2025-11-05 @ CHESS 7b2

Our fourth CHESS beam time of the 2025-3 run cycle.

Goals

• Training new members
• Standardize methods across beamlines (Kara visiting)
• Data processing on voltage park (Joseph, Justin visiting)
• Mac1 +/- ADP-ribose
• Continue data collection on NrdE, DNA crystals
• Screen ATCase R-state crystals (PALA, +/- ATP and GTP)
• Test next prototype guard aperture for on-axis camera setup

Participants

Steve M, Xiaokun P, Katie L, Sarah H, Marcus G, Shaheer K (from Ando lab), Kara Z (from Fraser lab), Joseph L and Justin B (from Astera).

• 
  Participants during the afternoon of day 1 (left to right): Joseph, Steve, Justin, Kara, Sarah, and Xiaokun. Marcus, Shaheer, and Katie arrived later (not shown).

• 
  Justin and Joseph running mdx2 on a Voltage Park node during data collection.

• 
  Kara obtained difference density showing ADP-ribose bound to Mac1 after soaking.

• 
  Katie and Sarah collecting a dataset.

• 
  Shaheer operating the beamline.

• 
  Shaheer, Katie, and Sarah at 7b2. Katie is looping a crystal in the humid chamber.

• 
  Shaheer inspecting the mounted crystal under a stereo microscope.

• 
  Marcus in the 7b2 hutch, placing a sample on the goniometer.

• 
  Xiaokun teaching Shaheer crystal looping technique.

Data

Root directory at CHESS: /nfs/chess/raw/2025-3/id7b2/meisburger/20251105

Root directory on OSN: s3://diffuse-chess-public/20251105

Beamline setup

The beamline setup was similar to 9/24/2025 (no in-line camera, no temperature control). A new SPEC macro was developed to automatically take crystal images every 30 degrees.

parameter	value	notes
X-ray energy	14 keV @ 0.01% bandwidth	Si 111 channel cut mono inserted
Beam size	100 µm x 100 µm, top-hat profile	Defined using slits, CRL bypassed (toroidal mirror focusing only)
Flux	5.6 x 1010 ph/s unattenuated	See station notebook: Steve Meisburger CHESS notebook #3 p. 52
Background reduction	300 µm aperture placed close to the sample	The prototype aperture tests were not successful, so we reverted to the default setup.
Centering camera	top-view camera	1.713 µm / pixel at 4x zoom ratio. A new spec macro was used to automatically save crystal images every 30 degrees.
Beamstop	700 µm diameter Mo disk suspended on mylar sheet, semi-transparent	At this energy, the bleedthrough was more intense than usual, and there were some faint diffraction rings visible in the image
Data collection software	"MX Collect" (python) & SPEC	The main GUI was the same as last time. A <prefix>_scan.json file was created for each data collection. Counter values and motor positions were recorded in a <prefix>.spec file. Images in h5 format were generated using Eiger filewriter.
Temperature control	none	All datasets collected at ambient temperature

• 
  Mike C and Zhongwu W created this new version of the on-axis centering mirror with integrated cleanup aperture. Zhongwu drilled a 250 µm hole in tantalum to create an X-ray aperture, and this was epoxied to the end of the Mo tube. During commisioning time, we were able to align the aperture, and it worked as expected (no slit scatter around the beamstop). However, it was apparent from the diffraction images that epoxy had gotten into the aperture hole. Mike C will work on gluing jig and create a new version for our next visit.

• 
  Goniometer setup that was used for data collection, showing the beamstop and guard aperture close to the sample.

Samples

All protein crystals were grown by vapor diffusion using 24-well hanging drop trays.

Name	Sample	Well composition	Drop composition	Notes
Lysozyme	50 mg/mL lysozyme in 20 mM NaOAc pH 4.6	0.7-1.0 M NaCl, 0.1 M NaOAc pH 4.4	2 µL protein + 2 µL well solution	Shaheer's lysozyme tray (9/16/25).
Mac1	SARS CoV2 NSP3 macrodomain and seed stock from UCSF. 40 mg/mL Mac1 in 150 mM NaCl, 20 mM Tris pH 8, 5% glycerol	28-38% (w/vol) PEG 3000 + 100 mM CHES (pH 9.5)	2 µl protein solution + 1 ul well solution + 1 ul seeds (diluted 10x)	Mac1 tray #1 row C (10/2/2025) and tray #2 (10/30/2025). See Katie L Ando Lab notebook pp. 12, 15, 18, 19.
ADPr	200 mM ADP-ribose in water			from UCSF (source: Sigma A0752)
BsNrdE	Bacillus subtilis class Ib ribonucleotide reductase alpha subunit. 4.5 mg/mL Holo-NrdE protein in 50 mM HEPES pH 7.6, 50 mM NaCl, 5 mM MgCl2, 2 mM TCEP, 1% glycerol, 5 mM ATP, 1 mM ADP, 0.5 mM dGTP	50 mM HEPES pH 7, 5-6% (w/vol) PEG 3350, 1% tryptone	1:1 protein:well solution (4-5 µL total volume)	Marcus' BsNrdE tray #2, set up on 10/25/2025. See Marcus' Ando Lab notebook pp. 55-59.
DNA	S2T7-1, S2T7-2, and S2T7-3 single strands at 83 µM in 40 mM tris acetate, 2 mM EDTA	40 mM tris acetate, 37.5 mM Mg acetate, 1.75 M ammonium sulfate	3:1 ratio of DNA + well solution, total volume 3 µL (row A) or 5 µL (row B)	DNA tray #2. See Steve Meisburger Ando Lab notebook #3, pp. 50, 51.
ATCase	Escherichia coli Aspartate Transcarbamoylase and seed stock. 16 µM holoenzyme in 40 mM Tris pH 7.5, 15 mM MgCl2, 1 mM TCEP, 2 mM PALA, +/- nucleotides (10 mM ATP and 2 mM GTP)	8% v/v Tacsimate pH 7.0, 5-15% PEG-3350	2 µL protein + 1 µL well solution + 1 µL seed stock	RAG-ATCase tray #2 (10/7/2025) and R-ATCase tray #1 (10/29/2025). See Sarah H Ando Lab notebook pp. 95, 103.

