2025-11-25 @ ALS 8.3.1
Overall Goals
- Further optimize diffuse collection at 8.3.1
- Lysozyme, Mac1 (P43 and C2), Huwe1 data collection
- Mac1 soaks in both P43 and C2 spacegroups
Participants
- In person: Kara, James, Mehagan
- Remote: Justin and Joseph for processing, Steve for setting up OSN sync
Data
- Root directory at ALS:
/data/mcfuser/ucsf/fraser/25Nov2025/ - Root directory on OSN:
s3://diffuse-als-public/20251125/
Beamline Settings
Day 1 Parameters
| Parameter | Value | Notes |
|---|---|---|
| X-ray energy | 14000 eV @ 0.014 % bandwidth | |
| Beam size | 100 µm x 100 µm, circle | pinhole.com 300 Defocused Beam_defocused.jpg for profile M1 moved from 2660 to 1980 M2 to compensate Divergence - 0.6 x 0.1 mrad |
| Flux | 1.73 x 10¹⁰ ph/s | 860 Gy/s But we are in decay mode of the ring so will drop over time |
| Beamstop | standard 8.3.1 | |
| Data collection software | Blu-Ice | |
| Temperature control | None, turned cryo stream off and moved out |
Samples
| Name | Sample | Well composition | Drop composition | Notes |
|---|---|---|---|---|
| Mac1 (P43) | SARS CoV2 NSP3 macrodomain and seed stock. 40 mg/mL Mac1 in 150 mM NaCl, 20 mM Tris pH 8, 5% glycerol | 34% (w/vol) PEG 3000 + 100 mM CHES (pH 9.5) | 200 nL protein solution + 100 nL well solution + 100 nL seeds | Galen setup Reference PDB: 7TWF |
| Huwe1 (P43 21 2) | 88 mg/mL | NaCl 2-3.5M NaOac 50-200mM pH 4.84 | 200 nL protein solution + 200 nL well solution | |
| Lysozyme | 20 mg/mL | From Hampton kit 30% PEG 5000, 1 M NaCl, 0.05 M Na Acetate trihydrate pH 4.6 |
||
| Mac1 (C2) | SARS CoV2 NSP3 macrodomain, 15 mg/mL Mac1 in 150 mM NaCl, 20 mM Tris pH 8, 5% glycerol | 100 mM Tris pH 8.5, 100 mM Na Acetate, 28% PEG 4000 | 200 nL protein solution + 200 nL well solution | Kara purified protein and setup 2 trays on 11/23 Reference PDB: 7KR0 |
Data collection, day 1
1. Lysozyme
Subdirectory: lysozyme/lysozyme_1
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| lysozyme_1 | 30 | 750 | 0.1 | 7200 | 0.01 | 175 | Crystal slipped, aborted run |
2. Mac1
Subdirectory: mac1/mac1_1
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_1 | 30 | 750 | 0.1 | 7200 | 0.01 | 175 | Diffraction weaker than expected and some extra background rings, may need to clean beam path up here and possibly increase dose Detector still paused around 7000 frames but eventually finished after writing out the data |
| mac1_1_bg | 30 | 750 | 1 | 720 | 0.1 | 175 | Went ~180 um away, rings still present - seems to be coming from collimator |
Need to get rid of collimator rings - try lowering energy to see if that helps as a start
Note
Switching to 11.145 keV - will be higher dose rate
| Parameter | Value | Notes |
|---|---|---|
| X-ray energy | 11145 eV @ 0.014 % bandwidth | |
| Beam size | 100 µm x 100 µm, circle | Divergence the same - 0.6 x 0.1 mrad Some tune up after changing energy |
| Flux | 3.26 x 10¹⁰ ph/s | 2573 Gy/s But we are in decay mode of the ring so will drop over time |
3. Mac1
Subdirectory: mac1/mac1_2
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_2_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | Increased exposure time to help the detector Much better diffraction Data got broken up into sweeps still All data got written with no pause |
| mac1_2_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Moved 150 away |
Based on XDS plots - for sure damaging beyond 440 deg (based on Garman limit), damaging more like 180 deg for R_d, might be okay
4. Mac1
Subdirectory: mac1/mac1_3
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_3_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | Looking good |
| mac1_3_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Moved 150 away |
5. Huwe1
Subdirectory: huwe1/huwe1_1
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| huwe1_1_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | Looking good |
| huwe1_1_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Moved 250 away Also looks like it survives to about 360 |
6. Huwe1
Subdirectory: huwe1/huwe1_2
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| huwe1_2_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | Looking good |
