous difference Patterson maps was improved by omitting certain images near the end of the data collection run.

Reference Case 4:   Ba/K SAD; 91 nt RNA-chromophore complex.
Adrian Ferré-D'Amaré Laboratory -- Dataset 111
2.5 Å dataset collected at ALS BL 5.0.2 using 6.0 keV X-rays from a crystal of 'Spinach' a fluorescent RNA analog of GFP. Although anomalous signal was very weak, a heavy atom substructure comprised of one barium and six potassium ions resulted in good quality SAD electron density maps.

Reference Case 5:   Zn SAD; 4 Zn/ASU protein/RNA complex.
Piotr Sliz's Laboratory -- Dataset 3
2.9 Å Zn SAD dataset was sufficient to determine a crystal structure of Lin28/let-7d protein-microRNA complex. X-ray beam size was adjusted to maximize flux and minimize radiation damage. One swapped-dimer is located in each asymmetric unit. Two native zinc atoms are located in each tandem CCHC zinc knuckles domain.

Reference Case 6:   3.29-Å SeMet SAD 9 Se/ASU.
Katya Heldwein's Laboratory -- Dataset 123
This 3.29-Å selenomethionine SAD data set, collected at 0.9789 Å wavelength at BNL X25 beamline, was sufficient to determine the phases and to trace the structure of HSV-2 gH/gL complex. There are 9 Se sites in the ASU. During integration in HKL2000, χ2 appeared very large for some sectors of the data set. These correlated with crystal orientation and likely resulted from a large difference in cell edges (a=b=88 Å vs c=333 Å).

Reference Case 7:   MR-SAD at 7.0 Å.
Thomas Schwartz's Laboratory -- Dataset 179, Dataset 180
Contaminating E.coli protein 4FCC_A, acting as a crystallization chaperone, was found readily by MR. Using these MR phases seven (Ta6Br12)2+-positions could be found in the 8.8 Å derivative dataset 180. The combined MR-SAD phases were sufficient to position two copies of Nup37 (4FHL) and two copies of Nup120 in the asymmetric unit.

Reference Case 8:   MR-SAD at 2.65 Å (44 Se atoms/ASU).
Gabriella Rudenko's Laboratory -- Dataset 78, Dataset 218
3.25 Å dataset (#218) from a crystal of the selenomethionyl neurexin 1alpha ectodomain and 2.65 Å higher resolution native dataset (#78), both collected at APS using multiple settings. The structure has 2 molecules/ASU with a total of 14 ordered domains and ~2000 residues. Molecular replacement successfully placed 8 LNS domains (using a single LNS domain as a search model, i.e. ~9% of the scattering mass) generating phases which could be used to reveal 37 out of 44 Se atoms/ASU in the 3.25 Å SeMet SAD data set. Refinement was completed using dataset #78.

Reference Case 9:   3.25 Å dataset used for MR with a 9-Å cryo-EM envelope.
Yizhi Jane Tao's Laboratory -- Dataset 9
A 3.25-Å resolution dataset was collected at APS LS-CAT. The structure was determined by molecular replacement using a 9-Å resolution cryo-EM reconstruction as a phasing model. Solvent flattening and 15-fold noncrystallographic symmetry averaging were applied during phase extension.

Reference Case 10:   MR/large unit cell, anisotropic.
Catherine Drennan's Laboratory -- Dataset 83
Diffraction data from different regions of a crystal of Isobutyryl-coenzyme A mutase fused, a 250 kDa dimeric enzyme. This crystal had a large unit cell (a,b = 319 Å, c = 344 Å) and the data were anisotropic. Separate integration of the 6 wedges with individually adjusted resolution limits and scaling together yields a complete 3.35 Å dataset that can be used for molecular replacement.

Reference Case 11:   MR, lipidic cubic phase.
Andrew Kruse's Laboratory (data collected in Kobilka Laboratory) -- Dataset 125
Diffraction data for lipidic cubic phase crystals of human M2 muscarinic acetylcholine receptor bound to the agonist iperoxo, the allosteric modulator LY2119620, and the conformationally-selective nanobody Nb9-8.

Reference Case 12:   X-ray diffuse scattering.
James Fraser's Laboratory -- Dataset 68
1.2 Å dataset collected at SSRL provides a high-resolution standard dataset of the enzyme Cyclophilin to examine the influence of data collection temperature to compare to XFEL data, and to measure X-ray diffuse scattering.

Reference Case 13:   Fe-MAD; 1 Fe / ASU
C.S. Raman's Laboratory -- Dataset 97
A highly challenging three-wavelength MAD collected at ALS BL8.3.1 from recalcitrant crystals diffracting 7 keV X-rays to a Bragg spacing of 2.88 Å (complete data set) facilitated the identification of heme iron, as well as good quality electron density suitable for tracing the entire polypeptide chain, cofactor, and heme-bound gaseous ligand.