• 
  Well B2 of Shaheer's lysozyme tray (9/16/2025) Well solution: 0.7 M NaCl, 0.1 M NaOAc pH = 4.4. Drop: 2 uL of lysozyme + 2 uL well solution

• 
  Well B5 of Shaheer's lysozyme tray (9/16/2025). Well solution: 1.0 M NaCl, 0.1 M NaOAc pH = 4.4. Drop: 2 uL of lysozyme + 2 uL well solution

• 
  Well A3 of Mac1 tray #2 (10/30/2025). Well solution: 30% (w/vol) PEG 3000 + 100 mM CHES (pH 9.5). Drop: 2 µl protein solution + 1 ul well solution + 1 ul seeds (diluted 10x in water).

• 
  Well B1 of Mac1 tray #2 (10/30/2025). Well solution: 30% (w/vol) PEG 3000 + 100 mM CHES (pH 9.5). Drop: 2 µl protein solution + 1 ul well solution + 1 ul seeds (diluted 10x in water).

• 
  Well B4 of Mac1 tray #2 (10/30/2025). Well solution: 30% (w/vol) PEG 3000 + 100 mM CHES (pH 9.5). Drop: 2 µl protein solution + 1 ul well solution + 1 ul seeds (diluted 10x in water).

• 
  Well C1 of Mac1 tray #1 (10/2/2025). Well solution: 28% (w/vol) PEG 3000 + 100 mM CHES (pH 9.5). Drop: 2 µl protein solution + 1 ul well solution + 1 ul seeds (diluted 10x in well solution).

• 
  Well C2 of Mac1 tray #1 (10/2/2025). Well solution: 30% (w/vol) PEG 3000 + 100 mM CHES (pH 9.5). Drop: 2 µl protein solution + 1 ul well solution + 1 ul seeds (diluted 10x in well solution).

• 
  Well C3 of Mac1 tray #1 (10/2/2025). Well solution: 32% (w/vol) PEG 3000 + 100 mM CHES (pH 9.5). Drop: 2 µl protein solution + 1 ul well solution + 1 ul seeds (diluted 10x in well solution).

• 
  Well A4 (right drop) of BsNrdE tray #2 (10/25/2025). Well solution: 50 mM HEPES (pH 7), 5.5% (w/vol) PEG 3350, 1% tryptone (total volume of 250 uL). Drop: 2 µl protein solution + 2 ul well solution.

• 
  Well A5 (right drop) of BsNrdE tray #2 (10/25/2025). Well solution: 50 mM HEPES (pH 7), 5% (w/vol) PEG 3350, 1% tryptone (total volume of 250 uL). Drop: 2 µl protein solution + 2 ul well solution.

• 
  Well B5 (right drop) of BsNrdE tray #2 (10/25/2025). Well solution: 50 mM HEPES (pH 7), 5% (w/vol) PEG 3350, 1% tryptone (total volume of 250 uL). Drop: 2 µl protein solution + 2 ul well solution.

• 
  Well C3 (right drop) of BsNrdE tray #2 (10/25/2025). Well solution: 50 mM HEPES (pH 7), 6% (w/vol) PEG 3350, 1% tryptone (total volume of 250 uL). Drop: 2.5 µl protein solution + 2.5 ul well solution.

• 
  Well C1 of RAG-ATCase tray #2 (10/7/2025). Well solution: 8% v/v Tacsimate pH 7.0, 12% PEG-3350. Drop: 2 µL protein + 1 µL well solution + 1 µL seed stock.

• 
  Well C2 of RAG-ATCase tray #2 (10/7/2025). Well solution: 8% v/v Tacsimate pH 7.0, 12.5% PEG-3350. Drop: 2 µL protein + 1 µL well solution + 1 µL seed stock.

• 
  Well B6 of RAG-ATCase tray #2 (10/7/2025). Well solution: 8% v/v Tacsimate pH 7.0, 15% PEG-3350. Drop: 2 µL protein + 1 µL well solution + 1 µL seed stock.

• 
  Well B6 of R-ATCase tray (10/29/25). Well solution: 8% v/v Tacsimate pH 7.0, 15% PEG-3350. Drop: 2 µL protein + 1 µL well solution + 1 µL seed stock.