| huwe1_2_data_2 | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | Moved 150 um away to take data on new spot on the crystal Detector a little unhappy, paused in the middle a few times - will give multiple sweeps |
| huwe1_2_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Moved 300 away from second spot |
James increased the ram limit to help
7. Huwe1
Subdirectory: huwe1/huwe1_3
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| huwe1_1_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | Looking good and detector happier, wrote as one sweep |
| huwe1_1_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Moved 300 away |
Mac1 - set up 3 soaks - 9 uL reservoir solution + 1 uL 200 mM ADPr (so final concentration is 20 mM) - First soak - starts at 0 min - Second soak - starts at 6 min - Third soak - starts at 11 min
8. Mac1
Subdirectory: mac1/mac1_4
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_4_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | |
| mac1_4_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Accidentally went into mac1_4_bg directory |
Now at 1700 Gy/s, ring keeps decaying
9. Mac1
Subdirectory: mac1/mac1_5
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_5_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | |
| mac1_5_bg | 0 | 720 | 1 | 720 | 0.2 | 175 |
Warning
Skip, crystal slipped
10. Mac1 + ADPr
Soaked with ADPr for ~50 min (9 uL + 1 uL)
Subdirectory: mac1/mac1_6
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_6_adpr_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | All good |
| mac1_6_adpr_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | All good, went 300 um away |
Info
Quick check with XDS/dimple - looks like high ADPr occupancy
Shutters closed by ALS - finally refilling the ring
Beam back - tuneup.com
Flux - 5.922 x 10^10 ph/s, dose is 4668 Gy/s
11. Mac1 + ADPr
Soaked with for ~60 min (9 uL + 1 uL)
Subdirectory: mac1/mac1_7
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_7_adpr_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | All good |
| mac1_7_adpr_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 250 um away |
Quick check with XDS/dimple
fairly high occupancy, but not as high as Mac1_6
12. Lysozyme
Subdirectory: lysozyme/lysozyme_2
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| lysozyme_2_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | All good |
| lysozyme_2_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 350 um away |
13. Lysozyme
Subdirectory: lysozyme/lysozyme_3
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| lysozyme_3_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | All good |
| lysozyme_3_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 200 um away |
14. Mac1 + ADPr
Soaked with ADPr for ~15min (9 uL + 1 uL)
Subdirectory: mac1/mac1_8
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_8_adpr_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | All good |
| mac1_8_adpr_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 200 um away |
Quick check with XDS/dimple
density is there
15. Mac1 + ADPr
Soaked with ADPr for ~30min (9 uL + 1 uL)
Subdirectory: mac1/mac1_9
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_9_adpr_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | All good |
| mac1_9_adpr_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 250 um away |
Quick check with XDS/dimple
density is there
16. Mac1 + ADPr
Soaked with ADPr for ~20min (9 uL + 1 uL)
Subdirectory: mac1/mac1_10
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_10_adpr_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | All good |
| mac1_10_adpr_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 200 um away |
Quick check with XDS/dimple
pretty good occupancy - maybe due to smaller crystal?
17. Mac1 + ADPr
Soaked with ADPr for ~50min (9 uL + 1 uL)
Subdirectory: mac1/mac1_11
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_11_adpr_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | All good |
| mac1_11_adpr_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 200 um away |
Quick check with XDS/dimple
lower occupancy, maybe due to larger crystal?