• 
  Well A6 of DNA tray #2. Well solution: 40 mM tris acetate, 37.5 mM Mg acetate, 1.75 M ammonium sulfate. Drop: 2.25 µl DNA solution + 0.75 µl of well solution

• 
  Well B6 of DNA tray #2. Well solution: 40 mM tris acetate, 37.5 mM Mg acetate, 1.75 M ammonium sulfate. Drop: 3.75 µl DNA solution + 1.25 µl of well solution

Data collection, day 1

Samples for room temperature data collection were harvested in the humidity chamber (100 % RH). Reusable MiTeGen bases are used with micro RT capillaries cut to length, with 10 µL of well solution in the tip.

With the large number of participants, we sometimes had multiple projects going at once. Note that the following is organized chronologically rather than by project.

1. Lysozyme

Steve looped at ~600 µm lysozyme crystal from well B5 of Shaheer's tray using a 500 µm loop, micro RT with 10 µL well solution in the tip. Harvested in humid chamber at ~100% RH.

Subdirectory: lysozyme/calibration_sample/lys_cal1

Saved 12 images (full rotation, every 30 degrees), top view, zoom 2: lys_cal1_160_50_tvc_zoom2_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
lys_cal1_3616	0	360	0.1	3600	0.01	100	185	14
lys_cal1_3617	0	360	0.1	3600	0.01	47.1	185	14
lys_cal1_bg_3618	0	360	1	360	0.1	100	185	14
lys_cal1_bg_3619	0	360	1	360	0.1	47.1	185	14

Note

3616 and 3617 were collected from different locations.

xia2 processing

	lys_cal1_3616	lys_cal1_3617
Mosaic spread	0.014	0.012
Resolution	1.08	1.09
Unit Cell	[78.44, 78.44, 37.61, 90.0, 90.0, 90.0]	[78.43, 78.43, 37.61, 90.0, 90.0, 90.0]
Image range	[1, 3600]	[1, 3600]
Completeness	97.0	97.6
Multiplicity	21.4	21.8
I/sigma	16.6	13.5
Rpim	0.022	0.024
Wilson B factor	16.38	16.1
Space group	P 43 21 2	P 43 21 2

2. Mac1 + ADPr

Kara opened well A3 from Katie's Mac1 tray #2. She pipetted 1 µL of 40 mM ADP-ribose (in water). We don't exactly know the drop volume, our target concentration is 20 mM. Kara set a timer for 10 minutes. She loaded 10 µL of well solution in the capillary.

Subdirectory: mac1/mac1_1_adpr

Saved images every 30 degrees: mac1_1_adpr_10_60_tvc_zoom2_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
mac1_1_adpr_3620	0	720	0.1	7200	0.01	100	185	14
mac1_1_adpr_bg_3621	0	720	1	720	0.1	100	185	14

xia2 processing (first 180 degrees)

	mac1_1_adpr_3620
Mosaic spread	0.009
Resolution	1.02
Unit Cell	[88.47, 88.47, 40.07, 90.0, 90.0, 90.0]
Image range	[1, 1800]
Completeness	81.3
Multiplicity	5.7
I/sigma	9.7
Rpim	0.052
Wilson B factor	11.97
Space group	P 43

Note

Ran a quick molecular replacement using dimple. No ADPr density observed.

3. BsNrdE

Marcus looped a NrdE crystal from well A4 (right drop) in NrdE tray #2. 400 µm loop, sleeve with 8 µL well solution.

Subdirectory: BsNrdE/MG_A4r

Saved 12 images (every 30˚): MG_A4r_10_60_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
MG_A4r_3622	0	720	0.1	7200	0.01	100	225	14
MG_A4r_bg_3623	0	720	1	720	0.1	100	225	14

Warning

Noticed lots of radiation damage. Next sample, try a vector scan or attenuation.

Saved a photo of the cracked crystal: MG_A4r_cracked_tvc_zoom4_1.png

xia2 processing (first 180 degrees)

	MG_A4r_3622
Mosaic spread	0.032
Resolution	1.97
Unit Cell	[120.0, 126.67, 127.1, 90.0, 90.0, 90.0]
Image range	[1, 1800]
Completeness	100.0
Multiplicity	6.9
I/sigma	11.6
Rpim	0.043
Wilson B factor	39.04
Space group	P 21 21 21

4. Mac1 + ADPr

Kara added 1 µL of 200 mM ADP-ribose to the crystal drop, pipetted up and down, and waited for ~10 minutes before looping. 10 µL of well solution was added to a new sleeve (re-used loop). The crystal came from Katie's Mac1 tray #2, but the particular well was not recorded.

Subdirectory: mac1/mac1_2_adpr

Saved images every 30˚: mac1_2_adpr_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
mac1_2_adpr_3624	0	720	0.1	7200	0.01	100	185	14
mac1_2_adpr_bg_3625	0	720	1	720	0.1	100	185	14

Warning

For scan 3624, the shutter was closed at the start of data collection. Frames 1-660 are blank.

xia2 processing (first 360 degrees)

	mac1_2_adpr_3624
Mosaic spread	0.021
Resolution	1.02
Unit Cell	[88.47, 88.47, 40.06, 90.0, 90.0, 90.0]
Image range	[671, 3600]
Completeness	78.9
Multiplicity	9.5
I/sigma	19.4
Rpim	0.018
Wilson B factor	12.86
Space group	P 43

5. BsNrdE

Marcus looped a NrdE crystal from well C3 (right drop) from NrdE tray #2 using a 400 µm loop with 8 µL well solution in sleeve.

Subdirectory: BsNrdE/MG_C3r

Saved 12 images per 30˚: MG_C3r_adpr_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	∆x (µm)	E (keV)
MG_C3r_3626	0	360	0.1	3600	0.01	100	225	347.76	14
MG_C3r_bg_3627	0	360	1	360	0.1	100	225	nan	14

3626 was a vector scan.

How to estimate dose for vector scans

∆x is the component of translation along the spindle axis. The maximum residence time of a part of the crystal in the beam can be estimated as (w/∆x)*images*∆t, where w is the beam width (100 µm). Here, 10.35 s.

xia2 processing

	MG_C3r_3626
Mosaic spread	0.023
Resolution	1.92
Unit Cell	[120.01, 126.72, 127.14, 90.0, 90.0, 90.0]
Image range	[1, 3600]
Completeness	99.8
Multiplicity	13.9
I/sigma	11.5
Rpim	0.033
Wilson B factor	38.71
Space group	P 21 21 21

6. Mac1 + ADPr

Kara made a drop of 9 µL reservoir solution and added 1 µL of 200 mM ADPr. She looped a few crystals into the drop, and let soak for ~10 minutes. The crystal came from Katie's Mac1 tray #1. The well was not recorded, but was one of C1, C2, or C3.

Subdirectory: mac1/mac1_3_adpr

Saved 12 images per 30˚: mac1_3_adpr_190_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
mac1_3_adpr_3628	0	720	0.1	7200	0.01	100	185	14
mac1_3_adpr_bg_3629	0	720	1	720	0.1	100	185	14

xia2 processing (first 360 degrees)

	mac1_3_adpr_3628
Mosaic spread	0.017
Resolution	1.04
Unit Cell	[88.41, 88.41, 39.86, 90.0, 90.0, 90.0]
Image range	[1, 3600]
Completeness	82.4
Multiplicity	11.8
I/sigma	14.9
Rpim	0.022
Wilson B factor	12.69
Space group	P 43

Note

Did a quick MR using dimple: density shows ~50% occupancy of ADPr!

7. ATCase (RAG)

Sarah looped an ATCase crystal from well C1 of RAG tray #2 using a 300 µm loop with 8 µL well solution in sleeve.

Subdirectory: RAG_ATCase

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
rag_1_3630	0	360	0.1	3600	0.01	100	300	14
rag_1_bg_3631	0	360	1	360	0.1	100	300	14

Evident radiation damage from spot fading. Next time use attenuation and/or vector scan.

xia2 processing (first 90 degrees)

	rag_1_3630
Mosaic spread	0.016
Resolution	3.02
Unit Cell	[126.54, 152.72, 206.18, 90.0, 90.0, 90.0]
Image range	[1, 900]
Completeness	89.5
Multiplicity	3.9
I/sigma	7.2
Rpim	0.081
Wilson B factor	80.68
Space group	P 21 21 21

8. ATCase (RAG)

Sarah looped another ATCase crystal from the same well (C1, RAG tray #2) using a 300 µm loop with 8 µL well solution in sleeve.

Subdirectory: RAG_ATCase

sample	prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	∆x (µm)	E (keV)
rag_2	rag_2_3632	0	360	0.1	3600	0.01	47.1	300	167.93	14
rag_2	rag_2_bg_3633	0	360	1	360	0.1	47.1	300	nan	14

Vector data collection was used to distribute the dose along the crystal.

xia2 processing (first 180 degrees)

	rag_2_3632
Mosaic spread	0.039
Resolution	3.02
Unit Cell	[126.23, 152.53, 206.32, 90.0, 90.0, 90.0]
Image range	[1, 1800]
Completeness	100.0
Multiplicity	6.9
I/sigma	6.5
Rpim	0.096
Wilson B factor	79.1
Space group	P 21 21 21

9. Mac1

Katie looped a Mac1 crystal from well B1 of Mac1 tray #2. 500 µm loop with 10 µL well solution in the sleeve.

Subdirectory: mac1/mac1_4

Saved images every 30˚: mac1_4_190_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
mac1_4_3634	0	720	0.1	7200	0.01	100	185	14
mac1_4_bg_3635	0	720	1	720	0.1	100	185	14

xia2 processing

	mac1_4_3634
Mosaic spread	0.009
Resolution	1.02
Unit Cell	[88.42, 88.42, 39.94, 90.0, 90.0, 90.0]
Image range	[1, 7200]
Completeness	80.7
Multiplicity	22.8
I/sigma	26.1
Rpim	0.015
Wilson B factor	13.03
Space group	P 43

10. Lysozyme

Shaheer looped a lysozyme crystal from well B2 (right drop) of 9/16/25 lysozyme tray. 500 µm loop with 10 µL well solution in sleeve.