Mac1 C2, 1 - test, skip
End of day 1
Data collection, day 2
11.26.2025
Starting with long Mac1 P43 soaks
- AVI-3731 (opener) - 2 uL + 0.5 uL (100 mM stock) - some phase separation in the drop, probably due to DMSO? Let soak go 1-2 hrs - crystals cracked at ~1 hr mark, try again with shorter time or lower concentration
- ADPr - 9 uL + 1 uL (200 mM stock) - let go 1-2 hrs
Dose rate: 4828 Gy/s
18. Mac1 + ADPr
Soaked with ADPr for ~90min (9 uL + 1 uL)
Subdirectory: mac1/mac1_12
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_12_adpr_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | All good |
| mac1_12_adpr_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 200 um away - accidentally has 824 images because the data collection was incorrectly set for 7200 images and aborted |
Quick check with XDS/dimple
ADPr density
Mac1, 13 - skip
19. Mac1 + AVI-3731
Added 100 nL directly to well H9 (multiple crystals) let sit for ~30 min
Subdirectory: mac1/mac1_14
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_14_avi3731_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | All good |
| mac1_14_avi3731_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 200 um away |
Quick check with XDS/dimple
Not sure what the compound is supposed to look like but there is positive density near V49, not many large sidechain motions though
20. Mac1 + AVI-3731
Added 100 nL directly to well H10 (multiple crystals) let sit for ~30 min
Subdirectory: mac1/mac1_15
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_15_avi3731_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | All good |
| mac1_15_avi3731_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 200 um away |
Quick check with XDS/dimple
for sure ligand density, not a ton of sidechain motions
At 60 min, crystals are dead for drops H9 and H10
For G10, added 200 nL of well solution and then 100 nL of AVI3731
Also made two drops - 4.5 uL each, transferred one crystal per drop, then added 0.5 uL of AVI3731 to the drop
21. Mac1 + AVI-3731
Added 200 nL reservoir + 100 nL AVI-3731 directly to well G10 (multiple crystals) let sit for ~45 min
Subdirectory: mac1/mac1_16
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_16_avi3731_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | All good |
| mac1_16_avi3731_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 200 um away |
Quick check with XDS/dimple
seeing large green density, not a lot of motions
22. Mac1 + AVI-3731
Made 4.5 uL drop of reservoir solution, added crystal, and then added 0.5 uL AVI-3731 (single crystal) let sit for ~45 min
Subdirectory: mac1/mac1_17
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_17_avi3731_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | Diffraction a bit weaker than typical - looks like I only got a piece of the crystal |
| mac1_17_avi3731_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 400 um away |
Quick check with XDS/dimple
larger green blob for ligand
23. Mac1 + AVI 3731
Made 4.5 uL drop of reservoir solution, added crystal, and then added 0.5 uL AVI-3731 (single crystal) let sit for ~60 min (second drop)
Subdirectory: mac1/mac1_18
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_18_avi3731_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | Was able to loop the entire crystal, all good |
| mac1_18_avi3731_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 400 um away |
Quick check with XDS/dimple
stronger ligand density and also some other peaks?
Made 2 more 4.5 uL drops, added crystal, and then added 0.5 uL of AVI 3731 - let sit for 1.5-2 hrs
Mac1 C2, 2 - lost it or not diffracting well, skip
24. Mac1 + AVI-3731
Made 4.5 uL drop of reservoir solution, added crystal, and then added 0.5 uL AVI-3731 (single crystal) let sit for ~120 min
Subdirectory: mac1/mac1_19
| Prefix | φ0 (deg.) | φ1 (deg.) | ∆φ (deg.) | Images | ∆t (s) | d (mm) | Notes |
|---|---|---|---|---|---|---|---|
| mac1_19_avi3731_data | 0 | 720 | 0.1 | 7200 | 0.02 | 175 | all good |
| mac1_19_avi3731_bg | 0 | 720 | 1 | 720 | 0.2 | 175 | Went 200 um away |
Quick check with XDS/dimple
strong ligand density
done!