Subdirectory: lysozyme/sk_B2r

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
sk_B2r_3636	0	360	0.1	3600	0.01	100	200	14
sk_B2r_bg_3637	0	360	1	360	0.1	100	200	14

xia2 processing

	sk_B2r_3636
Mosaic spread	0.013
Resolution	1.08
Unit Cell	[78.42, 78.42, 37.61, 90.0, 90.0, 90.0]
Image range	[1, 3600]
Completeness	88.4
Multiplicity	20.5
I/sigma	24.9
Rpim	0.012
Wilson B factor	16.04
Space group	P 43 21 2

11. Mac1

Katie looped a Mac1 crystal from well B4 of Mac1 Tray #2. 500 µm loop with 10 µL well solution in sleeve.

Subdirectory: mac1/mac1_5

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
mac1_5_3638	0	720	0.1	7200	0.01	100	185	14
mac1_5_bg_3639	0	720	1	720	0.1	100	185	14

xia2 processing

	mac1_5_3638
Mosaic spread	0.008
Resolution	1.02
Unit Cell	[88.44, 88.44, 39.97, 90.0, 90.0, 90.0]
Image range	[1, 7200]
Completeness	81.9
Multiplicity	22.5
I/sigma	19.7
Rpim	0.02
Wilson B factor	12.93
Space group	P 43

12. BsNrdE

Xiaokun looped a BsNrdE crystal from Macrus' crystal tray, well B5 (right drop). The rod-shaped crystal was oriented diagonally ~45˚ on the loop, in order to improve reciprocal space coverage after symmetry averaging.

Subdirectory: BsNrdE/MG_B5r

Saved images every 30˚: MG_B5r_190_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	∆x (µm)	E (keV)
MG_B5r_3640	190	550	0.1	3600	0.01	100	225	210.79	14
MG_B5r_bg_3641	190	550	1	360	0.1	100	225	nan	14

Vector scan along the length of the crystal was used to distribute the dose.

xia2 processing

	MG_B5r_3640
Mosaic spread	0.026
Resolution	1.94
Unit Cell	[120.04, 126.79, 127.16, 90.0, 90.0, 90.0]
Image range	[1, 3600]
Completeness	99.9
Multiplicity	13.9
I/sigma	11.2
Rpim	0.035
Wilson B factor	40.29
Space group	P 21 21 21

13. Mac1 + ADPr

Kara made a new drop with 9 µL reservoir solution + 1 µL 200 mM ADPr, looped mac1 crystal and put it into the new drop. Allowed to soak for 30 minutes before harvesting. The crystal came from Katie's Mac1 tray #1. The well was not recorded, but was one of C1, C2, or C3.

Subdirectory: mac1/mac1_6_adpr

Saved images every 30˚: mac1_adpr_6_190_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
mac1_6_adpr_3642	0	720	0.1	7200	0.01	100	185	14
mac1_6_adpr_bg_3643	0	720	1	720	0.1	100	185	14

xia2 processing

	mac1_6_adpr_3642
Mosaic spread	0.026
Resolution	1.04
Unit Cell	[88.37, 88.37, 39.84, 90.0, 90.0, 90.0]
Image range	[1, 3600]
Completeness	84.3
Multiplicity	11.5
I/sigma	14.6
Rpim	0.021
Wilson B factor	12.44
Space group	P 43

14. BsNrdE

Xiaokun looped another crystal from Marcus' NrdE tray #2, well A5 (right drop). The rod-shaped crystal was oriented diagonally on the loop, to improve coverage of reciprocal space after symmetry averaging.

Subdirectory: BsNrdE/MG_A5r

Saved images every 30˚: MG_A5r_6_190_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	∆x (µm)	E (keV)
MG_A5r_3644	10	550	0.1	5400	0.01	100	225	308.3	14
MG_A5r_bg_3645	10	550	1	540	0.1	100	225	nan	14

Vector scan along the length of the crystal to distribute dose.

xia2 processing

	MG_A5r_3644
Mosaic spread	0.027
Resolution	1.94
Unit Cell	[120.05, 126.81, 127.17, 90.0, 90.0, 90.0]
Image range	[1, 5400]
Completeness	100.0
Multiplicity	20.8
I/sigma	10.3
Rpim	0.04
Wilson B factor	39.41
Space group	P 21 21 21

15. Mac1 + ADPr

Kara looped mac1 crystal and put it into a drop with 9 µL reservoir solution + 1 µL 200 mM ADPr. Allowed to soak for 30 minutes before harvesting. The crystal came from Katie's Mac1 tray #1. The well was not recorded, but was one of C1, C2, or C3.

Subdirectory: mac1/mac1_7_adpr

Saved images very 30˚: mac1_7_adpr_190_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
mac1_7_adpr_3646	0	720	0.1	7200	0.01	100	185	14
mac1_7_adpr_bg_3647	0	720	1	720	0.1	100	185	14

xia2 processing (first 360 degrees)

	mac1_7_adpr_3646
Mosaic spread	0.022
Resolution	1.04
Unit Cell	[88.37, 88.37, 39.86, 90.0, 90.0, 90.0]
Image range	[1, 3600]
Completeness	84.5
Multiplicity	11.4
I/sigma	13.3
Rpim	0.023
Wilson B factor	12.24
Space group	P 43

16. Mac1 + ADPr

Kara looped mac1 crystal and put it into a drop with 9 µL reservoir solution + 1 µL 200 mM ADPr. Allowed to soak for 45 minutes before harvesting. The crystal came from Katie's Mac1 tray #1. The well was not recorded, but was one of C1, C2, or C3.

Subdirectory: mac1/mac1_8_adpr

Saved images every 30˚: mac1_8_adpr_190_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
mac1_8_adpr_3648	0	720	0.1	7200	0.01	100	185	14
mac1_8_adpr_bg_3649	0	720	1	720	0.1	100	185	14

xia2 processing (first 360 degrees)

	mac1_8_adpr_3648
Mosaic spread	0.028
Resolution	1.04
Unit Cell	[88.37, 88.37, 39.92, 90.0, 90.0, 90.0]
Image range	[1, 3600]
Completeness	82.7
Multiplicity	11.7
I/sigma	13.9
Rpim	0.023
Wilson B factor	12.08
Space group	P 43

17. Mac1 + ADPr

Kara looped mac1 crystal and put it into a drop with 9 µL reservoir solution + 1 µL 200 mM ADPr. Allowed to soak for 45 minutes before harvesting. The crystal came from Katie's Mac1 tray #1. The well was not recorded, but was one of C1, C2, or C3.

Subdirectory: mac1/mac1_9_adpr

Saved images every 30˚: mac1_9_adpr_175_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
mac1_9_adpr_3650	0	720	0.1	7200	0.01	100	185	14
mac1_9_adpr_bg_3651	0	720	1	720	0.1	100	185	14
mac1_9_adpr_3652	0	720	0.1	7200	0.01	100	185	14
mac1_9_adpr_bg_3653	0	720	1	720	0.1	100	185	14

Warning

Beamstop not in place for scans 3650, 3651. After replacing beamstop, the data collection was repeated (scans 3652, 3653) at a different (nominally undamaged) spot on the crystal.

xia2 processing (first 360 degrees)

	mac1_9_adpr_3652
Mosaic spread	0.021
Resolution	1.15
Unit Cell	[88.33, 88.33, 39.83, 90.0, 90.0, 90.0]
Image range	[1, 3600]
Completeness	98.3
Multiplicity	12.5
I/sigma	10.3
Rpim	0.034
Wilson B factor	12.66
Space group	P 43

18. ATCase (R)

Steve looped several R-state crystals from well B6 of Sarah's new tray (R-state with PALA, no nucleotides) in a 50 µm loop. The goal here is just to check for diffraction.

Subdirectory: R_ATCase/ratc_1

prefix	φ0 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
ratc_1_3658	0	0.1	1	1	100	300	14
ratc_1_3659	0	0.1	1	1	100	300	14

Crystals were not visible in the centering camera. Took snapshots at two locations on the loop.

There were powder-like rings at 3.03 Å and 1.9 Å. Are these salt crystals? Or is the loop dirty?

19. ATCase (R)

Steve scooped up a whole mess of crystals from the same drop using a micromesh and blotted from the back with a kimwipe.

Subdirectory: R_ATCase/ratc_2

Took snapshots to check for diffraction. Each snap is a different crystal.

prefix	φ0 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
ratc_2_3660	0	0.1	1	1	100	300	14
ratc_2_3661	335	0.1	1	1	100	300	14
ratc_2_3662	335	0.1	1	1	100	300	14
ratc_2_3663	335	0.1	1	1	100	300	14

Warning

Realized that the φ0 angle was set incorrectly: given the vertical orientation of the centering camera, I should subtract 90 degrees.

More snapshots:

prefix	φ0 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
ratc_2_3664	245	0.1	1	1	100	300	14

Decided to set up a grid scan: 6 x 8 steps, 1s per point, 0.5 degree rotation. Step pitch is 157 x 149 µm

prefix	φ0 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
ratc_2_3665	245	0.5	1	1	100	300	14

Observed some hits, mostly at ~8 Å resolution.

Question

Did something go wrong with the grid scan program? Several of the shots contained diffraction from the metal pin. Needs investigation / testing

Taking a few more manual snapshots

prefix	φ0 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
ratc_2_3666	245	0.5	1	1	100	300	14
ratc_2_3667	245	0.5	1	1	100	300	14
ratc_2_3668	245	0.5	1	1	100	300	14
ratc_2_3669	245	0.5	1	1	100	300	14
ratc_2_3670	245	0.5	1	1	100	300	14
ratc_2_3671	245	0.5	1	1	100	300	14
ratc_2_3672	245	0.5	1	1	100	300	14

Some spots visible to ~4 Å resolution.

Saved an image of the loop: ratc_2_335_tvc_zoom2_1.png

Done for the night.

Tomorrow continue with Mac1 + ADPr, ATCase, and DNA

Data collection, day 2

November 6, 2025.

Kara, Steve, Joseph, & Justin arrived in the morning.

Here's the plan:

• First, collect another Mac1+ADPr dataset at 45 minutes soak time to repeat the earlier ~100% occupancy dataset.
• Next, collect some DNA data from the new tray. Goal is to improve S/N and background subtraction by using larger crystals.
• Collect more crystals of ATCase (RAG) to get a complete (hopefully undamaged) map with good enough S/N for processing.

20. Mac1 + ADPr

Kara looped another mac1, and soaked for 45 minutes in a drop containing 9 µL of reservoir solution + 1 µL of ADPr (200 mM).

Subdirectory: mac1/mac1_10_adpr

Saved images every 30˚: mac1_10_adpr_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
mac1_10_adpr_3673	0	720	0.1	7200	0.01	100	185	14
mac1_10_adpr_bg_3674	0	720	1	720	0.1	100	185	14

xia2 processing

	mac1_10_adpr_3673
Mosaic spread	0.026
Resolution	0.0
Unit Cell	[88.38, 88.38, 39.83, 90.0, 90.0, 90.0]
Image range	[1, 7200]
Completeness	82.9
Multiplicity	22.2
I/sigma	23.2
Rpim	0.012
Wilson B factor	12.91
Space group	P 43

21. DNA

Steve harvested a large DNA crystal from well B6 of tray #2 using a 200 µm loop.

Subdirectory: dna/dna_1

Saved images every 30˚: dna_1_75_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
dna_1_3675	0	360	0.1	3600	0.01	28.6	450	14
dna_1_3676	220	230	0.1	100	0.1	100	450	14
dna_1_bg_3677	220	230	0.1	100	0.1	100	450	14
dna_1_bg_3678	0	360	1	360	0.1	28.6	450	14

Datasets 3675 and 3676 are collected from different spots on the crystal.

Warning

The first time I processed this using xia2, the moasicity blew up and it got stuck in integration. Possibly the crystal slipped or cracked during data collection? I repeated processing just on the first 15 degrees. Need to revisit this more carefully.

xia2 processing (first 15 degrees)

	dna_1_3675
Mosaic spread	0.022
Resolution	4.33
Unit Cell	[107.34, 107.34, 92.78, 90.0, 90.0, 120.0]
Image range	[1, 150]
Completeness	42.6
Multiplicity	1.9
I/sigma	2.6
Rpim	0.105
Wilson B factor	248.0
Space group	R 3 2

22. DNA

Steve looped another, smaller crystal from the same drop.

Subdirectory: dna/dna_2

Saved images every 30˚: dna_2_75_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
dna_2_3679	0	360	0.1	3600	0.01	28.6	450	14
dna_2_3680	70	80	0.1	100	0.1	100	450	14
dna_2_bg_3681	70	80	0.1	100	0.1	100	450	14
dna_2_bg_3682	0	360	1	360	0.1	28.6	450	14

Datasets 3679 and 3680 are collected from the same spot

xia2 processing

	dna_2_3679
Mosaic spread	0.034
Resolution	4.0
Unit Cell	[107.2, 107.2, 93.44, 90.0, 90.0, 120.0]
Image range	[1, 3600]
Completeness	100.0
Multiplicity	19.7
I/sigma	2.2
Rpim	0.047
Wilson B factor	182.9
Space group	R 3 2

23. DNA

Looped another from the same drop.

Subdirectory: dna/dna_3

Saved images every 30˚: dna_3_25_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
dna_3_3683	0	360	0.1	3600	0.01	28.6	450	14
dna_3_3684	200	210	0.1	100	0.1	100	450	14
dna_3_bg_3685	200	210	0.1	100	0.1	100	450	14
dna_3_bg_3686	0	360	1	360	0.1	28.6	450	14

Datasets 3683 and 3684 are collected from the same spot

xia2 processing

	dna_3_3683
Mosaic spread	0.031
Resolution	4.66
Unit Cell	[107.23, 107.23, 93.1, 90.0, 90.0, 120.0]
Image range	[1, 3600]
Completeness	100.0
Multiplicity	19.4
I/sigma	5.9
Rpim	0.035
Wilson B factor	307.34
Space group	R 3 2

24. DNA

Looped the last crystal present in the drop. Used a smaller, 150 µm loop.

Subdirectory: dna/dna_4

Saved images every 30˚: dna_4_55_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
dna_4_3687	0	360	0.1	3600	0.01	28.6	450	14
dna_4_3688	50	60	0.1	100	0.1	100	450	14
dna_4_bg_3689	50	60	0.1	100	0.1	100	450	14
dna_4_bg_3690	0	360	1	360	0.1	28.6	450	14

Datasets 3687 and 3688 are collected from the same spot

xia2 processing

	dna_4_3687
Mosaic spread	0.036
Resolution	4.03
Unit Cell	[107.2, 107.2, 93.48, 90.0, 90.0, 120.0]
Image range	[1, 3600]
Completeness	100.0
Multiplicity	19.7
I/sigma	2.9
Rpim	0.05
Wilson B factor	180.65
Space group	R 3 2

25. DNA

Steve harvested a large crystal from well A6 of DNA tray #2, 200 µm loop.

Subdirectory: dna/dna_5

Saved images every 30˚: dna_5_0_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
dna_5_3691	0	360	0.1	3600	0.01	28.6	450	14
dna_5_3692	0	10	0.1	100	0.1	100	450	14
dna_5_bg_3693	0	10	0.1	100	0.1	100	450	14
dna_5_bg_3694	0	360	1	360	0.1	28.6	450	14

xia2 processing

	dna_5_3691
Mosaic spread	0.045
Resolution	4.02
Unit Cell	[107.28, 107.28, 93.04, 90.0, 90.0, 120.0]
Image range	[1, 3600]
Completeness	100.0
Multiplicity	19.8
I/sigma	2.1
Rpim	0.044
Wilson B factor	179.84
Space group	R 3 2

26. DNA

Harvested the large crystal from well A6, 200 µm loop.

Subdirectory: dna/dna_6

Saved images every 30˚: dna_6_160_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
dna_6_3695	0	360	0.1	3600	0.01	28.6	450	14
dna_6_3696	160	170	0.1	100	0.1	100	450	14
dna_6_bg_3697	160	170	0.1	100	0.1	100	450	14
dna_6_bg_3698	0	360	1	360	0.1	28.6	450	14

xia2 processing

	dna_6_3695
Mosaic spread	0.038
Resolution	4.02
Unit Cell	[107.21, 107.21, 93.01, 90.0, 90.0, 120.0]
Image range	[1, 3600]
Completeness	100.0
Multiplicity	19.5
I/sigma	4.0
Rpim	0.036
Wilson B factor	216.61
Space group	R 3 2

27. ATCase (RAG)

Steve looped a long ATCase (RAG) crystal from Sarah's tray RAG #2 (dated 10/7/2025) well B6. A 200 µm loop was used, but the crystal is long, and hit hangs off the end. Strategy is to use a vector scan, and then filter frame ranges by diffraction quality if needed.

Subdirectory: RAG_ATCase

Saved images every 30˚: rag_3_40_30_tvc_zoom4_{01..12}.png

sample	prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	∆x (µm)	E (keV)
rag_3	rag_3_3699	0	720	0.1	7200	0.01	100	300	630.15	14
rag_3	rag_3_bg_3700	0	720	1	720	0.1	100	300	nan	14

Warning

After initial processing with xia2, observed that the R-factor explodes after frame ~4500. Should be reprocessed using a truncated frame range.

xia2 processing

	rag_3_3699
Mosaic spread	0.073
Resolution	3.01
Unit Cell	[126.72, 151.88, 206.09, 90.0, 90.0, 90.0]
Image range	[1, 7200]
Completeness	100.0
Multiplicity	27.1
I/sigma	7.5
Rpim	0.443
Wilson B factor	82.22
Space group	P 21 21 21

28. ATCase (RAG)

Steve looped another long RAG crystal from well C1 (same tray). This one is oriented vertically on the loop, so vector scan is not effective. Lets try low dose instead (use attenuation).

Subdirectory: RAG_ATCase

Saved images every 30˚: rag_4_220_30_tvc_zoom4_{01..12}.png

sample	prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
rag_4	rag_4_3701	150	510	0.1	3600	0.01	28.6	300	14
rag_4	rag_4_bg_3702	150	510	1	360	0.1	28.6	300	14

xia2 processing

	rag_4_3701
Mosaic spread	0.032
Resolution	3.19
Unit Cell	[126.64, 152.24, 206.22, 90.0, 90.0, 90.0]
Image range	[1, 3600]
Completeness	100.0
Multiplicity	14.0
I/sigma	4.0
Rpim	0.116
Wilson B factor	84.67
Space group	P 21 21 21

29. ATCase (RAG)

Steve opened well C2 of Sarah's RAG tray #2. The right-hand drop has ~5 large single-looking crystals. Katie is attempting to fish them.

Katie looped a crystal from well C2 using a 200 µm loop.

Subdirectory: RAG_ATCase

sample	prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
rag_5	rag_5_3703	0	360	0.1	3600	0.01	28.6	300	14

Warning

no diffraction

30. ATCase (RAG)

Katie looped another from well C2

Subdirectory: RAG_ATCase

Saved images every 30˚: rag_6_55_30_tvc_zoom4_{01..12}.png

sample	prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
rag_6	rag_6_3704	0	360	0.1	3600	0.01	28.6	300	14
rag_6	rag_6_bg_3705	0	360	1	360	0.1	28.6	300	14

Warning

poor diffraction (see warning below)

31. ATCase (RAG)

Katie looped another from well C2

Subdirectory: RAG_ATCase

Saved images every 30˚: rag_7_225_30_tvc_zoom4_{01..12}.png

prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
rag_7_3706	0	360	1	360	0.1	100	300	14

Warning

Katie noticed that the drop is staring to dry out. It turned out that the humid box had dropped to 60 % RH --> needed a refresh. This could explain why the last several datasets had poor diffraction.

32. ATCase (RAG)

Refreshed the humid chamber, waited until it reached 100%, then opened well C3. Katie looped a crystal and used a fresh sleeve with 10 µL of well solution.

Subdirectory: RAG_ATCase

sample	prefix	φ0 (deg.)	φ1 (deg.)	∆φ (deg.)	images	∆t (s)	tf (%)	d (mm)	E (keV)
rag_8	rag_8_3707	0	360	0.1	3600	0.01	28.6	300	14
rag_8	rag_8_3708	120	130	0.1	100	0.1	100	300	14
rag_8	rag_8_bg_3709	120	130	0.1	100	0.1	100	300	14
rag_8	rag_8_bg_3710	0	360	1	360	0.1	28.6	300	14

xia2 processing

	rag_8_3707
Mosaic spread	0.041
Resolution	3.45
Unit Cell	[129.64, 148.25, 209.35, 90.0, 90.0, 90.0]
Image range	[1, 3600]
Completeness	100.0
Multiplicity	14.0
I/sigma	2.0
Rpim	0.193
Wilson B factor	63.03
Space group	P 21 21 21

Done

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